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Academic literature on the topic 'Viable but non-culturable bacteria (VBNC)'
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Journal articles on the topic "Viable but non-culturable bacteria (VBNC)"
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Hamabata, Takashi, Mitsutoshi Senoh, Masaaki Iwaki, Ayae Nishiyama, Akihiko Yamamoto, and Keigo Shibayama. "Induction and Resuscitation of Viable but Nonculturable Corynebacterium diphtheriae." Microorganisms 9, no. 5 (April 26, 2021): 927. http://dx.doi.org/10.3390/microorganisms9050927.
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Many pathogenic bacteria, including Escherichia coli and Vibrio cholerae, can become viable but nonculturable (VBNC) following exposure to specific stress conditions. Corynebacterium diphtheriae, a known human pathogen causing diphtheria, has not previously been shown to enter the VBNC state. Here, we report that C. diphtheriae can become VBNC when exposed to low temperatures. Morphological differences in culturable and VBNC C. diphtheriae were examined using scanning electron microscopy. Culturable cells presented with a typical rod-shape, whereas VBNC cells showed a distorted shape with an expanded center. Cells could be transitioned from VBNC to culturable following treatment with catalase. This was further evaluated via RNA sequence-based transcriptomic analysis and reverse-transcription quantitative PCR of culturable, VBNC, and resuscitated VBNC cells following catalase treatment. As expected, many genes showed different behavior by resuscitation. The expression of both the diphtheria toxin and the repressor of diphtheria toxin genes remained largely unchanged under all four conditions (culturable, VBNC, VBNC after the addition of catalase, and resuscitated cells). This is the first study to demonstrate that C. diphtheriae can enter a VBNC state and that it can be rescued from this state via the addition of catalase. This study helps to expand our general understanding of VBNC, the pathogenicity of VBNC C. diphtheriae, and its environmental survival strategy.
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Fleischmann, Susanne, Christian Robben, Thomas Alter, Peter Rossmanith, and Patrick Mester. "How to Evaluate Non-Growing Cells—Current Strategies for Determining Antimicrobial Resistance of VBNC Bacteria." Antibiotics 10, no. 2 (January 26, 2021): 115. http://dx.doi.org/10.3390/antibiotics10020115.
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Thanks to the achievements in sanitation, hygiene practices, and antibiotics, we have considerably improved in our ongoing battle against pathogenic bacteria. However, with our increasing knowledge about the complex bacterial lifestyles and cycles and their plethora of defense mechanisms, it is clear that the fight is far from over. One of these resistance mechanisms that has received increasing attention is the ability to enter a dormancy state termed viable but non-culturable (VBNC). Bacteria that enter the VBNC state, either through unfavorable environmental conditions or through potentially lethal stress, lose their ability to grow on standard enrichment media, but show a drastically increased tolerance against antimicrobials including antibiotics. The inability to utilize traditional culture-based methods represents a considerable experimental hurdle to investigate their increased antimicrobial resistance and impedes the development and evaluation of effective treatments or interventions against bacteria in the VBNC state. Although experimental approaches were developed to detect and quantify VBNCs, only a few have been utilized for antimicrobial resistance screening and this review aims to provide an overview of possible methodological approaches.
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Wideman, Nathan E., James D. Oliver, Philip Glen Crandall, and Nathan A. Jarvis. "Detection and Potential Virulence of Viable but Non-Culturable (VBNC) Listeria monocytogenes: A Review." Microorganisms 9, no. 1 (January 19, 2021): 194. http://dx.doi.org/10.3390/microorganisms9010194.
