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Gatta E, Abd El E, Brunoldi M, Irfan M, Isolabella T, Massabò D, Parodi F, Prati P, Vernocchi V, Mazzei F. Viability studies of bacterial strains exposed to nitrogen oxides and light in controlled atmospheric conditions. Sci Rep 2025; 15:10320. [PMID: 40133562 PMCID: PMC11937341 DOI: 10.1038/s41598-025-94898-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2024] [Accepted: 03/18/2025] [Indexed: 03/27/2025] Open
Abstract
Airborne biological particles, such as pollen, fungi, bacteria, viruses, and plant or animal detritus, are known as bioaerosols. Understanding bioaerosols' behavior, especially their reaction to pollutants and atmospheric conditions, is crucial for addressing environmental and health issues related to air quality. Such complex investigations can benefit from experiments in controlled but realistic environments, such as the Atmospheric Simulation Chamber facility ChAMBRe (Chamber for Aerosol Modeling and Bio-aerosol Research). In this work, we report on the results of several experiments that were conducted at ChAMBRe using three strains of bacteria: E. coli, B. subtilis, and P. fluorescens. The goal of these experiments was to quantitively study how the culturability of these bacteria is affected by exposure to NO, NO2, and light. The experimental approach was simple but carefully controlled: before being introduced into ChAMBRe, the bacteria samples were characterized using three different methods to determine the ratio of viable to total bacteria. The bacteria suspension was then aerosolized and introduced into ChAMBRe, where it was exposed to two different concentrations of NO and NO2, in dark conditions and with simulated solar radiation. The culturability of the bacteria was assessed by collecting bacteria samples directly onto Petri dishes by an Andersen impactor at various time intervals after the end of injection. Finally, the formed bacteria colonies were counted after 24-48 h of incubation to measure their culturability and the temporal trend. The results show a reduction of culturability for all bacteria strains when exposed to NO2 (from 50 to 70%) and to high concentrations of NO (i.e. around 30% at more than 1200 ppb) at concentration values higher than the typical urban ambient values. Even higher effects were observed exposing the bacteria strain to a proxy of solar light. The findings show how atmospheric simulation chambers help the comprehension of interactions between pollutants and bioaerosols in controlled atmospheric environments.
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Affiliation(s)
- Elena Gatta
- Dipartimento di Fisica, Università di Genova, Via Dodecaneso 33, 16143, Genoa, Italy
| | - Elena Abd El
- Dipartimento di Fisica, Università di Genova, Via Dodecaneso 33, 16143, Genoa, Italy
- INFN - Sezione di Genova, Via Dodecaneso 33, 16143, Genoa, Italy
| | - Marco Brunoldi
- Dipartimento di Fisica, Università di Genova, Via Dodecaneso 33, 16143, Genoa, Italy
- INFN - Sezione di Genova, Via Dodecaneso 33, 16143, Genoa, Italy
| | - Muhammad Irfan
- Dipartimento di Fisica, Università di Genova, Via Dodecaneso 33, 16143, Genoa, Italy
| | - Tommaso Isolabella
- Dipartimento di Fisica, Università di Genova, Via Dodecaneso 33, 16143, Genoa, Italy
- INFN - Sezione di Genova, Via Dodecaneso 33, 16143, Genoa, Italy
| | - Dario Massabò
- Dipartimento di Fisica, Università di Genova, Via Dodecaneso 33, 16143, Genoa, Italy
- INFN - Sezione di Genova, Via Dodecaneso 33, 16143, Genoa, Italy
| | - Franco Parodi
- INFN - Sezione di Genova, Via Dodecaneso 33, 16143, Genoa, Italy
| | - Paolo Prati
- Dipartimento di Fisica, Università di Genova, Via Dodecaneso 33, 16143, Genoa, Italy
- INFN - Sezione di Genova, Via Dodecaneso 33, 16143, Genoa, Italy
| | | | - Federico Mazzei
- Dipartimento di Fisica, Università di Genova, Via Dodecaneso 33, 16143, Genoa, Italy
- INFN - Sezione di Genova, Via Dodecaneso 33, 16143, Genoa, Italy
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Nammoura Neto GM, Schneider RP. Variation of gene ratios in mock communities constructed with purified 16S rRNA during processing. Sci Rep 2024; 14:31577. [PMID: 39738093 PMCID: PMC11686170 DOI: 10.1038/s41598-024-61614-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2022] [Accepted: 05/07/2024] [Indexed: 01/01/2025] Open
Abstract
16S ribosomal nucleic acid (16S rRNA) analysis allows to specifically target the metabolically active members of microbial communities. The stability of the ratios between target genes in the workflow, which is essential for the bioprocess-relevance of the data derived from this analysis, was investigated using synthetic mock communities constructed by mixing purified 16S rRNA from Bacillus subtilis (Bs), Staphylococcus aureus (Sa), Pseudomonas aeruginosa (Pa), Klebsiella pneumoniae (Kp) and Burkholderia cepacia (Bc) in different proportions. The RT reaction yielded one copy of cDNA per rRNA molecule for Pa, Bc and Sa but only 2/3 of the expected cDNA from 16S rRNAs of Bs and Kp. The combination of Taq DNA Platinum polymerase with subcycling PCR (scPCR) produced uniform yields of approximately 70% for second strand PCR synthesis from all target cDNAs. The proportion between templates in multicycle PCR was best preserved after 10 cycle scPCR followed by cloning. With MiSeq sequencing, correct proportions for about two thirds of templates were recovered after 10 cycle scPCR with Taq Platinum. 30 cycles standard PCR (stdPCR) or scPCR proved particularly harmful to proportion data and should be avoided.
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Affiliation(s)
- Georges Mikhael Nammoura Neto
- Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, Av. Professor Lineu Prestes, 1374, São Paulo, 05508-900, Brazil
| | - René Peter Schneider
- Department of Chemical Engineering, Polytechnic School, University of São Paulo, Av. Prof. Luciano Gualberto, Travessa 3, n. 380., São Paulo, SP, CEP 05508-900, Brazil.
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Lopes KF, Freire ML, Murta SMF, Oliveira E. Efficacy of vaccines based on chimeric or multiepitope antigens for protection against visceral leishmaniasis: A systematic review. PLoS Negl Trop Dis 2024; 18:e0012757. [PMID: 39739955 PMCID: PMC11753665 DOI: 10.1371/journal.pntd.0012757] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2024] [Revised: 01/22/2025] [Accepted: 12/04/2024] [Indexed: 01/02/2025] Open
Abstract
BACKGROUND Visceral leishmaniasis (VL) is an infectious parasitic disease caused by the species Leishmania (Leishmania) infantum in the Mediterranean Basin, the Middle East, Central Asia, South America, and Central America, and Leishmania (Leishmania) donovani in Asia and Africa. VL represents the most severe and systemic form of the disease and is fatal if left untreated. Vaccines based on chimeric or multiepitope antigens hold significant potential to induce a highly effective and long-lasting immune response against infections by these parasites. This review systematically compiles data on the efficacy and protective capabilities of chimeric and multiepitope antigens, while also identifying potential immunogenic targets for vaccine development. METHODOLOGY A systematic search was conducted by independent reviewers across four databases to assess the efficacy of vaccines based on chimeric or multiepitope antigens against VL. The review included original studies that reported parasite load or positivity rates in animals immunized with these vaccines and subsequently challenged or exposed to L. infantum infection in preclinical and clinical studies. Key information was extracted, tabulated, and analyzed, with the risk of bias being assessed using the SYRCLE Risk Tool. PRINCIPAL FINDINGS A total of 22 studies were selected, with only one being a randomized clinical trial. Most of the studies were conducted with mice, followed by dogs and hamsters. The reduction in parasite load varied from 14% to 99.6% and from 1.7 to 9.0 log orders. Limiting dilution was the most used method for assessing parasite load, followed by quantitative real-time polymerase chain reaction (qPCR). Most domains had an uncertain risk of bias due to insufficient information described. CONCLUSIONS Vaccine formulations containing various chimeric or multiepitope antigens have been developed and evaluated in different preclinical trials, with only one advancing to clinical trials and commercialization. However, the findings of this review highlight the promising potential of chimeric and multiepitope antigens as vaccine candidates against VL. The evidence presented could play a crucial role in guiding the rational development of new studies focused on using these antigens for vaccination against VL.
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Affiliation(s)
- Karine Ferreira Lopes
- Genômica Funcional de Parasitos, Instituto René Rachou–Oswaldo Cruz Foundation, Belo Horizonte, Minas Gerais, Brazil
| | - Mariana Lourenço Freire
- Pesquisa Clínica e Políticas Públicas em Doenças Infecciosas e Parasitárias, Instituto René Rachou—Oswaldo Cruz Foundation, Belo Horizonte, Minas Gerais, Brazil
| | - Silvane Maria Fonseca Murta
- Genômica Funcional de Parasitos, Instituto René Rachou–Oswaldo Cruz Foundation, Belo Horizonte, Minas Gerais, Brazil
| | - Edward Oliveira
- Genômica Funcional de Parasitos, Instituto René Rachou–Oswaldo Cruz Foundation, Belo Horizonte, Minas Gerais, Brazil
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Taye B, Melkamu R, Tajebe F, Ibarra-Meneses AV, Adane D, Atnafu S, Adem M, Adane G, Kassa M, Asres MS, van Griensven J, van Henten S, Pareyn M. Evaluation of Loopamp Leishmania detection kit for the diagnosis of cutaneous leishmaniasis in Ethiopia. Parasit Vectors 2024; 17:431. [PMID: 39407317 PMCID: PMC11481786 DOI: 10.1186/s13071-024-06475-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2024] [Accepted: 09/03/2024] [Indexed: 10/19/2024] Open
Abstract
BACKGROUND Cutaneous leishmaniasis (CL) in Ethiopia and some parts of Kenya is predominantly caused by Leishmania aethiopica. While skin-slit (SS) microscopy is routinely used for CL diagnosis, more sensitive molecular tests are available. The Loopamp™ Leishmania detection kit (Loopamp) is a robust loop-mediated isothermal amplification (LAMP) assay with the potential for implementation in primary healthcare facilities. In this study, we comparatively assessed the diagnostic accuracy of four methods currently used to diagnose CL: Loopamp, kinetoplast DNA (kDNA) PCR, spliced leader RNA (SL-RNA) PCR and SS microscopy. METHODS A study on 122 stored tape disc samples of suspected CL patients was conducted in Gondar, northwestern Ethiopia. Routine SS microscopy results were obtained from all patients. Total nucleic acids were extracted from the tapes and subjected to PCR testing targeting kDNA and SL-RNA, and Loopamp. Diagnostic accuracy was calculated with SS microscopy as a reference test. The limit of detection (LoD) of Loopamp and kDNA PCR were determined for cultured L. aethiopica and Leishmania donovani. RESULTS Of the 122 patients, 64 (52.5%) were identified as CL cases based on SS microscopy. Although the PCR tests showed a sensitivity of 95.3% (95% confidence interval [CI] 91.6-99.1), Loopamp only had 48.4% (95% CI 39.6-57.3) sensitivity and 87.9% (95% CI 82.1-93.7) specificity. The LoD of Loopamp for L. donovani was 100-fold lower (20 fg/µl) than that for L. aethiopica (2 pg/µl). CONCLUSIONS The Loopamp™ Leishmania detection kit is not suitable for the diagnosis of CL in Ethiopia, presumably due to a primer mismatch with the L. aethiopica 18S rRNA target. Further research is needed to develop a simple and sensitive point-of-care test that allows the decentralization of CL diagnosis in Ethiopia.
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Affiliation(s)
- Behailu Taye
- Department of Immunology and Molecular Biology, University of Gondar, Gondar, Ethiopia.
- Department of Medical Laboratory Science, Dilla University, Dilla, Ethiopia.
| | - Roma Melkamu
- Leishmaniasis Research and Treatment Center, University of Gondar, Gondar, Ethiopia
| | - Fitsumbrhan Tajebe
- Department of Immunology and Molecular Biology, University of Gondar, Gondar, Ethiopia
| | - Ana Victoria Ibarra-Meneses
- Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada
| | - Desalegn Adane
- Leishmaniasis Research and Treatment Center, University of Gondar, Gondar, Ethiopia
| | - Saba Atnafu
- Leishmaniasis Research and Treatment Center, University of Gondar, Gondar, Ethiopia
| | - Mohammed Adem
- Department of Immunology and Molecular Biology, University of Gondar, Gondar, Ethiopia
| | - Gashaw Adane
- Department of Immunology and Molecular Biology, University of Gondar, Gondar, Ethiopia
| | - Mekibib Kassa
- Leishmaniasis Research and Treatment Center, University of Gondar, Gondar, Ethiopia
| | | | - Johan van Griensven
- Department of Clinical Sciences, Institute of Tropical Medicine, Antwerp, Belgium
| | - Saskia van Henten
- Department of Clinical Sciences, Institute of Tropical Medicine, Antwerp, Belgium
| | - Myrthe Pareyn
- Department of Clinical Sciences, Institute of Tropical Medicine, Antwerp, Belgium
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Mahmoudi S, García MJ, Drain PK. Current approaches for diagnosis of subclinical pulmonary tuberculosis, clinical implications and future perspectives: a scoping review. Expert Rev Clin Immunol 2024; 20:715-726. [PMID: 38879875 DOI: 10.1080/1744666x.2024.2326032] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2023] [Accepted: 02/28/2024] [Indexed: 06/18/2024]
Abstract
INTRODUCTION Subclinical tuberculosis (TB) is the presence of TB disease among people who are either asymptomatic or have minimal symptoms. AREAS COVERED Currently, there are no accurate diagnostic tools and clear treatment approaches for subclinical TB. In this study, a comprehensive literature search was conducted across major databases. This review aimed to uncover the latest advancements in diagnostic approaches, explore their clinical implications, and outline potential future perspectives. While innovative technologies are in development to enable sputum-free TB tests, there remains a critical need for precise diagnostic tools tailored to the unique characteristics of subclinical TB. Given the complexity of subclinical TB, a multidisciplinary approach involving clinicians, microbiologists, epidemiologists, and public health experts is essential. Further research is needed to establish standardized diagnostic criteria and treatment guidelines specifically tailored for subclinical TB, acknowledging the unique challenges posed by this elusive stage of the disease. EXPERT OPINION Efforts are needed for the detection, diagnosis, and treatment of subclinical TB. In this review, we describe the importance of subclinical TB, both from a clinical and public health perspective and highlight the diagnostic and treatment gaps of this stage.
