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1
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Shi J, Zhou X, Zhang S, Sun F, Shen C, Su X. Unveiling the distribution characteristics of rpf-like genes and indigenous resuscitation promoting factor production in PCB-contaminated soils. JOURNAL OF ENVIRONMENTAL MANAGEMENT 2024; 357:120803. [PMID: 38569268 DOI: 10.1016/j.jenvman.2024.120803] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/13/2023] [Revised: 02/17/2024] [Accepted: 03/30/2024] [Indexed: 04/05/2024]
Abstract
Resuscitation promoting factors (Rpfs), known for their anti-dormancy cytokine properties, have been extensively investigated in the medical field. Although the Rpf from Micrococcus luteus has been successfully utilized to resuscitate and stimulate microbial populations for the degradation of polychlorinated biphenyls (PCBs), the presence of indigenous Rpf homologs in PCB-contaminated soils has not been established. In this study, the distribution characteristics of rpf-like genes and indigenous strain capable of producing Rpf in PCB-contaminated soils were explored. The results revealed the widespread presence of Rpf-like domains and their associated genes, particularly in close association with heavy metals and PCBs. The rpf-like genes were predominantly found in Proteobacteria and displayed a positive correlation with genes involved in PCB degradation and viable but non-culturable (VBNC) formation. Notably, the recombinant Rpf-Ac protein derived from the indigenous strain Achromobacter sp. HR2 exhibited muralytic activity and demonstrated significant efficacy in resuscitating the growth of VBNC cells, while also stimulating the growth of normal cells. These findings shed light on the prevalent presence of Rpf homologs in PCB-contaminated soils and their potential to resuscitate functional populations in the VBNC state, thereby enhancing in situ bioremediation.
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Affiliation(s)
- Jie Shi
- College of Geography and Environmental Science, Zhejiang Normal University, Jinhua, 321004, China
| | - Xinru Zhou
- College of Geography and Environmental Science, Zhejiang Normal University, Jinhua, 321004, China
| | - Shusheng Zhang
- The Management Center of Wuyanling National Natural Reserve in Zhejiang, Wenzhou, 325500, China
| | - Faqian Sun
- College of Geography and Environmental Science, Zhejiang Normal University, Jinhua, 321004, China
| | - Chaofeng Shen
- Department of Environmental Engineering, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, 310058, China
| | - Xiaomei Su
- College of Geography and Environmental Science, Zhejiang Normal University, Jinhua, 321004, China.
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2
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Shi J, Yang Y, Zhang S, Lin Q, Sun F, Lin H, Shen C, Su X. New insights into survival strategies and PCB bioremediation potential of resuscitated strain Achromobacter sp. HR2 under combined stress conditions. JOURNAL OF HAZARDOUS MATERIALS 2024; 465:133242. [PMID: 38103289 DOI: 10.1016/j.jhazmat.2023.133242] [Citation(s) in RCA: 7] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/07/2023] [Revised: 12/08/2023] [Accepted: 12/10/2023] [Indexed: 12/19/2023]
Abstract
The resuscitated strains achieved through the addition of resuscitation promoting factor (Rpf) hold significant promise as bio-inoculants for enhancing the bioremediation of polychlorinated biphenyls (PCBs). Nevertheless, the potential of these resuscitated strains to transition into a viable but non-culturable (VBNC) state, along with the specific stressors that initiate this transformation, remains to be comprehensively elucidated. In this study, a resuscitated strain HR2, obtained through Rpf amendment, was employed to investigate its survival strategies under combined stress involving low temperature (LT), and PCBs, in the absence and presence of heavy metals (HMs). Whole-genome analysis demonstrated that HR2, affiliated with Achromobacter, possessed 107 genes associated with the degradation of polycyclic aromatic compounds. Remarkably, HR2 exhibited effective degradation of Aroclor 1242 and robust resistance to stress induced by LT and PCBs, while maintaining its culturability. However, when exposed to the combined stress of LT, PCBs, and HMs, HR2 entered the VBNC state. This state was characterized by significant decreases in enzyme activities and notable morphological, physiological, and molecular alterations compared to normal cells. These findings uncovered the survival status of resuscitated strains under stressful conditions, thereby offering valuable insights for the development of effective bioremediation strategies.
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Affiliation(s)
- Jie Shi
- College of Geography and Environmental Science, Zhejiang Normal University, Jinhua 321004, China
| | - Yingying Yang
- College of Geography and Environmental Science, Zhejiang Normal University, Jinhua 321004, China
| | - Shusheng Zhang
- The Management Center of Wuyanling National Natural Reserve in Zhejiang, Wenzhou 325500, China
| | - Qihua Lin
- College of Geography and Environmental Science, Zhejiang Normal University, Jinhua 321004, China
| | - Faqian Sun
- College of Geography and Environmental Science, Zhejiang Normal University, Jinhua 321004, China
| | - Hongjun Lin
- College of Geography and Environmental Science, Zhejiang Normal University, Jinhua 321004, China
| | - Chaofeng Shen
- Department of Environmental Engineering, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China
| | - Xiaomei Su
- College of Geography and Environmental Science, Zhejiang Normal University, Jinhua 321004, China.
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3
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Yang Y, Zhang Q, Lin Q, Sun F, Shen C, Lin H, Su X. Unveiling the PCB biodegradation potential and stress survival strategies of resuscitated strain Pseudomonas sp. HR1. ENVIRONMENTAL POLLUTION (BARKING, ESSEX : 1987) 2024; 344:123320. [PMID: 38185359 DOI: 10.1016/j.envpol.2024.123320] [Citation(s) in RCA: 6] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/30/2023] [Revised: 01/03/2024] [Accepted: 01/04/2024] [Indexed: 01/09/2024]
Abstract
The exploration of resuscitated strains, facilitated by the resuscitation promoting factor (Rpf), has substantially expanded the pool of cultivated degraders, enhancing the screening of bio-inoculants for bioremediation applications. However, it remains unknown whether these resuscitated strains can re-enter the viable but non-culturable (VBNC) state and the specific stress conditions that trigger such a transition. In this work, the whole genome, and polychlorinated biphenyl (PCB)-degrading capabilities of a resuscitated strain HR1, were investigated. Notably, the focus of this exploration was on elucidating whether HR1 would undergo a transition into the VBNC state when exposed to low temperature and PCBs, with and without the presence of heavy metals (HMs). The results suggested that the resuscitated strain Pseudomonas sp. HR1 harbored various functional genes related to xenobiotic biodegradation, demonstrating remarkable efficiency in Aroclor 1242 degradation and strong resistance against stress induced by low temperature and PCBs. Nevertheless, when exposed to the combined stress of low temperature, PCBs, and HMs, HR1 underwent a transition into the VBNC state. This transition was characterized by significant decreases in enzyme activities and notable changes in both morphological and physiological traits when compared to normal cells. Gene expression analysis revealed molecular shifts underlying the VBNC state, with down-regulated genes showed differential expression across multiple pathways and functions, including oxidative phosphorylation, glycolysis, tricarboxylic acid cycle, amino acid metabolism, translation and cytoplasm, while up-regulated genes predominantly associated with transcription regulation, membrane function, quorum sensing, and transporter activity. These findings highlighted the great potential of resuscitated strains as bio-inoculants in bioaugmentation and shed light on the survival mechanisms of functional strains under stressful conditions, which should be carefully considered during bioremediation processes.
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Affiliation(s)
- Yingying Yang
- College of Geography and Environmental Science, Zhejiang Normal University, Jinhua, 321004, China
| | - Qian Zhang
- College of Geography and Environmental Science, Zhejiang Normal University, Jinhua, 321004, China
| | - Qihua Lin
- College of Geography and Environmental Science, Zhejiang Normal University, Jinhua, 321004, China
| | - Faqian Sun
- College of Geography and Environmental Science, Zhejiang Normal University, Jinhua, 321004, China
| | - Chaofeng Shen
- Department of Environmental Engineering, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, 310058, China
| | - Hongjun Lin
- College of Geography and Environmental Science, Zhejiang Normal University, Jinhua, 321004, China
| | - Xiaomei Su
- College of Geography and Environmental Science, Zhejiang Normal University, Jinhua, 321004, China.
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4
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Tang X, Li Y, Liu M, Hou L, Han P. Abundance, diversity and physiological preferences of comammox Nitrospira in urban groundwater. THE SCIENCE OF THE TOTAL ENVIRONMENT 2023; 904:167333. [PMID: 37748616 DOI: 10.1016/j.scitotenv.2023.167333] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/25/2023] [Revised: 09/01/2023] [Accepted: 09/22/2023] [Indexed: 09/27/2023]
Abstract
Complete ammonia oxidizer (comammox Nitrospira), catalyze complete nitrification process in a single organism, are frequently detected in groundwater ecosystem. However, the ecological niches and environmental driving factors of comammox Nitrospira in urban groundwater are largely unknown. Here we investigated the communities of ammonia oxidizers in urban groundwater located in Shanghai city, China. Quantitative analysis demonstrated the dominance of comammox Nitrospira over classical ammonia oxidizers (ammonia-oxidizing archaea and bacteria, AOA and AOB). Phylogenetic analysis showed clades B and A2 comprise the majority of comammox Nitrospira groups. Temperature was one of the most vital factors affecting comammox Nitrospira community. Furthermore, clade A comammox Nitrospira can be enriched by urea substrate, which was in line with the ability of utilizing urea by the pure clade A comammox culture Nitrospira inopinata. In addition, we observed that relatively low temperature (<20 °C) and high copper levels (>0.04 mg L-1) can stimulate the growth of comammox Nitrospira. Overall, this study revealed the presence, diversity and physiological preferences of comammox Nitrospira in urban groundwater nitrification, shedding insights on the ecological roles of comammox Nitrospira in subsurface environment.