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The detection, enumeration, and virulence potential of viable but non-culturable (VBNC) pathogens continues to be a topic of discussion. While there is a lack of definitive evidence that VBNC Listeria monocytogenes (Lm) pose a public health risk, recent studies suggest that Lm in its VBNC state remains virulent. VBNC bacteria cannot be enumerated by traditional plating methods, so the results from routine Lm testing may not demonstrate a sample’s true hazard to public health. We suggest that supplementing routine Lm testing methods with methods designed to enumerate VBNC cells may more accurately represent the true level of risk. This review summarizes five methods for enumerating VNBC Lm: Live/Dead BacLightTM staining, ethidium monoazide and propidium monoazide-stained real-time polymerase chain reaction (EMA- and PMA-PCR), direct viable count (DVC), 5-cyano-2,3-ditolyl tetrazolium chloride-4′,6-diamidino-2-phenylindole (CTC-DAPI) double staining, and carboxy-fluorescein diacetate (CDFA) staining. Of these five supplementary methods, the Live/Dead BacLightTM staining and CFDA-DVC staining currently appear to be the most accurate for VBNC Lm enumeration. In addition, the impact of the VBNC state on the virulence of Lm is reviewed. Widespread use of these supplemental methods would provide supporting data to identify the conditions under which Lm can revert from its VBNC state into an actively multiplying state and help identify the environmental triggers that can cause Lm to become virulent. Highlights: Rationale for testing for all viable Listeria (Lm) is presented. Routine environmental sampling and plating methods may miss viable Lm cells. An overview and comparison of available VBNC testing methods is given. There is a need for resuscitation techniques to recover Lm from VBNC. A review of testing results for post VBNC virulence is compared
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Wagley, Sariqa, Helen Morcrette, Andrea Kovacs-Simon, Zheng R. Yang, Ann Power, Richard K. Tennant, John Love, Neil Murray, Richard W. Titball, and Clive S. Butler. "Bacterial dormancy: A subpopulation of viable but non-culturable cells demonstrates better fitness for revival." PLOS Pathogens 17, no. 1 (January 13, 2021): e1009194. http://dx.doi.org/10.1371/journal.ppat.1009194.
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The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V. parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella. Our results showed that two subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:ΔlldD) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:ΔlldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state.
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Grey, Brian E., and Todd R. Steck. "The Viable But Nonculturable State ofRalstonia solanacearum May Be Involved in Long-Term Survival and Plant Infection." Applied and Environmental Microbiology 67, no. 9 (September 1, 2001): 3866–72. http://dx.doi.org/10.1128/aem.67.9.3866-3872.2001.
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ABSTRACT The role of the dormant-like viable but nonculturable (VBNC) condition in the etiology of bacterial infection was examined using a plant system. The plant-pathogenic bacterium Ralstonia solanacearum was first shown to enter into the VBNC state both in response to cupric sulfate when in a saline solution and when placed in autoclaved soil. To determine if the VBNC condition is related to pathogenesis, the physiological status of bacteria recovered from different regions of inoculated tomato plants was determined at different stages of infection. The fraction of in planta bacteria that were VBNC increased during infection and became greater than 99% by the late stage of disease. The possibility that soil-dwelling VBNC bacteria may resuscitate and infect plants was also examined. When tomato seeds were germinated in sterile soil that contained VBNC but no detectable culturable forms of R. solanacearumcells, resuscitation was observed to occur in soil adjacent to plant roots; these resuscitated bacteria were able to infect plants. This is the first report of R. solanacearum entering the VBNC state and of resuscitation of any VBNC plant-pathogenic bacteria and provides evidence that the VBNC state may be involved in explaining the persistent nature of some infections.
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Zhong, Junliang, and Xihong Zhao. "Transcriptomic Analysis of Viable but Non-Culturable Escherichia coli O157:H7 Formation Induced by Low Temperature." Microorganisms 7, no. 12 (November 30, 2019): 634. http://dx.doi.org/10.3390/microorganisms7120634.
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Escherichia coli O157:H7 is one of the most common pathogenic bacteria that pose a threat to food safety. The aim of this study was to investigate the mechanisms of the formation of viable but non-culturable (VBNC) E. coli O157:H7 induced by low temperature (−20 °C) using RNA sequencing (RNA-Seq) transcriptomics analysis. The results of the present investigation revealed the presence of 2298 differentially expressed genes in VBNC cells, accounting for 46.03% of the total number of genes. Additionally, GO function and KEGG pathway enrichment analysis were performed to investigate the functional and related metabolic pathways of the differentially expressed genes. We found that the ion transport, protein synthesis, and protein transmembrane transport activities were significantly improved in the VBNC cells, indicating that E. coli O157:H7 cells synthesized a considerable amount of protein to maintain the levels of their functional metabolic processes and life activities in the VBNC state. In conclusion, we suggest that the increased synthesis of proteins such as SecY, FtsY, and Ffh might indicate that they are the key proteins involved in the improvement of the transmembrane transport activities in VBNC E. coli O157:H7 cells, maintaining their functional metabolism in the VBNC state and enhancing their survival ability under low temperatures.
