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Zhuang L, Gong J, Zhao Y, Yang J, Liu G, Zhao B, Song C, Zhang Y, Shen Q. Progress in methods for the detection of viable Escherichia coli. Analyst 2024; 149:1022-1049. [PMID: 38273740 DOI: 10.1039/d3an01750h] [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: 01/27/2024]
Abstract
Escherichia coli (E. coli) is a prevalent enteric bacterium and a necessary organism to monitor for food safety and environmental purposes. Developing efficient and specific methods is critical for detecting and monitoring viable E. coli due to its high prevalence. Conventional culture methods are often laborious and time-consuming, and they offer limited capability in detecting potentially harmful viable but non-culturable E. coli in the tested sample, which highlights the need for improved approaches. Hence, there is a growing demand for accurate and sensitive methods to determine the presence of viable E. coli. This paper scrutinizes various methods for detecting viable E. coli, including culture-based methods, molecular methods that target DNAs and RNAs, bacteriophage-based methods, biosensors, and other emerging technologies. The review serves as a guide for researchers seeking additional methodological options and aiding in the development of rapid and precise assays. Moving forward, it is anticipated that methods for detecting E. coli will become more stable and robust, ultimately contributing significantly to the improvement of food safety and public health.
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Affiliation(s)
- Linlin Zhuang
- School of Animal Husbandry and Veterinary Medicine, Jiangsu Vocational College of Agriculture and Forestry, Jurong 212400, P. R. China.
- State Key Laboratory of Digital Medical Engineering, Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Science and Medical Engineering & Basic Medicine Research and Innovation Center of Ministry of Education, Zhongda Hospital, Southeast University, Nanjing 211102, P. R. China.
| | - Jiansen Gong
- Poultry Institute, Chinese Academy of Agricultural Sciences, Yangzhou 225125, P. R. China
| | - Ying Zhao
- State Key Laboratory of Digital Medical Engineering, Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Science and Medical Engineering & Basic Medicine Research and Innovation Center of Ministry of Education, Zhongda Hospital, Southeast University, Nanjing 211102, P. R. China.
| | - Jianbo Yang
- School of Animal Husbandry and Veterinary Medicine, Jiangsu Vocational College of Agriculture and Forestry, Jurong 212400, P. R. China.
| | - Guofang Liu
- School of Animal Husbandry and Veterinary Medicine, Jiangsu Vocational College of Agriculture and Forestry, Jurong 212400, P. R. China.
| | - Bin Zhao
- School of Animal Husbandry and Veterinary Medicine, Jiangsu Vocational College of Agriculture and Forestry, Jurong 212400, P. R. China.
| | - Chunlei Song
- School of Animal Husbandry and Veterinary Medicine, Jiangsu Vocational College of Agriculture and Forestry, Jurong 212400, P. R. China.
| | - Yu Zhang
- State Key Laboratory of Digital Medical Engineering, Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Science and Medical Engineering & Basic Medicine Research and Innovation Center of Ministry of Education, Zhongda Hospital, Southeast University, Nanjing 211102, P. R. China.
| | - Qiuping Shen
- School of Animal Husbandry and Veterinary Medicine, Jiangsu Vocational College of Agriculture and Forestry, Jurong 212400, P. R. China.
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Zhang F, Zhang F, Dong Y, Li L, Pang Y. New Insights into Biomarkers for Evaluating Therapy Efficacy in Pulmonary Tuberculosis: A Narrative Review. Infect Dis Ther 2023; 12:2665-2689. [PMID: 37938418 PMCID: PMC10746651 DOI: 10.1007/s40121-023-00887-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2023] [Accepted: 10/20/2023] [Indexed: 11/09/2023] Open
Abstract
Evaluating therapy efficacy is crucial for patients with tuberculosis (TB), especially those with drug-resistant tuberculosis (DR-TB). The World Health Organization currently recommends sputum smear and culture as the standard methods for evaluating pulmonary tuberculosis (PTB) therapy efficacy. However, these approaches have limitations including low sensitivity, lengthy culture periods, and susceptibility to contamination. There is an urgent need for dependable biomarkers to evaluate therapy efficacy in patients with PTB. Numerous new biomarkers of Mycobacterium tuberculosis (MTB) and the host have been used in recent studies to evaluate PTB therapy efficacy. A systematic review and update of these biomarkers can facilitate the discovery of novel biomarkers and assessment models, as well as provide a solid scientific basis for alternative indicators of evaluating therapy efficacy. In this review we summarize the recent advancements and limitations of biomarkers used to monitor therapy efficacy, highlighting the importance of utilizing a combination of biomarkers. Although some biomarkers have potential in evaluating the efficacy of therapy in patients with PTB, they also have some limitations. Further research, validation, and optimization are required to identify the most reliable and effective alternative biomarkers and apply them to clinical practice.
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Affiliation(s)
- Fuzhen Zhang
- Department of Bacteriology and Immunology, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, No. 97, Machang, Tongzhou District, Beijing, 101149, People's Republic of China
- Department of Epidemiology, School of Public Health, Cheeloo College of Medicine, Shandong University, Jinan, 250012, People's Republic of China
| | - Fan Zhang
- Department of Epidemiology, School of Public Health, Cheeloo College of Medicine, Shandong University, Jinan, 250012, People's Republic of China
| | - Yu Dong
- Department of Bacteriology and Immunology, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, No. 97, Machang, Tongzhou District, Beijing, 101149, People's Republic of China
| | - Liang Li
- Department of Bacteriology and Immunology, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, No. 97, Machang, Tongzhou District, Beijing, 101149, People's Republic of China.
- Department of Epidemiology, School of Public Health, Cheeloo College of Medicine, Shandong University, Jinan, 250012, People's Republic of China.
| | - Yu Pang
- Department of Bacteriology and Immunology, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, No. 97, Machang, Tongzhou District, Beijing, 101149, People's Republic of China.
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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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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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Surger MJ, Blank LM. Assessment of microbial activity by CO 2 production during heating oil storage. Eng Life Sci 2022; 22:508-518. [PMID: 35936071 PMCID: PMC9349135 DOI: 10.1002/elsc.202100144] [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: 12/18/2021] [Revised: 03/02/2022] [Accepted: 04/05/2022] [Indexed: 11/09/2022] Open
Abstract
Microbial activity is the driving force of the carbon cycle, including the digestion of biomass in the soil, oceans, and oil deposits. This natural diversity of microbial carbon sources poses challenges for humans. Contamination monitoring can be difficult in oil tanks and similar settings. To assess microbial activity in such industrial settings, off-gas analysis can be employed by considering growth and non-growth-associated metabolic activity. In this work, we describe the monitoring of CO2 as a method for measuring microbial activity. We revealed that the CO2 signal corresponds to classical growth curves, exemplified by Pseudomonas fluorescens, Yarrowia lipolytica, and Penicillium chrysogenum. Deviations of the CO2 signal from the growth curves occurred when the yield of biomass on the substrate changed (i.e., the non-growth-associated metabolic activities). We monitored CO2 to track the onset of microbial contamination in an oil tank. This experimental setup was applied to determine the susceptibility of heating oil and biodiesel to microbial contamination long before the formation of problematic biofilms. In summary, the measurement of CO2 production by bacteria, yeasts, and molds allowed the permanent monitoring of microbial activity under oil storage conditions without invasive sampling.
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Affiliation(s)
- Maximilian J. Surger
- Institute of Applied Microbiology (iAMB)Aachen Biology and Biotechnology (ABBt)RWTH Aachen UniversityAachenGermany
| | - Lars M. Blank
- Institute of Applied Microbiology (iAMB)Aachen Biology and Biotechnology (ABBt)RWTH Aachen UniversityAachenGermany
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Xia X, Yang H, Cao J, Zhang J, He Q, Deng R. Isothermal nucleic acid amplification for food safety analysis. Trends Analyt Chem 2022. [DOI: 10.1016/j.trac.2022.116641] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
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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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Baymiev AK, Baymiev AK, Kuluev BR, Shvets KY, Yamidanov RS, Matniyazov RT, Chemeris DA, Zubov VV, Alekseev YI, Mavzyutov AR, Ivanenkov YA, Chemeris AV. Modern Approaches to Differentiation of Live and Dead Bacteria Using Selective Amplification of Nucleic Acids. Microbiology (Reading) 2020. [DOI: 10.1134/s0026261720010038] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
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Kumar SS, Ghosh AR. Assessment of bacterial viability: a comprehensive review on recent advances and challenges. Microbiology (Reading) 2019; 165:593-610. [DOI: 10.1099/mic.0.000786] [Citation(s) in RCA: 55] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Affiliation(s)
- Shravanthi S. Kumar
- Department of Integrative Biology, School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore-632014, Tamil Nadu, India
| | - Asit Ranjan Ghosh
- Department of Integrative Biology, School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore-632014, Tamil Nadu, India
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Ramezani R, Forouzandeh Moghadam M, Rasaee MJ. Development of Sensitive and Rapid RNA Transcription-based Isothermal Amplification Method for Detection of Mycobacterium tuberculosis. Avicenna J Med Biotechnol 2019; 11:169-175. [PMID: 31057719 PMCID: PMC6490409] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
BACKGROUND The accurate and early diagnosis of tuberculosis is important for its effective management. During the last decade, several molecular methods for detection of Tuberculosis (TB) have been developed. Since RNA especially mRNA has a generally much shorter half-life than DNA, its detection may be useful for the assessment of viability of bacteria. This research is a Nucleic Acid Sequence Based Amplification-Enzyme Linked Immunosorbent Assay (NASBA-ELISA) which was designed and developed for rapid detection of viable Mycobacterium tuberculosis (M. tuberculosis). METHODS Oligonucleotide primers targeting tuf gene encoding viability marker EF-Tu mRNAs were selected and used for the amplification of mycobacterial RNA by the isothermal NASBA Digoxigenin (DIG) labeling process and incorporated with DIG-UTP, reverse transcriptase and T7 RNA polymerase. RESULTS Using the NASBA-ELISA system, as little as 17.5 pg of RNA of M. tuberculosis was detected within 4 hr and no interference was encountered in the amplification and detection of viable M. tuberculosis in the presence of non-target RNA or DNA. Results obtained from the clinical specimens showed 97 and 75% of sensitivity and specificity, respectively. CONCLUSION The NASBA-ELISA system offers several advantages in terms of sensitivity, rapidity and simplicity for detection of M. tuberculosis. Furthermore, due to its simplicity and high sensitivity feature, it could be used in limited access laboratories in a cost-effective manner.
