Characterization and potential mechanisms of highly antibiotic tolerant VBNC Escherichia coli induced by low level chlorination.
Ye Chengsong,Lin Huirong,Zhang Menglu,Chen Sheng,Yu Xin
Scientific reports
Escherichia coli is an important pathogenic indicator in drinking water. Viable but non-culturable (VBNC) E. coli induced by low level chlorination was found to have higher antibiotic tolerance. The emerging of VBNC bacteria in drinking water systems is posing challenges to the control of bio-safety. It is necessary to study the underlying mechanisms of VBNC state E. coli under actual residual chlorine condition of drinking water pipe network. In this study, we investigated the changes of morphology and gene expressions that might present such state. The results indicated that the size of VBNC E. coli was not remarkably changed or recovered culturability under favorable environmental conditions. Results from transcriptomic analysis revealed that the regulated genes related to fimbrial-like adhesin protein, putative periplasmic pilin chaperone, regulators of the transcriptional regulation, antibiotic resistance genes and stress-induced genes, rendering VBNC cells more tolerant to adverse environmental conditions. In total of 16 genes were significantly up-regulated under the VBNC state, including three genes encoding toxic protein (ygeG, ibsD, shoB), indicating that VBNC E. coil was still a threat to human. The work is of great relevance in the context of better understanding this poorly understood physiological state.
10.1038/s41598-020-58106-3
Modelling the effect of chlorination/chloramination on induction of viable but non-culturable (VBNC) .
Chen Sheng,Zeng Jie,Wang Yahong,Ye Chengsong,Zhu Shuai,Feng Lin,Zhang Shenghua,Yu Xin
Environmental technology
Many bacteria, including , are known to enter into a viable but non-culturable (VBNC) state when exposed to harsh environmental stresses. The VBNC cells introduced by chlorination/chloramination have raised increasing concern about biological safety of drinking water. A quantitative relationship between chlorination/chloramination and number of VBNC cells has not been found. In this study, a mathematical model was developed to quantify the effect of chlorination/chloramination on induction of viable but non-culturable (VBNC) . the model was generated based on a first order kinetics of chlorination/chloramination using the data collected from laboratory disinfection experiments. The disinfection rates of culturable cells ( ) and viable cells ( ) were dose-dependent, and they were also modelled in different initial concentrations by regression analysis to overcome the shortcoming of dose-dependent. In general, the and values for chlorination ( , 2.59-29.89 h; , 19.52-26.74 h) was 2-58 times greater than that for chloramination ( , 0.5446-10.81 h; , 0.3398-14.57 h), suggesting that chlorine was more effective than chloramine in reducing the number of culturable and VBNC cells at same dose of disinfectant. Ultimately, the generated models, which could describe the dynamics of VBNC cells formation in chlorination/chloramination, can provide practical guidance in drinking water treatment and it can also be applied to risk assessment of drinking water management systems.
10.1080/09593330.2019.1611939
Detection and distribution of vbnc/viable pathogenic bacteria in full-scale drinking water treatment plants.