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Affiliation(s)
- Shima Mahmoudi
- Biotechnology Centre, Silesian University of Technology, Gliwice, Poland
| | - Maria J García
- Department of Preventive Medicine and Public Health and Microbiology, Autonoma University of Madrid, Madrid, Spain
| | - Paul K Drain
- International Clinical Research Center, Department of Global Health, Schools of Medicine and Public Health, University of Washington, Seattle, WA, USA
- Department of Epidemiology, School of Public Health, University of Washington, Seattle, WA, USA
- Division of Allergy and Infectious Diseases, Department of Medicine, School of Medicine, University of Washington, Seattle, WA, USA
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Faier-Pereira A, Finamore-Araujo P, Brito CRDN, Peres EG, de Lima Yamaguchi KK, de Castro DP, Moreira OC. The Development of a One-Step RT-qPCR for the Detection and Quantification of Viable Forms of Trypanosoma cruzi in Açai Samples from Areas at Risk of Chagas Disease through Oral Transmission. Int J Mol Sci 2024; 25:5531. [PMID: 38791565 PMCID: PMC11122307 DOI: 10.3390/ijms25105531] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2024] [Revised: 05/06/2024] [Accepted: 05/10/2024] [Indexed: 05/26/2024] Open
Abstract
Currently, approximately 70% of new cases of Chagas disease (CD) in Brazil are attributed to oral transmission, particularly through foods such as açaí, bacaba, and sugarcane juice, primarily in the northern and northeastern regions of the country. This underscores the imperative need to control the spread of the disease. The methods utilized to conduct quality control for food associated with outbreaks and to assess the potential for the oral transmission of CD through consuming açaí primarily rely on isolating the parasite or inoculating food into experimental animals, restricting the analyses to major research centers. While there are existing studies in the literature on the detection and quantification of T. cruzi DNA in açaí, the evaluation of parasites' viability using molecular methods in this type of sample and differentiating between live and dead parasites in açaí pulp remain challenging. Consequently, we developed a molecular methodology based on RT-qPCR for detecting and quantifying viable T. cruzi in açaí pulp samples. This protocol enables the stabilization and preservation of nucleic acids in açaí, along with incorporating an exogenous internal amplification control. The standardization of the RNA extraction method involved a simple and reproducible approach, coupled with a one-step RT-qPCR assay. The assay underwent validation with various T. cruzi DTUs and demonstrated sensitivity in detecting up to 0.1 viable parasite equivalents/mL in açaí samples. Furthermore, we investigated the effectiveness of a bleaching method in eliminating viable parasites in açaí samples contaminated with T. cruzi by comparing the detection of DNA versus RNA. Finally, we validated this methodology using açaí pulp samples positive for T. cruzi DNA, which were collected in a municipality with a history of oral CD outbreaks (Coari-AM). This validation involved comparing the detection and quantification of total versus viable T. cruzi. Collectively, our findings demonstrate the feasibility of this methodology in detecting viable forms of T. cruzi in açaí pulp samples, emerging as a crucial tool for monitoring oral outbreaks of Chagas disease resulting from açaí consumption.
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Affiliation(s)
- Amanda Faier-Pereira
- Laboratory of Molecular Virology and Parasitology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro 21040-360, Brazil
| | - Paula Finamore-Araujo
- Laboratory of Molecular Virology and Parasitology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro 21040-360, Brazil
| | | | - Eldrinei Gomes Peres
- Departament of Chemistry, Federal University of Amazonas, Manaus 69067-005, Brazil
| | | | - Daniele Pereira de Castro
- Laboratory of Biochemistry and Physiology of Insects, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro 21040-360, Brazil
| | - Otacilio C. Moreira
- Laboratory of Molecular Virology and Parasitology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro 21040-360, Brazil
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Wang Y, Zheng T, Li X, Wu P. Integrating Recombinase Polymerase Amplification and Photosensitization Colorimetric Detection in One Tube for Fast Screening of C. sakazakii in Formula Milk Powder. Anal Chem 2024; 96:5727-5733. [PMID: 38546834 DOI: 10.1021/acs.analchem.4c01130] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/10/2024]
Abstract
Cronobacter sakazakii (C. sakazakii) is a widely existing opportunistic pathogen and thus threatens people with low immunity, especially infants. To prevent the outbreak, a rapid and accurate on-site testing method is required. The current standard culture-based method is time-consuming (3-4 days), while the nucleic acid amplification (PCR)-based detection is mostly carried out in central laboratories. Herein, isothermal recombinase polymerase amplification (RPA) coupled with a photosensitization colorimetric assay (PCA) was adopted for the on-site detection of C. sakazakii in powdered infant formulas (PIFs). The lowest visual detection concentration of C. sakazakii is 800 cfu/mL and 2 cfu/g after 8 h bacteria pre-enrichment. Furthermore, to avoid typical cap opening-resulted aerosol pollution, the PCA reagents were lyophilized onto the cap of the RPA tube (containing lyophilized RPA reagents). After amplification, the tube was subjected to simple shaking to mix the PCA reagents with the amplification products for light-driven color development. Such a one-tube assay offered a lowest concentration of 1000 copies of genomic DNA of C. sakazakii within 1 h. After 8 h of bacterial enrichment, the lowest detecting concentration could be pushed down to 5 cfu/g bacteria in PIF. To facilitate on-site monitoring, a portable, battery-powered PCA device was designed to mount the typical RPA 8-tube strip, and a color analysis cellphone APP was further employed for facile readout.
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Affiliation(s)
- Yanying Wang
- Analytical & Testing Center, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064, China
| | - Ting Zheng
- Analytical & Testing Center, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064, China
| | - Xianming Li
- Department of Rheumatology and Immunology, West China Hospital, Sichuan University, Chengdu 610041, China
| | - Peng Wu
- Analytical & Testing Center, State Key Laboratory of Hydraulics and Mountain River Engineering, Sichuan University, Chengdu 610064, China
- School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang 453007, China
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Mobed A, Darvishi M, Kohansal F, Dehfooli FM, Alipourfard I, Tahavvori A, Ghazi F. Biosensors; nanomaterial-based methods in diagnosing of Mycobacterium tuberculosis. J Clin Tuberc Other Mycobact Dis 2024; 34:100412. [PMID: 38222862 PMCID: PMC10787265 DOI: 10.1016/j.jctube.2023.100412] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2024] Open
Abstract
Diagnosis of Mycobacterium tuberculosis (Mtb) before the progression of pulmonary infection can be very effective in its early treatment. The Mtb grows so slowly that it takes about 6-8 weeks to be diagnosed even using sensitive cell culture methods. The main opponent in tuberculosis (TB) and nontuberculous mycobacterial (NTM) epidemiology, like in all contagious diseases, is to pinpoint the source of infection and reveal its transmission and dispersion ways in the environment. It is crucial to be able to distinguish and monitor specific mycobacterium strains in order to do this. In food analysis, clinical diagnosis, environmental monitoring, and bioprocess, biosensing technologies have been improved to manage and detect Mtb. Biosensors are progressively being considered pioneering tools for point-of-care diagnostics in Mtb discoveries. In this review, we present an epitome of recent developments of biosensing technologies for M. tuberculosis detection, which are categorized on the basis of types of electrochemical, Fluorescent, Photo-thermal, Lateral Flow, Magneto-resistive, Laser, Plasmonic, and Optic biosensors.
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Affiliation(s)
- Ahmad Mobed
- Infectious and Tropical Diseases Research Center, Clinical Research Institute, Tabriz University of Medical Sciences, Iran
| | - Mohammad Darvishi
- Infectious Diseases and Tropical Medicine Research Center (IDTMRC), Department of Aerospace and Subaquatic Medicine, AJA University of Medical Sciences, Tehran, Iran
| | - Fereshteh Kohansal
- Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | | | - Iraj Alipourfard
- Institute of Medical Science and Technology, Tehran University of Medical Sciences, Tehran, Iran
| | - Amir Tahavvori
- Internal Department, Medical Faculty, Urmia University of Medical Sciences, Iran
| | - Farhood Ghazi
- Internal Department, Medical Faculty, Urmia University of Medical Sciences, Iran
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Ahlat M, Aydin C, Kaya S, Baysallar M. Identification of root canal microbiota profiles of periapical tissue diseases using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anaerobe 2023; 84:102791. [PMID: 37925063 DOI: 10.1016/j.anaerobe.2023.102791] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2023] [Revised: 10/20/2023] [Accepted: 10/23/2023] [Indexed: 11/06/2023]
Abstract
OBJECTIVES The purpose of this study was to identify microorganisms isolated from various periapical tissue diseases using Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) and classify them via an unsupervised machine learning approach. METHODS A total of 150 patients with various apical conditions and teeth in need of endodontic retreatment were divided into five groups, including Retreatment, Acute Apical Abscess, Chronic Apical Abscess, Acute Apical Periodontitis, and Chronic Apical Periodontitis. Samples were collected from root canals using paper points after agitating with a #10 K file then microorganisms were identified using MALDI-TOF-MS. Data were analyzed using a hierarchical clustering method. Quadruple clusters and dendrograms were formed according to similarities and dissimilarities. RESULTS A total of 80 species were identified representative of six different phyla. The most similar microorganism species identified were: ''Enterococcus faecalis'' between 21 and 23-year-old female cases in Retreatment group; ''Lactobacillus rhamnosus'' between 20 and 18-year-old male cases in Symptomatic Apical Abscess cases; ''Lactobacillus paracasei'' between 26 and 40-year-old male cases in Asymptomatic Apical Abscess cases; ''Enterococcus faecalis'' between 48 and 50-year-old female cases in Symptomatic Apical Periodontitis cases; ''Lactobacillus rhamnosus'' between 48 and 60-year-old male cases in Asymptomatic Apical Periodontitis cases. CONCLUSIONS MALDI-TOF MS can be considered a fast and high-throughput screening technique for microbial species identification in endodontics. Thus, it will provide valuable data for future research designs regarding periapical tissue diseases. As the MALDI-TOF MS database expands and comprehensive data becomes available, the relationship between microbial profiles and disease progression will become increasingly apparent.
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Affiliation(s)
- Mete Ahlat
- University of Health Sciences, Gulhane Faculty of Dentistry, Department of Endodontics, Ankara, Turkey.
| | - Cumhur Aydin
- University of Health Sciences, Gulhane Faculty of Dentistry, Department of Endodontics, Ankara, Turkey.
| | - Sinem Kaya
- University of Health Sciences, Gulhane Faculty of Medicine, Department of Medical Microbiology, Ankara, Turkey.
| | - Mehmet Baysallar
- University of Health Sciences, Gulhane Faculty of Medicine, Department of Medical Microbiology, Ankara, Turkey.
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Zmerli O, Bellali S, Haddad G, Hisada A, Ominami Y, Raoult D, Bou Khalil J. Rapid microbial viability assay using scanning electron microscopy: a proof-of-concept using Phosphotungstic acid staining. Comput Struct Biotechnol J 2023; 21:3627-3638. [PMID: 37501704 PMCID: PMC10371768 DOI: 10.1016/j.csbj.2023.07.010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2023] [Revised: 06/27/2023] [Accepted: 07/09/2023] [Indexed: 07/29/2023] Open
Abstract
Multiple stains have been historically utilized in electron microscopy to provide proper contrast and superior image quality enabling the discovery of ultrastructures. However, the use of these stains in microbiological viability assessment has been limited. Phosphotungstic acid (PTA) staining is a common negative stain used in scanning electron microscopy (SEM). Here, we investigate the feasibility of a new SEM-PTA assay, aiming to determine both viable and dead microbes. The optimal sample preparation was established by staining bacteria with different PTA concentrations and incubation times. Once the assay conditions were set, we applied the protocol to various samples, evaluating bacterial viability under different conditions, and comparing SEM-PTA results to culture. The five minutes 10% PTA staining exhibited a strong distinction between viable micro-organisms perceived as hypo-dense, and dead micro-organisms displaying intense internal staining which was confirmed by high Tungsten (W) peak on the EDX spectra. SEM-PTA viability count after freezing, freeze-drying, or oxygen exposure, were concordant with culture. To our knowledge, this study is the first contribution towards PTA staining of live and dead bacteria. The SEM-PTA strategy demonstrated the feasibility of a rapid, cost-effective and efficient viability assay, presenting an open-view of the sample, and providing a potentially valuable tool for applications in microbiome investigations and antimicrobial susceptibility testing.
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Affiliation(s)
- Omar Zmerli
- Institut Hospitalo-Universitaire Méditerranée Infection 19-21 Boulevard Jean Moulin 13005 Marseille, France
- Aix-Marseille Université, Institut de Recherche pour le Développement (IRD), UMR Microbes Evolution Phylogeny and Infections (MEPHI), Marseille, France
| | - Sara Bellali
- Institut Hospitalo-Universitaire Méditerranée Infection 19-21 Boulevard Jean Moulin 13005 Marseille, France
| | - Gabriel Haddad
- Institut Hospitalo-Universitaire Méditerranée Infection 19-21 Boulevard Jean Moulin 13005 Marseille, France
- Aix-Marseille Université, Institut de Recherche pour le Développement (IRD), UMR Microbes Evolution Phylogeny and Infections (MEPHI), Marseille, France
| | - Akiko Hisada
- Hitachi, Ltd. Research & Development Group, 2520, Akanuma, Hatoyama, Saitama, 350- 0395, Japan
| | - Yusuke Ominami
- Hitachi High-Tech Corporation, 882 Ichige, Hitachinaka-shi, Ibaraki-ken 312-8504, Japan
| | | | - Jacques Bou Khalil
- Institut Hospitalo-Universitaire Méditerranée Infection 19-21 Boulevard Jean Moulin 13005 Marseille, France
- Aix-Marseille Université, Institut de Recherche pour le Développement (IRD), UMR Microbes Evolution Phylogeny and Infections (MEPHI), Marseille, France
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11
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Alshammari A, Abdulmawla ST, Alsaigh R, Alarjani KM, Aldosari NS, Muthuramamoorthy M, Assaifan AK, Albrithen H, Alzahrani KE, Alodhayb AN. Toward the Real-Time and Rapid Quantification of Bacterial Cells Utilizing a Quartz Tuning Fork Sensor. MICROMACHINES 2023; 14:1114. [PMID: 37374699 DOI: 10.3390/mi14061114] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/01/2023] [Revised: 05/20/2023] [Accepted: 05/21/2023] [Indexed: 06/29/2023]
Abstract
The quantitative evaluation of bacterial populations is required in many studies, particularly in the field of microbiology. The current techniques can be time-consuming and require a large volume of samples and trained laboratory personnel. In this regard, on-site, easy-to-use, and direct detection techniques are desirable. In this study, a quartz tuning fork (QTF) was investigated for the real-time detection of E. coli in different media, as well as the ability to determine the bacterial state and correlate the QTF parameters to the bacterial concentration. QTFs that are commercially available can also be used as sensitive sensors of viscosity and density by determining the QTFs' damping and resonance frequency. As a result, the influence of viscous biofilm adhered to its surface should be detectable. First, the response of a QTF to different media without E. coli was investigated, and Luria-Bertani broth (LB) growth medium caused the largest change in frequency. Then, the QTF was tested against different concentrations of E. coli (i.e., 102-105 colony-forming units per milliliter (CFU/mL)). As the E. coli concentration increased, the frequency decreased from 32.836 to 32.242 kHz. Similarly, the quality factor decreased with the increasing E. coli concentration. With a coefficient (R) of 0.955, a linear correlation between the QTF parameters and bacterial concentration was established with a 26 CFU/mL detection limit. Furthermore, a considerable change in frequency was observed against live and dead cells in different media. These observations demonstrate the ability of QTFs to distinguish between different bacterial states. QTFs allow real-time, rapid, low-cost, and non-destructive microbial enumeration testing that requires only a small volume of liquid sample.