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Affiliation(s)
- Reihaneh Ramezani
- Department of Biomedical Sciences, Women Research Center, Alzahra University, Tehran, Iran,Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
| | - Mahdi Forouzandeh Moghadam
- Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran,Corresponding author: Mahdi Forouzandeh Moghadam, Ph.D., Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat, Modares University, Tehran, Iran, Tel: +98 912 3937320, Fax: +98 21 8288386, E-mail:
| | - Mohammad Javad Rasaee
- Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
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Santos Ferreira I, Kikhney J, Kursawe L, Kasper S, Gonçalves LMD, Trampuz A, Moter A, Bettencourt AF, Almeida AJ. Encapsulation in Polymeric Microparticles Improves Daptomycin Activity Against Mature Staphylococci Biofilms-a Thermal and Imaging Study. AAPS PharmSciTech 2018; 19:1625-1636. [PMID: 29488195 DOI: 10.1208/s12249-018-0974-7] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2017] [Accepted: 02/11/2018] [Indexed: 02/07/2023] Open
Abstract
Eradication of Gram-positive biofilms is a critical aspect in implant-associated infection treatment. Although antibiotic-containing particulate carriers may be a promising strategy for overcoming biofilm tolerance, the assessment of their interaction with biofilms has not been fully explored. In the present work, the antibiofilm activity of daptomycin- and vancomycin-loaded poly(methyl methacrylate) (PMMA) and PMMA-Eudragit RL 100 (EUD) microparticles against methicillin-resistant Staphylococcus aureus (MRSA) and polysaccharide intercellular adhesin-positive S. epidermidis biofilms was investigated using isothermal microcalorimetry (IMC) and fluorescence in situ hybridization (FISH). The minimal biofilm inhibitory concentrations (MBIC) of MRSA biofilms, as determined by IMC, were 5 and 20 mg/mL for daptomycin- and vancomycin-loaded PMMA microparticles, respectively. S. epidermidis biofilms were less susceptible, with a MBIC of 20 mg/mL for daptomycin-loaded PMMA microparticles. Vancomycin-loaded microparticles were ineffective. Adding EUD to the formulation caused a 4- and 16-fold reduction of the MBIC values of daptomycin-loaded microparticles for S. aureus and S. epidermidis, respectively. FISH corroborated the IMC results and provided additional insights on the antibiofilm effect of these particles. According to microscopic analysis, only daptomycin-loaded PMMA-EUD microparticles were causing a pronounced reduction in biofilm mass for both strains. Taken together, although IMC indicated that a biofilm inhibition was achieved, microscopy showed that the biofilm was not eradicated and still contained FISH-positive, presumably viable bacteria, thus indicating that combining the two techniques is essential to fully assess the effect of microparticles on staphylococcal biofilms.
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Affiliation(s)
- Inês Santos Ferreira
- Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal
| | - Judith Kikhney
- Biofilmcenter, Deutsches Herzzentrum Berlin, Charité-Universitätsmedizin Berlin, Hindenburgdamm 30, 12203, Berlin, Germany
- Institute for Microbiology and Hygiene, Charité-Universitätsmedizin Berlin, Hindenburgdamm 30, 12203, Berlin, Germany
| | - Laura Kursawe
- Biofilmcenter, Deutsches Herzzentrum Berlin, Charité-Universitätsmedizin Berlin, Hindenburgdamm 30, 12203, Berlin, Germany
| | - Stefanie Kasper
- Biofilmcenter, Deutsches Herzzentrum Berlin, Charité-Universitätsmedizin Berlin, Hindenburgdamm 30, 12203, Berlin, Germany
| | - Lídia M D Gonçalves
- Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal
| | - Andrej Trampuz
- Center for Musculoskeletal Surgery, Charité-Universitätsmedizin Berlin, Free and Humboldt-University of Berlin, Charitéplatz 1, 10117, Berlin, Germany
| | - Annette Moter
- Biofilmcenter, Deutsches Herzzentrum Berlin, Charité-Universitätsmedizin Berlin, Hindenburgdamm 30, 12203, Berlin, Germany
| | - Ana Francisca Bettencourt
- Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal.
| | - António J Almeida
- Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal.
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Juvonen R, Partanen T, Koivula T. Evaluation of Reverse-Transcription PCR Detection of 16S rRNA andTufmRNA for Viable/Dead Discrimination of Beer-Spoilage Lactic Acid Bacteria. JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 2018. [DOI: 10.1094/asbcj-2010-0416-01] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Affiliation(s)
| | | | - Teija Koivula
- VTT Technical Research Centre of Finland, VTT, Finland
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13
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Fang XY, Li WB, Zhang CF, Huang ZD, Zeng HY, Dong Z, Zhang WM. Detecting the Presence of Bacterial DNA and RNA by Polymerase Chain Reaction to Diagnose Suspected Periprosthetic Joint Infection after Antibiotic Therapy. Orthop Surg 2018; 10:40-46. [PMID: 29383856 DOI: 10.1111/os.12359] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/07/2017] [Accepted: 11/23/2017] [Indexed: 01/27/2023] Open
Abstract
OBJECTIVE To explore the diagnostic efficiency of DNA-based and RNA-based quantitative polymerase chain reaction (qPCR) analyses for periprosthetic joint infection (PJI). METHODS To determine the detection limit of DNA-based and RNA-based qPCR in vitro, Staphylococcus aureus and Escherichia coli strains were added to sterile synovial fluid obtained from a patient with knee osteoarthritis. Serial dilutions of samples were analyzed by DNA-based and RNA-based qPCR. Clinically, patients who were suspected of having PJI and eventually underwent revision arthroplasty in our hospital from July 2014 to December 2016 were screened. Preoperative puncture or intraoperative collection was performed on patients who met the inclusion and exclusion criteria to obtain synovial fluid. DNA-based and RNA-based PCR analyses and culture were performed on each synovial fluid sample. The patients' demographic characteristics, medical history, and laboratory test results were recorded. The diagnostic efficiency of both PCR assays was compared with culture methods. RESULTS The in vitro analysis demonstrated that DNA-based qPCR assay was highly sensitive, with the detection limit being 1200 colony forming units (CFU)/mL of S. aureus and 3200 CFU/mL of E. coli. Meanwhile, The RNA-based qPCR assay could detect 2300 CFU/mL of S. aureus and 11 000 CFU/mL of E. coli. Clinically, the sensitivity, specificity, and accuracy were 65.7%, 100%, and 81.6%, respectively, for the culture method; 81.5%, 84.8%, and 83.1%, respectively, for DNA-based qPCR; and 73.6%, 100%, and 85.9%, respectively, for RNA-based qPCR. CONCLUSIONS DNA-based qPCR could detect suspected PJI with high sensitivity after antibiotic therapy. RNA-based qPCR could reduce the false positive rates of DNA-based assays. qPCR-based methods could improve the efficiency of PJI diagnosis.
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Affiliation(s)
- Xin-Yu Fang
- Department of Orthopaedic Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.,Department of Orthopaedic Surgery, The Third Affiliated Hospital of Fujian Medical University, Fuzhou, China
| | - Wen-Bo Li
- Department of Orthopaedic Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China
| | - Chao-Fan Zhang
- Department of Orthopaedic Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China.,Li Ka Shing Faculty of Medicine, Department of Orthopaedics and Traumatology, The University of Hong Kong, Hong Kong SAR, China
| | - Zi-da Huang
- Department of Orthopaedic Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China
| | - Hui-Yi Zeng
- Department of Orthopaedic Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China
| | - Zheng Dong
- Department of Orthopaedic Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China
| | - Wen-Ming Zhang
- Department of Orthopaedic Surgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China
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de Knegt GJ, Dickinson L, Pertinez H, Evangelopoulos D, McHugh TD, Bakker-Woudenberg IAJM, Davies GR, de Steenwinkel JEM. Assessment of treatment response by colony forming units, time to culture positivity and the molecular bacterial load assay compared in a mouse tuberculosis model. Tuberculosis (Edinb) 2017; 105:113-118. [PMID: 28610782 DOI: 10.1016/j.tube.2017.05.002] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2016] [Revised: 05/01/2017] [Accepted: 05/07/2017] [Indexed: 01/03/2023]
Abstract
The aim of the study is to compare counting of colony forming units (CFU), the time to positivity (TTP) assay and the molecular bacterial load (MBL) assay, and explore whether the last assays can detect a subpopulation which is unable to grown on solid media. CFU counting, TTP and the MBL assay were used to determine the mycobacterial load in matched lung samples of a murine tuberculosis model. Mice were treated for 24 weeks with 4 treatment arms: isoniazid (H) - rifampicin (R) - pyrazinamide (Z), HRZ-Streptomycin (S), HRZ - ethambutol (E) or ZES. Inverse relationships were observed when comparing TPP with CFU or MBL. Positive associations were observed when comparing CFU with MBL. Description of the net elimination of bacteria was performed for CFU vs. time, MBL vs. time and 1/TTP vs. time and fitted by nonlinear regression. CFU vs. time and 1/TTP vs. time showed bi-phasic declines with the exception of HRZE. A similar rank order, based on the alpha slope, was found comparing CFU vs. time and TTP vs. time, respectively HRZE, HRZ, HRZS and ZES. In contrast, MBL vs. time showed a mono-phasic decline with a flat gradient of elimination and a different rank order respectively, ZES, HRZ, HRZE and HRZS. The correlations found between methods reflects the ability of each to discern the general mycobacterial load. Based on the description of net elimination, we conclude that the MBL assay can detect a subpopulation of Mycobacterium tuberculosis which is not detected by the CFU or TTP assays.