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Affiliation(s)
- Xiufeng Tang
- Key Laboratory of Geographic Information Science (Ministry of Education), School of Geographic Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China
| | - Ye Li
- Key Laboratory of Geographic Information Science (Ministry of Education), School of Geographic Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China
| | - Min Liu
- Key Laboratory of Geographic Information Science (Ministry of Education), School of Geographic Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China
| | - Lijun Hou
- Key Laboratory of Geographic Information Science (Ministry of Education), School of Geographic Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China
| | - Ping Han
- Key Laboratory of Geographic Information Science (Ministry of Education), School of Geographic Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China; State Key Laboratory of Estuarine and Coastal Research, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China; Institute of Eco-Chongming, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China.
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5
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Asavasuthiphan V, Nuisin R, Kraiya C, Sukwattanasinitt M, Rashatasakhon P. Ratiometric Fluorescent Sensor for Copper(II) and Phosphate Ions from Aminopyrene Derivatives. Photochem Photobiol 2021; 98:856-863. [PMID: 34861046 DOI: 10.1111/php.13569] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2021] [Accepted: 11/30/2021] [Indexed: 11/27/2022]
Abstract
Three derivatives of 1-aminopyrene are functionalized with 2-picolyl and 2-picolinyl groups and are tested as fluorescent sensors for metal ions. The target compounds are successfully synthesized in yields of 50-90% and characterized by 1 H-NMR, 13 C-NMR, and HRMS. The compound with an amino picolyl group (P1) exhibits an excellent selectivity toward Cu(II) ion as the fluorescent signal shifts from 433 to 630 nm. From a fluorescence titration experiment, the limit of detection for Cu(II) ion is estimated as 0.19 µm. The fluorescence spectral shift by Cu(II) ion is reliant on the use of acetonitrile as a co-solvent, and the results from cyclic voltammetry and UV-Vis spectroscopy suggest that the sensing mechanism involves a coordination complex between the P1, acetonitrile and Cu(II) ion. Furthermore, this P1-Cu complex can also be used as a selective fluorescent sensor for PO4 3- ion with a detection limit of 0.44 µm.
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Affiliation(s)
- Voravin Asavasuthiphan
- Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand
| | - Roongkan Nuisin
- Department of Environmental Science, Faculty of Science, Chulalongkorn University, Bangkok, Thailand
| | - Charoenkwan Kraiya
- Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.,Electrochemistry and Optical Spectroscopy Center of Excellence, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand
| | - Mongkol Sukwattanasinitt
- Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.,Nanotec-CU Center of Excellence on Food and Agriculture, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand
| | - Paitoon Rashatasakhon
- Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.,Nanotec-CU Center of Excellence on Food and Agriculture, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand
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6
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Maertens L, Matroule JY, Van Houdt R. Characteristics of the copper-induced viable-but-non-culturable state in bacteria. World J Microbiol Biotechnol 2021; 37:37. [PMID: 33544256 PMCID: PMC7864824 DOI: 10.1007/s11274-021-03006-5] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2020] [Accepted: 01/17/2021] [Indexed: 02/06/2023]
Abstract
The antimicrobial applications of copper (Cu) are exploited in several industries, such as agriculture and healthcare settings. While Cu is capable of efficiently killing microorganisms, sub-lethal doses can induce a viable-but-non-culturable (VBNC) state in bacteria of many distinct clades. VBNC cells cannot be detected by standard culture-based detection methods, and can become a threat to plants and animals as they often retain virulent traits upon resuscitation. Here we discuss the putative mechanisms of the Cu-induced VBNC state. Common observations in Cu-induced VBNC cells include a cellular response to reactive oxygen species, the exhaustion of energy reserves, and a reconfiguration of the proteome. While showing partial overlap with other VBNC state-inducing stressors, these changes seem to be part of an adaptive response to Cu toxicity. Furthermore, we argue that Cu resistance mechanisms such as P-type ATPases and multicopper oxidases may ward off entry into the VBNC state to some extent. The spread of these mechanisms across multi-species populations could increase population-level resistance to Cu antimicrobials. As Cu resistance mechanisms are often co-selected with antibiotic resistance mechanisms, this threat is exacerbated.
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Affiliation(s)
- Laurens Maertens
- Microbiology Unit, Interdisciplinary Biosciences, Belgian Nuclear Research Centre (SCK CEN), Mol, Belgium.,Research Unit in Microorganisms Biology (URBM), Narilis Institute, University of Namur, Namur, Belgium
| | - Jean-Yves Matroule
- Research Unit in Microorganisms Biology (URBM), Narilis Institute, University of Namur, Namur, Belgium
| | - Rob Van Houdt
- Microbiology Unit, Interdisciplinary Biosciences, Belgian Nuclear Research Centre (SCK CEN), Mol, Belgium.
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Moreira Martins PM, Gong T, de Souza AA, Wood TK. Copper Kills Escherichia coli Persister Cells. Antibiotics (Basel) 2020; 9:antibiotics9080506. [PMID: 32806704 PMCID: PMC7459663 DOI: 10.3390/antibiotics9080506] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2020] [Revised: 08/07/2020] [Accepted: 08/10/2020] [Indexed: 02/04/2023] Open
Abstract
Due to their reduced metabolism, persister cells can survive most antimicrobial treatments, which usually rely on corrupting active biochemical pathways. Therefore, molecules that kill bacterial persisters should function in a metabolism-independent manner. Some anti-persister compounds have been found previously, such as the DNA-crosslinkers mitomycin C and cisplatin, but more effective and lower cost alternatives are needed. Copper alloys have been used since ancient times due to their antimicrobial properties, and they are still used in agriculture to control plant bacterial diseases. By stopping transcription with rifampicin and by treating with ampicillin to remove non-persister cells, we created a population that consists solely of Escherichia coli persister cells. Using this population of persister cells, we demonstrate that cupric compounds kill E. coli persister cells. Hence, copper ions may be used in controlling the spread of important bacterial strains that withstand treatment with conventional antimicrobials by forming persister cells.
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Affiliation(s)
- Paula Maria Moreira Martins
- Department of Chemical Engineering, Pennsylvania State University, University Park, PA 16802, USA; (P.M.M.M.); (T.G.)
- Biotechnology Lab, Centro de Citricultura Sylvio Moreira, Instituto Agronômico de Campinas, Cordeirópolis-SP 13490-970, Brazil;
| | - Ting Gong
- Department of Chemical Engineering, Pennsylvania State University, University Park, PA 16802, USA; (P.M.M.M.); (T.G.)
| | - Alessandra A. de Souza
- Biotechnology Lab, Centro de Citricultura Sylvio Moreira, Instituto Agronômico de Campinas, Cordeirópolis-SP 13490-970, Brazil;
| | - Thomas K. Wood
- Department of Chemical Engineering, Pennsylvania State University, University Park, PA 16802, USA; (P.M.M.M.); (T.G.)
- Correspondence:
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8
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Giachino A, Waldron KJ. Copper tolerance in bacteria requires the activation of multiple accessory pathways. Mol Microbiol 2020; 114:377-390. [DOI: 10.1111/mmi.14522] [Citation(s) in RCA: 64] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2020] [Revised: 04/18/2020] [Accepted: 04/20/2020] [Indexed: 12/17/2022]
Affiliation(s)
- Andrea Giachino
- Biosciences Institute Faculty of Medical Sciences Newcastle University Newcastle upon Tyne UK
| | - Kevin J. Waldron
- Biosciences Institute Faculty of Medical Sciences Newcastle University Newcastle upon Tyne UK
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9
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Rtimi S, Kiwi J. Recent advances on sputtered films with Cu in ppm concentrations leading to an acceleration of the bacterial inactivation. Catal Today 2020. [DOI: 10.1016/j.cattod.2018.06.016] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
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10
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Wagner FB, Diwan V, Dechesne A, Fowler SJ, Smets BF, Albrechtsen HJ. Copper-Induced Stimulation of Nitrification in Biological Rapid Sand Filters for Drinking Water Production by Proliferation of Nitrosomonas spp. ENVIRONMENTAL SCIENCE & TECHNOLOGY 2019; 53:12433-12441. [PMID: 31593453 DOI: 10.1021/acs.est.9b03885] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/10/2023]
Abstract
Copper is a cofactor of the ammonia monooxygenase, an essential enzyme for the activity of ammonia oxidizing prokaryotes (AOP). Copper dosing at less than 1 μg/L stimulated ammonium removal in the poorly nitrifying biological filters of three full-scale drinking water treatment plants. Upon copper dosing, the ammonium concentration in the effluent decreased from up to 0.18 to less than 0.01 mg NH4+-N/L. To investigate how copper dosing affected the filter microbial community, we applied amplicon sequencing and qPCR targeting key nitrifying groups, including complete ammonia oxidizing (comammox) Nitrospira. Copper dosing increased the abundance of different nitrifiers. Multiple Nitrosomonas variants (betaproteobacterial ammonia oxidizers), which initially collectively represented 1% or less of the total community, increased almost 10-fold. Comammox Nitrospira were abundant and increased too, but their relative abundance within the AOP decreased because of Nitrosomonas proliferation. No other consistent change in the filter communities was detected, as well as no adverse effect of copper on the filters functionality. Our results show that copper dosing in three independent treatment plants was associated with consistent growth of AOP and that efficient nitrification was achieved through the joint contribution of comammox Nitrospira and an increasing fraction of betaproteobacterial ammonia oxidizers.