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Affiliation(s)
- Abeer Alshammari
- Department of Physics and Astronomy, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
| | - Sabaa T Abdulmawla
- Department of Physics and Astronomy, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
| | - Reem Alsaigh
- Department of Physics and Astronomy, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
| | - Khaloud Mohammed Alarjani
- Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
| | - Norah Salim Aldosari
- Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
| | | | - Abdulaziz K Assaifan
- Department of Biomedical Technology, College of Applied Medical Sciences, King Saud University, Riyadh 11451, Saudi Arabia
| | - Hamad Albrithen
- Department of Physics and Astronomy, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
| | - Khalid E Alzahrani
- Department of Physics and Astronomy, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
| | - Abdullah N Alodhayb
- Department of Physics and Astronomy, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
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12
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Verschoor YL, Vrijlandt A, Spijker R, van Hest RM, ter Hofstede H, van Kempen K, Henningsson AJ, Hovius JW. Persistent Borrelia burgdorferi Sensu Lato Infection after Antibiotic Treatment: Systematic Overview and Appraisal of the Current Evidence from Experimental Animal Models. Clin Microbiol Rev 2022; 35:e0007422. [PMID: 36222707 PMCID: PMC9769629 DOI: 10.1128/cmr.00074-22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
Lyme borreliosis is caused by spirochetes belonging to the Borrelia burgdorferi sensu lato group, which are transmitted by Ixodes tick species living in the temperate climate zones of the Northern Hemisphere. The clinical manifestations of Lyme borreliosis are diverse and treated with oral or intravenous antibiotics. In some patients, long-lasting and debilitating symptoms can persist after the recommended antibiotic treatment. The etiology of such persisting symptoms is under debate, and one hypothesis entails persistent infection by a subset of spirochetes after antibiotic therapy. Here, we review and appraise the experimental evidence from in vivo animal studies on the persistence of B. burgdorferi sensu lato infection after antibiotic treatment, focusing on the antimicrobial agents doxycycline and ceftriaxone. Our review indicates that some in vivo animal studies found sporadic positive cultures after antibiotic treatment. However, this culture positivity often seemed to be related to inadequate antibiotic treatment, and the few positive cultures in some studies could not be reproduced in other studies. Overall, current results from animal studies provide insufficient evidence for the persistence of viable and infectious spirochetes after adequate antibiotic treatment. Borrelial nucleic acids, on the contrary, were frequently detected in these animal studies and may thus persist after antibiotic treatment. We put forward that research into the pathogenesis of persisting complaints after antibiotic treatment for Lyme borreliosis in humans should be a top priority, but future studies should most definitely also focus on explanations other than persistent B. burgdorferi sensu lato infection after antibiotic treatment.
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Affiliation(s)
- Y. L. Verschoor
- Amsterdam UMC, Location University of Amsterdam, Department of Internal Medicine, Section of Infectious Diseases, Amsterdam UMC Multidisciplinary Lyme Borreliosis Center, Amsterdam, The Netherlands
- Amsterdam UMC, Location University of Amsterdam, Center for Experimental and Molecular Medicine, Amsterdam, The Netherlands
| | - A. Vrijlandt
- Amsterdam UMC, Location University of Amsterdam, Department of Internal Medicine, Section of Infectious Diseases, Amsterdam UMC Multidisciplinary Lyme Borreliosis Center, Amsterdam, The Netherlands
- Amsterdam UMC, Location University of Amsterdam, Center for Experimental and Molecular Medicine, Amsterdam, The Netherlands
| | - R. Spijker
- Amsterdam UMC, Location University of Amsterdam, Amsterdam Public Health, Medical Library, Amsterdam, The Netherlands
| | - R. M. van Hest
- Amsterdam UMC, Location University of Amsterdam, Department of Hospital Pharmacy and Clinical Pharmacology, Amsterdam, The Netherlands
| | - H. ter Hofstede
- Department of Internal Medicine, Section of Infectious Diseases, Lyme Borreliosis Outpatient Clinic, Radboudumc, Nijmegen, The Netherlands
| | | | - A. J. Henningsson
- Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden
- Department of Clinical Microbiology in Jönköping, Region Jönköping County, Linköping University, Linköping, Sweden
| | - J. W. Hovius
- Amsterdam UMC, Location University of Amsterdam, Department of Internal Medicine, Section of Infectious Diseases, Amsterdam UMC Multidisciplinary Lyme Borreliosis Center, Amsterdam, The Netherlands
- Amsterdam UMC, Location University of Amsterdam, Center for Experimental and Molecular Medicine, Amsterdam, The Netherlands
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13
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Yalley AK, Ahiatrogah S, Kafintu-Kwashie AA, Amegatcher G, Prah D, Botwe AK, Adusei-Poku MA, Obodai E, Nii-Trebi NI. A Systematic Review on Suitability of Molecular Techniques for Diagnosis and Research into Infectious Diseases of Concern in Resource-Limited Settings. Curr Issues Mol Biol 2022; 44:4367-4385. [PMID: 36286015 PMCID: PMC9601131 DOI: 10.3390/cimb44100300] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2022] [Revised: 09/15/2022] [Accepted: 09/16/2022] [Indexed: 11/16/2022] Open
Abstract
Infectious diseases significantly impact the health status of developing countries. Historically, infectious diseases of the tropics especially have received insufficient attention in worldwide public health initiatives, resulting in poor preventive and treatment options. Many molecular tests for human infections have been established since the 1980s, when polymerase chain reaction (PCR) testing was introduced. In spite of the substantial innovative advancements in PCR technology, which currently has found wide application in most viral pathogens of global concern, the development and application of molecular diagnostics, particularly in resource-limited settings, poses potential constraints. This review accessed data from sources including PubMed, Google Scholar, the Web of Knowledge, as well as reports from the World Health Organization’s Annual Meeting on infectious diseases and examined these for current molecular approaches used to identify, monitor, or investigate some neglected tropical infectious diseases. This review noted some growth efforts in the development of molecular techniques for diagnosis of pathogens that appear to be common in resource limited settings and identified gaps in the availability and applicability of most of these molecular diagnostics, which need to be addressed if the One Health goal is to be achieved.
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Affiliation(s)
- Akua K. Yalley
- Department of Medical Laboratory Sciences, School of Biomedical and Allied Health Sciences, University of Ghana, Accra P.O. Box KB 143, Ghana
| | - Selasie Ahiatrogah
- Department of Obstetrics and Gynaecology, College of Medicine, Pan African University of Life and Earth Sciences Institute, University of Ibadan, Ibadan P.O. Box 22133, Nigeria
| | - Anna A. Kafintu-Kwashie
- Department of Medical Microbiology, University of Ghana Medical School, Accra GA-221-1570, Ghana
| | - Gloria Amegatcher
- Department of Medical Laboratory Sciences, School of Biomedical and Allied Health Sciences, University of Ghana, Accra P.O. Box KB 143, Ghana
| | - Diana Prah
- West African Centre for Cell Biology of Infectious Pathogens, University of Ghana, Accra P.O. Box LG 54, Ghana
| | - Akua K. Botwe
- Molecular Biology Unit, Kintampo Health Research Centre, Ghana Health Service, Kintampo P.O. Box 200, Ghana
| | - Mildred A. Adusei-Poku
- Department of Medical Microbiology, University of Ghana Medical School, Accra GA-221-1570, Ghana
| | - Evangeline Obodai
- Department of Virology, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra P.O. Box LG 581, Ghana
| | - Nicholas I. Nii-Trebi
- Department of Medical Laboratory Sciences, School of Biomedical and Allied Health Sciences, University of Ghana, Accra P.O. Box KB 143, Ghana
- Correspondence: ; Tel.: +233-54-827-6424
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14
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A Plasma-Based Decontamination Process Reveals Potential for an in-Process Surface-Sanitation Method. PLASMA 2022. [DOI: 10.3390/plasma5030027] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Methods, which use an indirect plasma treatment for the inactivation of microorganisms in foods, claim a vastly growing field of research. This paper presents a method that uses plasma-processed air (PPA) as a sanitizer. In addition to a sanitation concept for the decontamination of produce in the value chain, the presented method offers a possible application as an “in-process” surface sanitation. PPA provides antimicrobial-potent species, which are predominantly reactive nitrogen species (RNS); this has an outstanding groove penetration property. In an experimental approach, surfaces, made from materials, which are frequently used for the construction of food-processing plants, were inoculated with different microorganisms. Listeria monocytogenes (ATCC 15313), Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 10538), Salmonella enterica subsp. enterica serovar Typhimurium (ATCC 43971), and Salmonella enterica subsp. enterica serovar Enteritidis (ATCC 13076) are all microorganisms that frequently appear in foods and possess the risk for cross-contamination from the plant to the produce or vice versa. The contaminated samples were treated for various treatment times (1–5 min) with PPA of different antimicrobial potencies. Subsequently, the microbial load on the specimens was determined and compared with the load of untreated samples. As a result, reduction factors (RF) up to several log10-steps were obtained. Although surface and the bacterial strain showed an influence on the RF, the major influence was seen by a prolongation of the treatment time and an increase in the potency of the PPA.
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15
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Haddad G, Takakura T, Bellali S, Fontanini A, Ominami Y, Khalil JB, Raoult D. A preliminary investigation into bacterial viability using scanning electron microscopy–energy-dispersive X-ray analysis: The case of antibiotics. Front Microbiol 2022; 13:967904. [PMID: 36003945 PMCID: PMC9393632 DOI: 10.3389/fmicb.2022.967904] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2022] [Accepted: 07/20/2022] [Indexed: 11/13/2022] Open
Abstract
The metabolic stages of bacterial development and viability under different stress conditions induced by disinfection, chemical treatments, temperature, or atmospheric changes have been thoroughly investigated. Here, we aim to evaluate early metabolic modifications in bacteria following induced stress, resulting in alterations to bacterial metabolism. A protocol was optimized for bacterial preparation using energy-dispersive X-ray (EDX) microanalysis coupled with scanning electron microscopy (SEM), followed by optimizing EDX data acquisition and analysis. We investigated different preparation methods aiming to detect modifications in the bacterial chemical composition at different states. We first investigated Escherichia coli, acquiring data from fresh bacteria, after heat shock, and after contact with 70% ethanol, in order to prove the feasibility of this new strategy. We then applied the new method to different bacterial species following 1 h of incubation with increasing doses of antibiotics used as a stress-inducing agent. Among the different materials tested aiming to avoiding interaction with bacterial metabolites, phosphorous-doped silicon wafers were selected for the slide preparation. The 15 kV acceleration voltage ensured all the chemical elements of interest were excited. A thick layer of bacterial culture was deposited on the silicon wafer providing information from multiple cells and intra-cellular composition. The EDX spectra of fresh, heat-killed, and alcohol-killed E. coli revealed important modifications in magnesium, potassium, and sodium. Those same alterations were detected when applying this strategy to bacteria exposed to antibiotics. Tests based on SEM–EDX acquisition systems would provide early predictions of the bacterial viability state in different conditions, yielding earlier results than culture.
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Affiliation(s)
- Gabriel Haddad
- Institut Hospitalo-Universitaire Méditerranée Infection, Marseille, France
- Aix-Marseille Univ, IRD, APHM, MEPHI, Marseille, France
| | | | - Sara Bellali
- Institut Hospitalo-Universitaire Méditerranée Infection, Marseille, France
| | - Anthony Fontanini
- Institut Hospitalo-Universitaire Méditerranée Infection, Marseille, France
| | | | - Jacques Bou Khalil
- Institut Hospitalo-Universitaire Méditerranée Infection, Marseille, France
- Jacques Bou Khalil,
| | - Didier Raoult
- Institut Hospitalo-Universitaire Méditerranée Infection, Marseille, France
- Aix-Marseille Univ, IRD, APHM, MEPHI, Marseille, France
- *Correspondence: Didier Raoult,
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16
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Glaciers as microbial habitats: current knowledge and implication. J Microbiol 2022; 60:767-779. [DOI: 10.1007/s12275-022-2275-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2022] [Revised: 07/04/2022] [Accepted: 07/04/2022] [Indexed: 10/16/2022]
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17
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Zhan Y, Gao X, Li S, Si Y, Li Y, Han X, Sun W, Li Z, Ye F. Development and Evaluation of Rapid and Accurate CRISPR/Cas13-Based RNA Diagnostics for Pneumocystis jirovecii Pneumonia. Front Cell Infect Microbiol 2022; 12:904485. [PMID: 35782118 PMCID: PMC9240425 DOI: 10.3389/fcimb.2022.904485] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2022] [Accepted: 05/16/2022] [Indexed: 12/01/2022] Open
Abstract
Background Pneumocystis jirovecii can result in a serious pulmonary infection, Pneumocystis jirovecii pneumonia, in immunocompetent hosts. The diagnosis of Pneumocystis jirovecii pneumonia has long been a major clinical concern, and there are limitations with the currently utilized immunostaining and polymerase chain reaction diagnosis/detection technologies (e.g., insufficient sensitivity and accuracy). Hence, we sought to establish a rapid and RNA-specific transcription mediated amplification and CRISPR/Cas13a-based diagnostics targeted P. jirovecii-mitochondrial large subunit ribosomal RNA. Methods The procedure of the diagnostics included amplification of the extracted RNA samples by transcription mediated amplification, followed by CRISPR/Cas13 detection, and ultimately, the judgment of the results after 30 minutes of fluorescence signal. Later, the diagnostic performance of the CRISPR/Cas13-based diagnostics were tested on the 62 surplus clinical samples. Results This CRISPR/Cas13-based diagnostics achieved limits of detection of approximately 2 copies/µL transcribed RNA templates, with no cross reaction to other respiratory pathogens, including bacteria and fungi. Similar to in-house quantitative real-time polymerase chain reaction, CRISPR/Cas13-based diagnostics was still positive in 243-fold diluted bronchial alveolar lavage fluid. A preliminary evaluation of 62 surplus bronchial alveolar lavage fluid samples from patients suspected of Pneumocystis jirovecii pneumonia showed that CRISPR/Cas13-based diagnostics achieved a 78.9% sensitivity and a 97.7% specificity in the diagnosis of Pneumocystis jirovecii pneumonia. Conclusion Our study demonstrates that the CRISPR/Cas13-based diagnostics technique has good performance for the accurate and specific diagnosis of Pneumocystis jirovecii pneumonia.