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Affiliation(s)
- Gerjo J de Knegt
- Erasmus MC, University Medical Centre Rotterdam, Department of Medical Microbiology & Infectious Diseases, Rotterdam, The Netherlands.
| | - Laura Dickinson
- Department of Molecular & Clinical Pharmacology, University of Liverpool, Liverpool, United Kingdom
| | - Henry Pertinez
- Department of Molecular & Clinical Pharmacology, University of Liverpool, Liverpool, United Kingdom
| | | | - Timothy D McHugh
- Centre for Clinical Microbiology, University College London, London, United Kingdom
| | - Irma A J M Bakker-Woudenberg
- Erasmus MC, University Medical Centre Rotterdam, Department of Medical Microbiology & Infectious Diseases, Rotterdam, The Netherlands
| | - Gerry R Davies
- Department of Molecular & Clinical Pharmacology, University of Liverpool, Liverpool, United Kingdom
| | - Jurriaan E M de Steenwinkel
- Erasmus MC, University Medical Centre Rotterdam, Department of Medical Microbiology & Infectious Diseases, Rotterdam, The Netherlands
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Loonen AJM, Wolffs PFG, de Bresser M, Habraken M, Bruggeman CA, Hermans MHA, van den Brule AJC. Tuf mRNA rather than 16S rRNA is associated with culturable Staphylococcus aureus. World J Clin Infect Dis 2015; 5:86-93. [DOI: 10.5495/wjcid.v5.i4.86] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/09/2015] [Revised: 04/20/2015] [Accepted: 06/11/2015] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the presence of various nucleic acids targets of Staphylococcus aureus (S. aureus) during bacterial growth and antibiotic induced killing in relation to viability.
METHODS: S. aureus was cultured to log phase and spiked in Todd Hewitt (TH) broth and whole blood of healthy human volunteers. Viability of S. aureus after flucloxacillin treatment (0, 1, 3 and 6 d) was assessed by culture on bloodagar plates. DNA and RNA were isolated from 200 μL. cDNA synthesis was performed by using random primers. The presence of S. aureus DNA, rRNA, and mRNA were determined by real-time polymerase chain reaction of the 16S rDNA and tuf gene (elongation factor Tu).
RESULTS: S. aureus spiked in TH broth without antibiotics grew from day 0-6 and DNA (tuf and 16S), and 16S rRNA remained detectable during this whole period. During flucloxacillin treatment S. aureus lost viability from day 3 onwards, while the 16S rRNA-gene and its RNA transcripts remained detectable. DNA and rRNA can be detected in flucloxacillin treated S. aureus cultures that do not further contain culturable bacteria. However, tuf mRNA became undetectable from day 3 onwards. Tuf mRNA can only be detected from samples with culturable bacteria. When spiking S. aureus in whole blood instead of broth no bacterial growth was seen, neither in the absence nor in the presence of flucloxacillin. Accordingly, no increase in DNA and RNA levels of both 16S rDNA and the tuf gene were detected.
CONCLUSION: Tuf mRNA expression is associated with culturable S. aureus and might be used to monitor antibiotic effects.
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Branco P, Monteiro M, Moura P, Albergaria H. Survival rate of wine-related yeasts during alcoholic fermentation assessed by direct live/dead staining combined with fluorescence in situ hybridization. Int J Food Microbiol 2012; 158:49-57. [PMID: 22819715 DOI: 10.1016/j.ijfoodmicro.2012.06.020] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2012] [Revised: 06/21/2012] [Accepted: 06/30/2012] [Indexed: 10/28/2022]
Abstract
Real-time detection of microorganisms involved in complex microbial process, such as wine fermentations, and evaluation of their physiological state is crucial to predict whether or not those microbial species will be able to impact the final product. In the present work we used a direct live/dead staining (LDS) procedure combined with fluorescence in situ hybridization (FISH) to simultaneously assess the identity and viability of Saccharomyces cerevisiae (Sc) and Hanseniaspora guilliermondii (Hg) during fermentations performed with single and mixed cultures. The population evolution of both yeasts was determined by plating and by LDS combined with species-specific FISH-probes labeled with Fluorescein. Since the FISH method involves the permeabilization of the cell membrane prior to hybridization and that it may influence the free diffusion of PI in and out of the cells, we optimized the concentration of this dye (0.5 μg of PI per 10(6) cells) for minimal diffusion (less than 2%). Fluorescent cells were enumerated by hemocytometry and flow cytometry. Results showed that the survival rate of Sc during mixed cultures was high throughout the entire process (60% of viable cells at the 9th day), while Hg began to die off at the 2nd day, exhibited 98% of dead cells at the 3rd day (45 g/l of ethanol) and became completely unculturable at the 4th day. However, under single culture fermentation the survival rate and culturability of Hg decreased at a much slower pace, exhibiting at the 7th day (67 g/l of ethanol) 8.7×10(4) CFU/ml and 85% of dead cells. Thus, our work demonstrated that the LDS-FISH method is able to simultaneously assess the viability and identity of these wine-related yeast species during alcoholic fermentation in a fast and reliable way. In order to validate PI-staining as a viability marker during alcoholic fermentation, we evaluated the effect of ethanol on the membrane permeability of Sc and Hg cells, as well as their capacity to recover membrane integrity after being exposed to different levels of ethanol (1%, 6%, 10%, 12% v/v). Results showed that while Sc cells were able to recover membrane integrity after ethanol exposure, Hg cells were not. However, under alcoholic fermentation Sc cells didn't recover membrane integrity after the mid-term (4-5 days) of alcoholic fermentation.
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Affiliation(s)
- Patrícia Branco
- Unidade Bioenergia, LNEG, Estrada do Paço do Lumiar, 22, 1649-038 Lisboa, Portugal
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Aarthi P, Bagyalakshmi R, Therese KL, Malathi J, Mahalakshmi B, Madhavan HNR. Optimization and application of a reverse transcriptase polymerase chain reaction to determine the bacterial viability in infectious endophthalmitis. Curr Eye Res 2012; 37:1114-20. [PMID: 22757687 DOI: 10.3109/02713683.2012.704476] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
PURPOSE To develop RNA based assay - reverse transcriptase polymerase chain reaction (RT-PCR) to detect viable bacteria in intraocular specimens obtained from patients with infectious endophthalmitis. MATERIALS AND METHODS Thirty-five intraocular specimens (19 vitreous fluid and 16 aqueous humor) collected from patients with typical infectious endophthalmitis were subjected to conventional and molecular microbiological investigations. Culture negative, eubacterial genome PCR positive intraocular specimens were subjected to denaturing high performance liquid chromatography (dHPLC) for separation of mixed genomes and subsequently identified by PCR based DNA sequencing. In parallel, RT-PCR was performed to detect the presence of viable bacteria in intraocular specimens. RESULTS Among 35 intraocular specimens, single bacterial genome was detected in 9 (25.7%) and two or more genomes in 26 (74.28%) intraocular specimens. Eubacterial genome was detected by RT-PCR in 29 (82.85%) specimens. PCR based dHPLC followed by PCR based DNA sequencing revealed the presence of 65 bacterial genomes in 35 intraocular specimens. Five novel genera namely Terrabacter species, Facklamia species, Xylella fastidiosa, Duganella species and Synechococcus species were detected. CONCLUSION RT-PCR serves as a rapid and reliable tool to detect viable bacteria causing endophthalmitis.
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Affiliation(s)
- Pasupathi Aarthi
- Larsen and Toubro Microbiology Research Centre, Kamal Nayan Bajaj Institute for Research in Vision and Ophthalmology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India
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Andorra I, Monteiro M, Esteve-Zarzoso B, Albergaria H, Mas A. Analysis and direct quantification of Saccharomyces cerevisiae and Hanseniaspora guilliermondii populations during alcoholic fermentation by fluorescence in situ hybridization, flow cytometry and quantitative PCR. Food Microbiol 2011; 28:1483-91. [DOI: 10.1016/j.fm.2011.08.009] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2010] [Revised: 07/29/2011] [Accepted: 08/07/2011] [Indexed: 10/17/2022]
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Hao X, Wang Q, Cao Y, van Loosdrecht MCM. Evaluating sludge minimization caused by predation and viral infection based on the extended activated sludge model No. 2d. WATER RESEARCH 2011; 45:5130-5140. [PMID: 21821271 DOI: 10.1016/j.watres.2011.07.013] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/27/2011] [Revised: 07/11/2011] [Accepted: 07/12/2011] [Indexed: 05/31/2023]
Abstract
The Activated Sludge Model No. 2d (ASM2d) was extended to incorporate the processes of both predation and viral infection. The extended model was used to evaluate the contributions of predation and viral infection to sludge minimization in a sequencing batch reactor (SBR) system enriching polyphosphate-accumulating organisms (PAOs). Three individual decay processes formulated according to the general model rules were used in the extended model. The model was firstly calibrated and validated by different experimental results. It was used to evaluate the potential extent of predation and viral infection on sludge minimization. Simulations indicate that predation contributes roughly two times more to sludge minimization than viral infection in the SBR system enriching PAOs. The sensitivity analyses of the selected key parameters reveal that there are thresholds on both predation and viral infection rates, if they are too large a minimal sludge retention time is obtained and the effluent quality is deteriorating. Due to the thresholds, the contributions of predation and viral infection to sludge minimization are limited to a maximal extent of about 21% and 9%, respectively. However, it should be noted that the parameters concerning predation and viral infection were not calibrated separately by independent experiment in our study due to the lack of an effective method, especially for the parameters regarding viral infection. Therefore, it is essential to better evaluate these parameters in the future.
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Affiliation(s)
- Xiaodi Hao
- Key Laboratory of Urban Stormwater System and Water Environment/R & D Centre for Sustainable Environmental Biotechnology, Beijing University of Civil Engineering and Architecture, Ministry of Education, 1 Zhanlanguan Road, Xicheng District, Beijing 100044, PR China.