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Affiliation(s)
- Florian B Wagner
- Department of Environmental Engineering , Technical University of Denmark , Bygningstorvet Building 115 , Kgs. Lyngby , 2800 , Denmark
| | - Vaibhav Diwan
- Department of Environmental Engineering , Technical University of Denmark , Bygningstorvet Building 115 , Kgs. Lyngby , 2800 , Denmark
| | - Arnaud Dechesne
- Department of Environmental Engineering , Technical University of Denmark , Bygningstorvet Building 115 , Kgs. Lyngby , 2800 , Denmark
| | - S Jane Fowler
- Department of Environmental Engineering , Technical University of Denmark , Bygningstorvet Building 115 , Kgs. Lyngby , 2800 , Denmark
| | - Barth F Smets
- Department of Environmental Engineering , Technical University of Denmark , Bygningstorvet Building 115 , Kgs. Lyngby , 2800 , Denmark
| | - Hans-Jørgen Albrechtsen
- Department of Environmental Engineering , Technical University of Denmark , Bygningstorvet Building 115 , Kgs. Lyngby , 2800 , Denmark
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The Use of Copper as an Antimicrobial Agent in Health Care, Including Obstetrics and Gynecology. Clin Microbiol Rev 2019; 32:32/4/e00125-18. [PMID: 31413046 DOI: 10.1128/cmr.00125-18] [Citation(s) in RCA: 83] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Health care-associated infections (HAIs) are a global problem associated with significant morbidity and mortality. Controlling the spread of antimicrobial-resistant bacteria is a major public health challenge, and antimicrobial resistance has become one of the most important global problems in current times. The antimicrobial effect of copper has been known for centuries, and ongoing research is being conducted on the use of copper-coated hard and soft surfaces for reduction of microbial contamination and, subsequently, reduction of HAIs. This review provides an overview of the historical and current evidence of the antimicrobial and wound-healing properties of copper and explores its possible utility in obstetrics and gynecology.
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12
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Halmenschelager PT, da Rocha JBT. Biochemical CuSO 4 Toxicity in Drosophila melanogaster Depends on Sex and Developmental Stage of Exposure. Biol Trace Elem Res 2019; 189:574-585. [PMID: 30178145 DOI: 10.1007/s12011-018-1475-y] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/15/2018] [Accepted: 08/07/2018] [Indexed: 01/01/2023]
Abstract
Copper is a transition metal that exists in different chemical forms (e.g., Cu2+,Cu+, and Cu0) and at high concentrations it is toxic. Here, we investigated the Cu2+-induced toxicity in Drosophila melanogaster, evaluating the survival, locomotion, and the activity of acetylcholinesterase (AChE) and glutathione S-transferase (GST) enzymes. Flies were exposed to Cu2+(0.1-1 mmol CuSO4/kg of diet or approximately 0.1-1 mM Cu2+) and allowed to mate during 24 h. GST and AChE enzymes were evaluated in the larvae and in the head and the body (thorax + abdomen) of the adult male and females flies. The total number of adult females (0.4-1 mM) and males (0.75 and 1 mM) was decreased by CuSO4. The climbing ability was hampered in flies exposed to 1 mM Cu2+. In larvae, Cu2+(0.4-1 mM) increased AChE activity (P < 0.002). In males' heads, 0.4 mM Cu2+ increased the AChE activity (P < 0.01). In adults' bodies, Cu2+inhibited the activity in both sexes, but with greater effectiveness in males (0.1 to 1 mM) than in females (1 mM). Regarding GST activity, 0.1 mM Cu2+increased, but 1 mM decrease GST in larvae. In the head of flies, Cu2+decreased the GST activity at intermediate (0.4 mM) and increased GST at the highest concentration (1 mM) in males. In the bodies, the effect of Cu2+was similar. In conclusion, Cu2+exposure in D. melanogaster disrupted locomotion and enzymatic parameters that can be related to changes in AChE and in the detoxifying GST enzyme.
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Affiliation(s)
- Paula Tais Halmenschelager
- Department of Biochemistry and Molecular Biology, Center for Natural and Exact Sciences, Federal University of Santa Maria, Santa Maria, Rio Grande do Sul, Brazil
| | - João Batista Teixeira da Rocha
- Department of Biochemistry and Molecular Biology, Center for Natural and Exact Sciences, Federal University of Santa Maria, Santa Maria, Rio Grande do Sul, Brazil.
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13
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Schnabel U, Handorf O, Yarova K, Zessin B, Zechlin S, Sydow D, Zellmer E, Stachowiak J, Andrasch M, Below H, Ehlbeck J. Plasma-Treated Air and Water-Assessment of Synergistic Antimicrobial Effects for Sanitation of Food Processing Surfaces and Environment. Foods 2019; 8:foods8020055. [PMID: 30717375 PMCID: PMC6406376 DOI: 10.3390/foods8020055] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2019] [Revised: 01/28/2019] [Accepted: 01/29/2019] [Indexed: 01/28/2023] Open
Abstract
The synergistic antimicrobial effects of plasma-processed air (PPA) and plasma-treated water (PTW), which are indirectly generated by a microwave-induced non-atmospheric pressure plasma, were investigated with the aid of proliferation assays. For this purpose, microorganisms (Listeria monocytogenes, Escherichia coli, Pectobacterium carotovorum, sporulated Bacillus atrophaeus) were cultivated as monocultures on specimens with polymeric surface structures. Both the distinct and synergistic antimicrobial potential of PPA and PTW were governed by the plasma-on time (5⁻50 s) and the treatment time of the specimens with PPA/PTW (1⁻5 min). In single PTW treatment of the bacteria, an elevation of the reduction factor with increasing treatment time could be observed (e.g., reduction factor of 2.4 to 3.0 for P. carotovorum). In comparison, the combination of PTW and subsequent PPA treatment leads to synergistic effects that are clearly not induced by longer treatment times. These findings have been valid for all bacteria (L. monocytogenes > P. carotovorum = E. coli). Controversially, the effect is reversed for endospores of B. atrophaeus. With pure PPA treatment, a strong inactivation at 50 s plasma-on time is detectable, whereas single PTW treatment shows no effect even with increasing treatment parameters. The use of synergistic effects of PTW for cleaning and PPA for drying shows a clear alternative for currently used sanitation methods in production plants. Highlights: Non-thermal atmospheric pressure microwave plasma source used indirect in two different modes-gaseous and liquid; Measurement of short and long-living nitrite and nitrate in corrosive gas PPA (plasma-processed air) and complex liquid PTW (plasma-treated water); Application of PTW and PPA in single and combined use for biological decontamination of different microorganisms.
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Affiliation(s)
- Uta Schnabel
- Plasma Bioengineering, Leibniz Institute for Plasma Science and Technology e.V., Felix-Hausdorff-Straße 2, 17491 Greifswald, Germany.
- School of Food Science and Environmental Health, College of Sciences and Health, Technological University Dublin, Cathal Brugha Street, D01 HV58 Dublin, Ireland.
| | - Oliver Handorf
- Plasma Bioengineering, Leibniz Institute for Plasma Science and Technology e.V., Felix-Hausdorff-Straße 2, 17491 Greifswald, Germany.
| | - Kateryna Yarova
- Plasma Bioengineering, Leibniz Institute for Plasma Science and Technology e.V., Felix-Hausdorff-Straße 2, 17491 Greifswald, Germany.
| | - Björn Zessin
- Plasma Bioengineering, Leibniz Institute for Plasma Science and Technology e.V., Felix-Hausdorff-Straße 2, 17491 Greifswald, Germany.
| | - Susann Zechlin
- Plasma Bioengineering, Leibniz Institute for Plasma Science and Technology e.V., Felix-Hausdorff-Straße 2, 17491 Greifswald, Germany.
| | - Diana Sydow
- Plasma Bioengineering, Leibniz Institute for Plasma Science and Technology e.V., Felix-Hausdorff-Straße 2, 17491 Greifswald, Germany.
| | - Elke Zellmer
- Institute for Hygiene and Environmental Medicine, Faculty of Medicine, University of Greifswald, Walter-Rathenau-Straße 49A, 17475 Greifswald, Germany.
| | - Jörg Stachowiak
- Plasma Bioengineering, Leibniz Institute for Plasma Science and Technology e.V., Felix-Hausdorff-Straße 2, 17491 Greifswald, Germany.
| | - Mathias Andrasch
- Plasma Bioengineering, Leibniz Institute for Plasma Science and Technology e.V., Felix-Hausdorff-Straße 2, 17491 Greifswald, Germany.
| | - Harald Below
- Institute for Hygiene and Environmental Medicine, Faculty of Medicine, University of Greifswald, Walter-Rathenau-Straße 49A, 17475 Greifswald, Germany.
| | - Jörg Ehlbeck
- Plasma Bioengineering, Leibniz Institute for Plasma Science and Technology e.V., Felix-Hausdorff-Straße 2, 17491 Greifswald, Germany.