Guo Lizheng,Wan Kun,Zhu Jianwen,Ye Chengsong,Chabi Kassim,Yu Xin
Journal of hazardous materials
Viable but non-culturable (VBNC) bacteria have attracted widespread attention since they are inherently undetected by traditional culture-dependent methods. Importantly, VBNC bacteria could resuscitate under favorable conditions leading to significant public health concerns. Although the total number of viable bacteria has been theorized to be far greater than those that can be cultured, there have been no reports quantifying VBNC pathogenic bacteria in full-scale drinking water treatment plants (DWTPs). In this work, we used both culture-dependent and quantitative PCR combination with propidium monoazide (PMA) dye approaches to characterize cellular viability. Further, we established a method to quantify viable pathogens by relating specific gene copies to viable cell numbers. Ratios of culturable bacteria to viable 16S rRNA gene copies in water and biological activated carbon (BAC) biofilms were 0-4.75% and 0.04-56.24%, respectively. The VBNC E. coli, E. faecalis, P. aeruginosa, Salmonella sp., and Shigella sp. were detected at levels of 0-10 cells/100 mL in source water, 0-10 cells/100 mL in chlorinated water, and 0-10 cells/g in BAC biofilms. In addition, differences between the total and viable community structures after ozonation and chlorination were investigated. The relative abundance of opportunistic pathogens such as Mycobacterium, Sphingomonas, etc. increased in final water, likely due to their chlorine resistance. In summary, we detected significant quantities of viable/VBNC opportunistic pathogens in full-scale DWTPs, confirming that traditional, culture-dependent methods are inadequate for detecting VBNC bacteria. These findings suggest a need to develop and implement rapid, accurate methods for the detection of VBNC pathogenic bacteria in DWTPs to ensure the safety of drinking water.
10.1016/j.jhazmat.2020.124335
High pressure processing combined with selected hurdles: Enhancement in the inactivation of vegetative microorganisms.
Yang Peiqing,Rao Lei,Zhao Liang,Wu Xiaomeng,Wang Yongtao,Liao Xiaojun
Comprehensive reviews in food science and food safety
High pressure processing (HPP) as a nonthermal processing (NTP) technology can ensure microbial safety to some extent without compromising food quality. However, for vegetative microorganisms, the existence of pressure-resistant subpopulations, the revival of sublethal injury (SLI) state cells, and the resuscitation of viable but nonculturable (VBNC) state cells may constitute potential food safety risks and pose challenges for the further development of HPP application. HPP combined with selected hurdles, such as moderately elevated or low temperature, low pH, natural antimicrobials (bacteriocin, lactate, reuterin, endolysin, lactoferrin, lactoperoxidase system, chitosan, essential oils), or other NTP (CO , UV-TiO photocatalysis, ultrasound, pulsed electric field, ultrafiltration), have been highlighted as feasible alternatives to enhance microbial inactivation (synergistic or additive effect). These combinations can effectively eliminate the pressure-resistant subpopulation, reduce the population of SLI or VBNC state cells and inhibit their revival or resuscitation. This review provides an updated overview of the microbial inactivation by the combination of HPP and selected hurdles and restructures the possible inactivation mechanisms.
10.1111/1541-4337.12724
The diagnostic tools for viable but nonculturable pathogens in the food industry: Current status and future prospects.
Gao Rui,Liao Xinyu,Zhao Xihong,Liu Donghong,Ding Tian
Comprehensive reviews in food science and food safety
Viable but nonculturable (VBNC) microorganisms have been recognized as pathogenic contaminants in foods and environments. The failure of VBNC cells to form the visible colonies hinders the ability to use conventional media for their detection. Efficient and rapid detection of pathogens in the VBNC state is a prerequisite to ensure the food safety and public health. Despite their nonculturability, VBNC cells have distinct characteristics, such as morphology, metabolism, chemical composition, and gene and protein expression, that have been used as the basis for the development of abundant diagnostic tools. This review covers the current status and advances in various approaches for examining microorganisms in the VBNC state, including but not limited to the methodological aspects, advantages, and drawbacks of each technique. Existing methods, such as direct viable count, SYTO/PI dual staining, and propidium monoazide quantitative polymerase chain reaction (PCR), as well as some techniques with potential to be applied in the future, such as digital PCR, enhanced-surface Raman spectroscopy, and impedance-based techniques, are summarized in depth. Finally, future prospects for the one-step detection of VBNC bacteria are proposed and discussed. We believe that this review can provide more optional methods for researchers and promote the development of rapid, accurate detecting methods, and for inspectors, the diagnostic tools can provide data to undertake risk analysis of VBNC cells.
10.1111/1541-4337.12695
Induction, detection, formation, and resuscitation of viable but non-culturable state microorganisms.