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Molecular bacterial load assay, a culture-free biomarker for rapid and accurate quantification of sputum Mycobacterium tuberculosis bacillary load during treatment. J Clin Microbiol 2011; 49:3905-11. [PMID: 21900522 DOI: 10.1128/jcm.00547-11] [Citation(s) in RCA: 86] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
A molecular assay to quantify Mycobacterium tuberculosis is described. In vitro, 98% (n = 96) of sputum samples with a known number of bacilli (10(7) to 10(2) bacilli) could be enumerated within 0.5 log(10). In comparison to culture, the molecular bacterial load (MBL) assay is unaffected by other microorganisms present in the sample, results are obtained more quickly (within 24 h) and are seldom inhibited (0.7% samples), and the MBL assay critically shows the same biphasic decline as observed longitudinally during treatment. As a biomarker of treatment response, the MBL assay responds rapidly, with a mean decline in bacterial load for 111 subjects of 0.99 log(10) (95% confidence interval [95% CI], 0.81 to 1.17) after 3 days of chemotherapy. There was a significant association between the rate of bacterial decline during the same 3 days and bacilli ml(-1) sputum at day 0 (linear regression, P = 0.0003) and a 3.62 increased odds ratio of relapse for every 1 log(10) increase in pretreatment bacterial load (95% CI, 1.53 to 8.59).
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Almeida C, Azevedo NF, Santos S, Keevil CW, Vieira MJ. Discriminating multi-species populations in biofilms with peptide nucleic acid fluorescence in situ hybridization (PNA FISH). PLoS One 2011; 6:e14786. [PMID: 21479268 PMCID: PMC3066202 DOI: 10.1371/journal.pone.0014786] [Citation(s) in RCA: 115] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2010] [Accepted: 10/11/2010] [Indexed: 11/25/2022] Open
Abstract
BACKGROUND Our current understanding of biofilms indicates that these structures are typically composed of many different microbial species. However, the lack of reliable techniques for the discrimination of each population has meant that studies focusing on multi-species biofilms are scarce and typically generate qualitative rather than quantitative data. METHODOLOGY/PRINCIPAL FINDINGS We employ peptide nucleic acid fluorescence in situ hybridization (PNA FISH) methods to quantify and visualize mixed biofilm populations. As a case study, we present the characterization of Salmonella enterica/Listeria monocytogenes/Escherichia coli single, dual and tri-species biofilms in seven different support materials. Ex-situ, we were able to monitor quantitatively the populations of ∼56 mixed species biofilms up to 48 h, regardless of the support material. In situ, a correct quantification remained more elusive, but a qualitative understanding of biofilm structure and composition is clearly possible by confocal laser scanning microscopy (CLSM) at least up to 192 h. Combining the data obtained from PNA FISH/CLSM with data from other established techniques and from calculated microbial parameters, we were able to develop a model for this tri-species biofilm. The higher growth rate and exopolymer production ability of E. coli probably led this microorganism to outcompete the other two [average cell numbers (cells/cm(2)) for 48 h biofilm: E. coli 2,1 × 10(8) (± 2,4 × 10(7)); L. monocytogenes 6,8 × 10(7) (± 9,4 × 10(6)); and S. enterica 1,4 × 10(6) (± 4,1 × 10(5))]. This overgrowth was confirmed by CSLM, with two well-defined layers being easily identified: the top one with E. coli, and the bottom one with mixed regions of L. monocytogenes and S. enterica. SIGNIFICANCE While PNA FISH has been described previously for the qualitative study of biofilm populations, the present investigation demonstrates that it can also be used for the accurate quantification and spatial distribution of species in polymicrobial communities. Thus, it facilitates the understanding of interspecies interactions and how these are affected by changes in the surrounding environment.
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Affiliation(s)
- Carina Almeida
- Institute for Biotechnology and Bioengineering (IBB), Centre of Biological Engineering, Universidade do Minho, Campus de Gualta, Braga, Portugal
- Environmental Healthcare Unit, School of Biological Sciences, University of Southampton, Southampton, United Kingdom
| | - Nuno F. Azevedo
- Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal
| | - Sílvio Santos
- Institute for Biotechnology and Bioengineering (IBB), Centre of Biological Engineering, Universidade do Minho, Campus de Gualta, Braga, Portugal
| | - Charles W. Keevil
- Environmental Healthcare Unit, School of Biological Sciences, University of Southampton, Southampton, United Kingdom
| | - Maria J. Vieira
- Institute for Biotechnology and Bioengineering (IBB), Centre of Biological Engineering, Universidade do Minho, Campus de Gualta, Braga, Portugal
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Mitchell RM, Gollnick NS, Sreevatsan S, Russell DG, Schukken YH. Quantification of Mycobacterium avium subsp. paratuberculosis (MAP) survival in monocyte-derived macrophages. Vet Immunol Immunopathol 2010; 139:73-8. [PMID: 20832125 DOI: 10.1016/j.vetimm.2010.08.003] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2010] [Revised: 07/06/2010] [Accepted: 08/09/2010] [Indexed: 10/19/2022]
Abstract
Real-time PCR assays were developed to quantitate Mycobacterium avium subsp. paratuberculosis (MAP) in bovine monocyte-derived macrophages. We measured the absolute number of both host cells and bacteria in in vitro challenge assays. Results obtained from real-time quantitative PCR (qPCR) DNA copy counts were compared to visual quantitation of fluorescent-stained MAP in macrophages. Conclusions from our original visual analysis were supported by the second (qPCR) methodology; however, the qPCR assay proved to be more consistent between samples and was easier to perform. There was a strain-to-strain difference in growth curves between fluorescent quantitation (FQ) and qPCR that we believe to be a consequence of bacterial growth characteristics in FQ. In summary, real-time PCR assays provided a more accurate and precise method for evaluating intracellular growth dynamics when comparing strains of MAP.
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Affiliation(s)
- Rebecca M Mitchell
- Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
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Kiraz N, Saglik I, Kiremitci A, Kasifoglu N, Akgun Y. Evaluation of the GenoType Mycobacteria Direct assay for direct detection of the Mycobacterium tuberculosis complex obtained from sputum samples. J Med Microbiol 2010; 59:930-934. [PMID: 20448064 DOI: 10.1099/jmm.0.013490-0] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
An increase in the prevalence of tuberculosis (TB) in recent years has accelerated the search for novel tools for the rapid diagnosis of TB infection. This study evaluated the GenoType Mycobacteria Direct (GTMD) assay (Hain Lifescience) for direct detection of the Mycobacterium tuberculosis complex (MTBC) from sputum samples and compared it with conventional methods. The GTMD test is a commercial assay produced using strip techniques and works based on a nucleic acid sequence-based amplification technique. This test allows 23S rRNA amplification-based detection of MTBC, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii and Mycobacterium malmoense directly from decontaminated clinical samples within 6 h. In the present study, 115 sputum samples were processed to detect acid-fast bacilli (AFB) using two microscopy methods (carbol fuchsin and fluorescent staining), two culture methods [Löwenstein-Jensen (LJ) and BACTEC 12B media] and the GTMD test. The results showed that 86 of the samples were positive by direct microscopy, 84 were positive by BACTEC 12B culture, 73 were positive by LJ culture and 95 were positive by the GTMD test. All of the isolates turned out to be MTBC. Moreover, the sensitivity and specificity of the GTMD test for MTBC in patients were 97 and 58 %, respectively, taking the culture combination as the gold standard. When the test was compared with culture of samples from anti-TB-treated patients, the sensitivity and specificity for the test were 100 and 15 %, respectively. Low specificity in treated people might arise from depressed proliferation of AFB. As the two methods target the same living bacilli, the difference is obviously notable. When the culture results and clinical findings of the patients were evaluated together (true-positive specimens), the sensitivity and specificity values of the GTMD test for all patients were 97 and 90 %, respectively. However, both of these values increased to 100 % for the patients receiving anti-TB treatment. These results implied that, to determine whether the patient's sputum contains living AFB, more sensitive techniques should be employed during the follow-up of the patients. These observations suggest that the GTMD method can be useful for early diagnosis of clinically and radiologically suspicious TB cases where smears are negative for Mycobacterium. In addition, the use of a GTMD test in smear-positive cases is helpful and practical in order to identify MTBC quickly. This allows more rapid treatment decisions and infection control precautions.
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Affiliation(s)
- Nuri Kiraz
- Eskisehir Osmangazi University, Faculty of Medicine, Department of Microbiology, Eskisehir, Turkey
| | - Imran Saglik
- Eskisehir Osmangazi University, Faculty of Medicine, Department of Microbiology, Eskisehir, Turkey
| | - Abdurrahman Kiremitci
- Eskisehir Osmangazi University, Faculty of Medicine, Department of Microbiology, Eskisehir, Turkey
| | - Nilgun Kasifoglu
- Eskisehir Osmangazi University, Faculty of Medicine, Department of Microbiology, Eskisehir, Turkey
| | - Yurdanur Akgun
- Eskisehir Osmangazi University, Faculty of Medicine, Department of Microbiology, Eskisehir, Turkey
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Kao MC, Durst RA. Detection of Escherichia coliUsing Nucleic Acid Sequence-Based Amplification and Oligonucleotide Probes for 16S Ribosomal RNA. ANAL LETT 2010. [DOI: 10.1080/00032711003654005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
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Rôças IN, Siqueira JF. Identification of bacteria enduring endodontic treatment procedures by a combined reverse transcriptase-polymerase chain reaction and reverse-capture checkerboard approach. J Endod 2010; 36:45-52. [PMID: 20003934 DOI: 10.1016/j.joen.2009.10.022] [Citation(s) in RCA: 82] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2009] [Revised: 10/13/2009] [Accepted: 10/18/2009] [Indexed: 11/15/2022]
Abstract
INTRODUCTION This study identified the bacterial taxa enduring endodontic treatment procedures by using a combined 16S ribosomal RNA-based reverse-transcriptase polymerase chain reaction (RT-PCR) and reverse-capture checkerboard hybridization approach. METHODS Samples were taken from infected canals of 15 teeth with apical periodontitis before treatment (S1), after chemomechanical preparation with NaOCl as the irrigant (S2), and after interappointment medication with a calcium hydroxide paste (S3). Bacterial presence was first screened by a DNA-based single PCR assay. RNA extracts were subjected to RT-PCR, and the resulting products were surveyed for the presence of 28 targeted taxa by using the checkerboard method. RESULTS Bacteria were found in all S1 samples. Detectable levels of bacterial ribosomal RNA, used as an indicator of viability, were observed in 60% of the cases after chemomechanical preparation and 53% after intracanal medication. The most prevalent taxa in S1 were Olsenella uli (67%), Pyramidobacter piscolens (60%), Streptococcus species (53%), and Bacteroidetes clone X083 (53%). Streptococcus species (47%), Fusobacterium nucleatum (40%), and O. uli (33%) prevailed in S2, whereas Streptococcus species (47%), Propionibacterium acnes (27%), and O. uli (27%) were the most frequent taxa in S3. CONCLUSIONS The present study with a combined molecular approach revealed that bacterial diversity was overall markedly reduced by treatment procedures. Although bacterial taxa more frequently identified in post-treatment samples emerge as potential risk factors for persistent disease, this remains to be determined by longitudinal studies.