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14
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Role of food sanitising treatments in inducing the ‘viable but nonculturable’ state of microorganisms. Food Control 2018. [DOI: 10.1016/j.foodcont.2018.04.016] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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15
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Ece S, Lambertz C, Fischer R, Commandeur U. Heterologous expression of a Streptomyces cyaneus laccase for biomass modification applications. AMB Express 2017; 7:86. [PMID: 28439850 PMCID: PMC5403781 DOI: 10.1186/s13568-017-0387-0] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2017] [Accepted: 04/11/2017] [Indexed: 11/24/2022] Open
Abstract
Laccases are used for the conversion of biomass into fermentable sugars but it is difficult to produce high yields of active laccases in heterologous expression systems. We overcame this challenge by expressing Streptomyces cyaneus CECT 3335 laccase in Escherichia coli (ScLac) and we achieved a yield of up to 104 mg L-1 following purification by one-step affinity chromatography. Stability and activity assays using simple lignin model substrates showed that the purified enzyme preparation was active over a broad pH range and at high temperatures, suggesting it would be suitable for biomass degradation. The reusability of ScLac was also demonstrated by immobilizing the enzyme on agarose beads with a binding yield of 33%, and by the synthesis of cross-linked enzyme aggregates with an initial activity recovery of 72%.
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Affiliation(s)
- Selin Ece
- Institute for Molecular Biotechnology (Biology VII), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany
| | - Camilla Lambertz
- Institute for Molecular Biotechnology (Biology VII), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany
| | - Rainer Fischer
- Institute for Molecular Biotechnology (Biology VII), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany
- Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, 52074 Aachen, Germany
| | - Ulrich Commandeur
- Institute for Molecular Biotechnology (Biology VII), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany
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16
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Liu J, Zhou R, Li L, Peters BM, Li B, Lin CW, Chuang TL, Chen D, Zhao X, Xiong Z, Xu Z, Shirtliff ME. Viable but non-culturable state and toxin gene expression of enterohemorrhagic Escherichia coli O157 under cryopreservation. Res Microbiol 2017; 168:188-193. [DOI: 10.1016/j.resmic.2016.11.002] [Citation(s) in RCA: 78] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2016] [Revised: 11/08/2016] [Accepted: 11/14/2016] [Indexed: 10/20/2022]
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17
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Recent Developments in Accelerated Antibacterial Inactivation on 2D Cu-Titania Surfaces under Indoor Visible Light. COATINGS 2017. [DOI: 10.3390/coatings7020020] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
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18
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Using adjuvants and environmental factors to modulate the activity of antimicrobial peptides. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2016; 1858:926-35. [PMID: 26751595 DOI: 10.1016/j.bbamem.2015.12.034] [Citation(s) in RCA: 45] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/16/2015] [Revised: 12/22/2015] [Accepted: 12/29/2015] [Indexed: 12/12/2022]
Abstract
The increase in antibiotic resistant and multi-drug resistant bacterial infections has serious implications for the future of health care. The difficulty in finding both new microbial targets and new drugs against existing targets adds to the concern. The use of combination and adjuvant therapies are potential strategies to counter this threat. Antimicrobial peptides (AMPs) are a promising class of antibiotics (ABs), particularly for topical and surface applications. Efforts have been directed toward a number of strategies, including the use of conventional ABs combined with AMPs, and the use of potentiating agents to increase the performance of AMPs. This review focuses on combination strategies such as adjuvants and the manipulation of environmental variables to improve the efficacy of AMPs as potential therapeutic agents. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.
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Kwon MJ, Yang JS, Lee S, Lee G, Ham B, Boyanov MI, Kemner KM, O'Loughlin EJ. Geochemical characteristics and microbial community composition in toxic metal-rich sediments contaminated with Au-Ag mine tailings. JOURNAL OF HAZARDOUS MATERIALS 2015; 296:147-157. [PMID: 25917692 DOI: 10.1016/j.jhazmat.2015.04.035] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/25/2014] [Revised: 03/17/2015] [Accepted: 04/12/2015] [Indexed: 06/04/2023]
Abstract
The effects of extreme geochemical conditions on microbial community composition were investigated for two distinct sets of sediment samples collected near weathered mine tailings. One set (SCH) showed extraordinary geochemical characteristics: As (6.7-11.5%), Pb (1.5-2.1%), Zn (0.1-0.2%), and pH (3.1-3.5). The other set (SCL) had As (0.3-1.2%), Pb (0.02-0.22%), and Zn (0.01-0.02%) at pH 2.5-3.1. The bacterial communities in SCL were clearly different from those in SCH, suggesting that extreme geochemical conditions affected microbial community distribution even on a small spatial scale. The clones identified in SCL were closely related to acidophilic bacteria in the taxa Acidobacterium (18%), Acidomicrobineae (14%), and Leptospirillum (10%). Most clones in SCH were closely related to Methylobacterium (79%) and Ralstonia (19%), both well-known metal-resistant bacteria. Although total As was extremely high, over 95% was in the form of scorodite (FeAsO4·2H2O). Acid-extractable As was only ∼118 and ∼14 mg kg(-1) in SCH and SCL, respectively, below the level known to be toxic to bacteria. Meanwhile, acid-extractable Pb and Zn in SCH were above toxic concentrations. Because As was present in an oxidized, stable form, release of Pb and/or Zn (or a combination of toxic metals in the sediment) from the sediment likely accounts for the differences in microbial community structure. The results also suggest that care should be taken when investigating mine tailings, because large differences in chemical/biological properties can occur over small spatial scales.
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Affiliation(s)
- Man Jae Kwon
- Korea Institute of Science and Technology, Gangneung, South Korea.
| | - Jung-Seok Yang
- Korea Institute of Science and Technology, Gangneung, South Korea.
| | - Seunghak Lee
- Korea Institute of Science and Technology, Seoul, South Korea
| | | | - Baknoon Ham
- Korea Institute of Science and Technology, Gangneung, South Korea
| | - Maxim I Boyanov
- Biosciences Division, Argonne National Laboratory, Argonne, IL, USA; Bulgarian Academy of Sciences, Institute of Chemical Engineering, Sofia, Bulgaria
| | - Kenneth M Kemner
- Biosciences Division, Argonne National Laboratory, Argonne, IL, USA
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20
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Tuteja N, Mishra P, Yadav S, Tajrishi M, Baral S, Sabat SC. Heterologous expression and biochemical characterization of a highly active and stable chloroplastic CuZn-superoxide dismutase from Pisum sativum. BMC Biotechnol 2015; 15:3. [PMID: 25887674 PMCID: PMC4333176 DOI: 10.1186/s12896-015-0117-0] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2014] [Accepted: 01/27/2015] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND CuZn-Superoxide dismutase (SOD) is a unique enzyme, which can catalyzes the dismutation of inevitable metabolic product i.e.; superoxide anion into molecular oxygen and hydrogen peroxide. The enzyme has gained wide interest in pharmaceutical industries due to its highly acclaimed antioxidative properties. The recombinant expression of this protein in its enzymatically active and stable form is highly desired and hence optimization of culture conditions and characterization of the related biochemical properties are essential to explore the significance of the enzyme in physiological, therapeutic, structural and transgenic research. RESULTS High-level expression of the chloroplastic isoform of Pisum sativum CuZn-SOD was achieved at 18°C, upon isopropyl β-D-1-thiogalactopyranoside induction and the process was optimized for maximum recovery of the protein in its soluble (enzymatically active) form. Both crude and purified protein fractions display significant increase in activity following supplementation of defined concentration Cu (CuSO4) and Zn (ZnSO4). Yield of the purified recombinant protein was ~ 4 mg L(-1) of culture volume and the bacterial biomass was ~ 4.5 g L(-1). The recombinant pea chloroplastic SOD was found to possess nearly 6 fold higher superoxide dismutase activity and the peroxidase activity was also 5 fold higher as compared to commercially available CuZn-superoxide dismutase. The computational, spectroscopic and biochemical characterization reveals that the protein harbors all the characteristics features of this class of enzyme. The enzyme was found to be exceptionally stable as evident from pH and temperature incubation studies and maintenance of SOD activity upon prolonged storage. CONCLUSIONS Overexpression and purification strategy presented here describes an efficient protocol for the production of a highly active and stable CuZn-superoxide dismutase in its recombinant form in E. coli system. The strategy can be utilized for the large-scale preparation of active CuZn-superoxide dismutase and thus it has wide application in pharmaceutical industries and also for elucidating the potential of this protein endowed with exceptional stability and activity.
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Affiliation(s)
- Narendra Tuteja
- Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi, 110067, India.
| | - Panchanand Mishra
- Stress Biology Laboratory, Gene Function and Regulation, Institute of Life Sciences, Bhubaneswar, 751023, Odisha, India.
| | - Sandep Yadav
- Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi, 110067, India.
| | - Marjan Tajrishi
- Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi, 110067, India.
| | - Sudhir Baral
- Stress Biology Laboratory, Gene Function and Regulation, Institute of Life Sciences, Bhubaneswar, 751023, Odisha, India.
| | - Surendra Chandra Sabat
- Stress Biology Laboratory, Gene Function and Regulation, Institute of Life Sciences, Bhubaneswar, 751023, Odisha, India.