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Hati, Revita Permata, Ratih Dewanti-Hariyadi, and L. Nuraida. "Cronobacter sakazakii local isolates response to acid stress and their resuscitability." Food Research 4, no. 1 (September 16, 2019): 244–53. http://dx.doi.org/10.26656/fr.2017.4(1).257.
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Cronobacter spp. has been reported to cause meningitis, necrotizing enterocolitis, and septicemia in a group of infants through the consumption of powder infant formula. These bacteria are reported to withstand various stress conditions such as heating, drying, low water activity, low pH, etc. A local isolate of Cronobacter sakazakii YRt2a was reportedly survived and entered Viable But Non-Culturable (VBNC) conditions during desiccation stress. This study aims to study the behavior of local isolates of Cronobacter spp. in response to acid stress and its resuscitability. C. sakazakii E2 and YRt2a were grown in TSB at pH 3.0±0.2 or 3.5±0.2. The number of culturable cells and viable cells were enumerated by the Total Plate Count and Direct Viable Count methods, respectively. Resuscitation was done by growing the stress or VBNC cells in TSB with or without sodium pyruvate, catalase, Tween 20, or Cronobacter autoinducer. The results showed that C. sakazakii E2 and YRt2a entered VBNC state after 60 mins of exposure to pH 3.0±0.2, while remained culturable after 120 minutes exposure to pH 3.5±0.2. TSB with or without sodium pyruvate, catalase, Tween 20, or Cronobacter autoinducer could resuscitate the stress or VBNC cells of C. sakazakii. Stress or VBNC state experienced by C. sakazakii in response to acid tends to be transient and can be resuscitated. C. sakazakii experiencing stress or VBNC may pose a risk for food safety.
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Highmore, Callum J., Jennifer C. Warner, Steve D. Rothwell, Sandra A. Wilks, and C. William Keevil. "Viable-but-NonculturableListeria monocytogenesandSalmonella entericaSerovar Thompson Induced by Chlorine Stress Remain Infectious." mBio 9, no. 2 (April 17, 2018): e00540-18. http://dx.doi.org/10.1128/mbio.00540-18.
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ABSTRACTThe microbiological safety of fresh produce is monitored almost exclusively by culture-based detection methods. However, bacterial food-borne pathogens are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses such as chlorine, which is commonly used for fresh produce decontamination. Here, complete VBNC induction of green fluorescent protein-taggedListeria monocytogenesandSalmonella entericaserovar Thompson was achieved by exposure to 12 and 3 ppm chlorine, respectively. The pathogens were subjected to chlorine washing following incubation on spinach leaves. Culture data revealed that total viableL. monocytogenesandSalmonellaThompson populations became VBNC by 50 and 100 ppm chlorine, respectively, while enumeration by direct viable counting found that chlorine caused a <1-log reduction in viability. The pathogenicity of chlorine-induced VBNCL. monocytogenesandSalmonellaThompson was assessed by usingCaenorhabditis elegans. Ingestion of VBNC pathogens byC. elegansresulted in a significant life span reduction (P= 0.0064 andP< 0.0001), and no significant difference between the life span reductions caused by the VBNC and culturableL. monocytogenestreatments was observed.L. monocytogeneswas visualized beyond the nematode intestinal lumen, indicating resuscitation and cell invasion. These data emphasize the risk that VBNC food-borne pathogens could pose to public health should they continue to go undetected.IMPORTANCEMany bacteria are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses. VBNC cells cannot be detected by standard laboratory culture techniques, presenting a problem for the food industry, which uses these techniques to detect pathogen contaminants. This study found that chlorine, a sanitizer commonly used for fresh produce, induces a VBNC state in the food-borne pathogensListeria monocytogenesandSalmonella enterica. It was also found that chlorine is ineffective at killing total populations of the pathogens. A life span reduction was observed inCaenorhabditis elegansthat ingested these VBNC pathogens, with VBNCL. monocytogenesas infectious as its culturable counterpart. These data show that VBNC food-borne pathogens can both be generated and avoid detection by industrial practices while potentially retaining the ability to cause disease.