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Affiliation(s)
- Isabela N Rôças
- Department of Endodontics and Molecular Microbiology Laboratory, Faculty of Dentistry, Estácio de Sá University, Rio de Janeiro, RJ, Brazil
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Bergin PF, Doppelt JD, Hamilton WG, Mirick GE, Jones AE, Sritulanondha S, Helm JM, Tuan RS. Detection of periprosthetic infections with use of ribosomal RNA-based polymerase chain reaction. J Bone Joint Surg Am 2010; 92:654-63. [PMID: 20194324 PMCID: PMC2827826 DOI: 10.2106/jbjs.i.00400] [Citation(s) in RCA: 80] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
BACKGROUND Previously described molecular biology techniques used to detect periprosthetic infections have been complicated by false-positive results. We have reported the development of a messenger RNA (mRNA)-based procedure to reduce these false-positive results. The limitations of this procedure are the lack of a universal target and reduced sensitivity due to a low concentration of bacterial mRNAs in test samples. The objective of the present study was to determine whether reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using universal primers can be used to detect the more abundant bacterial ribosomal RNA (rRNA) as an indicator of periprosthetic infection. METHODS Serial dilutions of simulated synovial fluid infections were analyzed with rRNA RT-qPCR to determine the detection limit of this assay. Escherichia coli cultures treated with gentamicin were analyzed with RT-qPCR over a twenty-day time course to determine the degradation of rRNA as compared with the decrease in the viable cell count as determined by means of cell plating. As a proof of concept, group-specific polymerase chain reaction primers were developed for Streptococcus species and were tested against fifteen orthopaedically relevant organisms to show the potential for speciation with this assay. Sixty-four patients with a symptomatic effusion at the site of a total knee arthroplasty were enrolled, and complete patient information was documented in a prospective manner. Synovial fluid analysis with rRNA RT-qPCR was performed in a blind fashion. RESULTS The rRNA RT-qPCR assay was able to detect as few as 590 colony forming units/mL of Staphylococcus aureus and 2900 colony forming units/mL of Escherichia coli. The rRNA RT-qPCR signal closely followed cell death, pointing to its potential use as a viability marker. Three group-specific primer sets correctly identified their intended targets without amplifying closely related species. Clinically, the test correctly identified all six patients with a confirmed infection and all fifty patients who clearly did not have an infection. Eight patients had some laboratory or clinical signs of infection, but their status could not be confirmed. Infection was indicated by rRNA RT-qPCR in three of these patients who had elevated synovial fluid white blood-cell counts but negative results on culture. For statistical purposes, all patients who were categorized as indeterminate were considered to have an infection for the purpose of analysis, for a prevalence of 22% in this cohort. CONCLUSIONS With respect to current diagnostic tests, rRNA-based RT-qPCR demonstrated 100% specificity and positive predictive value with a sensitivity equivalent to that of intraoperative culture. The RT-qPCR signal followed bacterial culture trends but exhibited detectable level for seven days after sterilization, allowing for the detection of infection after the antibiotic administration. These findings indicate that rRNA RT-qPCR is a sensitive and reliable test that retains the universal detection and speciation of DNA-based methods while functioning as a viability indicator.
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Affiliation(s)
- Patrick F. Bergin
- Department of Orthopaedic Surgery, George Washington University, 2150 Pennsylvania Avenue N.W., Washington, DC 20037
| | - Jason D. Doppelt
- Department of Orthopaedic Surgery, George Washington University, 2150 Pennsylvania Avenue N.W., Washington, DC 20037
| | - William G. Hamilton
- Anderson Orthopaedic Research Institute, 2501 Parker's Lane, Alexandria, VA 22306
| | - Gudrun E. Mirick
- Department of Orthopaedic Surgery, George Washington University, 2150 Pennsylvania Avenue N.W., Washington, DC 20037
| | - Angela E. Jones
- Department of Orthopaedic Surgery, George Washington University, 2150 Pennsylvania Avenue N.W., Washington, DC 20037
| | | | - Jeannine M. Helm
- Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Health and Human Services, Room 1140, Building 50, MSC 8022, Bethesda, MD 20892-8022
| | - Rocky S. Tuan
- Department of Orthopaedic Surgery, Center for Cellular and Molecular Engineering, University of Pittsburgh School of Medicine, 450 Technology Drive, Room 221, Pittsburgh, PA 15219. E-mail address for R.S. Tuan:
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Cenciarini-Borde C, Courtois S, La Scola B. Nucleic acids as viability markers for bacteria detection using molecular tools. Future Microbiol 2009; 4:45-64. [PMID: 19207099 DOI: 10.2217/17460913.4.1.45] [Citation(s) in RCA: 69] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
A large set of nucleic acid detection methods with good sensitivity and specificity are now available for the detection of pathogens in clinical, food and environmental samples. Given increasing demand, many efforts have been made to combine these methods to assess viability. Genomic DNA PCR amplification has been shown to be inappropriate for distinguishing viable from dead bacteria owing to DNA stability. Many authors have tried to bypass this difficulty by switching to RNA amplification methods such as reverse transcription-PCR and nucleic acid sequence-based amplification. More recently, researchers have developed methods combining specific sample pretreatment with nucleic acid detection methods, notably ethidium or propidium monoazide pretreatment coupled with PCR DNA detection or direct viable count methods and subsequent fluorescent in situ hybridization of 16S rRNA. This review evaluates the performance of these different methods for viability assessment.
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Affiliation(s)
- Claire Cenciarini-Borde
- CIRSEE (Centre International de Recherche Sur l'Eau et l'Environnement) - Suez Environment, 38 Rue Du Président Wilson 78230 Le Pecq, France.
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Cenciarini C, Courtois S, Raoult D, La Scola B. Influence of long time storage in mineral water on RNA stability of Pseudomonas aeruginosa and Escherichia coli after heat inactivation. PLoS One 2008; 3:e3443. [PMID: 18941615 PMCID: PMC2566809 DOI: 10.1371/journal.pone.0003443] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2008] [Accepted: 09/18/2008] [Indexed: 12/18/2022] Open
Abstract
Background Research of RNA viability markers was previously studied for many bacterial species. Few and different targets of each species have been checked and motley results can be found in literature. No research has been done about Pseudomonas aeruginosa in this way. Methodology/Principal Findings Disappearance of 48 transcripts was analyzed by two-steps reverse transcription and real time polymerase chain reaction (RT-PCR) after heat-killing of Pseudomonas aeruginosa previously stored in mineral water or not. Differential results were obtained for each target. 16S rRNA, 23S rRNA, groEL, and rpmE were showed as the most persistent transcripts and rplP, rplV, rplE and rpsD were showed as the most labile transcripts after P. aeruginosa death. However, the labile targets appeared more persistent in bacteria previously stored in mineral water than freshly cultivated (non stored). These nine transcripts were also analyzed in Escherichia coli after heat-killing and different to opposite results were obtained, notably for groEL which was the most labile transcript of E. coli. Moreover, opposite results were obtained between mineral water stored and freshly cultivated E. coli. Conclusions and Significance This study highlights four potential viability markers for P. aeruginosa and four highly persistent transcripts. In a near future, these targets could be associated to develop an efficient viability kit. The present study also suggests that it would be difficult to determine universal RNA viability markers for environmental bacteria, since opposite results were obtained depending on the bacterial species and the physiological conditions.
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Affiliation(s)
- Claire Cenciarini
- CIRSEE (Centre International de Recherche Sur l'Eau et l'Environnement) – Suez Environnement, Le Pecq, France
- URMITE, CNRS-IRD UMR 6236, Université de la Méditerranée, Faculté de Médecine, Marseille, France
| | - Sophie Courtois
- CIRSEE (Centre International de Recherche Sur l'Eau et l'Environnement) – Suez Environnement, Le Pecq, France
| | - Didier Raoult
- URMITE, CNRS-IRD UMR 6236, Université de la Méditerranée, Faculté de Médecine, Marseille, France
| | - Bernard La Scola
- URMITE, CNRS-IRD UMR 6236, Université de la Méditerranée, Faculté de Médecine, Marseille, France
- * E-mail:
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Lahtinen S, Ahokoski H, Reinikainen J, Gueimonde M, Nurmi J, Ouwehand A, Salminen S. Degradation of 16S rRNA and attributes of viability of viable but nonculturable probiotic bacteria. Lett Appl Microbiol 2008; 46:693-8. [DOI: 10.1111/j.1472-765x.2008.02374.x] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
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GRACIAS KIEVS, MCKILLIP JOHNL. NUCLEIC ACID SEQUENCE-BASED AMPLIFICATION (NASBA) IN MOLECULAR BACTERIOLOGY: A PROCEDURAL GUIDE. ACTA ACUST UNITED AC 2007. [DOI: 10.1111/j.1745-4581.2007.00099.x] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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Aellen S, Que YA, Guignard B, Haenni M, Moreillon P. Detection of live and antibiotic-killed bacteria by quantitative real-time PCR of specific fragments of rRNA. Antimicrob Agents Chemother 2006; 50:1913-20. [PMID: 16723545 PMCID: PMC1479112 DOI: 10.1128/aac.00869-05] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Assessing bacterial viability by molecular markers might help accelerate the measurement of antibiotic-induced killing. This study investigated whether rRNA could be suitable for this purpose. Cultures of penicillin-susceptible and penicillin-tolerant (Tol1 mutant) Streptococcus gordonii were exposed to mechanistically different penicillin and levofloxacin. Bacterial survival was assessed by viable counts and compared to quantitative real-time PCR amplification of either the 16S rRNA genes or the 16S rRNA, following reverse transcription. Penicillin-susceptible S. gordonii lost > or =4 log(10) CFU/ml of viability over 48 h of penicillin treatment. In comparison, the Tol1 mutant lost < or =1 log(10) CFU/ml. Amplification of a 427-bp fragment of 16S rRNA genes yielded amplicons that increased proportionally to viable counts during bacterial growth but did not decrease during drug-induced killing. In contrast, the same 427-bp fragment amplified from 16S rRNA paralleled both bacterial growth and drug-induced killing. It also differentiated between penicillin-induced killing of the parent and the Tol1 mutant (> or =4 log(10) CFU/ml and < or =1 log(10) CFU/ml, respectively) and detected killing by mechanistically unrelated levofloxacin. Since large fragments of polynucleotides might be degraded faster than smaller fragments, the experiments were repeated by amplifying a 119-bp region internal to the original 427-bp fragment. The amount of 119-bp amplicons increased proportionally to viability during growth but remained stable during drug treatment. Thus, 16S rRNA was a marker of antibiotic-induced killing, but the size of the amplified fragment was critical for differentiation between live and dead bacteria.