Dong Kai,Pan Hanxu,Yang Dong,Rao Lei,Zhao Liang,Wang Yongtao,Liao Xiaojun
Comprehensive reviews in food science and food safety
The viable but nonculturable (VBNC) state has been recognized as a strategy for bacteria to cope with stressful environments; in this state, bacteria fail to grow on routine culture medium but are actually alive and can resuscitate into a culturable state under favorable conditions. The VBNC state may pose a great threat to food safety and public health. To date, more than 100 VBNC microorganism species have been proven to exist in fields of food safety, environmental application, and agricultural diseases. Most harsh conditions can induce these microorganisms into the VBNC state, including food processing and preservation methods, adverse environmental conditions, and plant-disease controlling means. The characteristics of VBNC state cells differ from those of normally growing cells and dead cells, based on which of the various detection methods are developed, and they are of great significance for potential risk assessment. To provide molecular level insights into this state, many studies on induction and resuscitation mechanisms have emerged over the past three decades, including research on omics, specific genes, or proteins involved in VBNC state formation and the roles of promoters in resuscitation from the VBNC state. In this review, microorganism species, induction and resuscitation factors, detection methods, and formation and resuscitation mechanisms of the VBNC state are comprehensively and systematically summarized.
10.1111/1541-4337.12513
Resistance and induction of viable but non culturable states (VBNC) during inactivation of E. coli and Klebsiella pneumoniae by addition of HO to natural well water under simulated solar irradiation.
Alvear-Daza John J,García-Barco Alejandra,Osorio-Vargas Paula,Gutiérrez-Zapata Héctor M,Sanabria Janeth,Rengifo-Herrera Julián A
Water research
Inactivation of E. coli and Klebsiella pneumoniae by addition of HO 10 mg L into natural well water samples containing natural total iron concentrations (around 0.3 mg L) under simulated solar light was followed by bacterial culturability (plate count) and viability (DVC-FISH). Results showed that culturability of both bacteria was totally reduced while viability was only completely depleted for E. coli in well water samples depending of total iron concentration. Post-irradiation effects in presence of residual HO showed that viability of both bacteria kept dropping being totally reduced for E. coli cells while K. pneumoniae decreased only 1-log. SEM micrographs showed that E. coli and K. pneumoniae cells underwent morphological changes and size reduction according to VBNC states. Different dark and photo-induced processes where physical-chemical features of groundwater samples play an important role could be responsible of bacteria abatement.
10.1016/j.watres.2020.116499
A hidden risk: Survival and resuscitation of Escherichia coli O157:H7 in the viable but nonculturable state after boiling or microwaving.
Liu Yanming,Kumblathan Teresa,Uppal Gursharan K,Zhou Angela,Moe Birget,Hrudey Steve E,Li Xing-Fang
Water research
We report the existence and resuscitation of viable but nonculturable (VBNC) Escherichia coli O157:H7 cells in drinking water induced by the common point-of-use disinfection treatments of boiling or microwaving. Tap water and saline samples containing E. coli O157:H7 culturable cells from a bovine isolate or two clinical isolates were boiled (1, 10, or 15 min) on a hot plate or microwaved (1.5 min) to reach boiling. No culturable E. coli O157:H7 cells were observed in the treated samples using conventional plating methods. In samples boiled for 1 or 10 min, two viability assays separately detected that 2-5.5% of the cells retained an intact membrane, while 28 to 87 cells out of the initial 10 cells retained both measurable intracellular esterase activity and membrane integrity. In samples boiled for 15 min, no viable cells were detected. The microwaved samples contained 6-10% of cells with an intact membrane, while 21 to 108 cells out of the initial 10 cells retained both membrane integrity and esterase activity. The number of viable cells retaining both metabolic activity and membrane integrity were consistent in all samples, supporting the survival of a small number of E. coli O157:H7 cells in the VBNC state after boiling for 1 or 10 min or microwaving. Furthermore, the VBNC E. coli O157:H7 cells regained growth at 37 °C in culture media containing autoinducers produced by common non-pathogenic E. coli, commonly present in the human intestine, and norepinephrine. The resuscitated cells were culturable on conventional plates and expressed mRNA encoding the E. coli O157 lipopolysaccharide gene (rfbE) and the H7 flagellin gene (fliC). This study highlights potential concerns for public health risk management of VBNC E. coli O157:H7 in drinking water disinfected by heat treatment at point-of-use. The public health significance of these concerns warrants further investigation.