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Dusserre, Eric, Christophe Ginevra, Sylvie Hallier-Soulier, François Vandenesch, Gabriel Festoc, Jerome Etienne, Sophie Jarraud, and Maëlle Molmeret. "A PCR-Based Method for Monitoring Legionella pneumophila in Water Samples Detects Viable but Noncultivable Legionellae That Can Recover Their Cultivability." Applied and Environmental Microbiology 74, no. 15 (May 30, 2008): 4817–24. http://dx.doi.org/10.1128/aem.02899-07.
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ABSTRACT Legionella pneumophila is the causative agent of Legionnaires' disease. This bacterium is ubiquitous in aqueous environments and uses amoebae as an intracellular replicative niche. Real-time PCR has been developed for rapid detection of Legionella DNA in water samples. In addition to culturable bacteria, this method may also detect dead and viable but noncultivable (VBNC) legionellae. In order to understand the significance of positive PCR results in this setting, we prepared water samples containing known concentrations of L. pneumophila and analyzed them comparatively by means of conventional culture, real-time PCR, viability labeling, and immunodetection (solid-phase cytometry). We also examined the influence of chlorination on the results of the four methods. The different techniques yielded similar results for nonchlorinated water samples but not for chlorinated samples. After treatment for 24 h with 0.5 and 1 ppm chlorine, all cultures were negative, PCR and immunodetection showed about 106 genome units and bacteria/ml, and total-viable-count (TVC) labeling detected 105 and 102 metabolically active bacteria/ml, respectively. Thus, PCR also detected bacteria that were VBNC. The recoverability of VBNC forms was confirmed by 5 days of coculture with Acanthamoeba polyphaga. Therefore, some TVC-positive bacteria were potentially infective. These data show that L. pneumophila PCR detects not only culturable bacteria but also VBNC forms and dead bacterial DNA at low chlorine concentrations.
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Li, Yanlin, Jixiang Chen, Yonggang Wang, Dan Ma, and Wenhong Rui. "The effects of the recombinant YeaZ of Vibrio harveyi on the resuscitation and growth of soil bacteria in extreme soil environment." PeerJ 8 (December 21, 2020): e10342. http://dx.doi.org/10.7717/peerj.10342.
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Numerous bacteria entered the viable but non-culturable state due to the stresses of dry and salt in soils. YeaZ of Gram-negative bacteria is a resuscitation promoting factor (Rpf) homologous protein could resuscitate bacteria of natural environment in VBNC state. To investigate the promoting effect of YeaZ on the isolation of viable but non-culturable (VBNC) bacteria from soil samples in extreme environments, the recombinant YeaZ of Vibrio harveyi was prepared and added to the soil samples from volcanic soil and saline soil in Northwest China. The study has shown that YeaZ can promote the recovery and growth of soil microorganisms, and the number of cultivable bacteria in volcanic and saline soil has increased from 0.17 × 103 and 2.03 × 103 cfu⋅ml−1 to 1.00 × 103 and 5.55 × 103 cfu⋅ml−1, respectively. The 16S rDNA gene sequencing and phylogenetic analysis showed that YeaZ played an essential role in the increase of composition and diversity of bacteria. A total of 13 bacterial strains were isolated from the volcanic soil samples, which belong to phyla Actinobacteria, Firmicutes and Gamma-proteobacteria. Four species, including Ornithinimicrobium kibberense, Agrococcus citreus, Stenotrophomonas rhizophila and Pseudomonas zhaodongensis were found in the control group, while Micrococcus antarcticus, Kocuria rose, Salinibacterium xinjiangense, Planococcus antarcticus, Ornithinimicrobium kibberense and Pseudomonas zhaodongensis were isolated from the treatment groups (addition of YeaZ). Twenty-one strains were isolated from the saline soil samples, including eight species from the control group and thirteen species from the treatment groups, among which nine species were only found, including Bacillus oceanisediminis, Brevibacillus brevis, Paenibacillus xylanilyticus, Microbacterium maritypicum, B. subtilis, B. alcalophilus, B. niabensis, Oceanimonas doudoroffii and Zobellella taiwanensis. The results suggest that addition of YeaZ to soil samples can promote the recovery of VBNC. This method has the implications for the discovery of VBNC bacteria that have potential environmental functions.
Dissertations / Theses on the topic "Viable but non-culturable bacteria (VBNC)"
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Ameh, Ekwu Mark. "The use of bacteriophages as natural biocontrol agents against bacterial pathogens." Thesis, Cranfield University, 2016. http://dspace.lib.cranfield.ac.uk/handle/1826/11331.