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Affiliation(s)
- Yangqing Zhan
- The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Institute of Respiratory Health, State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou, China
| | - Xiaoqing Gao
- R&D Department, Hangzhou MatriDx Biotechnology Co., Ltd., Hanzhou, China
| | - Shaoqiang Li
- The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Institute of Respiratory Health, State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou, China
| | - Yeqi Si
- R&D Department, Hangzhou MatriDx Biotechnology Co., Ltd., Hanzhou, China
| | - Yuanxiang Li
- The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Institute of Respiratory Health, State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou, China
| | - Xu Han
- R&D Department, Hangzhou MatriDx Biotechnology Co., Ltd., Hanzhou, China
| | - Wenjun Sun
- The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Institute of Respiratory Health, State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou, China
| | - Zhengtu Li
- The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Institute of Respiratory Health, State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou, China
| | - Feng Ye
- The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Institute of Respiratory Health, State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou, China
- *Correspondence: Feng Ye,
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18
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Ali Q, Zheng H, Rao MJ, Ali M, Hussain A, Saleem MH, Nehela Y, Sohail MA, Ahmed AM, Kubar KA, Ali S, Usman K, Manghwar H, Zhou L. Advances, limitations, and prospects of biosensing technology for detecting phytopathogenic bacteria. CHEMOSPHERE 2022; 296:133773. [PMID: 35114264 DOI: 10.1016/j.chemosphere.2022.133773] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/18/2021] [Revised: 01/23/2022] [Accepted: 01/25/2022] [Indexed: 05/22/2023]
Abstract
Phytopathogenic bacteria cause severe economic losses in agricultural production worldwide. The spread rates, severity, and emerging plant bacterial diseases have become serious threat to the sustainability of food sources and the fruit industry. Detection and diagnosis of plant diseases are imperative in order to manage plant diseases in field conditions, greenhouses, and food storage conditions as well as to maximize agricultural productivity and sustainability. To date, various techniques including, serological, observation-based, and molecular methods have been employed for plant disease detection. These methods are sensitive and specific for genetic identification of bacteria. However, these methods are specific for genetic identification of bacteria. Currently, the innovative biosensor-based disease detection technique is an attractive and promising alternative. A biosensor system involves biological recognition and transducer active receptors based on sensors used in plant-bacteria diagnosis. This system has been broadly used for the rapid diagnosis of plant bacterial pathogens. In the present review, we have discussed the conventional methods of bacterial-disease detection, however, the present review mainly focuses on the applications of different biosensor-based techniques along with point-of-care (POC), robotics, and cell phone-based systems. In addition, we have also discussed the challenges and limitations of these techniques.
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Affiliation(s)
- Qurban Ali
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Agro-product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou, China; Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Key Laboratory of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Education, Nanjing, 210095, China.
| | - Hongxia Zheng
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Agro-product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou, China
| | - Muhammad Junaid Rao
- Guangxi Key Laboratory of Sugarcane Biology, College of Agriculture, Guangxi University, 100 Daxue Rd., 8, Nanning, Guangxi, 530004, PR China
| | - Mohsin Ali
- College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China
| | - Amjad Hussain
- College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China
| | - Muhammad Hamzah Saleem
- College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China
| | - Yasser Nehela
- Department of Plant Pathology, Citrus Research and Education Center, University of Florida, 700 Experiment Station Rd, Lake Alfred, FL, 33850, USA; Department of Agricultural Botany, Faculty of Agriculture, Tanta University, Tanta, Egypt
| | - Muhammad Aamir Sohail
- College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China
| | - Agha Mushtaque Ahmed
- Department of Entomology, Faculty of Crop Protection, Sindh Agriculture University Tando Jam, Sindh, Pakistan
| | - Kashif Ali Kubar
- Faculty of Agriculture, Lasbela University of Agriculture, Water and Marine Sciences, Uthal, 90150, Balochistan, Pakistan
| | - Shafaqat Ali
- Department of Environmental Sciences and Engineering, Government College University Allama Iqbal Road, 38000, Faisalabad, Pakistan
| | - Kamal Usman
- Agricultural Research Station, Office of VP for Research & Graduate Studies, Qatar University, 2713, Doha, Qatar
| | - Hakim Manghwar
- Lushan Botanical Garden, Chinese Academy of Sciences, Jiujiang, Jiangxi, 332900, China.
| | - Lei Zhou
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Agro-product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou, China.
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19
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Exploiting the Anti-Biofilm Effect of the Engineered Phage Endolysin PM-477 to Disrupt In Vitro Single- and Dual-Species Biofilms of Vaginal Pathogens Associated with Bacterial Vaginosis. Antibiotics (Basel) 2022; 11:antibiotics11050558. [PMID: 35625202 PMCID: PMC9137943 DOI: 10.3390/antibiotics11050558] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2022] [Revised: 03/30/2022] [Accepted: 04/01/2022] [Indexed: 12/10/2022] Open
Abstract
Bacterial vaginosis (BV) is the most frequent vaginal infection in women of reproductive age. It is caused by the overgrowth of anaerobic vaginal pathogens, such as Gardnerella vaginalis, Fannyhessea vaginae, and Prevotella bivia, which are vaginal pathogens detected during the early stages of incident BV and have been found to form multi-species biofilms. Treatment of biofilm-associated infections, such as BV, is challenging. In this study, we tested the role of an investigational engineered phage endolysin, PM-477, in the eradication of dual-species biofilms composed of G. vaginalis–F. vaginae or G. vaginalis–P. bivia. Single-species biofilms formed by these species were also analysed as controls. The effect of PM-477 on biomass and culturability of single- and dual-species biofilms was assessed in vitro using a microtiter plate assay, epifluorescence microscopy, confocal laser scanning microscopy, and quantitative PCR. The results showed that PM-477 was particularly effective in the disruption and reduction of culturability of G. vaginalis biofilms. In dual-species biofilms, PM-477 exhibited lower efficiency but was still able to selectively and significantly eliminate G. vaginalis. Since polymicrobial interactions have been shown to strongly affect the activity of various antibiotics, the activity of PM-477 in dual-species biofilms is a potentially promising result that should be further explored, aiming to completely eradicate multi-species biofilms associated with BV.
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20
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Freitas BL, Leach L, Chaturvedi V, Chaturvedi S. Reverse Transcription-Quantitative Real-Time PCR (RT-qPCR) Assay for the Rapid Enumeration of Live Candida auris Cells from the Health Care Environment. J Clin Microbiol 2022; 60:e0077921. [PMID: 34878804 PMCID: PMC8849214 DOI: 10.1128/jcm.00779-21] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2021] [Accepted: 12/01/2021] [Indexed: 01/24/2023] Open
Abstract
Ongoing health care-associated outbreaks of the multidrug-resistant yeast Candida auris have prompted the development of several rapid DNA-based molecular diagnostic tests. These tests do not distinguish between live and dead C. auris cells, limiting their use for environmental surveillance and containment efforts. We addressed this critical gap by developing a reverse transcription (RT)-quantitative real-time PCR (RT-qPCR) assay to rapidly detect live C. auris in health care environments. This assay targeted the internal transcribed spacer 2 (ITS2) ribosomal gene by obtaining pure RNA followed by reverse transcription (ITS2 cDNA) and qPCR. ITS2 cDNA was not detectable in bleach-killed cells but was detectable in heat- and ethanol-killed C. auris cells. The assay was highly sensitive, with a detection limit of 10 CFU per RT-qPCR. Validation studies yielded positive cycle threshold (CT) values from sponge matrix samples spiked with 102 to 105 CFU of live C. auris, while dead (bleach-killed) C. auris (105/mL) or other live Candida species (105/mL) had no CT values. Finally, 33 environmental samples positive for C. auris DNA but negative by culture were all negative by RT-qPCR assay, confirming the concordance between culture and the PCR assay. The RT-qPCR assay appears highly reproducible, robust, and specific for detecting live C. auris from environmental samples. The Candida auris RT-qPCR assay could be an invaluable tool in surveillance efforts to control the spread of live C. auris in health care environments.
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Affiliation(s)
- Bryanna Lexus Freitas
- Mycology Laboratory, Wadsworth Center, New York State Department of Health, Albany, New York, USA
| | - Lynn Leach
- Mycology Laboratory, Wadsworth Center, New York State Department of Health, Albany, New York, USA
| | - Vishnu Chaturvedi
- Mycology Laboratory, Wadsworth Center, New York State Department of Health, Albany, New York, USA
| | - Sudha Chaturvedi
- Mycology Laboratory, Wadsworth Center, New York State Department of Health, Albany, New York, USA
- Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, New York, USA
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21
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Wendel U. Assessing Viability and Stress Tolerance of Probiotics—A Review. Front Microbiol 2022; 12:818468. [PMID: 35154042 PMCID: PMC8829321 DOI: 10.3389/fmicb.2021.818468] [Citation(s) in RCA: 48] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2021] [Accepted: 12/27/2021] [Indexed: 11/26/2022] Open
Abstract
The interest in probiotics has increased rapidly the latest years together with the global market for probiotic products. Consequently, establishing reliable microbiological methods for assuring the presence of a certain number of viable microorganisms in probiotic products has become increasingly important. To assure adequate numbers of viable cells, authorities are enquiring for information on viability rates within a certain shelf-life in colony forming units (CFU). This information is obtained from plate count enumeration, a method that enables detection of bacterial cells based on their ability to replicate. Although performing plate count enumeration is one manner of assessing viability, cells can still be viable without possessing the ability to replicate. Thus, to properly assess probiotic viability, further analysis of a broader group of characteristics using several types of methods is proposed. In addition to viability, it is crucial to identify how well the cells in a probiotic product can survive in the gastrointestinal tract (GIT) and thus be able to mediate the desired health benefit while passing through the human body. A broad spectrum of different assay designs for assessing probiotic gastric tolerance have been used in research and quality control. However, the absence of any consensus on how to assess these qualities makes it difficult to compare between laboratories and to translate the results into in vivo tolerance. This review presents and discusses the complexity of assuring that a probiotic is suitable for beneficial consumption. It summarizes the information that can be subtracted from the currently available methods for assessment of viability and stress tolerance of a probiotic, hereby altogether defined as “activity.” Strengths and limitations of the different methods are presented together with favorable method combinations. Finally, the importance of choosing a set of analyses that reveals the necessary aspects of probiotic activity for a certain product or application is emphasized.
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22
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Wen J, Liu J, Wu J, He D. Rapid measurement of waterborne bacterial viability based on difunctional gold nanoprobe. RSC Adv 2022; 12:1675-1681. [PMID: 35425161 PMCID: PMC8978865 DOI: 10.1039/d1ra07287k] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2021] [Accepted: 12/15/2021] [Indexed: 12/18/2022] Open
Abstract
Rapid measurement of waterborne bacterial viability is crucial for ensuring the safety of public health. Herein, we proposed a colorimetric assay for rapid measurement of waterborne bacterial viability based on a difunctional gold nanoprobe (dGNP). This versatile dGNP is composed of bacteria recognizing parts and signal indicating parts, and can generate color signals while recognizing bacterial suspensions of different viabilities. This dGNP-based colorimetric assay has a fast response and can be accomplished within 10 min. Moreover, the proposed colorimetric method is able to measure bacterial viability between 0% and 100%. The method can also measure the viability of other bacteria including Staphylococcus aureus, Shewanella oneidensis, and Escherichia coli O157H7. Furthermore, the proposed method has acceptable recovery (95.5–104.5%) in measuring bacteria-spiked real samples. This study offers a simple and effective method for the rapid measurement of bacterial viability and therefore should have application potential in medical diagnosis, food safety, and environmental monitoring. A colorimetric method is proposed to measure waterborne bacterial viability by using a difunctional gold nanoprobe that can generate color signals while recognizing bacterial suspensions of different viabilities.![]()
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Affiliation(s)
- Junlin Wen
- Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, School of Environmental Science and Engineering, Institute of Environmental Health and Pollution Control, Guangdong University of Technology Guangzhou 510006 P. R. China
| | - Jianbo Liu
- Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, School of Environmental Science and Engineering, Institute of Environmental Health and Pollution Control, Guangdong University of Technology Guangzhou 510006 P. R. China
| | - Jialin Wu
- Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, School of Environmental Science and Engineering, Institute of Environmental Health and Pollution Control, Guangdong University of Technology Guangzhou 510006 P. R. China
| | - Daigui He
- College of Artificial Intelligence, Guangdong Mechanical & Electrical Polytechnic Guangzhou 510550 P. R. China +86-20-36552429 +86-20-36552429
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23
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Advances in aggregation induced emission (AIE) materials in biosensing and imaging of bacteria. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2021. [PMID: 34749976 PMCID: PMC8292011 DOI: 10.1016/bs.pmbts.2021.06.015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/22/2023]
Abstract
With their ubiquitous nature, bacteria have had a significant impact on human health and evolution. Though as commensals residing in/on our bodies several bacterial communities support our health in many ways, bacteria remain one of the major causes of infectious diseases that plague the human world. Adding to this, emergence of antibiotic resistant strains limited the use of available antibiotics. The current available techniques to prevent and control such infections remain insufficient. This has been proven during one of greatest pandemic of our generation, COVID-19. It has been observed that bacterial coinfections were predominantly observed in COVID-19 patients, despite antibiotic treatment. Such higher rates of coinfections in critical patients even after antibiotic treatment is a matter of concern. Owing to many reasons across the world drug resistance in bacteria is posing a major problem i. According to Center for Disease control (CDC) antibiotic report threats (AR), 2019 more than 2.8 million antibiotic resistant cases were reported, and more than 35,000 were dead among them in USA alone. In both normal and pandemic conditions, failure of identifying infectious agent has played a major role. This strongly prompts the need to improve upon the existing techniques to not just effective identification of an unknown bacterium, but also to discriminate normal Vs drug resistant strains. New techniques based on Aggregation Induced Emission (AIE) are not only simple and rapid but also have high accuracy to visualize infection and differentiate many strains of bacteria based on biomolecular variations which has been discussed in this chapter.
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24
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Siqueira JF, Rôças IN. A critical analysis of research methods and experimental models to study the root canal microbiome. Int Endod J 2021; 55 Suppl 1:46-71. [PMID: 34714548 DOI: 10.1111/iej.13656] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2021] [Revised: 10/22/2021] [Accepted: 10/27/2021] [Indexed: 12/15/2022]
Abstract
Endodontic microbiology deals with the study of the microbial aetiology and pathogenesis of pulpal and periradicular inflammatory diseases. Research in endodontic microbiology started almost 130 years ago and since then has mostly focussed on establishing and confirming the infectious aetiology of apical periodontitis, identifying the microbial species associated with the different types of endodontic infections and determining the efficacy of treatment procedures in eradicating or controlling infection. Diverse analytical methods have been used over the years, each one with their own advantages and limitations. In this review, the main features and applications of the most used technologies are discussed, and advice is provided to improve study designs in order to properly address the scientific questions and avoid setbacks that can compromise the results. Finally, areas of future research are described.