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MESH Headings
- Anti-Bacterial Agents/pharmacology
- Colony Count, Microbial
- DNA, Bacterial/genetics
- DNA, Bacterial/isolation & purification
- Genes, Bacterial
- Genetic Markers
- Kinetics
- Levofloxacin
- Microbial Sensitivity Tests
- Mutation
- Ofloxacin/pharmacology
- Penicillins/pharmacology
- Polymerase Chain Reaction
- RNA, Bacterial/analysis
- RNA, Bacterial/genetics
- RNA, Bacterial/isolation & purification
- RNA, Ribosomal/analysis
- RNA, Ribosomal/chemistry
- RNA, Ribosomal, 16S/analysis
- RNA, Ribosomal, 16S/chemistry
- RNA, Ribosomal, 16S/genetics
- RNA, Ribosomal, 16S/isolation & purification
- Streptococcus/drug effects
- Streptococcus/genetics
- Streptococcus/growth & development
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Affiliation(s)
- Steve Aellen
- Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
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Makepeace BL, Rodgers L, Trees AJ. Rate of elimination of Wolbachia pipientis by doxycycline in vitro increases following drug withdrawal. Antimicrob Agents Chemother 2006; 50:922-7. [PMID: 16495252 PMCID: PMC1426454 DOI: 10.1128/aac.50.3.922-927.2006] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Wolbachia pipientis is an obligate intracellular bacterium within the family Anaplasmataceae that infects many terrestrial arthropods and arthropod-transmitted nematodes (filariae). Several filarial species are major human pathogens, and antibiotics with activity against Wolbachia offer a promising new therapeutic approach, since the adult worms are relatively refractory to conventional anthelmintics but depend on Wolbachia for reproduction and viability. In a natural filarial parasite of cattle, Onchocerca ochengi, intermittent chemotherapy is adulticidal whereas the equivalent dose administered as a continuous treatment is not. To investigate this further and to aid the design of efficacious regimens for human therapy, we used Wolbachia-infected Aedes albopictus mosquito cells in vitro. Here, we describe for the first time the accelerated depletion of bacteria after antibiotic withdrawal relative to the rate of elimination in the continuous presence of the drug. Mosquito cells were incubated with doxycycline while changes in 16S (bacterial) and 18S (host) rRNA and rRNA genes were determined by quantitative PCR assays. In cultures treated for 7 or 14 days followed by 7 days of drug withdrawal, the Wolbachia-to-Aedes rRNA ratio declined by approximately 6 log, whereas immediately after 14 or 21 days of continuous treatment, the reduction was only approximately 4 log (P < 0.05). However, low levels of 16S rRNA remained after 21 days of treatment, irrespective of whether doxycycline was withdrawn. Application of similar methodology to related intracellular bacteria may reveal that this posttreatment effect is not restricted to Wolbachia and could have wider implications for the design of intermittent regimens for antibiotic chemotherapy.
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Affiliation(s)
- Benjamin L Makepeace
- Liverpool School of Tropical Medicine and Faculty of Veterinary Science, Pembroke Place, Liverpool L3 5QA, United Kingdom.
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Heyndrickx M, Rijpens N, Herman L. Molecular Detection and Typing of Foodborne Bacterial Pathogens: A Review. Appl Microbiol 2005. [DOI: 10.1007/0-306-46888-3_15] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
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van der Meide WF, Schoone GJ, Faber WR, Zeegelaar JE, de Vries HJC, Ozbel Y, Lai A Fat RFM, Coelho LIARC, Kassi M, Schallig HDFH. Quantitative nucleic acid sequence-based assay as a new molecular tool for detection and quantification of Leishmania parasites in skin biopsy samples. J Clin Microbiol 2005; 43:5560-6. [PMID: 16272487 PMCID: PMC1287793 DOI: 10.1128/jcm.43.11.5560-5566.2005] [Citation(s) in RCA: 71] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2005] [Revised: 05/12/2005] [Accepted: 08/17/2005] [Indexed: 11/20/2022] Open
Abstract
Currently available methods for the diagnosis of cutaneous leishmaniasis (CL) have low sensitivities or are unable to quantify the number of viable parasites. This constitutes a major obstacle for the diagnosis of the disease and for the study of the effectiveness of treatment schedules and urges the development of improved detection methods. In this study, quantitative nucleic acid sequence-based amplification (QT-NASBA) technology was used to detect and quantify Leishmania parasites in skin biopsy samples from CL patients. The assay is based on the detection of a small subunit rRNA (18S rRNA), which may allow for the detection of viable parasites. The QT-NASBA assay was evaluated using in vitro-cultured promastigotes and amastigotes and 2-mm skin biopsy samples from Old and New World CL patients. The study demonstrated that the lower detection limit of the QT-NASBA was two parasites per biopsy sample. Parasites could be quantified in a range of 2 to 11,300,000 parasites per biopsy sample. The QT-NASBA could detect levels of parasites 100-fold lower than those detected by conventional PCR. Test evaluation revealed that the QT-NASBA had a sensitivity of 97.5% and a specificity of 100% in the present study. The QT-NASBA is a highly sensitive and specific method that allows quantification of both Old and New World Leishmania parasites in skin biopsy samples and may provide an important tool for diagnosis as well as for monitoring the therapy of CL patients.
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Affiliation(s)
- Wendy F van der Meide
- Koninklijk Instituut voor de Tropen/Royal Tropical Institute, KIT Biomedical Research, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands.
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Sánchez JI, Rossetti L, Martínez B, Rodríguez A, Giraffa G. Application of reverse transcriptase PCR-based T-RFLP to perform semi-quantitative analysis of metabolically active bacteria in dairy fermentations. J Microbiol Methods 2005; 65:268-77. [PMID: 16181692 DOI: 10.1016/j.mimet.2005.07.018] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2005] [Revised: 07/21/2005] [Accepted: 07/28/2005] [Indexed: 11/29/2022]
Abstract
A method consisting of reverse transcriptase (RT)-PCR amplification of 16S rRNA from the total microbial community, coupled with T-RFLP, was optimized for semi-quantitative characterization of the metabolically active population in defined strain cultures of Lactococcus lactis ssp. lactis and Leuconostoc citreum, two mesophilic lactic acid bacteria (LAB) species routinely used in cheese manufacture. The set of PCR primers selected efficiently amplified the 16S rRNA from both bacterial species. The digestion of the PCR products with DdeI yielded different terminal restriction fragments (T-RFs) for each species. Nevertheless, additional T-RFs due to formation of chimeric molecules and pseudo-T-RFs derived from partly single-stranded 16S rRNA amplicons were observed in both species, although in minor amounts. Twenty PCR cycles were determined as the optimum to minimize the presence of artifactual fragments and to avoid underestimation of populations due to the saturation effect on DNA quantification caused by a PCR product excess. T-RFLP analysis showed a good repeatability when applied to mixed dairy cultures. Dynamics of two defined mixed starters consisting of a L. lactis ssp. lactis strain and a L. citreum strain were studied by this method and results compared to those obtained by a culture-dependent technique. The data indicated the suitability of T-RFLP to perform semi-quantitative analyses of microbial populations. Some slight differences could be explained by the presence of metabolically active cells that could not be detected by colony counting. RT-PCR-based T-RFLP can be an alternative to classical methods in order to study dynamics of metabolically active populations in relatively simple microbial ecosystems, such as defined dairy starter cultures.
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Affiliation(s)
- Jorge I Sánchez
- Instituto de Productos Lácteos de Asturias (IPLA-CSIC), ctra. Infiesto, s/n, 33300 Villaviciosa, Asturias, Spain
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McKillip JL, Drake M. Real-time nucleic acid-based detection methods for pathogenic bacteria in food. J Food Prot 2004; 67:823-32. [PMID: 15083739 DOI: 10.4315/0362-028x-67.4.823] [Citation(s) in RCA: 84] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Quality assurance in the food industry in recent years has involved the acceptance and implementation of a variety of nucleic acid-based methods for rapid and sensitive detection of food-associated pathogenic bacteria. Techniques such as polymerase chain reaction have greatly expedited the process of pathogen detection and have in some cases replaced traditional methods for bacterial enumeration in food. Conventional PCR, albeit sensitive and specific under optimized conditions, obligates the user to employ agarose gel electrophoresis as the means for endpoint analysis following sample processing. For the last few years, a variety of real-time PCR chemistries and detection instruments have appeared on the market, and many of these lend themselves to applications in food microbiology. These approaches afford a user the ability to amplify DNA or RNA, as well as detect and confirm target sequence identity in a closed-tube format with the use of a variety of fluorophores, labeled probes, or both, without the need to run gels. Such real-time chemistries also offer greater sensitivity than traditional gel visualization and can be semiquantitative and multiplexed depending on the specific experimental objectives. This review emphasizes the current systems available for real-time PCR-based pathogen detection, the basic mechanisms and requirements for each, and the prospects for development over the next few years in the food industry.