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Feio MJ, Sousa I, Ferreira M, Cunha-Silva L, Saraiva RG, Queirós C, Alexandre JG, Claro V, Mendes A, Ortiz R, Lopes S, Amaral AL, Lino J, Fernandes P, Silva AJ, Moutinho L, de Castro B, Pereira E, Perelló L, Gameiro P. Fluoroquinolone–metal complexes: A route to counteract bacterial resistance? J Inorg Biochem 2014; 138:129-143. [DOI: 10.1016/j.jinorgbio.2014.05.007] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2014] [Revised: 05/16/2014] [Accepted: 05/20/2014] [Indexed: 10/25/2022]
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Navarrete F, De La Fuente L. Response of Xylella fastidiosa to zinc: decreased culturability, increased exopolysaccharide production, and formation of resilient biofilms under flow conditions. Appl Environ Microbiol 2014; 80:1097-107. [PMID: 24271184 PMCID: PMC3911211 DOI: 10.1128/aem.02998-13] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2013] [Accepted: 11/20/2013] [Indexed: 12/30/2022] Open
Abstract
The bacterial plant pathogen Xylella fastidiosa produces biofilm that accumulates in the host xylem vessels, affecting disease development in various crops and bacterial acquisition by insect vectors. Biofilms are sensitive to the chemical composition of the environment, and mineral elements being transported in the xylem are of special interest for this pathosystem. Here, X. fastidiosa liquid cultures were supplemented with zinc and compared with nonamended cultures to determine the effects of Zn on growth, biofilm, and exopolysaccharide (EPS) production under batch and flow culture conditions. The results show that Zn reduces growth and biofilm production under both conditions. However, in microfluidic chambers under liquid flow and with constant bacterial supplementation (closer to conditions inside the host), a dramatic increase in biofilm aggregates was seen in the Zn-amended medium. Biofilms formed under these conditions were strongly attached to surfaces and were not removed by medium flow. This phenomenon was correlated with increased EPS production in stationary-phase cells grown under high Zn concentrations. Zn did not cause greater adhesion to surfaces by individual cells. Additionally, viability analyses suggest that X. fastidiosa may be able to enter the viable but nonculturable state in vitro, and Zn can hasten the onset of this state. Together, these findings suggest that Zn can act as a stress factor with pleiotropic effects on X. fastidiosa and indicate that, although Zn could be used as a bactericide treatment, it could trigger the undesired effect of stronger biofilm formation upon reinoculation events.
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Affiliation(s)
- Fernando Navarrete
- Department of Entomology and Plant Pathology, Auburn University, Auburn, Alabama, USA
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23
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Zou R, Zhou K, Stephanopoulos G, Too HP. Combinatorial engineering of 1-deoxy-D-xylulose 5-phosphate pathway using cross-lapping in vitro assembly (CLIVA) method. PLoS One 2013; 8:e79557. [PMID: 24223968 PMCID: PMC3818232 DOI: 10.1371/journal.pone.0079557] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2013] [Accepted: 10/02/2013] [Indexed: 11/18/2022] Open
Abstract
The ability to assemble multiple fragments of DNA into a plasmid in a single step is invaluable to studies in metabolic engineering and synthetic biology. Using phosphorothioate chemistry for high efficiency and site specific cleavage of sequences, a novel ligase independent cloning method (cross-lapping in vitro assembly, CLIVA) was systematically and rationally optimized in E. coli. A series of 16 constructs combinatorially expressing genes encoding enzymes in the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway were assembled using multiple DNA modules. A plasmid (21.6 kb) containing 16 pathway genes, was successfully assembled from 7 modules with high efficiency (2.0 x 10(3) cfu/ µg input DNA) within 2 days. Overexpressions of these constructs revealed the unanticipated inhibitory effects of certain combinations of genes on the production of amorphadiene. Interestingly, the inhibitory effects were correlated to the increase in the accumulation of intracellular methylerythritol cyclodiphosphate (MEC), an intermediate metabolite in the DXP pathway. The overexpression of the iron sulfur cluster operon was found to modestly increase the production of amorphadiene. This study demonstrated the utility of CLIVA in the assembly of multiple fragments of DNA into a plasmid which enabled the rapid exploration of biological pathways.
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Affiliation(s)
- Ruiyang Zou
- Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, Singapore, Singapore
| | - Kang Zhou
- Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, Singapore, Singapore
| | - Gregory Stephanopoulos
- Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, Singapore, Singapore
- Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America
| | - Heng Phon Too
- Chemical and Pharmaceutical Engineering, Singapore-MIT Alliance, Singapore, Singapore
- Department of Biochemistry, National University of Singapore, Singapore, Singapore
- * E-mail:
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Kushneruk MA, Tugarova AV, Il’chukova AV, Slavkina EA, Starichkova NI, Bogatyrev VA, Antonyuk LP. Factors inducing transition from growth to dormancy in rhizobacteria Azospirillum brasilense. Microbiology (Reading) 2013. [DOI: 10.1134/s0026261713050081] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
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Moyne AL, Harris LJ, Marco ML. Assessments of total and viable Escherichia coli O157:H7 on field and laboratory grown lettuce. PLoS One 2013; 8:e70643. [PMID: 23936235 PMCID: PMC3728298 DOI: 10.1371/journal.pone.0070643] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2013] [Accepted: 06/19/2013] [Indexed: 02/07/2023] Open
Abstract
Leafy green produce has been associated with numerous outbreaks of foodborne illness caused by strains of Escherichia coli O157:H7. While the amounts of culturable E. coli O157:H7 rapidly decline after introduction onto lettuce in the field, it remains to be determined whether the reduction in cell numbers is due to losses in cell viability, cell injury and a subsequent inability to be detected by standard laboratory culturing methods, or a lack of adherence and hence rapid removal of the organism from the plants during application. To assess which of these options is most relevant for E. coli O157:H7 on leafy green produce, we developed and applied a propidium monoazide (PMA) real-time PCR assay to quantify viable (with PMA) and total (without PMA) E. coli O157:H7 cells on growth chamber and field-grown lettuce. E. coli O157:H7, suspended in 0.1% peptone, was inoculated onto 4-week-old lettuce plants at a level of approximately 10(6) CFU/plant. In the growth chamber at low relative humidity (30%), culturable amounts of the nontoxigenic E. coli O157:H7 strain ATCC 700728 and the virulent strain EC4045 declined 100 to 1000-fold in 24 h. Fewer E. coli O157:H7 cells survived when applied onto plants in droplets with a pipette compared with a fine spray inoculation. Total cells for both strains were equivalent to inoculum levels for 7 days after application, and viable cell quantities determined by PMA real-time PCR were approximately 10(4) greater than found by colony enumeration. Within 2 h after application onto plants in the field, the number of culturable E. coli ATCC 700728 was reduced by up to 1000-fold, whereas PCR-based assessments showed that total cell amounts were equivalent to inoculum levels. These findings show that shortly after inoculation onto plants, the majority of E. coli O157:H7 cells either die or are no longer culturable.
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Affiliation(s)
- Anne-Laure Moyne
- Department of Food Science and Technology, University of California Davis, Davis, California, United States of America
- Western Center for Food Safety, University of California Davis, Davis, California, United States of America
| | - Linda J. Harris
- Department of Food Science and Technology, University of California Davis, Davis, California, United States of America
- Western Center for Food Safety, University of California Davis, Davis, California, United States of America
| | - Maria L. Marco
- Department of Food Science and Technology, University of California Davis, Davis, California, United States of America
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Nowakowska J, Oliver JD. Resistance to environmental stresses by Vibrio vulnificus in the viable but nonculturable state. FEMS Microbiol Ecol 2013; 84:213-22. [PMID: 23228034 DOI: 10.1111/1574-6941.12052] [Citation(s) in RCA: 108] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2012] [Revised: 11/05/2012] [Accepted: 12/03/2012] [Indexed: 11/29/2022] Open
Abstract
Vibrio vulnificus is responsible for 95% of all seafood-associated fatalities in the United States. When water temperatures drop below c. 13 °C, the cells enter into the viable but nonculturable (VBNC) state wherein they are unable to grow on routine media but retain viability and the ability to return to the culturable state. The aim of this study was to determine whether V. vulnificus cells in the VBNC state are protected against a variety of potentially lethal challenges (heat, oxidative, osmotic, pH, ethanol, antibiotic and heavy metal) and to examine genetic regulators that might underlie such protection. The data presented here indicate that VBNC cells of this pathogen are protected against a wide variety of stresses and retain the ability to return to the culturable state. Surprisingly, we found no significant difference in the expression of relA and spoT between VBNC and logarithmic cells, nor any significant difference in the expression of rpoS in the case of the clinical (C) genotype of this pathogen. However, expression of relA was significantly different in VBNC cells of the environmental (E) genotype compared with logarithmic cells. This might account for findings indicating an enhanced ability for E-genotype cells to withstand environmental changes better than C-genotype cells.
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Affiliation(s)
- Joanna Nowakowska
- Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223, USA.
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27
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Formation and resuscitation of viable but nonculturable Salmonella typhi. BIOMED RESEARCH INTERNATIONAL 2012; 2013:907170. [PMID: 23509799 PMCID: PMC3591152 DOI: 10.1155/2013/907170] [Citation(s) in RCA: 62] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/25/2012] [Revised: 11/20/2012] [Accepted: 11/25/2012] [Indexed: 01/27/2023]
Abstract
Salmonella typhi is a pathogen that causes the human disease of typhoid fever. The aim of this study was to investigate the viable but nonculturable (VBNC) state of S. typhi. Some samples were stimulated at 4°C or −20°C, while others were induced by different concentrations of CuSO4. Total cell counts remained constant throughout several days by acridine orange direct counting; however, plate counts declined to undetectable levels within 48 hours by plate counting at −20°C. The direct viable counts remained fairly constant at this level by direct viable counting. Carbon and nitrogen materials slowly decreased which indicated that a large population of cells existed in the VBNC state and entered the VBNC state in response to exposure to 0.01 or 0.015 mmol/L CuSO4 for more than 14 or 12 days, respectively. Adding 3% Tween 20 or 1% catalase enabled cells to become culturable again, with resuscitation times of 48 h and 24 h, respectively. The atomic force microscope results showed that cells gradually changed in shape from short rods to coccoids, and decreased in size when they entered the VBNC state. Further animal experiments suggested that resuscitated cells might regain pathogenicity.