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Affiliation(s)
- José F Siqueira
- Department of Endodontics and Molecular Microbiology Laboratory, Faculty of Dentistry, Grande Rio University, Rio de Janeiro, Brazil.,Department of Dental Research, Faculty of Dentistry, Iguaçu University (UNIG), Nova Iguaçu, Brazil
| | - Isabela N Rôças
- Department of Endodontics and Molecular Microbiology Laboratory, Faculty of Dentistry, Grande Rio University, Rio de Janeiro, Brazil.,Department of Dental Research, Faculty of Dentistry, Iguaçu University (UNIG), Nova Iguaçu, Brazil
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25
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Campelo TA, Cardoso de Sousa PR, Nogueira LDL, Frota CC, Zuquim Antas PR. Revisiting the methods for detecting Mycobacterium tuberculosis: what has the new millennium brought thus far? Access Microbiol 2021; 3:000245. [PMID: 34595396 PMCID: PMC8479963 DOI: 10.1099/acmi.0.000245] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2021] [Accepted: 06/17/2021] [Indexed: 01/07/2023] Open
Abstract
Tuberculosis (TB) affects around 10 million people worldwide in 2019. Approximately 3.4 % of new TB cases are multidrug-resistant. The gold standard method for detecting Mycobacterium tuberculosis, which is the aetiological agent of TB, is still based on microbiological culture procedures, followed by species identification and drug sensitivity testing. Sputum is the most commonly obtained clinical specimen from patients with pulmonary TB. Although smear microscopy is a low-cost and widely used method, its sensitivity is 50-60 %. Thus, owing to the need to improve the performance of current microbiological tests to provide prompt treatment, different methods with varied sensitivity and specificity for TB diagnosis have been developed. Here we discuss the existing methods developed over the past 20 years, including their strengths and weaknesses. In-house and commercial methods have been shown to be promising to achieve rapid diagnosis. Combining methods for mycobacterial detection systems demonstrates a correlation of 100 %. Other assays are useful for the simultaneous detection of M. tuberculosis species and drug-related mutations. Novel approaches have also been employed to rapidly identify and quantify total mycobacteria RNA, including assessments of global gene expression measured in whole blood to identify the risk of TB. Spoligotyping, mass spectrometry and next-generation sequencing are also promising technologies; however, their cost needs to be reduced so that low- and middle-income countries can access them. Because of the large impact of M. tuberculosis infection on public health, the development of new methods in the context of well-designed and -controlled clinical trials might contribute to the improvement of TB infection control.
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Affiliation(s)
- Thales Alves Campelo
- Faculdade de Medicina, Departamento de Patologia e Medicina Legal, Federal University of Ceará, Fortaleza, Brazil
| | | | - Lucas de Lima Nogueira
- Faculdade de Medicina, Departamento de Patologia e Medicina Legal, Federal University of Ceará, Fortaleza, Brazil
| | - Cristiane Cunha Frota
- Faculdade de Medicina, Departamento de Patologia e Medicina Legal, Federal University of Ceará, Fortaleza, Brazil
| | - Paulo Renato Zuquim Antas
- Laboratório de Imunologia Clínica, Instituto Oswaldo Cruz, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil
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26
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Khan MAA, Faisal K, Chowdhury R, Nath R, Ghosh P, Ghosh D, Hossain F, Abd El Wahed A, Mondal D. Evaluation of molecular assays to detect Leishmania donovani in Phlebotomus argentipes fed on post-kala-azar dermal leishmaniasis patients. Parasit Vectors 2021; 14:465. [PMID: 34503557 PMCID: PMC8428120 DOI: 10.1186/s13071-021-04961-6] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2021] [Accepted: 08/17/2021] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Post-kala-azar dermal leishmaniasis (PKDL) caused by Leishmania donovani (LD) is a skin disorder that often appears after treatment of visceral leishmaniasis (VL) patients. PKDL patients are potential reservoirs of LD parasites, which can initiate a new epidemic of anthroponotic VL. Therefore, host infectiousness to its sand fly vector is a critical factor for transmission, and its accurate estimation can facilitate control strategies. At present, conventional microscopy serves as the reference method to detect parasites in its vector. However, low sensitivity of microscopy can be a limiting factor. METHODS In this study, real-time quantitative PCR (LD-qPCR) and recombinase polymerase amplification (LD-RPA) assays were evaluated against microscopy for the detection of LD DNA extracted from live sand flies five days after controlled feeding on PKDL cases. RESULTS The sensitivity of LD-qPCR and LD-RPA assays were found to be 96.43 and 100%, respectively, against microscopy for the selected fed sand flies (n = 28), and an absolute specificity of both molecular tools for apparently unfed sand flies (n = 30). While the proportion of infectious cases among 47 PKDL patients was estimated as 46.81% as defined by microscopic detection of LD in at least one fed sand fly per case, LD-RPA assay evaluation of only the microscopy negative sand flies fed to those 47 PKDL cases estimated an even greater proportion of infectious cases (51.06%). In overall estimation of the infectious cases in retrospective manner, discordance in positivity rate was observed (p < 0.05) between LD-RPA (59.57%) assay and microscopy (46.81%), while LD-RPA had slightly better positivity rate than LD-qPCR (55.32%) as well. CONCLUSIONS Considering the sensitivity, cost, detection time, and field applicability, RPA assay can be considered as a promising single molecular detection tool for investigations pertaining to LD infections in sand flies and/or host infectiousness in PKDL, while it can also be useful in confirmation of microscopy negative sand fly samples.
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Affiliation(s)
- Md Anik Ashfaq Khan
- Institute of Animal Hygiene and Veterinary Public Health, University of Leipzig, An den Tierkliniken 43, 04103, Leipzig, Germany.,Nutrition and Clinical Services Division, International Centre for Diarrheal Disease Research Bangladesh, 1212, Dhaka, Bangladesh
| | - Khaledul Faisal
- Nutrition and Clinical Services Division, International Centre for Diarrheal Disease Research Bangladesh, 1212, Dhaka, Bangladesh
| | - Rajashree Chowdhury
- Nutrition and Clinical Services Division, International Centre for Diarrheal Disease Research Bangladesh, 1212, Dhaka, Bangladesh
| | - Rupen Nath
- Nutrition and Clinical Services Division, International Centre for Diarrheal Disease Research Bangladesh, 1212, Dhaka, Bangladesh
| | - Prakash Ghosh
- Nutrition and Clinical Services Division, International Centre for Diarrheal Disease Research Bangladesh, 1212, Dhaka, Bangladesh
| | - Debashis Ghosh
- Nutrition and Clinical Services Division, International Centre for Diarrheal Disease Research Bangladesh, 1212, Dhaka, Bangladesh
| | - Faria Hossain
- Nutrition and Clinical Services Division, International Centre for Diarrheal Disease Research Bangladesh, 1212, Dhaka, Bangladesh
| | - Ahmed Abd El Wahed
- Institute of Animal Hygiene and Veterinary Public Health, University of Leipzig, An den Tierkliniken 43, 04103, Leipzig, Germany.
| | - Dinesh Mondal
- Nutrition and Clinical Services Division, International Centre for Diarrheal Disease Research Bangladesh, 1212, Dhaka, Bangladesh. .,Laboratory Sciences and Services Division, International Centre for Diarrheal Disease Research Bangladesh, 1212, Dhaka, Bangladesh.
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27
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Van Genechten W, Van Dijck P, Demuyser L. Fluorescent toys 'n' tools lighting the way in fungal research. FEMS Microbiol Rev 2021; 45:fuab013. [PMID: 33595628 PMCID: PMC8498796 DOI: 10.1093/femsre/fuab013] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2020] [Accepted: 02/14/2021] [Indexed: 12/13/2022] Open
Abstract
Although largely overlooked compared to bacterial infections, fungal infections pose a significant threat to the health of humans and other organisms. Many pathogenic fungi, especially Candida species, are extremely versatile and flexible in adapting to various host niches and stressful situations. This leads to high pathogenicity and increasing resistance to existing drugs. Due to the high level of conservation between fungi and mammalian cells, it is hard to find fungus-specific drug targets for novel therapy development. In this respect, it is vital to understand how these fungi function on a molecular, cellular as well as organismal level. Fluorescence imaging allows for detailed analysis of molecular mechanisms, cellular structures and interactions on different levels. In this manuscript, we provide researchers with an elaborate and contemporary overview of fluorescence techniques that can be used to study fungal pathogens. We focus on the available fluorescent labelling techniques and guide our readers through the different relevant applications of fluorescent imaging, from subcellular events to multispecies interactions and diagnostics. As well as cautioning researchers for potential challenges and obstacles, we offer hands-on tips and tricks for efficient experimentation and share our expert-view on future developments and possible improvements.
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Affiliation(s)
- Wouter Van Genechten
- VIB-KU Leuven Center for Microbiology, Kasteelpark Arenberg 31, 3001 Leuven-heverlee, Belgium
- Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Kasteelpark Arenberg 31, 3001 Leuven-Heverlee, Belgium
- Laboratory for Nanobiology, Department of Chemistry, KU Leuven, Celestijnenlaan 200g, 3001 Leuven-Heverlee, Belgium
| | - Patrick Van Dijck
- VIB-KU Leuven Center for Microbiology, Kasteelpark Arenberg 31, 3001 Leuven-heverlee, Belgium
- Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Kasteelpark Arenberg 31, 3001 Leuven-Heverlee, Belgium
| | - Liesbeth Demuyser
- VIB-KU Leuven Center for Microbiology, Kasteelpark Arenberg 31, 3001 Leuven-heverlee, Belgium
- Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Kasteelpark Arenberg 31, 3001 Leuven-Heverlee, Belgium
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28
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Gomes SC, Fachin S, da Fonseca JG, Angst PDM, Lamers ML, da Silva ISB, Nunes LN. Dental biofilm of symptomatic COVID-19 patients harbours SARS-CoV-2. J Clin Periodontol 2021; 48:880-885. [PMID: 33899251 PMCID: PMC8251434 DOI: 10.1111/jcpe.13471] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2020] [Revised: 04/08/2021] [Accepted: 04/09/2021] [Indexed: 12/12/2022]
Abstract
Aims SARS‐CoV‐2 RNA has been recovered from different sites in the human body, including the mouth. The present study aimed to investigate the presence of SARS‐CoV‐2 RNA in the dental biofilm of symptomatic patients who tested positive in nasopharyngeal and oropharyngeal (NASO/ORO) samples. Materials & Methods An observational clinical study of individuals with flu‐like symptoms was conducted between July and September 2020. Dental biofilm (BIO) samples were collected and analysed using real‐time quantitative polymerase chain reaction (RT‐qPCR) to determine the virus's presence. Results Seventy participants (40 ± 9.8 years of age, 71.4% female) tested positive for SARS‐CoV‐2 RNA in NASO/ORO samples and were included in the study. Among them, 13 tested positive in BIO samples (18.6%; 95% CI: [9.5, 27.7]). The median and interquartile range of cycle quantification (Cq) for NASO/ORO and BIO samples were 15.9 [6.9] and 35.9 [4.0] (p = .001), respectively. BIO‐positive participants showed a higher virus load in NASO/ORO samples (p = .012) than those testing negative (Cq = 20.4 [6.1]). Conclusions Dental biofilms from symptomatic COVID‐19 patients harbour SARS‐CoV‐2 RNA and might be a potential reservoir with an essential role in COVID‐19 transmission.
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Affiliation(s)
- Sabrina Carvalho Gomes
- Department of Conservative Dentistry, Dentistry School, Federal University of Rio Grande do Sul, Porto Alegre, Brazil.,Hospital de Clínicas de Porto Alegre, Federal University of Rio Grande do Sul, Porto Alegre, Brazil
| | - Sabrina Fachin
- Resident in Periodontology, Dental Faculty, Federal University of Rio Grande do Sul, Porto Alegre, Brazil
| | | | - Patrícia Daniela Melchiors Angst
- Department of Conservative Dentistry, Dentistry School, Federal University of Rio Grande do Sul, Porto Alegre, Brazil.,Hospital de Clínicas de Porto Alegre, Federal University of Rio Grande do Sul, Porto Alegre, Brazil
| | - Marcelo Lazzaron Lamers
- Department of Morphological Sciences, Dentistry School, Federal University of Rio Grande do Sul, Porto Alegre, Brazil
| | - Ilma Simoni Brum da Silva
- Department of Physiology, Basic Health Science Institute, Federal University of Rio Grande do Sul, Porto Alegre, Brazil
| | - Luciana Neves Nunes
- Mathematics and Statistics Institute, Federal University of Rio Grande do Sul, Porto Alegre, Brazil
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Thapa N, Danyluk MD, Gerberich KM, Johnson EG, Dewdney MM. Assessment of the Effect of Thermotherapy on ' Candidatus Liberibacter asiaticus' Viability in Woody Tissue of Citrus via Graft-Based Assays and RNA Assays. PHYTOPATHOLOGY 2021; 111:808-818. [PMID: 32976056 DOI: 10.1094/phyto-04-20-0152-r] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/11/2023]
Abstract
In 2019, citrus production in Florida declined by more than 70%, mostly because of Huanglongbing (HLB), which is caused by the bacterium 'Candidatus Liberibacter asiaticus' (CLas). Thermotherapy for HLB-affected trees was proposed as a short-term management solution to maintain field productivity. It was hypothesized that thermotherapy could eliminate HLB from affected branches; therefore, the study objectives were to show which time-temperature combinations eliminated CLas from woody tissues. Hardening, rounded Valencia twigs collected from HLB-affected field trees were treated in a steam chamber at different time-temperature combinations (50°C for 60 s; 55°C for 0, 30, 60, 90, and 120 s; 60°C for 30 s; and an untreated control). Three independent repetitions of 13 branches per treatment were grafted onto healthy rootstocks and tested to detect CLas after 6, 9, and 12 months. For the RNA-based CLas viability assay, three branches per treatment were treated and bark samples were peeled for RNA extraction and subsequent gene expression analyses. During the grafting study, at 12 months after grafting, a very low frequency of trees grafted with twigs treated at 55°C for 90 s and 55°C for 120 s had detectable CLas DNA. In the few individuals with CLas, titers were significantly lower (P ≤ 0.0001) and could have been remnants of degrading DNA. Additionally, there was a significant decrease (P ≤ 0.0001) in CLas 16S rRNA expression at 55°C for 90 s, 55°C for 120 s, and 60°C for 30 s (3.4-fold change, 3.4-fold change, and 2.3-fold change, respectively) in samples 5 days after treatment. Heat injury, not total CLas kill, could explain the limited changes in transcriptional activity; however, failed recovery and eventual death of CLas resulted in no CLas detection in most of the grafted trees treated with the highest temperatures or longest durations.
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Affiliation(s)
- Naweena Thapa
- Plant Pathology Department, Citrus Research and Education Center, University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850
| | - Michelle D Danyluk
- Food Science and Human Nutrition, Citrus Research and Education Center, University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850
| | - Kayla M Gerberich
- Plant Pathology Department, Citrus Research and Education Center, University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850
| | - Evan G Johnson
- Plant Pathology Department, Citrus Research and Education Center, University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850
| | - Megan M Dewdney
- Plant Pathology Department, Citrus Research and Education Center, University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850
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Bailoni L, Carraro L, Cardin M, Cardazzo B. Active Rumen Bacterial and Protozoal Communities Revealed by RNA-Based Amplicon Sequencing on Dairy Cows Fed Different Diets at Three Physiological Stages. Microorganisms 2021; 9:754. [PMID: 33918504 PMCID: PMC8066057 DOI: 10.3390/microorganisms9040754] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2021] [Revised: 03/29/2021] [Accepted: 04/01/2021] [Indexed: 12/12/2022] Open
Abstract
Seven Italian Simmental cows were monitored during three different physiological stages, namely late lactation (LL), dry period (DP), and postpartum (PP), to evaluate modifications in their metabolically-active rumen bacterial and protozoal communities using the RNA-based amplicon sequencing method. The bacterial community was dominated by seven phyla: Proteobacteria, Bacteroidetes, Firmicutes, Spirochaetes, Fibrobacteres, Verrucomicrobia, and Tenericutes. The relative abundance of the phylum Proteobacteria decreased from 47.60 to 28.15% from LL to DP and then increased to 33.24% in PP. An opposite pattern in LL, DP, and PP stages was observed for phyla Verrucomicrobia (from 0.96 to 4.30 to 1.69%), Elusimicrobia (from 0.32 to 2.84 to 0.25%), and SR1 (from 0.50 to 2.08 to 0.79%). The relative abundance of families Succinivibrionaceae and Prevotellaceae decreased in the DP, while Ruminococcaceae increased. Bacterial genera Prevotella and Treponema were least abundant in the DP as compared to LL and PP, while Ruminobacter and Succinimonas were most abundant in the DP. The rumen eukaryotic community was dominated by protozoal phylum Ciliophora, which showed a significant decrease in relative abundance from 97.6 to 93.9 to 92.6 in LL, DP, and PP, respectively. In conclusion, the physiological stage-dependent dietary changes resulted in a clear shift in metabolically-active rumen microbial communities.