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Affiliation(s)
- John L McKillip
- Department of Biology, Ball State University, Muncie, Indiana 47306, USA
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Batté M, Mathieu L, Laurent P, Prévost M. Influence of phosphate and disinfection on the composition of biofilms produced from drinking water, as measured by fluorescence in situ hybridization. Can J Microbiol 2003; 49:741-53. [PMID: 15162199 DOI: 10.1139/w03-094] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Biofilms were grown in annular reactors supplied with drinking water enriched with 235 µg C/L. Changes in the biofilms with ageing, disinfection, and phosphate treatment were monitored using fluorescence in situ hybridization. EUB338, BET42a, GAM42a, and ALF1b probes were used to target most bacteria and the alpha (α), beta (β), and gamma (γ) subclasses of Proteobacteria, respectively. The stability of biofilm composition was checked after the onset of colonization between T = 42 days and T = 113 days. From 56.0% to 75.9% of the cells detected through total direct counts with DAPI (4'-6-diamidino-2-phenylindole) were also detected with the EUB338 probe, which targets the 16S rRNA of most bacteria. Among these cells, 16.9%–24.7% were targeted with the BET42a probe, 1.8%–18.3% with the ALF1b probe, and <2.5% with the GAM42a probe. Phosphate treatment induced a significant enhancement to the proportion of γ-Proteobacteria (detected with the GAM42a probe), a group that contains many health-related bacteria. Disinfection with monochloramine for 1 month or chlorine for 3 days induced a reduction in the percentage of DAPI-stained cells that hybridized with the EUB338 probe (as expressed by percentages of EUB338 counts/DAPI) and with any of the ALF1b, BET42a, and GAM42a probes. The percentage of cells detected by any of the three probes (ALF1b+BET42a+GAM42a) tended to decrease, and reached in total less than 30% of the EUB338-hybridized cells. Disinfection with chlorine for 7 days induced a reverse shift; an increase in the percentage of EUB338 counts targeted by any of these three probes was noted, which reached up to 87%. However, it should be noted that the global bacterial densities (heterotrophic plate counts and total direct counts) tended to decrease over the duration of the experiment. Therefore, those bacteria that could be considered to resist 7 days of chlorination constituted a small part of the initial biofilm community, up to the point at which the other bacterial groups were destroyed by chlorination. The results suggest that there were variations in the kinetics of inactivation by disinfectant, depending on the bacterial populations involved.Key words: biofilm, phosphate, chlorine, monochloramine, FISH, drinking water.
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Affiliation(s)
- M Batté
- Ecole Polytechnique de Montréal, C.P. 6079, Succ. Centre Ville, Montréal, QC H3C 3A7, Canada
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Baeumner AJ, Cohen RN, Miksic V, Min J. RNA biosensor for the rapid detection of viable Escherichia coli in drinking water. Biosens Bioelectron 2003; 18:405-13. [PMID: 12604258 DOI: 10.1016/s0956-5663(02)00162-8] [Citation(s) in RCA: 92] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
A highly sensitive and specific RNA biosensor was developed for the rapid detection of viable Escherichia coli as an indicator organism in water. The biosensor is coupled with protocols developed earlier for the extraction and amplification of mRNA molecules from E. coli [Anal. Biochem. 303 (2002) 186]. However, in contrast to earlier detection methods, the biosensor allows the rapid detection and quantification of E. coli mRNA in only 15-20 min. In addition, the biosensor is portable, inexpensive and very easy to use, which makes it an ideal detection system for field applications. Viable E. coli are identified and quantified via a 200 nt-long target sequence from mRNA (clpB) coding for a heat shock protein. For sample preparation, a heat shock is applied to the cells prior to disruption. Then, mRNA is extracted, purified and finally amplified using the isothermal amplification technique Nucleic acid sequence-based amplification (NASBA). The amplified RNA is then quantified with the biosensor. The biosensor is a membrane-based DNA/RNA hybridization system using liposome amplification. The various biosensor components such as DNA probe sequences and concentration, buffers, incubation times have been optimized, and using a synthetic target sequence, a detection limit of 5 fmol per sample was determined. An excellent correlation to a much more elaborate and expensive laboratory based detection system was demonstrated, which can detect as few as 40 E. coli cfu/ml. Finally, the assay was tested regarding its specificity; no false positive signals were obtained from other microorganisms or from nonviable E. coli cells.
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Affiliation(s)
- Antje J Baeumner
- Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, USA.
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van Beckhoven JRCM, Stead DE, van der Wolf JM. Detection of Clavibacter michiganensis subsp. sepedonicus by AmpliDet RNA, a new technology based on real time monitoring of NASBA amplicons with a molecular beacon. J Appl Microbiol 2003; 93:840-9. [PMID: 12392531 DOI: 10.1046/j.1365-2672.2002.01765.x] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
AIMS To develop a procedure for direct detection of viable cells of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, based on AmpliDet RNA, in which amplicons generated by nucleic acid sequence based amplification (NASBA) are monitored in real time with a molecular beacon. METHODS AND RESULTS Five methods were evaluated and fine-tuned for extraction of RNA from Cms. The most efficient non-commercial RNA extraction method included an enzymatic breakdown of the cell wall followed by a phenol extraction. AmpliDet RNA enabled detection of 10,000 molecules of purified rRNA per reaction and 100 cfu of Cms per reaction in more complex samples. Two primer pairs were tested with DNA and RNA purified from Cms. One primer pair was able to distinguish live from dead cells. CONCLUSIONS An AmpliDet RNA was developed which enabled fast and specific detection of viable cells of Cms in complex substrates at a detection limit of 100 cfu per reaction. SIGNIFICANCE AND IMPACT OF THE STUDY This novel AmpliDet RNA is carried out in sealed tubes, thus reducing the risk of carry-over contamination. The method will be particularly suitable for studies on the epidemiology of Cms in which viable cells should be exclusively detected.
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Bentsink L, Leone GOM, van Beckhoven JRCM, van Schijndel HB, van Gemen B, van der Wolf JM. Amplification of RNA by NASBA allows direct detection of viable cells of Ralstonia solanacearum in potato. J Appl Microbiol 2002; 93:647-55. [PMID: 12234348 DOI: 10.1046/j.1365-2672.2002.01725.x] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
AIMS The objective of this study was to develop a Nucleic Acid Sequence Based Amplification (NASBA) assay, targeting 16S rRNA sequences, for direct detection of viable cells of Ralstonia solanacearum, the causal organism of bacterial wilt. The presence of intact 16S rRNA is considered to be a useful indicator for viability, as a rapid degradation of this target molecule is found upon cell death. METHODS AND RESULTS It was demonstrated by RNase treatment of extracted nucleic acids from R. solanacearum cell suspensions that NASBA exclusively detected RNA and not DNA. The ability of NASBA to assess viability was demonstrated in two sets of experiments. In the first experiment, viable and chlorine-killed cells of R. solanacearum were added to a potato tuber extract and tested in NASBA and PCR. In NASBA, only extracts spiked with viable cells resulted in a specific signal after Northern blot analysis, whereas in PCR, targeting 16S rDNA sequences, both extracts with viable and killed cells resulted in specific signals. In the second experiment, the survival of R. solanacearum on metal strips was studied using NASBA, PCR-amplification and dilution plating on the semiselective medium SMSA. A positive correlation was found between NASBA and dilution plating detecting culturable cells, whereas PCR-amplification resulted in positive reactions also long after cells were dead. The detection level of NASBA for R. solanacearum added to potato tuber extracts was determined at 104 cfu per ml of extract, equivalent to 100 cfu per reaction. With purified RNA a detection level of 104 rRNA molecules was found. This corresponds with less than one bacterial cell, assuming that a metabolically active cell contains ca 105 copies of rRNA. Preliminary experiments demonstrated the potential of NASBA to detect R. solanacearum in naturally infected potato tuber extracts. CONCLUSIONS NASBA specifically amplifies RNA from viable cells of R. solanacearum even present in complex substrates at a level of 100 cfu per reaction. SIGNIFICANCE AND IMPACT OF THE STUDY The novel NASBA assay will be particularly valuable for detection of R. solanacearum in ecological studies in which specifically viable cells should be determined.
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MESH Headings
- Base Sequence
- Betaproteobacteria/genetics
- Betaproteobacteria/growth & development
- Betaproteobacteria/isolation & purification
- Culture Media
- DNA, Ribosomal/analysis
- DNA, Ribosomal/genetics
- Genes, rRNA/genetics
- Iron/metabolism
- Molecular Sequence Data
- Plant Diseases/microbiology
- Polymerase Chain Reaction
- RNA, Bacterial/analysis
- RNA, Bacterial/genetics
- RNA, Ribosomal, 16S/analysis
- RNA, Ribosomal, 16S/genetics
- Self-Sustained Sequence Replication/methods
- Sequence Analysis, DNA
- Solanum tuberosum/microbiology
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Affiliation(s)
- L Bentsink
- Plant Research International, Wageningen, The Netherlands
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Fenhalls G, Stevens-Muller L, Warren R, Carroll N, Bezuidenhout J, Van Helden P, Bardin P. Localisation of mycobacterial DNA and mRNA in human tuberculous granulomas. J Microbiol Methods 2002; 51:197-208. [PMID: 12133612 DOI: 10.1016/s0167-7012(02)00076-3] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
In situ hybridisation was used to detect the presence of Mycobacterium tuberculosis in paraffin-embedded lung tissue of nine patients diagnosed with tuberculosis (TB). Mycobacterial DNA was found in all nine patients and in 175 out of 191 granulomas examined. A combination of in situ hybridisation and immunohistochemistry techniques demonstrated that mycobacterial DNA was associated with CD68-positive cells with the morphology of macrophages and giant cells. Mycobacterial DNA was also found within the necrotic regions of some granulomas. mRNA for the mycobacterial RNA polymerase beta subunit (rpoB) was detected by RNA: RNA in situ hybridisation. The rpoB mRNA was also localised to CD68-positive cells with the morphology of macrophages and to giant cells of certain necrotic granulomas. No rpoB mRNA was found in the necrotic regions of granulomas. Mycobacterial DNA was detected in 92% of patient granulomas of which 8% were positive for rpoB mRNA. The ability to identify mycobacterial RNA transcripts within human tuberculous granulomas affords us the opportunity to analyse the interplay between pathogen gene expression and the human immune response and should provide valuable insight into the mechanisms used by M. tuberculosis to persist within the human host.