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28
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Borkow G, Monk A. Fighting nosocomial infections with biocidal non-intrusive hard and soft surfaces. World J Clin Infect Dis 2012; 2:77-90. [DOI: 10.5495/wjcid.v2.i4.77] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Approximately 7 million people worldwide acquire a healthcare associated infection each year. Despite aggressive monitoring, hand washing campaigns and other infection control measures, nosocomial infections (NI) rates, especially those caused by antibiotic resistant pathogens, are unacceptably high worldwide. Additional ways to fight these infections need to be developed. A potential overlooked and neglected source of nosocomial pathogens are those found in non-intrusive soft and hard surfaces located in clinical settings. Soft surfaces, such as patient pyjamas and beddings, can be an excellent substrate for bacterial and fungal growth under appropriate temperature and humidity conditions as those present between patients and the bed. Bed making in hospitals releases large quantities of microorganisms into the air, which contaminate the immediate and non-immediate surroundings. Microbes can survive on hard surfaces, such as metal trays, bed rails and door knobs, for very prolonged periods of time. Thus soft and hard surfaces that are in direct or indirect contact with the patients can serve as a source of nosocomial pathogens. Recently it has been demonstrated that copper surfaces and copper oxide containing textiles have potent intrinsic biocidal properties. This manuscript reviews the recent laboratory and clinical studies, which demonstrate that biocidal surfaces made of copper or containing copper can reduce the microbiological burden and the NI rates.
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29
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Sousa I, Claro V, Pereira JL, Amaral AL, Cunha-Silva L, de Castro B, Feio MJ, Pereira E, Gameiro P. Synthesis, characterization and antibacterial studies of a copper(II) levofloxacin ternary complex. J Inorg Biochem 2012; 110:64-71. [PMID: 22469700 DOI: 10.1016/j.jinorgbio.2012.02.003] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2011] [Revised: 02/03/2012] [Accepted: 02/03/2012] [Indexed: 10/14/2022]
Abstract
Solution behavior of levofloxacin (lvx) complexes with copper(II) in the presence and absence of phen was studied in aqueous solution, by potentiometry. The results obtained show that under physiological conditions (micromolar concentration range and pH 7.4) only copper(II):lvx:phen ternary complexes are stable. Hence, a novel copper(II) ternary complex of fluoroquinolone levofloxacin with nitrogen donor heterocyclic ligand phen was synthesized and characterized by means of UV-Visible and IR spectroscopy, elemental analysis and X-Ray crystallography. In the synthesized complex (1), [Cu(lvx)(phen)(H(2)O)](NO(3)).2H(2)O, levofloxacin acts as a bidentate ligand coordinating to the metal, in its anionic form, through the carbonyl and carboxyl oxygens and phen coordinates through two N-atoms forming the equatorial plane of a distorted square-pyramidal geometry. The fifth ligand of the penta-coordinated Cu(II) centre is occupied axially by an oxygen atom from a water molecule. Minimum inhibitory concentration (MIC) determinations of the complex and comparison with free levofloxacin in various E. coli strains indicated that the Cu-complex is as efficient an antimicrobial as the free antibiotic (both in the case of the dissolved synthesized complex and the complex formed following stoichiometric mixture of the individual components in solution). Moreover, results strongly suggest that the cell intake route of both species is different supporting, therefore, the complex's suitability as a candidate for further biological testing in fluoroquinolone-resistant microorganisms.
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Affiliation(s)
- Isabel Sousa
- Requimte, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre, 4169-007 Porto, Portugal
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Dwidjosiswojo Z, Richard J, Moritz MM, Dopp E, Flemming HC, Wingender J. Influence of copper ions on the viability and cytotoxicity of Pseudomonas aeruginosa under conditions relevant to drinking water environments. Int J Hyg Environ Health 2011; 214:485-92. [DOI: 10.1016/j.ijheh.2011.06.004] [Citation(s) in RCA: 67] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2011] [Revised: 06/10/2011] [Accepted: 06/15/2011] [Indexed: 10/18/2022]
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Aurass P, Prager R, Flieger A. EHEC/EAEC O104:H4 strain linked with the 2011 German outbreak of haemolytic uremic syndrome enters into the viable but non-culturable state in response to various stresses and resuscitates upon stress relief. Environ Microbiol 2011; 13:3139-48. [DOI: 10.1111/j.1462-2920.2011.02604.x] [Citation(s) in RCA: 82] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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Pinto D, Almeida V, Almeida Santos M, Chambel L. Resuscitation of Escherichia coli VBNC cells depends on a variety of environmental or chemical stimuli. J Appl Microbiol 2011; 110:1601-11. [DOI: 10.1111/j.1365-2672.2011.05016.x] [Citation(s) in RCA: 101] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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Furkert FH, Sörensen JH, Arnoldi J, Robioneck B, Steckel H. Antimicrobial Efficacy of Surface-Coated External Fixation Pins. Curr Microbiol 2011; 62:1743-51. [DOI: 10.1007/s00284-011-9923-3] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2010] [Accepted: 03/04/2011] [Indexed: 11/25/2022]
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Saraiva R, Lopes S, Ferreira M, Novais F, Pereira E, Feio MJ, Gameiro P. Solution and biological behaviour of enrofloxacin metalloantibiotics: A route to counteract bacterial resistance? J Inorg Biochem 2010; 104:843-50. [DOI: 10.1016/j.jinorgbio.2010.03.017] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2010] [Accepted: 03/26/2010] [Indexed: 12/22/2022]
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Palmieri ACB, do Amaral AM, Homem RA, Machado MA. Differential expression of pathogenicity- and virulence-related genes of Xanthomonas axonopodis pv. citri under copper stress. Genet Mol Biol 2010; 33:348-53. [PMID: 21637493 PMCID: PMC3036875 DOI: 10.1590/s1415-47572010005000030] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2009] [Accepted: 10/14/2009] [Indexed: 11/21/2022] Open
Abstract
In this study, we used real-time quantitative PCR (RT-qPCR) to evaluate the expression of 32 genes of Xanthomonas axonopodis pv. citri related to pathogenicity and virulence that are also involved in copper detoxification. Nearly all of the genes were up-regulated, including copA and copB. Two genes homologous to members of the type II secretion system (xcsH and xcsC) and two involved in the degradation of plant cell wall components (pglA and pel) were the most expressed in response to an elevated copper concentration. The type II secretion system (xcs operon) and a few homologues of proteins putatively secreted by this system showed enhanced expression when the bacteria were exposed to a high concentration of copper sulfate. The enhanced expression of the genes of secretion II system during copper stress suggests that this pathway may have an important role in the adaptative response of X. axonopodis pv. citri to toxic compounds. These findings highlight the potential role of these genes in attenuating the toxicity of certain metals and could represent an important means of bacterial resistance against chemicals used to control diseases.
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Díaz M, Herrero M, García LA, Quirós C. Application of flow cytometry to industrial microbial bioprocesses. Biochem Eng J 2010. [DOI: 10.1016/j.bej.2009.07.013] [Citation(s) in RCA: 182] [Impact Index Per Article: 12.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
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Harrison JJ, Tremaroli V, Stan MA, Chan CS, Vacchi-Suzzi C, Heyne BJ, Parsek MR, Ceri H, Turner RJ. Chromosomal antioxidant genes have metal ion-specific roles as determinants of bacterial metal tolerance. Environ Microbiol 2009; 11:2491-509. [PMID: 19555372 DOI: 10.1111/j.1462-2920.2009.01973.x] [Citation(s) in RCA: 80] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
Microbiological metal toxicity involves redox reactions between metal species and cellular molecules, and therefore, we hypothesized that antioxidant systems might be chromosomal determinants affecting the susceptibility of bacteria to metal toxicity. Here, survival was quantified in metal ion-exposed planktonic cultures of several Escherichia coli strains, each bearing a mutation in a gene important for redox homeostasis. This characterized approximately 250 gene-metal combinations and identified that sodA, sodB, gor, trxA, gshA, grxA and marR have distinct roles in safeguarding or sensitizing cells to different toxic metal ions (Cr(2)O(7)(2-), Co(2+), Cu(2+), Ag(+), Zn(2+), AsO(2)(-), SeO(3)(2-) or TeO(3)(2-)). To shed light on these observations, fluorescent sensors for reactive oxygen species (ROS) and reduced thiol (RSH) quantification were used to ascertain that different metal ions exert oxidative toxicity through disparate modes-of-action. These oxidative mechanisms of metal toxicity were categorized as involving ROS and thiol-disulfide chemistry together (AsO(2)(-), SeO(3)(2-)), ROS predominantly (Cu(2+), Cr(2)O(7)(2-)) or thiol-disulfide chemistry predominantly (Ag(+), Co(2+), Zn(2+), TeO(3)(2-)). Corresponding to this, promoter-luxCDABE fusions showed that toxic doses of different metal ions up- or downregulate the transcription of gene sets marking distinct pathways of cellular oxidative stress. Altogether, our findings suggest that different metal ions are lethal to cells through discrete pathways of oxidative biochemistry, and moreover, indicate that chromosomally encoded antioxidant systems may have metal ion-specific physiological roles as determinants of bacterial metal tolerance.