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Bacteriophages are viruses that specifically infect bacteria. The bactericidal nature of lytic bacteriophages has been exploited by scientists for decades with the hope to utilise them in the fight against bacterial infections and antibiotic resistant bacteria in medical settings. More recently, the potential applications of bacteriophages for biocontrol in the agrifood and environmental sectors have been investigated in an attempt to develop ‘natural’ antimicrobial products. Bacteriophages have a couple of decisive advantages over conventional methods of controlling pathogenic bacteria, such as high host specificity, the ability to self-replicate, and the ability to evolve with their hosts. However, more research is needed to optimise the parameters for phage applications, including the impact of environmental conditions on lysis efficiency, multiplicity of infection, and to significantly minimise the emergence of bacterial resistance to phages. Temperature plays a key role in every biological activity in nature. It is also assumed that temperature has an effect on phage lysis efficiency. A comprehensive study of it and how it affects both the host cells and their corresponding phages is crucial to ensure the efficient removal of bacterial pathogens. In this thesis, temperature (as selected parameter) was investigated to determine its influence on the lysis effectiveness of the three different phages belonging to the family of the Myoviridea that were isolated and purified from a single water sample taken from a brook receiving treated wastewater. We used the multiplicity of infection of 1 in all of our study in this project. Temperature was found to have a significant impact on phage-mediated lysis efficiency. Both the temperature of incubation of the phage-bacteria mixture (incubation temperature) and the temperature history of bacterial hosts were found to have profound effects on plaque sizes as well as plaque numbers. Plaque size and number decreased with increasing temperature. For the phages examined, bacterial lysis was more efficient at 20°C compared to 30 or 37°C. Phages were suggested to be well adapted to the environment where they were isolated from with general implications for use in biological disinfection. Furthermore, the temperature history of the bacteria (prior to phage encounter) was found to have a modulating effect on their susceptibility to lysis. A second part of this study compared the performance of the three phages in regard to bacterial resistance. The emergence of bacterial resistance is a major obstacle to the success of bacteriophages applications. The use of multiple phages is typically recommended and has proven better than the use of a single phage. However, the bestway to perform phage treatment is still very unclear. This study therefore compared simultaneous addition of multiple phages (in form of a cocktail) with the sequential addition of the individual phages at different time points in trying to delay the emergence of bacterial resistance. The data obtained from this work suggest that lysis effectiveness can be adjusted to optimize any treatment goal. For fast initial bacterial clearance the use of a single phage with short time maximal lysis efficiency proved most efficient, while the simultaneous addition of phages in the form of a cocktail was most successful strategy in our study. Addition of selected phages sequentially can be normalized in such a way that is just as effective as a cocktail. A third part of this thesis looked into the susceptibility of bacteria that had undergone sublethal disinfection. We addressed the question whether bacteria subjected to sublethal doses of chlorine and UV are still susceptible to phage-mediated lysis. The chlorine treatments indicated the development of a phage-insensitive phenotype for a critical chlorine dose in the transition zone between live and dead. The remaining live (and culturable) bacteria were shown insensitive to the selected phage. The lowest UV exposure at 2.8 mJ/cm2 eliminated bacteria susceptibility to the phages. This phage- resistant phenotype may have serious consequences for the application of phages on foods or water that have previously undergone a weak disinfection regime.
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Barrett, Tanya. "An investigation into the molecular basis of the viable but non-culturable response in bacteria." Thesis, University of Aberdeen, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265678.
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The viable but non-culturable (VBNC) state is outstanding among bacterial stress responses as being completely uncharacterised at the molecular level. The aim of this investigation was to gain an insight into the molecular basis of the condition by identifying genes whose expression was up-regulated in response to VBNC-inducing stimuli. First, a model experimental system was established where bacteria were induced to enter the state in a routine and predictable manner. Escherichia coli HB101 exhibited a partial viable but non-culturable phenotype when inoculated into microcosms of artificial seawater at 37°C, Pseudomonas fluorescens 10586 became viable but non-culturable in microcosms of drinking water incubated at 37°C, and Vibrio vulnificus MO6-24/T entered a viable, non-culturable state in artificial seawater at 5°C. A transposon mutagenesis strategy utilising a promoter-less bioluminescent reporter cassette, luxAB, was employed in the search for VBNC-associated genes. The mini-Tn5 luxAB transposon was induced to transform into arbitrary positions of the P. fluorescens 10586 chromosome, thus creating a library of P. fluorescens luxAB mutants. This library (consisting of over 1200 transformants) was screened for those which were dark under normal circumstances, but luminesced in response to VBNC stimuli, indicating that the transposon had integrated downstream of a gene up-regulated during the VBNC response. Unfortunately, no mutant examined exhibited such a bioluminescence profile. Differential display of RNA technology was employed subsequently and resulted in the cloning and sequencing of several V. vulnificus transcripts thought to be associated with the VBNC state. Although absolute verification of the involvement of these transcripts was not achieved, hints as to what mechanisms lay at the basis of the VBNC state were gained. Some findings indicated that VBNC cells experience considerable levels of oxidative stress, and it was proposed that this physiological state may lie at the crux of the VBNC phenotype.
