Multiple factors trigger the formation and resuscitation of the VBNC state in alcohol-producing Klebsiella pneumoniae.

Klebsiella pneumoniae can enter a viable but nonculturable (VBNC) state to survive in unfavorable environments. Our research found that high-, medium-, and low-alcohol-producing K. pneumoniae strains are associated with nonalcoholic fatty liver disease. However, the presence of the three Kpn strains has not been reported in the VBNC state or during resuscitation. In this study, the effects of different strains, salt concentrations, oxygen concentrations, temperatures, and nutrients in K. pneumoniae VBNC state were evaluated. The results showed that high-alcohol-producing K. pneumoniae induced a slower VBNC state than medium-alcohol-producing K. pneumoniae, and low-alcohol-producing K. pneumoniae. A high-salt concentration and micro-oxygen environment accelerated the loss of culturability. Simultaneously, both real-time quantitative PCR and droplet digital PCR were developed to compare the quantitative comparison of three Kpn strain VBNC states by counting single-copy gene numbers. At 22°C or 37°C, the number of culturable cells decreased significantly from about 108 to 105-106 CFU/mL. In addition, imipenem, ciprofloxacin, polymyxin, and phiW14 inhibited cell resuscitation but could not kill VBNC-state cells. These results revealed that the different environments evaluated play different roles in the VBNC induction process, and new effective strategies for eliminating VBNC-state cells need to be further studied. These findings provide a better understanding of VBNC-state occurrence, maintenance, detection, and absolute quantification, as well as metabolic studies of resuscitation resistance and ethanol production.IMPORTANCEBacteria may enter VBNC state under different harsh environments. Pathogenic VBNC bacteria cells in clinical and environmental samples pose a potential threat to public health because cells cannot be found by routine culture. The alcohol-producing Kpn VBNC state was not reported, and the influencing factors were unknown. The formation and recovery of VBNC state is a complete bacterial escape process. We evaluated the influence of multiple induction conditions on the formation of VBNC state and recovery from antibiotic and bacteriophage inhibition, and established a sensitive molecular method to enumerate the VBNC cells single-copy gene. The method can improve the sensitivity of pathogen detection in clinical, food, and environmental contamination monitoring, and outbreak warning. The study of the formation and recovery of VBNC-state cells under different stress environments will also promote the microbiological research on the development, adaptation, and resuscitation in VBNC-state ecology.

Induction into viable but non culturable state and outgrowth heterogeneity of Listeria monocytogenes is affected by stress history and type of growth.

Exposure to sublethal stresses related to food-processing may induce a heterogenous mixture of cells that co-exist, comprising healthy, sublethally injured, dormant and dead cells. Heterogeneity in survival capacity and dormancy of single cells may impede the detection of foodborne pathogens. In this study, we exposed Listeria monocytogenes Scott A strain, to peracetic acid (PAA; 20-40 ppm) and to acidic conditions (hydrochloric (HCl) and acetic (AA) acid, adjusted to pH 2.7-3.0, to evaluate the resuscitation capacity and outgrowth kinetics of metabolically active cells in two different media. Injury and the viable-but-non-culturable (VBNC) status of cells were assessed by flow cytometry using CFDA (metabolically active) and PI (dead) staining. Stressed CFDA+PI- cells were sorted on Tryptic Soy (TS) Agar or in TS broth, both supplemented with 0.6 % Yeast Extract (TSAYE or TSBYE), to evaluate culturability. Resuscitation capacity of CFDA+PI-sorted cells (10 events/well) was monitored by visual inspection on TSAYE and by optical density measurement in TSBYE for 5 days. Sorting of L. monocytogenes viable cells (CFDA+PI-) in Ringer's solution on TSAYE and TSBYE showed 100 % recovery in both media (control condition), while the mean lag time in TSBYE was 9.6 h. Treatment with 20 ppm PAA for 90 and 180 min resulted in 74.79 % and 85.82 % of non-culturable cells in TSBYE and increased the average lag time to 41.7 h and 43.8 h, respectively, compared to the control (9.6 h). The longest average lag time (79.5 h) was detected after treatment with 30 ppm PAA for 90 min, while at the same condition sorting of CFDA+PI- cells resulted in 95.05 % and 93.94 % non-culturable cells on TSAYE and TSBYE, respectively. The highest percentage of wells with non-culturable cells (96.17 %) was detected on TSAYE after treatment with 40 ppm PAA for 30 min. Fractions of VBNC cells were detected in TSBYE after treatment with HCl pH 3.0 for 60 and 240 min, and in TSAYE and TSBYE after exposure to AA pH 2.7. Treatment with AA pH 2.7 for 150-300 min increased the range of recorded lag time values compared to 60 min, from 8.6 h up to 13.3 h, as well as the mean lag times in TSBYE. Modelling of the outgrowth kinetics comparing the two types of stress (oxidative vs acid) and the two systems of growth (colonial vs planktonic) revealed that low starting concentrations hindered the detection of viable L. monocytogenes cells, either due to VBNC induction or cell heterogeneity.

Persistence of viable but nonculturable Legionella pneumophila state in hospital water systems: A hidden enemy?