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Affiliation(s)
- Lucia Bailoni
- Department of Comparative Biomedicine and Food Science (BCA), University of Padova, Viale dell’Universitá 16, 35020 Legnaro, PD, Italy; (L.C.); (M.C.); (B.C.)
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Label-free chemiresistor biosensor based on reduced graphene oxide and M13 bacteriophage for detection of coliforms. Anal Chim Acta 2021; 1150:338232. [PMID: 33583547 DOI: 10.1016/j.aca.2021.338232] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Revised: 01/12/2021] [Accepted: 01/14/2021] [Indexed: 12/31/2022]
Abstract
Coliform bacteria are well known as informative indicators for bacterial contamination in water. This study presents a novel chemiresistor biosensor using M13 phage-modified reduced graphene oxide (rGO) for detection of Escherichia coli (E. coli), as coliform bacteria. M13 phage, as a biorecognition element, was immobilized on the rGO channel, so that it can bind to negatively charged E. coli bacteria, allowing the gating effect on the biosensor and the change in its resistance. The prepared materials and device were characterized using spectroscopic, microscopic, and electrical measurements. FTIR and XRD results proved the successful fabrication of GO and rGO nanosheets. AFM results showed that the prepared nanosheets were monolayer. The SEM micrographs of the M13-functionalized devices, soaked in two different concentrations of E. coli, confirmed the successful capturing of E. coli and that the signal change is concentration-dependent. As a result, a linear and specific response towards E. coli was observed and the limit of detection was determined to be 45 CFU/mL. Further, the proposed sensor system showed selectivity towards the tested coliforms. These results suggested this sensing system could be a promising tool for detecting coliforms with an economic, accurate, rapid, and directly applicable process.
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32
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Liu W, Li R, Deng F, Yan C, Zhou X, Miao L, Li X, Xu Z. A Cell Membrane Fluorogenic Probe for Gram-Positive Bacteria Imaging and Real-Time Tracking of Bacterial Viability. ACS APPLIED BIO MATERIALS 2021; 4:2104-2112. [PMID: 35014338 DOI: 10.1021/acsabm.0c01269] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Bacterial infections are a global healthcare problem, resulting in serious clinical morbidities and mortality. Real-time monitoring of live bacteria by fluorescent imaging technology has potential in diagnosis of bacterial infections, elucidating antimicrobial agents' mode of action, assessing drug toxicity, and examining bacterial antimicrobial resistance. In this work, a naphthalimide-derived fluorescent probe ZTRS-BP was developed for wash-free Gram-positive bacteria imaging. The probe aggregated in aqueous solutions and exhibited aggregation-caused fluorescence quenching (ACQ). The interaction with Gram-positive bacteria cell walls would selectively disaggregate the probe and the liberated probes were dispersed on the outside of the bacteria cell walls to achieve surface fluorescence imaging. There were no such interactions with Gram-negative bacteria, which indicates that selective binding and imaging of Gram-positive bacteria was achieved. The binding of zinc ions by ZTRS-BP can enhance the fluorescent signals on the bacterial surface by inhibiting the process of photoinduced electron transfer. ZTRS-BP-Zn(II) complex was an excellent dye to discriminate mixed Gram-positive and Gram-negative bacteria. Also, live and dead bacteria can be differentially imaged by ZTRS-BP-Zn(II). Furthermore, ZTRS-BP-Zn(II) was used for real-time monitoring bacteria viability such as B. cereus treated with antibiotic vancomycin.
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Affiliation(s)
- Weiwei Liu
- State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012, China.,CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
| | - Ruihua Li
- The Second Affiliated Hospital of Dalian Medical University, Dalian 116023, China
| | - Fei Deng
- CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.,School of Chemistry and Chemical Engineering, Jinggangshan University, Ji'an, Jiangxi 343009, China
| | - Chunyu Yan
- CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.,Zhang Dayu Schoole of Chemistry, Dalian University of Technology, Dalian 116012, China
| | - Xuelian Zhou
- CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.,Zhang Dayu Schoole of Chemistry, Dalian University of Technology, Dalian 116012, China
| | - Lu Miao
- CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
| | - Xiaolian Li
- State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012, China
| | - Zhaochao Xu
- State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012, China.,CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.,Zhang Dayu Schoole of Chemistry, Dalian University of Technology, Dalian 116012, China
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Lake Sedimentary DNA Research on Past Terrestrial and Aquatic Biodiversity: Overview and Recommendations. QUATERNARY 2021. [DOI: 10.3390/quat4010006] [Citation(s) in RCA: 60] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The use of lake sedimentary DNA to track the long-term changes in both terrestrial and aquatic biota is a rapidly advancing field in paleoecological research. Although largely applied nowadays, knowledge gaps remain in this field and there is therefore still research to be conducted to ensure the reliability of the sedimentary DNA signal. Building on the most recent literature and seven original case studies, we synthesize the state-of-the-art analytical procedures for effective sampling, extraction, amplification, quantification and/or generation of DNA inventories from sedimentary ancient DNA (sedaDNA) via high-throughput sequencing technologies. We provide recommendations based on current knowledge and best practises.
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Bronzato JD, Bomfim RA, Hayasida GZP, Cúri M, Estrela C, Paster BJ, Gomes BPFA. Analysis of microorganisms in periapical lesions: A systematic review and meta-analysis. Arch Oral Biol 2021; 124:105055. [PMID: 33588190 DOI: 10.1016/j.archoralbio.2021.105055] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2020] [Revised: 01/05/2021] [Accepted: 01/06/2021] [Indexed: 12/30/2022]
Abstract
AIMS The aim of this study was to systematically review the literature on prevalence of microorganisms and their viability/activity in endodontic periapical lesions. DESIGN Literature research was performed on five electronic biomedical databases from their start dates to June 2020. Only studies evaluating the presence of microorganisms in periapical lesions in human permanent teeth with secondary/persistent infection were included. Two reviewers independently assessed the eligibility for inclusion, extracted data and evaluated the risk of bias. Meta-analysis and binominal tests were used to analyse the resulting data. RESULTS From the 1,313 records found, 23 full-texts were included for qualitative and quantitative analysis. The prevalence of microorganisms in endodontic periapical lesions was 87 % (95 % CI, 75-94) and the prevalence of viable/active microorganisms was 82 % (95 % CI, 66-91). There were statistical differences in the geographic area subgroup and between viable bacteria and active viruses. The most common detection method of microorganisms was the molecular one (69 %), and the most prevalent bacteria were the species Actinomyces, Fusobacterium and Prevotella (40 %). Most of the included studies had moderate risk of bias. CONCLUSIONS The prevalence of microorganisms in endodontic periapical lesions was 87 % and the prevalence of viable/active microorganisms was 82 %.
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Affiliation(s)
- Juliana D Bronzato
- Department of Restorative Dentistry, Endodontics Division, Piracicaba Dental School, State University of Campinas-UNICAMP, Piracicaba, SP, Brazil
| | - Rafael A Bomfim
- Department of Community Health, School of Dentistry, Federal University of Mato Grosso do Sul, Campo Grande, MS, Brazil
| | - Giovanna Z P Hayasida
- Department of Applied Math and Statistics, Institute of Math and Computing Sciences, University of São Paulo, São Carlos, SP, Brazil
| | - Mariana Cúri
- Department of Applied Math and Statistics, Institute of Math and Computing Sciences, University of São Paulo, São Carlos, SP, Brazil
| | - Carlos Estrela
- Department of Endodontics, School of Dentistry, Federal University of Goiás, Goiânia, GO, Brazil
| | - Bruce J Paster
- Department of Microbiology, Forsyth Institute, Cambridge, United States
| | - Brenda P F A Gomes
- Department of Restorative Dentistry, Endodontics Division, Piracicaba Dental School, State University of Campinas-UNICAMP, Piracicaba, SP, Brazil.
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Evaluation of conventional and four real-time PCR methods for the detection of Leishmania on field-collected samples in Ethiopia. PLoS Negl Trop Dis 2021; 15:e0008903. [PMID: 33434190 PMCID: PMC7802924 DOI: 10.1371/journal.pntd.0008903] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2020] [Accepted: 10/19/2020] [Indexed: 12/13/2022] Open
Abstract
In most low-resource settings, microscopy still is the standard method for diagnosis of cutaneous leishmaniasis, despite its limited sensitivity. In Ethiopia, the more sensitive molecular methods are not yet routinely used. This study compared five PCR methods with microscopy on two sample types collected from patients with a suspected lesion to advise on optimal diagnosis of Leishmania aethiopica. Between May and July 2018, skin scrapings (SS) and blood exudate from the lesion spotted on filter paper (dry blood spot, DBS) were collected for PCR from 111 patients of four zones in Southern Ethiopia. DNA and RNA were simultaneously extracted from both sample types. DNA was evaluated by a conventional PCR targeting ITS-1 and three probe-based real-time PCRs: one targeting the SSU 18S rRNA and two targeting the kDNA minicircle sequence (the 'Mary kDNA PCR' and a newly designed 'LC kDNA PCR' for improved L. aethiopica detection). RNAs were tested with a SYBR Green-based RT-PCR targeting spliced leader (SL) RNA. Giemsa-stained SS smears were examined by microscopy. Of the 111 SS, 100 were positive with at least two methods. Sensitivity of microscopy, ITS PCR, SSU PCR, Mary kDNA PCR, LC kDNA PCR and SL RNA PCR were respectively 52%, 22%, 64%, 99%, 100% and 94%. Microscopy-based parasite load correlated well with real-time PCR Ct-values. Despite suboptimal sample storage for RNA detection, the SL RNA PCR resulted in congruent results with low Ct-values. DBS collected from the same lesion showed lower PCR positivity rates compared to SS. The kDNA PCRs showed excellent performance for diagnosis of L. aethiopica on SS. Lower-cost SL RNA detection can be a complementary high-throughput tool. DBS can be used for PCR in case microscopy is negative, the SS sample can be sent to the referral health facility where kDNA PCR method is available.
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Guesmi S, Pujic P, Nouioui I, Dubost A, Najjari A, Ghedira K, Igual JM, Miotello G, Cherif A, Armengaud J, Klenk HP, Normand P, Sghaier H. Ionizing-radiation-resistant Kocuria rhizophila PT10 isolated from the Tunisian Sahara xerophyte Panicum turgidum: Polyphasic characterization and proteogenomic arsenal. Genomics 2020; 113:317-330. [PMID: 33279651 DOI: 10.1016/j.ygeno.2020.11.029] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2020] [Revised: 10/17/2020] [Accepted: 11/29/2020] [Indexed: 10/22/2022]
Abstract
A new strain belonging to the genus Kocuria, designed PT10, was isolated from irradiated roots of the xerophyte Panicum turgidum. Isolate PT10 is a Gram-positive, coccoid, aerobic and ionizing-radiation (IR)-resistant actinobacterium. PT10 has shown an ability to survive under extreme conditions, such as gamma irradiation, desiccation and high concentration of hydrogen peroxide. Phenotypic, chemotaxonomic and comparative genome analyses support the assignment of strain PT10 (LMG 31102 = DSM 108617) as Kocuria rhizophila. The complete genome sequence of PT10 consists of one chromosome (2,656,287 bps), with a 70.7% G + C content and comprises 2481 protein-coding sequences. A total of 1487 proteins were identified by LC-MS/MS profiling. In silico analyses revealed that the proteome of the oxidation-tolerant PT10 possesses several features explaining its IR-resistant phenotype and many adaptive pathways implicated in response to environmental pressures - desiccation, cold, reactive oxygen species and other stressors.
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Affiliation(s)
- Sihem Guesmi
- National Agronomy Institute of Tunisia, 43, Avenue Charles Nicolle, 1082 Tunis, Mahrajène, Tunisia; Laboratory ″Energy and Matter for Development of Nuclear Sciences″ (LR16CNSTN02), National Center for Nuclear Sciences and Technology, Sidi Thabet Technopark, 2020, Tunisia.
| | - Petar Pujic
- Université de Lyon, Université Lyon 1, Lyon, France; CNRS, UMR 5557, Ecologie Microbienne, 69622 Villeurbanne, Cedex, INRA, UMR1418, Villeurbanne, France
| | - Imen Nouioui
- School of Natural and Environmental Sciences, Newcastle University, Ridley Building 2, Newcastle upon Tyne NE1 7RU, UK; Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany
| | | | - Afef Najjari
- Université de Tunis El Manar, Faculté des Sciences de Tunis, LR03ES03 Microorganismes et Biomolécules Actives, 2092 Tunis, Tunisia
| | - Kais Ghedira
- Laboratory of Bioinformatics, Biomathematics and Biostatistics - LR16IPT09, Institut Pasteur de Tunis, 13, Place Pasteur, Tunis 1002, Tunisia
| | - José M Igual
- Instituto de Recursos Naturales y Agrobiología de Salamanca, Consejo Superior de Investigaciones Científicas (IRNASA-CSIC), c/Cordel de Merinas 40-52, 37008 Salamanca, Spain
| | - Guylaine Miotello
- Université Paris Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SPI, 30200 Bagnols-sur-Cèze, France
| | - Ameur Cherif
- Univ. Manouba, ISBST, BVBGR-LR11ES31, Biotechpole Sidi Thabet, 2020, Ariana, Tunisia
| | - Jean Armengaud
- Université Paris Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SPI, 30200 Bagnols-sur-Cèze, France
| | - Hans-Peter Klenk
- School of Natural and Environmental Sciences, Newcastle University, Ridley Building 2, Newcastle upon Tyne NE1 7RU, UK
| | | | - Haïtham Sghaier
- Laboratory ″Energy and Matter for Development of Nuclear Sciences″ (LR16CNSTN02), National Center for Nuclear Sciences and Technology, Sidi Thabet Technopark, 2020, Tunisia; Univ. Manouba, ISBST, BVBGR-LR11ES31, Biotechpole Sidi Thabet, 2020, Ariana, Tunisia.