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Shah, Manoj Kumar. "Characterizing the Impact of Stress Exposure on Survival of Foodborne Pathogens." Diss., North Dakota State University, 2019. https://hdl.handle.net/10365/29389.
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Bacterial pathogens transmitted by the fecal-oral route endure several stresses during survival/growth in host and non-host environments. For foodborne pathogens, understanding the range of phenotypic responses to stressors and the environmental factors that impact survival can provide insights for the development of control measures. For example, the gastrointestinal system presents acidic, osmotic, and cell-envelope stresses and low oxygen levels, but Listeria monocytogenes can withstand these stresses, causing illnesses in humans. Survival/growth characteristics may differ among L. monocytogenes strains under these stressors due to their genetic diversities. Our knowledge of such phenotypic characteristics under bile and salt stresses are inadequate. In this dissertation, variation in growth characteristics was observed among L. monocytogenes strains under bile and osmotic stresses with no evidence of cross-protection, but rather an antagonistic effect was observed with the formation of filaments when pre-exposed to 1% bile and treated with 6% NaCl. This shows that variation in stress adaptability exists among L. monocytogenes strains with the ability to form filaments under these conditions. Similarly, Salmonella survival in soil is dependent on several factors, such as soil, amendment types, moisture, irrigation, and desiccation stress. In this study, the use of HTPP (heat-treated poultry pellets) was investigated as a soil amendment in the survival/growth of Salmonella in soil extracts mimicking runoff events, and in soil cultivated with spinach plants to assess its safety for use for an organic fertilizer. The presence of HTPP in soil increased S. Newport survival with a greater likelihood of its transfer to and survival on spinach plants. Increased microbial loads and rpoS mutant showed decreased growth/survival in soil extracts, however, rpoS was not important for survival in soil under the tested conditions showing possible lack of desiccation stress. These results show that HTPP provided nutrients to the Salmonella for increased growth and survival in soil extracts and soil, respectively, which show that the use of treated BSAAO to soils may still require appropriate mitigation to minimize Salmonella Newport contamination of leafy greens in the pre-harvest environment. Overall, the results in this study increased our understanding of L. monocytogenes and Salmonella phenotypic adaptation to stressful environments.
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Taskin, Bilgin. "Selective Quantification Of Viable Escherichia Coli Cells In Biosolids Upon Propidium Monoazide Treatment By Quantitative Pcr." Phd thesis, METU, 2011. http://etd.lib.metu.edu.tr/upload/12612925/index.pdf.
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Density of fecal coliforms (FC) such as Escherichia coli is the most commonly used indicator of fecal pathogen content of biosolids. When biosolids are disposed off or used for soil amendment, they pose public health risks. So far anaerobic digesters have been considered to be an effective treatment option for pathogen and FC reduction in biosolids. However, recent studies revealed that there is a significant re-growth and reactivation of indicator organisms in biosolids upon dewatering by centrifugation. Although the exact mechanism of FC reactivation is yet to be understood, a few extensive recent studies strongly suggest that FC go into a viable but non-culturable (VBNC) state during anaerobic digestion.