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Affiliation(s)
- Joe J Harrison
- Department of Biological Sciences, University of Calgary, N. W. Calgary, Alberta, Canada
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Dinu LD, Delaquis P, Bach S. Nonculturable response of animal enteropathogens in the agricultural environment and implications for food safety. J Food Prot 2009; 72:1342-54. [PMID: 19610353 DOI: 10.4315/0362-028x-72.6.1342] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Concerns about animal enteropathogen contamination of fresh horticultural products have,increased worldwide and are mainly due to the ability of bacteria to survive under stress conditions in the agricultural environment and during raw-vegetable processing. This review challenges the idea that the viable but nonculturable phenomenon that has been proven to occur in plant-associated environments contributes to human pathogen survival and might be correlated with foodborne infection. Factors associated with the nonculturable response of bacteria in the field and during postharvest processing and distribution are discussed, specifically for the most common animal enteropathogens linked with the consumption of raw products: Escherichia coli O157:H7, Salmonella, Listeria monocytogenes, and Shigella spp. The accurate detection of live bacterial populations is essential for pathogen screening in food and environmental safety control and in epidemiological analysis and may have to be considered for identification of critical control points at the time of food inspection.
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Affiliation(s)
- Laura-Dorina Dinu
- Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, 4200 Highway 97 South, Summerland, British Columbia, Canada
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Copper and quaternary ammonium cations exert synergistic bactericidal and antibiofilm activity against Pseudomonas aeruginosa. Antimicrob Agents Chemother 2008; 52:2870-81. [PMID: 18519726 DOI: 10.1128/aac.00203-08] [Citation(s) in RCA: 122] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Biofilms are slimy aggregates of microbes that are likely responsible for many chronic infections as well as for contamination of clinical and industrial environments. Pseudomonas aeruginosa is a prevalent hospital pathogen that is well known for its ability to form biofilms that are recalcitrant to many different antimicrobial treatments. We have devised a high-throughput method for testing combinations of antimicrobials for synergistic activity against biofilms, including those formed by P. aeruginosa. This approach was used to look for changes in biofilm susceptibility to various biocides when these agents were combined with metal ions. This process identified that Cu(2+) works synergistically with quaternary ammonium compounds (QACs; specifically benzalkonium chloride, cetalkonium chloride, cetylpyridinium chloride, myristalkonium chloride, and Polycide) to kill P. aeruginosa biofilms. In some cases, adding Cu(2+) to QACs resulted in a 128-fold decrease in the biofilm minimum bactericidal concentration compared to that for single-agent treatments. In combination, these agents retained broad-spectrum antimicrobial activity that also eradicated biofilms of Escherichia coli, Staphylococcus aureus, Salmonella enterica serovar Cholerasuis, and Pseudomonas fluorescens. To investigate the mechanism of action, isothermal titration calorimetry was used to show that Cu(2+) and QACs do not interact in aqueous solutions, suggesting that each agent exerts microbiological toxicity through independent biochemical routes. Additionally, Cu(2+) and QACs, both alone and in combination, reduced the activity of nitrate reductases, which are enzymes that are important for normal biofilm growth. Collectively, the results of this study indicate that Cu(2+) and QACs are effective combinations of antimicrobials that may be used to kill bacterial biofilms.
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Delaedt Y, Daneels A, Declerck P, Behets J, Ryckeboer J, Peters E, Ollevier F. The impact of electrochemical disinfection on Escherichia coli and Legionella pneumophila in tap water. Microbiol Res 2008; 163:192-9. [PMID: 16793247 DOI: 10.1016/j.micres.2006.05.002] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2006] [Revised: 04/13/2006] [Accepted: 05/02/2006] [Indexed: 10/24/2022]
Abstract
In order to reduce the risks of Legionnaires' disease, caused by the bacterium Legionella pneumophila, disinfection of tap water systems contaminated with this bacterium is a necessity. This study investigates if electrochemical disinfection is able to eliminate such contamination. Hereto, water spiked with bacteria (10(4)CFU Escherichia coli or L. pneumophila/ml) was passed through an electrolysis cell (direct effect) or bacteria were added to tap water after passage through such disinfection unit (residual effect). The spiked tap water was completely disinfected, during passage through the electrolysis cell, even when only a residual free oxidant concentration of 0.07 mg/l is left (L. pneumophila). The residual effect leads to a complete eradication of cultivable E. coli, if after reaction time at least a free oxidant concentration of 0.08 mg/l is still present. Similar conditions reduce substantially L. pneumophila, but a complete killing is not realised.
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Affiliation(s)
- Yasmine Delaedt
- Laboratory of Aquatic Ecology, Katholieke Universiteit Leuven, Ch. Deberiotstraat 32, B-3000 Leuven, Belgium.
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Amorós I, Alonso JL, Romaguera S, Carrasco JM. Assessment of toxicity of a glyphosate-based formulation using bacterial systems in lake water. CHEMOSPHERE 2007; 67:2221-8. [PMID: 17270238 DOI: 10.1016/j.chemosphere.2006.12.020] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/19/2006] [Revised: 11/28/2006] [Accepted: 12/08/2006] [Indexed: 05/13/2023]
Abstract
A new Aeromonas bioassay is described to assess the potential harmful effects of the glyphosate-based herbicide, Roundup, in the Albufera lake, a protected area near Valencia. Viability markers as membrane integrity, culturability and beta-galactosidase production of Aeromonas caviae were studied to determine the influence of the herbicide in the bacterial cells. Data from the multifactor analysis of variance test showed no significant differences (P>0.05) between A. caviae counts of viability markers at the studied concentrations (0, 50 and 100 mg l-1 of glyphosate). The effects of Roundup on microbial biota present in the lake were assessed by measuring the number of indigenous mesophilic Aeromonas in presence of different amounts of the herbicide at 0, 50 and 100 mg l-1 of glyphosate. In samples containing 50 and 100 mg l-1 of glyphosate a significant (P<0.05) increase in Aeromonas spp. counts and accompanying flora was observed. The acute toxicity of Roundup and of Roundup diluted with Albufera lake water to Microtox luminescent bacterium (Vibrio fischeri) also was determined. The EC50 values obtained were 36.4 mg l-1 and 64.0 mgl-1 of glyphosate respectively. The acidity (pH 4.5) of the herbicide formulation was the responsible of the observed toxicity.
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Affiliation(s)
- I Amorós
- Instituto de Ingeniería del Agua y Medio Ambiente, Grupo De Química y Microbiología del Agua, Ciudad Politécnica de la Innovación, Edificio 8G, Acceso D, Universidad Politécnica de Valencia, Camino de Vera 14, 46022 Valencia, Spain.
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Higgins MJ, Chen YC, Murthy SN, Hendrickson D, Farrel J, Schafer P. Reactivation and growth of non-culturable indicator bacteria in anaerobically digested biosolids after centrifuge dewatering. WATER RESEARCH 2007; 41:665-73. [PMID: 17107701 DOI: 10.1016/j.watres.2006.09.017] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/17/2006] [Revised: 07/28/2006] [Accepted: 09/14/2006] [Indexed: 05/12/2023]
Abstract
Recent literature has reported that high concentrations of indicator bacteria such as fecal coliforms (FCs) were measured in anaerobically digested sludges immediately after dewatering even though low concentrations were measured prior to dewatering. This research hypothesized that the indicator bacteria can enter a non-culturable state during digestion, and are reactivated during centrifuge dewatering. Reactivation is defined as restoration of culturability. To examine this hypothesis, a quantitative polymerase chain reaction (qPCR) method was developed to enumerate Escherichia coli, a member of the FC group, during different phases of digestion and dewatering. For thermophilic digestion, the density of E. coli measured by qPCR could be five orders of magnitude greater than the density measured by standard culturing methods (SCMs), which is indicative of non-culturable bacteria. For mesophilic digestion, qPCR enumerated up to about one order of magnitude more E. coli than the SCMs. After centrifuge dewatering, the non-culturable organisms could be reactivated such that they are enumerated by SCMs, and the conditions in the cake allowed rapid growth of FCs and E. coli during cake storage.
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Affiliation(s)
- Matthew J Higgins
- Department of Civil and Environmental Engineering, Bucknell University, PA 17837, USA.