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Pareyn M, Hendrickx R, Girma N, Hendrickx S, Van Bockstal L, Van Houtte N, Shibru S, Maes L, Leirs H, Caljon G. Evaluation of a pan-Leishmania SL RNA qPCR assay for parasite detection in laboratory-reared and field-collected sand flies and reservoir hosts. Parasit Vectors 2020; 13:276. [PMID: 32487217 PMCID: PMC7268266 DOI: 10.1186/s13071-020-04141-y] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2020] [Accepted: 05/21/2020] [Indexed: 11/15/2022] Open
Abstract
Background In eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts. Methods Field-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve. Results The qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10−3 and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents. Conclusions This study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.![]()
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Affiliation(s)
- Myrthe Pareyn
- Evolutionary Ecology Group, University of Antwerp, Wilrijk, Belgium.
| | - Rik Hendrickx
- Laboratory of Microbiology, Parasitology and Hygiene, University of Antwerp, Wilrijk, Belgium
| | - Nigatu Girma
- Biology Department, Arba Minch University, Arba Minch, Ethiopia
| | - Sarah Hendrickx
- Laboratory of Microbiology, Parasitology and Hygiene, University of Antwerp, Wilrijk, Belgium
| | - Lieselotte Van Bockstal
- Laboratory of Microbiology, Parasitology and Hygiene, University of Antwerp, Wilrijk, Belgium
| | | | - Simon Shibru
- Biology Department, Arba Minch University, Arba Minch, Ethiopia
| | - Louis Maes
- Laboratory of Microbiology, Parasitology and Hygiene, University of Antwerp, Wilrijk, Belgium
| | - Herwig Leirs
- Evolutionary Ecology Group, University of Antwerp, Wilrijk, Belgium
| | - Guy Caljon
- Laboratory of Microbiology, Parasitology and Hygiene, University of Antwerp, Wilrijk, Belgium.
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Eberhardt E, Hendrickx R, Van den Kerkhof M, Monnerat S, Alves F, Hendrickx S, Maes L, Caljon G. Comparative evaluation of nucleic acid stabilizing reagents for RNA- and DNA-based Leishmania detection in blood as proxy for visceral burdens. J Microbiol Methods 2020; 173:105935. [PMID: 32376283 DOI: 10.1016/j.mimet.2020.105935] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2020] [Revised: 04/23/2020] [Accepted: 04/23/2020] [Indexed: 11/28/2022]
Abstract
BACKGROUND Molecular detection techniques using peripheral blood are preferred over invasive tissue aspiration for the diagnosis and post-treatment follow-up of visceral leishmaniasis (VL) patients. This study aims to identify suitable stabilizing reagents to prevent DNA and RNA degradation during storage and transport to specialized laboratories where molecular diagnosis is performed. METHODOLOGY The stabilizing capacities of different commercially available reagents were compared using promastigote-spiked human blood and peripheral blood of Syrian golden hamsters subjected to experimental infection, treatment (miltefosine or aminopyrazole DNDi-1044) and immunosuppression. The impact of various storage temperature conditions was tested in combination with an established kinetoplast DNA (kDNA) qPCR and a recently developed spliced leader RNA (SL-RNA) assay for Leishmania detection. PRINCIPAL FINDINGS Irrespective of the blood type and stabilizer used, threshold (cT) values obtained with the SL-RNA qPCR were systematically lower than those obtained with the kDNA assay, confirming the advantage of the SL-RNA assay over the widely used kDNA assay for low-level Leishmania detection. Peripheral blood parasite levels correlated relatively well with hepatic burdens. RNA protect cell reagent provided the most optimal simultaneous DNA and RNA stabilization in both human and hamster blood. However, this stabilizer requires an erythrocyte lysis step, which can be challenging under field conditions. DNA/RNA shield provides a good alternative for downstream kDNA and SL-RNA assays, especially if sample storage capacity at 4 °C can be guaranteed. CONCLUSIONS/SIGNIFICANCE The recommended stabilizing reagents are compatible with RNA- and DNA-based Leishmania detection in peripheral blood in the VL hamster model and spiked human blood. Since molecular detection techniques using peripheral blood are less invasive than microscopic assessment of tissue aspirates, the findings of this study may be applied to human VL clinical studies.
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Affiliation(s)
- Eline Eberhardt
- Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Wilrijk, Belgium
| | - Rik Hendrickx
- Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Wilrijk, Belgium
| | - Magali Van den Kerkhof
- Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Wilrijk, Belgium
| | | | - Fabiana Alves
- Drugs for Neglected Disease initiative (DNDi), Geneva, Switzerland
| | - Sarah Hendrickx
- Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Wilrijk, Belgium
| | - Louis Maes
- Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Wilrijk, Belgium
| | - Guy Caljon
- Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Wilrijk, Belgium.
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Skarbye AP, Thomsen PT, Krogh MA, Svennesen L, Østergaard S. Effect of automatic cluster flushing on the concentration of Staphylococcus aureus in teat cup liners. J Dairy Sci 2020; 103:5431-5439. [PMID: 32229116 DOI: 10.3168/jds.2019-17785] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2019] [Accepted: 01/27/2020] [Indexed: 11/19/2022]
Abstract
Automatic flushing of milking clusters between milking events is a control measure aimed at reducing transmission of mastitis pathogens from infectious milk to a subsequently milked cow. We evaluated the effect of flushing with cold water and flushing with water containing peracetic acid (PAA) on the concentration of Staphylococcus aureus in teat cup liners. Thirty-two clusters in a swing-over milking parlor (Dairymaster, Causeway, Ireland) were subjected to a simulated milking with S. aureus-contaminated milk. Sixteen clusters were not flushed (controls), whereas 8 clusters were flushed with cold water (966 ± 32 mL) and 8 clusters were flushed with water containing PAA (200 mL/mL). A random teat cup in each cluster was sampled by rinsing with a phosphate buffer. Teat cup samples were cultured on the day following collection on Baird-Parker plates to determine the concentration of S. aureus. In teat cup samples from control clusters, the mean concentration of S. aureus was 2.8 × 105 cfu/mL. The concentration of S. aureus was zero in teat cup samples from clusters flushed with cold water. In teat cup samples from clusters flushed with water containing PAA, the concentration of S. aureus was in general reduced compared with control clusters, but S. aureus was not removed completely. However, the automatic cluster flushing did not function properly when clusters were flushed with water containing PAA; thus, results reflected the effect of inadequate function rather than the effect of adding disinfectant to the flushing water. Before the main study, we conducted a pilot study to evaluate whether teat cup sampling with swabs and sample analysis with quantitative PCR were appropriate methods for the main study. Specifically, we evaluated the effect of swab sample mass on detection of S. aureus by quantitative PCR in the laboratory, Further, we compared PCR and bacterial culture on detection of S. aureus in a suspension following disinfection of the suspension with PAA. We sampled 20 identical S. aureus suspensions for culture and PCR by swabs before and after disinfection with PAA. Swab sample mass was determined by differential weighing and contributed to 46% of the variation observed in detection of S. aureus by PCR. Following disinfection with PAA, S. aureus remained detectable by PCR, although culturability ceased. Based on these results, we sampled teat cups in the main study with a buffer rinse and quantified S. aureus in the samples by bacterial culture. We concluded that automatic cluster flushing with cold water was effective in removing S. aureus from teat cup liners and that addition of PAA was therefore not necessary.
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Affiliation(s)
- Alice P Skarbye
- Department of Animal Science, Aarhus University, DK-8830 Tjele, Denmark.
| | - Peter T Thomsen
- Department of Animal Science, Aarhus University, DK-8830 Tjele, Denmark
| | - Mogens A Krogh
- Department of Animal Science, Aarhus University, DK-8830 Tjele, Denmark
| | - Line Svennesen
- Department of Veterinary and Animal Sciences, University of Copenhagen, DK-1870 Frederiksberg C, Denmark
| | - Søren Østergaard
- Department of Animal Science, Aarhus University, DK-8830 Tjele, Denmark
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Desbrousses C, Archer F, Colin A, Bobet-Erny A, Champavère A, Gros E, Beurdeley P, Cruveiller S, Tardy F, Eloit M. High-Throughput Sequencing (HTS) of newly synthetized RNAs enables one shot detection and identification of live mycoplasmas and differentiation from inert nucleic acids. Biologicals 2020; 65:18-24. [PMID: 32222272 DOI: 10.1016/j.biologicals.2020.03.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2019] [Revised: 02/19/2020] [Accepted: 03/11/2020] [Indexed: 11/26/2022] Open
Abstract
Mycoplasma contamination threatens both the safety of biologics produced in cell substrates as well as the quality of scientific results based on cell-culture observations. Methods currently used to detect contamination of cells include culture, enzymatic activity, immunofluorescence and PCR but suffer from some limitations. High throughput sequencing (HTS) can be used to identify microbes like mycoplasmas in biologics since it enables an unbiased approach to detection without the need to design specific primers to pre-amplify target sequences but it does not enable the confirmation of microbial infection since this could reflect carryover of inert sequences. In order to unambiguously differentiate the presence of live or dead mycoplasmas in biological products, the present method was developed based on metabolic RNA labelling of newly synthetized mycoplasmal RNAs. HTS of labelled RNA detected A549 cell infection with Acholeplasma laidlawii in a manner similar to both PCR and culture and demonstrated that this technique can unambiguously identify bacterial species and differentiates infected cells from cells exposed to a high inoculum of heat-inactivated mycoplasmas. This method therefore combines the advantage of culture (that detects only live microorganisms) with those of molecular tests (rapidity) together with a very broad range of bacterial detection and identification.
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Affiliation(s)
| | - Fabienne Archer
- University of Lyon, Université Claude Bernard Lyon1, INRAE, EPHE, IVPC, Viral Infections and Comparative Pathology, UMR754, F69007, Lyon, France
| | - Adélie Colin
- Université de Lyon, Anses Laboratoire de Lyon, VetAgro Sup, UMR Mycoplasmoses des Ruminants, F69364, Lyon, France
| | - Alexandra Bobet-Erny
- University of Lyon, Université Claude Bernard Lyon1, INRAE, EPHE, IVPC, Viral Infections and Comparative Pathology, UMR754, F69007, Lyon, France
| | - Angélique Champavère
- University of Lyon, Université Claude Bernard Lyon1, INRAE, EPHE, IVPC, Viral Infections and Comparative Pathology, UMR754, F69007, Lyon, France
| | | | | | | | - Florence Tardy
- Université de Lyon, Anses Laboratoire de Lyon, VetAgro Sup, UMR Mycoplasmoses des Ruminants, F69364, Lyon, France
| | - Marc Eloit
- PathoQuest, Paris, France; Ecole Nationale Vétérinaire d'Alfort, Université Paris-Est, Maisons-Alfort, 94704 Cedex, France; Pathogen Discovery Laboratory, Institut Pasteur, Paris, France.
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Zhao Z, Zhang K, Wu N, Li W, Xu W, Zhang Y, Niu Z. Estuarine sediments are key hotspots of intracellular and extracellular antibiotic resistance genes: A high-throughput analysis in Haihe Estuary in China. ENVIRONMENT INTERNATIONAL 2020; 135:105385. [PMID: 31855802 DOI: 10.1016/j.envint.2019.105385] [Citation(s) in RCA: 61] [Impact Index Per Article: 12.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/03/2019] [Revised: 10/17/2019] [Accepted: 12/02/2019] [Indexed: 05/25/2023]
Abstract
Estuaries lie between terrestrial/freshwater and marine ecosystems, receive considerable pollutant input from land-based sources, and are considerably influenced by human activities. However, little attention has been paid to combined research on extracellular antibiotic resistance genes (eARGs) and intracellular ARGs (iARGs) in the estuarine environment. In this study, we profiled eARGs and iARGs in sediments from Haihe Estuary, China by adopting high-throughput quantitative PCR and investigated their relationship with mobile genetic elements (MGEs), the bacterial community and environmental factors. The results showed that the abundance of eARGs ranged from 9.06 × 106 to 1.32 × 108 copies/g and that of iARGs ranged from 3.31 × 107 to 2.93 × 108 copies/g, indicating that estuarine sediments were key hotspots of eARGs and iARGs. Additionally, multidrug resistance genes were both highly diverse and abundant in Haihe Estuary, especially in coastal samples. The high abundance of vancomycin and carbapenemase resistance genes may pose a potential health risk to human. Salinity altered the composition and structure of the bacterial community. Partial redundancy analysis showed that the bacterial community and MGEs appeared to be the major drivers of ARG variance in estuarine sediment. This study provides an overview of the distribution of eARG and iARG along the Haihe Estuary and draws attention to the need to control pollutants in estuary ecosystems.
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Affiliation(s)
- Ze Zhao
- School of Marine Science and Technology, Tianjin University, Tianjin 300072, China
| | - Kai Zhang
- School of Environmental Science and Engineering, Tianjin University, Tianjin 300350, China; Henan Key Laboratory for Synergistic Prevention of Water and Soil Environmental Pollution, School of Geographic Sciences, Xinyang Normal University, Xinyang 464000, China
| | - Nan Wu
- School of Marine Science and Technology, Tianjin University, Tianjin 300072, China
| | - Wenjie Li
- School of Marine Science and Technology, Tianjin University, Tianjin 300072, China
| | - Weian Xu
- School of Marine Science and Technology, Tianjin University, Tianjin 300072, China
| | - Ying Zhang
- MOE Key Laboratory of Pollution Processes and Environmental Criteria, College of Environmental Science and Engineering, Nankai University, Tianjin 300350, China.
| | - Zhiguang Niu
- School of Marine Science and Technology, Tianjin University, Tianjin 300072, China; School of Environmental Science and Engineering, Tianjin University, Tianjin 300350, China.
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Hassan D, Omolo CA, Fasiku VO, Mocktar C, Govender T. Novel chitosan-based pH-responsive lipid-polymer hybrid nanovesicles (OLA-LPHVs) for delivery of vancomycin against methicillin-resistant Staphylococcus aureus infections. Int J Biol Macromol 2020; 147:385-398. [PMID: 31926237 DOI: 10.1016/j.ijbiomac.2020.01.019] [Citation(s) in RCA: 46] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2019] [Revised: 12/18/2019] [Accepted: 01/03/2020] [Indexed: 10/25/2022]
Abstract
The development of novel materials is necessary for adequate delivery of drugs to combat the Methicillin-resistant Staphylococcus aureus (MRSA) burden due to the limitations of conventional methods and challenges associated with antimicrobial resistance. Hence, this study aimed to synthesise a novel oleylamine based zwitterionic lipid (OLA) and explore its potential to formulate chitosan-based pH-responsive lipid-polymer hybrid nanovesicles (VM-OLA-LPHVs1) to deliver VM against MRSA. The OLA was synthesised, and the structure characterised by 1H NMR, 13C NMR, FT-IR and HR-MS. The preliminary biocompatibility of OLA and VM-OLA-LPHVs1 was evaluated on HEK-293, A-549, MCF-7 and HepG-2 cell lines using in vitro cytotoxicity assay. The VM-OLA-LPHVs1 were formulated by ionic gelation method and characterised in order to determine the hydrodynamic diameter (DH), morphology in vitro and in vivo antibacterial efficacy. The result of the in vitro cytotoxicity study revealed cell viability of above 75% in all cell lines when exposed to OLA and VM-OLA-LPHVs1, thus indicating their biosafety. The VM-OLA-LPHVs1 had a DH, polydispersity index (PDI), and EE% of 198.0 ± 14.04 nm, 0.137 ± 0.02, and 45.61 ± 0.54% respectively at physiological pH, with surface-charge (ζ) switching from negative at pH 7.4 to positive at pH 6.0. The VM release from the VM-OLA-LPHVs1 was faster at pH 6.0 compared to physiological pH, with 97% release after 72-h. The VM-OLA-LPHVs1 had a lower minimum inhibitory concentration (MIC) value of 0.59 μg/mL at pH 6.0 compared to 2.39 μg/mL at pH 7.4, against MRSA with 52.9-fold antibacterial enhancement. The flow cytometry study revealed that VM-OLA-LPHVs1 had similar bactericidal efficacy on MRSA compared to bare VM, despite an 8-fold lower VM concentration in the nanovesicles. Additionally, fluorescence microscopy study showed the ability of the VM-OLA-LPHVs1 to eliminate biofilms. The electrical conductivity, and protein/DNA concentration, increased and decreased respectively, as compared to bare VM which indicated greater MRSA membrane damage. The in vivo studies in a BALB/c mouse-infected skin model treated with VM-OLA-LPHVs1 revealed 95-fold lower MRSA burden compared to the group treated with bare VM. These findings suggest that OLA can be used as an effective novel material for complexation with biodegradable polymer chitosan (CHs) to form pH-responsive VM-OLA-LPHVs1 nanovesicles which show greater potential for enhancement and improvement of treatment of bacterial infections.