Therefore, quantitative detection of live cells among the total in biosolids samples, without using culturing-based approaches, is highly critical from a public health risk assessment perspective. Since recent investigations proved the significant re-growth and reactivation of indicator organisms. Persistence of DNA in the environment after cell death in the range of days to weeks limits the application of DNA-based approaches for the detection of live bacteria. Using selective nucleic acid intercalating dyes such as ethidium monoazide (EMA) and propidium monoazide (PMA) is one of the alternative approaches to detect and quantify the viable cells by quantitative PCR. These compounds have the ability to penetrate only into dead cells with compromised membrane integrity. They intercalate in the DNA via photo-inducible azide groups and in turn inhibit DNA amplification during PCR reactions. PMA has been successfully used in different studies and microorganisms but it has not been evaluated sufficiently for the complex environmental samples such as biosolids. In this study Escherichia coli ATCC 25922 and uidA gene were used as model organism and as target sequence respectively in absolute quantification method with real-time PCR. Experiments with the known quantities of live and dead cell mixtures showed that PMA treatment inhibits PCR amplification from dead cells with over 99% efficiency. The results of this study conclusively demonstrated that PMA-modified PCR could be successfully applied to the biosolids when total suspended solid (TSS) concentration is 2000 mg/L or below.
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Khamisse, Elissa. "Etude du microbiote susceptible de persister sur les surfaces d'un atelier de la filière viande bovine." Phd thesis, AgroParisTech, 2012. http://pastel.archives-ouvertes.fr/pastel-00770326.
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Ce travail de thèse concerne l'étude de l'écologie microbienne d'un atelier de découpe de viande bovine, dans le but de mieux comprendre la persistance bactérienne, c'est-à-dire, la présence répétée d'un même clone bactérien pendant une longue période malgré l'application bien conduite et régulière du nettoyage et de la désinfection (N-D). Des prélèvements par " chiffonnages " multiples de surfaces d'équipements ont été réalisés lors de trois campagnes de prélèvement espacées les unes des autres d'au moins six mois. Les prélèvements ont été réalisés sur un tapis convoyeur en polychlorure de vinyle (PVC) et sur des machines éplucheuses en acier inoxydable avant et après N-D. Nous avons quantifié les cellules totales (les cellules vivantes et les cellules mortes) par PCR quantitative en temps réel (qPCR), les cellules viables par EMA-qPCR, et les UFC (provenant de cellules cultivables) par dénombrement après incubation à 25°C sur gélose tryptone soja. Les résultats montrent qu'avant N-D, les cellules totales (en moyenne 5,6 - exprimé en log10 cellules/cm2 - sur PVC et 4,7 sur acier inoxydable) sont plus nombreuses que les cellules viables (4,5 sur PVC et 4,4 sur acier inoxydable) lesquelles sont plus nombreuses que les UFC (3,8 sur PVC et 2,9 sur acier inoxydable). Le N-D entraîne moins d'une réduction décimale (RD) des populations à l'exception des UFC sur acier inoxydable qui subissent 1,5 RD en moyenne. Ce dernier chiffre s'explique par des forces d'adhésion faibles. L'étude de la diversité des bactéries cultivables montre que sur un total de 51 genres identifiés, 13 seulement sont retrouvés lors des trois campagnes de prélèvements. Les isolats de ces 13 genres représentent 75, 72 et 62% des isolats des campagnes1, 2 et 3 respectivement. Parmi ces isolats, les plus fréquents sont (par ordre décroissant du nombre d'isolats) : Pseudomonas, Staphylococcus, Microbacterium, Acinetobacter, Chryseobacterium, Psychrobacter et Kocuria. Le génotypage d'isolats de 3 genres majoritaires (Staphylococcus, Pseudomonas et Acinetobacter) montre qu'une seule souche, Staphylococcus equorum, est sans aucun doute persistante. L'ensemble de ces observations montrent que l'écosystème varie d'une campagne à une autre. Ces modifications de la diversité bactérienne reflèteraient les modifications de flores des viandes traitées dans l'atelier, qui ont des origines multiples. En outre, il apparaît que, contrairement à ce qui est généralement admis, les bactéries à coloration de Gram négative cultivables sont plus facilement inactivées par le N-D que les bactéries à coloration de Gram positive. L'étude de l'écosystème par PCR-DGGE a permis d'identifier sept genres bactériens et montre que les espèces dominantes sont toutes sous forme vivante, autrement dit, aucune des espèces dominantes n'a été détectée uniquement sous forme de cellules mortes. Sur les sept genres identifiés six sont des Gram - dont majoritairement les genres Acinetobacter, Pseudomonas et Psychrobacter. Cette dominance montre que le N-D permet une forte perte de cultivabilité des bactéries Gram - mais qu'une grande partie n'est pas détachée. La dominance des bactéries Gram - observée par PCR-DGGE masque les staphylocoques qui ne sont pas détectés alors qu'ils sont majoritaires parmi la flore cultivable. Seul un genre bactérien, Propionibacterium, est identifié par PCR-DGGE uniquement mais il n'est trouvé qu'à une seule campagne et uniquement sur l'acier inoxydable avant N-D. En conclusion, l'avancée majeure de ce travail est la mise en évidence qu'une proportion importante de bactéries survit après les opérations très poussées de N-D mais pour une période transitoire.