10.1016/j.watres.2020.116102
Induction of Escherichia coli into a VBNC state through chlorination/chloramination and differences in characteristics of the bacterium between states.
Chen Sheng,Li Xi,Wang Yahong,Zeng Jie,Ye Chengsong,Li Xianping,Guo Lizheng,Zhang Shenghua,Yu Xin
Water research
Many pathogens can enter into a viable but nonculturable (VBNC) state in response to harsh environmental stresses. Bacteria in this state can retain certain features of viable cells, such as cellular integrity, metabolic activity, or virulence and may present health risks associated with drinking water. In this study, we investigated the ability of chlorination and chloramination, which are widely used methods to disinfect drinking water, to induce Escherichia coli into a VBNC state. After treatment with chlorine and chloramine at concentrations of 1, 2, 3, and 4 mg/L, the counts of culturable E. coli cells decreased from 10 CFU/mL to 0 CFU/mL at 5-60 min post treatment. Meanwhile, viable cell counts were still approximately 10-10 cells/mL. These viable E. coli cells may be induced into a VBNC state by chlorination and chloramination. Scanning electron microscopy and laser confocal microscopy showed that some bacteria maintained cellular integrity, but the average length of VBNC cells was less than that of culturable cells. Respiratory activity of VBNC cells decreased approximately 50% relative to that of culturable cells. We also used heavy water (DO) combined with Raman microspectroscopy to show that E. coli in a VBNC state retained metabolic activity involving water (e.g. condensation reactions) at the single-cell level. Furthermore, soxR, gadA, and katG genes remained highly expressed, suggesting that VBNC cells were physiologically active. Finally, resuscitation of VBNC cells induced by chlorine in Luria-Bertani (LB) broth was identified by calculating the generation time. Results of this study will facilitate a better understanding of the health risks associated with VBNC bacteria and the development of more effective strategies for drinking water disinfection.
10.1016/j.watres.2018.05.055
ATP-Dependent Dynamic Protein Aggregation Regulates Bacterial Dormancy Depth Critical for Antibiotic Tolerance.
Pu Yingying,Li Yingxing,Jin Xin,Tian Tian,Ma Qi,Zhao Ziyi,Lin Ssu-Yuan,Chen Zhanghua,Li Binghui,Yao Guang,Leake Mark C,Lo Chien-Jung,Bai Fan
Molecular cell
Cell dormancy is a widespread mechanism used by bacteria to evade environmental threats, including antibiotics. Here we monitored bacterial antibiotic tolerance and regrowth at the single-cell level and found that each individual survival cell shows different "dormancy depth," which in return regulates the lag time for cell resuscitation after removal of antibiotic. We further established that protein aggresome-a collection of endogenous protein aggregates-is an important indicator of bacterial dormancy depth, whose formation is promoted by decreased cellular ATP level. For cells to leave the dormant state and resuscitate, clearance of protein aggresome and recovery of proteostasis are required. We revealed that the ability to recruit functional DnaK-ClpB machineries, which facilitate protein disaggregation in an ATP-dependent manner, determines the lag time for bacterial regrowth. Better understanding of the key factors regulating bacterial regrowth after surviving antibiotic attack could lead to new therapeutic strategies for combating bacterial antibiotic tolerance.
10.1016/j.molcel.2018.10.022