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Affiliation(s)
- Gael Fenhalls
- MRC Center for Molecular and Cellular Biology, University of Stellenbosch Medical School, Cape Town, South Africa.
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de Magalhães VD, de Melo Azevedo FDP, Pasternak J, Valle Martino MD. Reliability of hsp65-RFLP analysis for identification of Mycobacterium species in cultured strains and clinical specimens. J Microbiol Methods 2002; 49:295-300. [PMID: 11869794 DOI: 10.1016/s0167-7012(01)00386-4] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
The purpose of this study was to evaluate the reliability of an amplification restriction analysis based method (hsp65-RFLP) to detect and identify mycobacterial species in clinical samples and cultures with low number of bacilli. We examined 247 clinical specimens and 88 culture vials, comparing hsp65-RFLP results with conventional culture/biochemical tests. The analytical sensitivity of the method was assessed with cerebrospinal fluid (CSF), broncho-alveolar lavage (BAL), sputum, water, and 12B medium containing defined amounts of mycobacterial chromosome. We detected the equivalent of 10(3) cells per ml in all samples, except sputum, the most common source of clinical sample for mycobacterial testing, which presented inhibition throughout. We investigated two purification procedures to overcome inhibition of DNA amplification: DNAzol and phenol/chloroform. The former was superior, eliminating inhibition in 93.7% of the clinical samples. The technique was effective for bacterial cultures, including those with very low growth indices (GIs), substantially abbreviating time for diagnosis, but showed low sensitivity (25%) when applied to clinical samples, an issue that has never been extensively assessed by other researchers.
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Affiliation(s)
- Vanda Dolabela de Magalhães
- Laboratório de Pesquisa e Desenvolvimento, Instituto de Ensino e Pesquisa, Hospital Israelita Albert Einstein, Av. Albert Einstein 627, 05651-901, São Paulo SP, Brazil.
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45
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Min J, Baeumner AJ. Highly sensitive and specific detection of viable Escherichia coli in drinking water. Anal Biochem 2002; 303:186-93. [PMID: 11950218 DOI: 10.1006/abio.2002.5593] [Citation(s) in RCA: 58] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
A highly sensitive and specific assay method was developed for the detection of viable Escherichia coli as an indicator organism in water, using nucleic acid sequence-based amplification (NASBA) and electrochemiluminescence (ECL) analysis. Viable E. coli were identified via a 200-nt-long target sequence from mRNA (clpB) coding for a heat shock protein. In the detection assay, a heat shock was applied to the cells prior to disruption to induce the synthesis of clpB mRNA and the mRNA was extracted, purified, and finally amplified using NASBA. The amplified mRNA was quantified with an ECL detection system after hybridization with specific DNA probes. Several disruption methods were investigated to maximize total RNA extracted from viable cells. Optimization was also carried out regarding the design of NASBA primer pairs and detection probes, as well as reaction and detection conditions. Finally, the assay was tested regarding sensitivity and specificity. Analysis of samples revealed that as few as 40 E. coli cells/mL can be detected, with no false positive signals resulting from other microorganisms or nonviable E. coli cells. Also, it was shown that a quantification of E. coli cells was possible with our assay method.
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Affiliation(s)
- Junhong Min
- Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York 14853, USA
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Nijhof MW, Fleer A, Hardus K, Vogely HC, Schouls LM, Verbout AJ, Dhert WJ. Tobramycin-containing bone cement and systemic cefazolin in a one-stage revision. Treatment of infection in a rabbit model. JOURNAL OF BIOMEDICAL MATERIALS RESEARCH 2002; 58:747-53. [PMID: 11745530 DOI: 10.1002/jbm.1073] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
The efficacy of tobramycin-containing bone cement with that of systemic cefazolin for treatment of infection in a one-stage revision model is compared. In addition, the value of detecting bacterial DNA after antibiotic treatment was investigated. An implant was inserted into the right tibia of rabbits after inoculation with Staphylococcus aureus. At 28 days, the implant was removed. Subsequently, either plain bone cement with or without systemic administration of cefazolin, or tobramycin-containing bone cement was injected into the medullary canal. The tibiae were cultured 14 days after revision (Day 42), and showed a significant decrease in bacterial counts for both antibiotic groups compared with the control group (p</=0.05). The rate of infection in the tobramycin-cement group was slightly higher (2/9) than in the cefazolin group (0/8), although the difference was not significant. Persistence of bacterial DNA after antibiotic treatment may be the result of delayed clearance of DNA and not a sign of active infection. This animal model shows that in a one-stage revision tobramycin-containing bone cement can reduce size and rate of infection, although systemic cefazolin may be more efficacious. Therefore, the use of antibiotic-containing bone cement combined with systemic antibiotic might provide optimal treatment.
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Affiliation(s)
- M W Nijhof
- Department of Orthopaedics, University Medical Center Utrecht, Utrecht, The Netherlands
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47
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Deiman B, van Aarle P, Sillekens P. Characteristics and applications of nucleic acid sequence-based amplification (NASBA). Mol Biotechnol 2002; 20:163-79. [PMID: 11876473 DOI: 10.1385/mb:20:2:163] [Citation(s) in RCA: 180] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Nucleic acid sequence-based amplification (NASBA) is a sensitive, isothermal, transcription-based amplification system specifically designed for the detection of RNA targets. In some NASBA systems, DNA is also amplified though very inefficiently and only in the absence of the corresponding RNA target or in case of an excess (>1,000-fold) of target DNA over RNA. As NASBA is primer-dependent and amplicon detection is based on probe binding, primer and probe design rules are included. An overview of various target nucleic acids that have been amplified successfully using NASBA is presented. For the isolation of nucleic acids prior to NASBA, the "Boom" method, based on the denaturing properties of guanidine isothiocyanate and binding of nucleic acid to silica particles, is preferred. Currently, electro-chemiluminescence (ECL) is recommended for the detection of the amplicon at the end of amplification. In the near future, molecular beacons will be introduced enabling "real-time detection," i.e., amplicon detection during amplification. Quantitative HIV-1 NASBA and detection of up to 48 samples can then be performed in only 90 min.
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48
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49
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Hu Y, Mangan JA, Dhillon J, Sole KM, Mitchison DA, Butcher PD, Coates AR. Detection of mRNA transcripts and active transcription in persistent Mycobacterium tuberculosis induced by exposure to rifampin or pyrazinamide. J Bacteriol 2000; 182:6358-65. [PMID: 11053379 PMCID: PMC94781 DOI: 10.1128/jb.182.22.6358-6365.2000] [Citation(s) in RCA: 122] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Mycobacterium tuberculosis can persist in an altered physiological state for many years after initial infection, and it may reactivate to cause active disease. An analogous persistent state, possibly consisting of several different subpopulations of bacteria, may arise during chemotherapy; this state is thought to be responsible for the prolonged period required for effective chemotherapy. Using two models of drug-induced persistence, we show that both microaerophilic stationary-phase M. tuberculosis treated with a high dose of rifampin in vitro and pyrazinamide-induced persistent bacteria in mice are nonculturable yet still contain 16S rRNA and mRNA transcripts. Also, the in vitro persistent, plate culture-negative bacteria incorporate radioactive uridine into their RNA in the presence of rifampin and can rapidly up-regulate gene transcription after the replacement of the drug with fresh medium and in response to heat shock. Our results show that persistent M. tuberculosis has transcriptional activity. This finding provides a molecular basis for the rational design of drugs targeted at persistent bacteria.
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Affiliation(s)
- Y Hu
- Department of Medical Microbiology, St. George's Hospital Medical School, London SW17 0RE, United Kingdom
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50
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Kempsell KE, Cox CJ, Hurle M, Wong A, Wilkie S, Zanders ED, Gaston JS, Crowe JS. Reverse transcriptase-PCR analysis of bacterial rRNA for detection and characterization of bacterial species in arthritis synovial tissue. Infect Immun 2000; 68:6012-26. [PMID: 10992514 PMCID: PMC101566 DOI: 10.1128/iai.68.10.6012-6026.2000] [Citation(s) in RCA: 87] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Onset of rheumatoid arthritis (RA) is widely believed to be preceded by exposure to some environmental trigger such as bacterial infectious agents. The influence of bacteria on RA disease onset or pathology has to date been controversial, due to inconsistencies between groups in the report of bacterial species isolated from RA disease tissue. Using a modified technique of reverse transcriptase-PCR amplification, we have detected bacterial rRNA in the synovial tissue of late-stage RA and non-RA arthritis controls. This may be suggestive of the presence of live bacteria. Sequencing of cloned complementary rDNA (crDNA) products revealed a number of bacterial sequences in joint tissue from each patient, and from these analyses a comprehensive profile of the organisms present was compiled. This revealed a number of different organisms in each patient, some of which are common to both RA and non-RA controls and are probably opportunistic colonizers of previously diseased tissue and others which are unique species. These latter organisms may be candidates for a specific role in disease pathology and require further investigation to exclude them as causative agents in the complex bacterial millieu. In addition, many of the detected bacterial species have not been identified previously from synovial tissue or fluid from arthritis patients. These may not be easily cultivable, since they were not revealed in previous studies using conventional in vitro bacterial culture methods. In situ hybridization analyses have revealed the joint-associated bacterial rRNA to be both intra- and extracellular. The role of viable bacteria or their nucleic acids as triggers in disease onset or pathology in either RA or non-RA arthritis controls is unclear and requires further investigation.
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Affiliation(s)
- K E Kempsell
- Glaxo Wellcome Medicines Research Centre, Stevenage SG2 1NY, United Kingdom.
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