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Affiliation(s)
- Daniel Hassan
- Discipline of Pharmaceutical Sciences, College of Health Sciences, University of KwaZulu-Natal, Private Bag X54001, Durban, South Africa
| | - Calvin A Omolo
- Discipline of Pharmaceutical Sciences, College of Health Sciences, University of KwaZulu-Natal, Private Bag X54001, Durban, South Africa; United States International University-Africa, School of Pharmacy and Health Sciences, Department of Pharmaceutics, P. O. Box 14634-00800, Nairobi, Kenya.
| | - Victoria Oluwaseun Fasiku
- Discipline of Pharmaceutical Sciences, College of Health Sciences, University of KwaZulu-Natal, Private Bag X54001, Durban, South Africa
| | - Chunderika Mocktar
- Discipline of Pharmaceutical Sciences, College of Health Sciences, University of KwaZulu-Natal, Private Bag X54001, Durban, South Africa
| | - Thirumala Govender
- Discipline of Pharmaceutical Sciences, College of Health Sciences, University of KwaZulu-Natal, Private Bag X54001, Durban, South Africa.
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Research and Technological Advances Regarding the Study of the Spread of Antimicrobial Resistance Genes and Antimicrobial-Resistant Bacteria Related to Animal Husbandry. INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH 2019; 16:ijerph16244896. [PMID: 31817253 PMCID: PMC6950033 DOI: 10.3390/ijerph16244896] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/23/2019] [Revised: 11/30/2019] [Accepted: 12/01/2019] [Indexed: 01/08/2023]
Abstract
The extensive use of antimicrobials in animal farms poses serious safety hazards to both the environment and public health, and this trend is likely to continue. Antimicrobial resistance genes (ARGs) are a class of emerging pollutants that are difficult to remove once introduced. Understanding the environmental transfer of antimicrobial-resistant bacteria (ARB) and ARGs is pivotal for creating control measures. In this review, we summarize the research progress on the spread and detection of ARB and ARG pollution related to animal husbandry. Molecular methods such as high-throughput sequencing have greatly enriched the information about ARB communities. However, it remains challenging to delineate mechanisms regarding ARG induction, transmission, and tempo-spatial changes in the whole process, from animal husbandry to multiple ecosystems. As a result, future research should be more focused on the mechanisms of ARG induction, transmission, and control. We also expect that future research will rely more heavily on metagenomic -analysis, metatranscriptomic sequencing, and multi-omics technologies
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Xu J, Zhao C, Chau Y, Lee YK. The synergy of chemical immobilization and electrical orientation of T4 bacteriophage on a micro electrochemical sensor for low-level viable bacteria detection via Differential Pulse Voltammetry. Biosens Bioelectron 2019; 151:111914. [PMID: 31999572 DOI: 10.1016/j.bios.2019.111914] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2019] [Revised: 11/07/2019] [Accepted: 11/21/2019] [Indexed: 01/02/2023]
Abstract
In this work, a wild-type T4 bacteriophage based micro electrochemical sensor (T4B-MES) was developed for specific and sensitive detection of viable pathogenic bacteria. Recently, bacteriophage has been widely applied as recognition elements for bacteria detection due to its low cost, high stability and specificity. Firstly, a systematic study was proposed in this paper to investigate the synergy of externally applied electric field and chemical functionalization on phage immobilization, involving several key factors such as Debye length. According to our experiments, the capture efficiency of the deposited phages had reached the maximum when the Debye length was comparable to the phage size. With the optimized immobilization protocol, the sensitivity of the T4B-MES was then determined with Differential Pulse Voltammetry (DPV), providing a quite low detection limit of 14 ± 5 cfu/mL and a wide dynamic range of 1.9 × 101-1.9 × 108 cfu/mL. In addition, the T4B-MES demonstrated the ability to distinguish viable and dead bacteria cells with high specificity, making it a promising solution in a variety of applications, e.g., water quality monitoring.
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Affiliation(s)
- Jingting Xu
- Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Hong Kong, China
| | - Cong Zhao
- Department of Mechanical and Aerospace Engineering, The Hong Kong University of Science and Technology, Hong Kong, China
| | - Ying Chau
- Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Hong Kong, China
| | - Yi-Kuen Lee
- Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Hong Kong, China; Department of Mechanical and Aerospace Engineering, The Hong Kong University of Science and Technology, Hong Kong, China.
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45
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Sedki M, Chen X, Chen C, Ge X, Mulchandani A. Non-lytic M13 phage-based highly sensitive impedimetric cytosensor for detection of coliforms. Biosens Bioelectron 2019; 148:111794. [PMID: 31678821 DOI: 10.1016/j.bios.2019.111794] [Citation(s) in RCA: 45] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2019] [Revised: 10/08/2019] [Accepted: 10/17/2019] [Indexed: 12/01/2022]
Abstract
A highly sensitive and selective non-lytic M13 phage-based electrochemical impedance spectroscopy (EIS) cytosensor for early detection of coliforms is introduced for the first time. Gold nanoparticles were electrochemically deposited on the surface of glassy carbon electrode, and the M13 phage particles were immobilized on them using 3-mercaptopropionic acid linker and zero-length crosslinking chemistry (EDC/NHS). Next, the sensor surface was blocked to avoid non-specific binding. The M13-EIS cytosensor was tested for detection of F+ pili Escherichia coli species, using XL1-Blue and K12 strains, as examples of coliforms. The selectivity against non-host strains was demonstrated using Pseudomonas Chlororaphis. The binding of E. coli to the M13 phage on the cytosensor surface increased the charge transfer resistance, enabling detection of coliforms. The biosensor achieved a limit of detection (LOD) of 14 CFU/mL, the lowest reported to-date using EIS-phage sensors, and exhibited a high selectivity towards the tested coliforms. The SEM micrographs confirmed the successful capturing of E. coli on the M13-based EIS cytosensor. Moreover, the sensor showed almost the same sensitivity in the simulated river water samples as in phosphate buffer, reflecting its applicability to real samples. On the other hand, this sensor system exhibited high stability under harsh environmental conditions of pH (3.0-10.0) and temperature as high as 45 °C for up to two weeks. Overall, this sensor system has excellent potential for real field detection of fecal coliforms.
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Affiliation(s)
- Mohammed Sedki
- Department of Materials Science and Engineering, University of California, Riverside, CA, 92521, USA
| | - Xingyu Chen
- Department of Chemical and Environmental Engineering, University of California, Riverside, CA, 92521, USA
| | - Chuan Chen
- Department of Chemical and Environmental Engineering, University of California, Riverside, CA, 92521, USA
| | - Xin Ge
- Department of Chemical and Environmental Engineering, University of California, Riverside, CA, 92521, USA
| | - Ashok Mulchandani
- Department of Chemical and Environmental Engineering, University of California, Riverside, CA, 92521, USA.
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RNA-based qPCR as a tool to quantify and to characterize dual-species biofilms. Sci Rep 2019; 9:13639. [PMID: 31541147 PMCID: PMC6754382 DOI: 10.1038/s41598-019-50094-3] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2019] [Accepted: 09/06/2019] [Indexed: 12/22/2022] Open
Abstract
While considerable research has focused on studying individual-species, we now face the challenge of determining how interspecies interactions alter bacterial behaviours and pathogenesis. Pseudomonas aeruginosa and Staphylococcus aureus are often found to co-infect cystic-fibrosis patients. Curiously, their interaction is reported as competitive under laboratory conditions. Selecting appropriate methodologies is therefore critical to analyse multi-species communities. Herein, we demonstrated the major biases associated with qPCR quantification of bacterial populations and optimized a RNA-based qPCR able not only to quantify but also to characterize microbial interactions within dual-species biofilms composed by P. aeruginosa and S. aureus, as assessed by gene expression quantification. qPCR quantification was compared with flow-cytometry and culture-based quantification. Discrepancies between culture independent and culture dependent methods could be the result of the presence of viable but not-cultivable bacteria within the biofilm. Fluorescence microscopy confirmed this. A higher sensitivity to detect viable cells further highlights the potentialities of qPCR approach to quantify biofilm communities. By using bacterial RNA and an exogenous mRNA control, it was also possible to characterize bacterial transcriptomic profile, being this a major advantage of this method.
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47
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de Oliveira França A, de Oliveira Ramos Pereira L, Ortiz Tanaka TS, Pereira de Oliveira M, Cavalheiros Dorval ME. Viability of Leishmania in blood donors: A tangible possibility of transfusion transmission. JOURNAL OF MICROBIOLOGY, IMMUNOLOGY, AND INFECTION = WEI MIAN YU GAN RAN ZA ZHI 2019; 53:176-178. [PMID: 31427110 DOI: 10.1016/j.jmii.2019.06.010] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/22/2018] [Revised: 04/05/2019] [Accepted: 06/20/2019] [Indexed: 01/09/2023]
Abstract
Asymptomatic individuals apparently able for blood donation, could be infected with Leishmania imposing risks for immunologically vulnerable recipients. Reverse transcribed conventional PCR targeting the 28S ribosomal subunit was conducted, in order to confirm the viability of the parasite in blood donors positive for Leishmania infection.
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Affiliation(s)
- Adriana de Oliveira França
- Laboratory of Clinical Parasitology, Graduate Program in Infectious and Parasitic Diseases, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brazil.
| | - Luiza de Oliveira Ramos Pereira
- Interdisciplinary Laboratory of Medical Research (LIPMed), Laboratory of Leishmaniasis Research, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
| | - Tayana Serpa Ortiz Tanaka
- Graduate Program in Infectious and Parasitic Diseases, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brazil
| | - Márcia Pereira de Oliveira
- Interdisciplinary Laboratory of Medical Research (LIPMed), Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brazil
| | - Maria Elizabeth Cavalheiros Dorval
- Laboratory of Clinical Parasitology, Graduate Program in Infectious and Parasitic Diseases, Universidade Federal de Mato Grosso do Sul, Campo Grande, MS, Brazil
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Dorn-In S, Gareis M, Schwaiger K. Differentiation of live and dead Mycobacterium tuberculosis complex in meat samples using PMA qPCR. Food Microbiol 2019; 84:103275. [PMID: 31421753 DOI: 10.1016/j.fm.2019.103275] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2019] [Revised: 07/15/2019] [Accepted: 07/16/2019] [Indexed: 01/28/2023]
Abstract
The causative agents of zoonotic bovine tuberculosis (bTB), Mycobacterium bovis and M. caprae, are members of the M. tuberculosis complex (MTC). Wildlife such as red deer infected with bTB are often without pathological findings, thus meat thereof may be classified as safe for human consumption. The culturing of MTC is time consuming and inappropriate to be applied with fresh meat and food. Therefore, a rapid method "PMA qPCR" to differentiate living and dead cells of MTC was developed in this study. By treating with 50 μM PMA™ dye, dead M. bovis BCG (≤104 cells/ml meat suspension) could be completely discriminated and was not detected by specific MTC PCR. The limit of detection of MTC without treatment with PMA™ dye was 10 cells/ml. All 50 venison samples obtained for field study purposes were negative for MTC. However, 40% were slightly PCR positive for non-TBC mycobacteria. By culturing using selective enrichment, one single colony of M. avium was isolated. This is the first report on the isolation of M. avium from venison. Considering the difficulties of diagnosing mycobacteria in various matrices, the developed PMA qPCR is applicable for the differentiation of dead and living cells of MTC in meat samples.
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Affiliation(s)
- Samart Dorn-In
- Chair of Food Safety, Faculty of Veterinary Medicine, LMU Munich, Schoenleutnerstr. 8, 85764, Oberschleissheim, Germany.
| | - Manfred Gareis
- Chair of Food Safety, Faculty of Veterinary Medicine, LMU Munich, Schoenleutnerstr. 8, 85764, Oberschleissheim, Germany
| | - Karin Schwaiger
- Chair of Food Safety, Faculty of Veterinary Medicine, LMU Munich, Schoenleutnerstr. 8, 85764, Oberschleissheim, Germany
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Liu TT, Yang H. An RNA-based quantitative and compositional study of ammonium-oxidizing bacteria and archaea in Lake Taihu, a eutrophic freshwater lake. FEMS Microbiol Ecol 2019; 95:5533317. [DOI: 10.1093/femsec/fiz117] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2019] [Accepted: 07/16/2019] [Indexed: 11/14/2022] Open
Abstract
ABSTRACTAmmonium-oxidizing archaea (AOA) and bacteria (AOB) play crucial roles in ammonium oxidation in freshwater lake sediment. However, previous reports on the predominance of AOA and AOB in the surface sediment of Lake Taihu have been based on DNA levels, detecting the total abundance of microbiota (including inactive cells), and have resulted in numerous contradictory conclusions. Existing RNA-level studies detecting active transcription are very limited. The current study, using RNA-based real-time quantification and clone library analysis, demonstrated that the amoA gene abundance of active AOB was higher than that of active AOA, despite conflicting results at the DNA level. Further exploration revealed a significant positive correlation between the potential nitrification rate (PNR) and the abundance of AOA and AOB at the RNA level, with irregular or contradictory correlation found at the DNA level. Ultimately, using quantitative analysis of RNA levels, we show AOB to be the active dominant contributor to ammonium oxidation. Our investigations also indicated that AOB were more diverse in high-ammonium lake regions, with Nitrosomonas being the active and dominating cluster, but that AOA had an advantage in the low-ammonium lake regions.
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Affiliation(s)
- Tong-tong Liu
- State Key Laboratory of Microbial metabolism, and School of Life Science & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, P.R. China
| | - Hong Yang
- State Key Laboratory of Microbial metabolism, and School of Life Science & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, P.R. China
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Abstract
Carbon dots (or carbon quantum dots) are small (less than 10 nm) and luminescent carbon nanoparticles with some form of surface passivation. As an emerging class of nanomaterials, carbon dots have found wide applications in medicine, bioimaging, sensing, electronic devices, and catalysis. In this review, we focus on the recent advancements of carbon dots for sensing and killing microorganisms, including bacteria, fungi, and viruses. Synthesis, functionalization, and a toxicity profile of these carbon dots are presented. We also discuss the underlying mechanisms of carbon dot-based sensing and killing of microorganisms.
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