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Kartz, Cory. "Transport of viable but non-culturable Escherichia coli O157:H7 in soil and groundwater." Master's thesis, 2011. http://hdl.handle.net/10048/1934.
Full textAbstract:
The influence of the viable but non-culturable (VBNC) state on specific phenotypic traits of Escherichia coli O157:H7 as well as its transport behaviour in porous media was examined in this study. E.coli O157:H7 is a human pathogen capable of entering a VBNC state following exposure to sublethal stress. In the VBNC state, E.coli O157:H7 is not detectable by culture assays; yet, is able to retain its ability to cause human illness. This study examined specific transport-related properties of culturable and VBNC E.coli O157:H7 cells. As well, transport behaviors of the two cellular states were compared using sand-packed columns under steady-state flow. When E.coli O157:H7 cells entered a VBNC state, significant decreases in the hydrophobicity and lengths/widths of the cells, and a significant increase in extracellular polymeric substances on the cell surfaces were measured. Transport experiments indicated significantly (p<0.05) greater mass transport of VBNC cells through unwashed sand compared to culturable cells. This research contributes to the current knowledge describing VBNC E.coli O157:H7 cells, raises questions concerning the accuracy of culture-based E.coli O157:H7 identification protocols, and suggests that bacteria transport in the subsurface is a truly dynamic process.Soil Science
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Al-Bana, Badii. "Characterization of The Viable but Non-Culturable Legionella pneumophila in Water and the Role of 3-Hydroxybutyrate Dehydrogenase in Its Formation." 2013. http://hdl.handle.net/10222/37024.
Full textAbstract:
Legionella pneumophila, the causative agent of Legionnaires’ disease (LD), is an intracellular pathogen of freshwater protozoa that can also persist in the environment as a free-living bacterium. L. pneumophila has many morphological forms that fit within a developmental cycle. In water, L. pneumophila enters into a viable but non-culturable (VBNC) state that is largely uncharacterized. VBNC cells were produced from two developmental L. pneumophila forms, stationary phase forms (SPFs) and mature infectious forms (MIFs) by suspension in double deionized (dd) or tap-water at 45°C. Electron microscopy results showed that VBNC cells have a unique morphology and that in tap water they lose their poly 3-hydroxybutyrate inclusion bodies. Both SPFs and MIFs lost culturability faster in dd- than in tap water, and addition of salts to dd-water prolonged L. pneumophila culturability and enhanced viability. However, MIFs retained higher viability in dd- and tap water (85% and 51%, respectively) than SPFs (5% and 20%, respectively) as determined by the BacLight vital stain. Only ~1 VBNC cell out of 105 of those produced from SPFs in tap water regained culturability via infection of Acanthamoeba. All VBNC cells, except for those produced from SPFs in dd-water, resisted both digestion inside Tetrahymena spp. and detergent-mediated lysis. SDS-PAGE analysis and shotgun proteomics revealed a number of VBNC cell specific proteins; one of these was 3-hydroxybutyrate dehydrogenase (BdhA), which is involved in the metabolism of poly 3-hydroxybutyrate inclusion bodies. A bdhA mutant showed an early loss of culturability and a dramatic decrease in viability as compared to the parent strain, and complementing the mutant with a functional bdhA gene restored the parent's strain phenotypes. In conclusion, VBNC L. pneumophila has a distinct morphology and physiology that varies according to the developmental stage and the environmental conditions used to produce such VBNC cells. VBNC cells have a different protein profile and morphology than the culturable cells, suggesting that this state constitutes a distinct differentiated form within the developmental cycle of L. pneumophila. BdhA seems to influence L. pneumophila survival and hence VBNC cell formation. Collectively, the results from this study provide a better understanding of L. pneumophila VBNC form and the factors influencing its formation.