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Tandon P, Chhibber S, Reed RH. Inactivation of Escherichia coli and coliform bacteria in traditional brass and earthernware water storage vessels. Antonie van Leeuwenhoek 2005; 88:35-48. [PMID: 15928975 DOI: 10.1007/s10482-004-7366-6] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/27/2004] [Accepted: 12/08/2004] [Indexed: 10/25/2022]
Abstract
The detection and enumeration of indicator bacteria such as Escherichia coli is used to assess the extent of faecal contamination of drinking water. On the basis of this approach, the effectiveness of storing water contaminated with faecal indicator bacteria in brass or earthern vessels (mutkas) of the type used in rural India have been investigated. Suspensions of bacteria in sterile distilled water were maintained for up to 48 h in each vessel and enumerated by surface plate counts on nutrient agar (non-selective) and several selective coliform media at 37 degrees C either under standard aerobic conditions, or under conditions designed to neutralise reactive oxygen species (ROS), e.g. using an anaerobic cabinet to prepare plates of pre-reduced growth medium or by inclusion of sodium pyruvate in the growth medium, with incubation of aerobically-prepared plates in an anaerobic jar. The counts obtained for E. coli decreased on short-term storage in a brass mutka; counts for selective media were lower than for equivalent counts for non-selective medium, with ROS-neutralised conditions giving consistently higher counts than aerobic incubation. However, after 48 h, no bacteria were cultivable under any conditions. Similar results were obtained using water from environmental sources in the Panjab, and from rural households where brass and earthern mutkas are used for storage of drinking water, with enumeration on selective coliform media (presumptive total coliforms). In all cases results indicated that, while storage of water in a brass mutka can inactivate E. coli and coliforms over a 48 h period, standard aerobic plate counting using selective media may not be fully effective in enumerating sub-lethally damaged bacteria.
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Affiliation(s)
- Puja Tandon
- Division of Biomedical Sciences, Northumbria University, Ellison Place, NE1 8ST, Newcastle upon Tyne, Tyne and Wear, UK
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Williams AP, Avery LM, Killham K, Jones DL. Persistence of Escherichia coli O157 on farm surfaces under different environmental conditions. J Appl Microbiol 2005; 98:1075-83. [PMID: 15836476 DOI: 10.1111/j.1365-2672.2004.02530.x] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
AIMS To compare the persistence of Escherichia coli O157 on a variety of common faecally contaminated farmyard material surfaces (wood and steel) under different moisture and temperature regimes. METHODS AND RESULTS Samples of field-conditioned farmyard materials (galvanized steel and wood) were cut into pieces and contaminated with fresh cattle faeces inoculated with nontoxigenic E. coli O157 (strain 3704). Thereafter, they were stored at four different environmental conditions; with temperature (5 and 20 degrees C) and moisture (moist or dry) as variables. Transfer of the pathogen to hands from the surfaces was also evaluated. Escherichia coli O157 numbers declined over time on all surfaces albeit at different rates according to the sample material and environmental conditions. Persistence was greatest on moist wood samples under cooler temperatures with large population numbers remaining after 28 days. Desiccation of surfaces resulted in a more rapid decline in E. coli O157 populations under both temperature regimes. Substantial numbers of colonies may also potentially be transferred to human hands from the surfaces during brief contact. CONCLUSIONS When environmental conditions are favourable, E. coli O157 may persist for considerable times on a range of surfaces. However, when exposed to higher temperatures and dehydration, survival is notably decreased. Overall, bacterial persistence was significantly greater on wood samples relative to steel. SIGNIFICANCE AND IMPACT OF THE STUDY Escherichia coli O157 is a prevalent pathogen, common in ruminant faeces. Contact with contaminated faeces may lead to human infection, resulting in possible severe illness. Although our study used only one strain of bacteria, our findings indicates that E. coli O157 has the potential to persist for long periods of time on gates, stiles and other farmyard surfaces under a range of environmental conditions. These farmyard surfaces therefore pose a potential infection pathway particularly where there is a high risk of direct human contact (e.g. child petting zoos, open farms).
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Affiliation(s)
- A P Williams
- School of Agricultural and Forest Sciences, University of Wales, Bangor, Gwynedd, UK
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Sachidanandham R, Gin KYH, Poh CL. Monitoring of active but non-culturable bacterial cells by flow cytometry. Biotechnol Bioeng 2005; 89:24-31. [PMID: 15540195 DOI: 10.1002/bit.20304] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Flow cytometric signatures (i.e., light scatter, red and green fluorescence) were obtained for the active but non-culturable (ABNC) cells of E. coli and a coliform isolate H03N1, in seawater microcosms using BacLight, a live-dead assay kit from Molecular Probes (Eugene/Portland, OR). Previous studies have reported that there are two major adaptations, which cells undergo during the formation of ABNC states: cell wall toughening and DNA condensation. Therefore, we hypothesized that the matured ABNC forms should be more resistant to extreme temperature treatments (i.e., by freezing in liquid nitrogen and thawing at room temperature) than the normal and transition populations. It was shown that the membrane-compromised cells (comprising of normal wild-type and dead cells which are less resistant to rapid freeze thaw) could be differentiated from the matured ABNC using BacLight staining and fluorescence detection by flow cytometry. The population of ABNC cells, which could not be cultured using m-FC media (for the enumeration of fecal coliforms), was resuscitated in phosphate buffer saline followed by growth in Luria broth. Flow cytometry was thus able to detect and differentiate the ABNC cells against a mixed population comprising of culturable cells, transition populations, and dead cells. The results also showed that the formation of ABNC is as early as 2 days in seawater microcosms. By directly comparing the coliform levels enumerated by the BacLight based flow cytometry assays and m-FC technique, it was shown that the presence of coliforms can be undetected by the membrane filtration method.
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Affiliation(s)
- Ramaiah Sachidanandham
- Department of Biotechnology, Food Resources & Marine Sciences Division, Kuwait Institute for Scientific Research, P.O. Box: 24885, 13109 Safat-Kuwait
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Anderson M, Bollinger D, Hagler A, Hartwell H, Rivers B, Ward K, Steck TR. Viable but nonculturable bacteria are present in mouse and human urine specimens. J Clin Microbiol 2004; 42:753-8. [PMID: 14766848 PMCID: PMC344478 DOI: 10.1128/jcm.42.2.753-758.2004] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The presence of viable but nonculturable bacteria in human clean-catch and mouse bladder-isolated urine specimens was investigated. Viable but nonculturable bacteria are alive but do not give rise to visible growth under nonselective growth conditions. Urine specimens obtained from human female volunteers with or without an active urinary tract infection were found to contain, on average, significantly more viable than culturable forms of bacteria. Additional support for the presence of viable but nonculturable cells in urine specimens considered sterile was obtained from examination of urine specimens obtained directly from the bladder of healthy mice. Because the viability assay used to study the viable but nonculturable condition is by necessity growth independent, and hence indirect, the accuracy of this assay that scores cells with intact cell membranes as being viable was studied. Greater than 95% of Escherichia coli cells exposed to lethal doses of UV irradiation were found to lose their membrane integrity within a day, a time frame similar to that used to examine urine specimens. These data suggest that viable but nonculturable cells can occur within regions of the urinary tract previously considered sterile.
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Affiliation(s)
- Marc Anderson
- Department of Biology, University of North Carolina at Charlotte, Charlotte, North Carolina 28223-0001, USA
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Abstract
A significant number of pathogenic microorganisms can be found in environmental reservoirs (air, water, soil). It is important to assess the viability status of these organisms to determine whether they pose a threat to public health. Classical methods for determining viability are time consuming. Hence, molecular methods have been developed to address this problem. Molecular methods offer speed, sensitivity and specificity. Both DNA and RNA have been analysed using molecular amplification methods such as polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR) and nucleic acid sequence-based amplification (NASBA). However, due to the variable persistence of nucleic acids in cells post-death, the correlation between presence of DNA and RNA and viability is not clear-cut. Similarly, the choice of target and sensitivity of the method can significantly affect the validity of the viability assay. This review assesses the molecular methods currently available and evaluates their ability to assess cell viability with emphasis on environmental pathogens.
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Affiliation(s)
- J T Keer
- BioAnalytical Innovation Team, LGC Limited, Queens Road, Teddington, Middlesex TW11 0LY, UK.
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Sachidanandham R, Gin KYH. Flow cytometric detection of beta-D-glucuronidase gene in wild-type bacterial cells using in-situ PCR. Biotechnol Bioeng 2003; 82:127-33. [PMID: 12584754 DOI: 10.1002/bit.10552] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
An in situ PCR-based flow cytometry method useful for monitoring the presence or absence of the beta-D-glucuronidase gene in Escherichia coli has been developed. A single-step fixation and permeabilization procedure, which maintained cell integrity at the elevated temperatures used during thermal cycling in the presence of PCR reagents, was demonstrated. We have chosen a shorter DNA sequence of length 147 bp for the PCR. Cells subjected to in situ PCR using fluorescein-12-dUTP as a label, showed the presence of uid both in epifluorescence microscopic examination and flow cytometric analysis. Multi-parametric analysis of flow cytometric profiles revealed that the efficiency of labeling was found to be high. The potential of in situ PCR for the detection of uid in intact coliform cells was then successfully tested with a fecal coliform isolated from the coastal waters of Singapore.
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Affiliation(s)
- Ramaiah Sachidanandham
- Tropical Marine Science Institute, 14 Kent Ridge Road, National University of Singapore, Singapore 119223.
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49
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Mukamolova GV, Kaprelyants AS, Kell DB, Young M. Adoption of the transiently non-culturable state — a bacterial survival strategy? Adv Microb Physiol 2003; 47:65-129. [PMID: 14560663 DOI: 10.1016/s0065-2911(03)47002-1] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
Microbial culturability can be ephemeral. Cells are not merely either dead or alive but can adopt physiological states in which they appear to be (transiently) non-culturable under conditions in which they are known normally to be able to grow and divide. The reacquisition of culturability from such states is referred to as resuscitation. We here develop the idea that this "transient non-culturability" is a consequence of a special survival strategy, and summarise the morphological, physiological and genetic evidence underpinning such behaviour and its adaptive significance.
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Affiliation(s)
- Galina V Mukamolova
- Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion SY23 3DD, UK
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