There is little evidence of the long-term consequences of maintaining sanitary hot water at high temperatures on the persistence of Legionella in the plumbing system. The aims of this study were to describe the persistence and genotypic variability of L. pneumophila in a hospital building with two entirely independent hot water distribution systems, and to estimate the thermotolerance of the genotypic variants by studying the quantity of VBNC L. pneumophila. Eighty isolates from 55 water samples obtained between the years 2012-2017 were analyzed. All isolates correspond to L. pneumophila serogroup 6. The isolates were discriminated in four restriction patterns by pulsed-field gel electrophoresis. In one installation, pattern A + Aa predominated, accounting for 75.8 % of samples, while the other installation exhibited pattern B as the most frequent (81.8 % of samples; p < 0.001). The mean temperature of the isolates was: 52.6 °C (pattern A + Aa) and 55.0 °C (pattern B), being significantly different. Nine strains were selected as representative among patterns to study their thermotolerance by flow-cytometry after 24 h of thermic treatment. VBNC bacteria were detected in all samples. After thermic treatment at 50 °C, 52.0 % of bacteria had an intact membrane, and after 55 °C this percentage decreased to 23.1 %. Each pattern exhibited varying levels of thermotolerance. These findings indicate that the same hospital building can be colonized with different predominant types of Legionella if it has independent hot water installations. Maintaining a minimum temperature of 50 °C at distal points of the system would allow the survival of replicative L. pneumophila. However, the presence of Legionella in hospital water networks is underestimated if culture is considered as the standard method for Legionella detection, because VBNC do not grow on culture plates. This phenomenon can carry implications for the Legionella risk management plans in hospitals that adjust their control measures based on the microbiological surveillance of water.

Seasonal Variability of Cultivable Nitrate-Reducing and Denitrifying Bacteria and Functional Gene Copy Number in Fresh Water Lake.

This study describes the seasonal course of denitrifying and nitrate-reducing bacteria in a dimictic mesotrophic lake (Lake Scharmützelsee, Brandenburg, Germany) within a three-year period from 2011 to 2013. The bacterial cell numbers were quantified by the fluorescence microscopy, most probable number (MPN) and PCR-dependent quantification of the chromosomal 16S rDNA and of the nirS and nirK gene copy number. The highest seasonal differences (up to three orders of magnitudes) have been measured using MPN in the epilimnion. This variation was not reflected by PCR-dependent approaches or direct microscopical enumeration. At adverse conditions (low temperature and/or low nitrate concentrations), the differences between MPN and gene copy numbers increased by up to five orders of magnitudes and decreased to one magnitude at favourable environmental conditions. These results can be explained best by an increasing ratio of viable but not cultivable (VBNC) cells or dead cells at impairing conditions. In the hypolimnion, the courses of MPN and nir gene copy numbers were similar. This can be explained by a higher feeding pressure and therefore smaller amounts of dormant cells. In the pelagial in general, the total cell numbers enumerated by either microscopical or molecular approaches were similar. In the sediment, more than 99% of the DNA was obviously not related to viable bacteria but was rather DNA in dead cells or adsorbed to particle surfaces.

New insights into survival strategies and PCB bioremediation potential of resuscitated strain Achromobacter sp. HR2 under combined stress conditions.

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.

A simple and rapid method for detecting fecal pollution in urban rivers by measuring the intrinsic ß-D-glucuronidase activity of Escherichia coli.

As urban rivers are domestic, industrial, and agricultural water resources, fecal pollution poses human health and environmental risks. In this study, we developed a simple and rapid method to detect fecal pollution in urban rivers. Water samples were mixed with liquid medium, including a fluorescent substrate and fluorescence intensity (F.I.) was measured using a microplate reader to determine Escherichia coli (E. coli) ß-D-glucuronidase (GUS) activity instead of E. coli concentration. GUS activities measurements in pure E. coli cultures revealed that E. coli incubated with a GUS substrate accumulated GUS enzymes in their cells, whereas those incubated without a GUS substrate did not. The increase in GUS activity corresponded to the proliferation of E. coli and the GUS activity increased linearly even during the lag growth phase of E. coli, indicating the presence of intrinsic GUS (iGUS) in E. coli cells before incubation. iGUS activity persisted at 81 % in the chlorinated samples, even though the E. coli concentration was reduced by a factor of 106. The iGUS activity persisted for approximately three days. Therefore, we assumed that E. coli present in fecal contaminants, in which GUS substrates are present, could be distinguished from those surviving in the natural environment for three days or longer by measuring iGUS activity. River water samples were collected upstream and downstream of the discharge outlets of municipal wastewater treatment plants and a combined sewer outlet. The iGUS activities were <0.24 mMFU/mL for the upstream samples and >0.21 mMFU/mL for the downstream samples. Interestingly, E. coli concentrations were not necessarily associated with fecal pollution. This indicates that by setting a threshold for iGUS activity, our method can be used as a simple and rapid method for detecting fecal pollution in urban rivers. Because the limit of detection for our method is 20 CFU/mL, our method is applicable to detecting high fecal pollution in a small river.

Comparative Analysis Using Raman Spectroscopy of the Cellular Constituents of Lacticaseibacillus paracasei Zhang in a Normal and Viable but Nonculturable State.

This study aimed to investigate the molecular composition of a viable but nonculturable (VBNC) state of a probiotic strain, Lacticaseibacillus paracasei Zhang (L. paracasei Zhang), using single-cell Raman spectroscopy (SCRS). Fluorescent microcopy with live/dead cell staining (propidium iodide and SYTO 9), plate counting, and scanning electron microscopy were used in combination to observe bacteria in an induced VBNC state. We induced the VBNC state by incubating the cells in de Man, Rogosa, and Sharpe broth (MRS) at 4 °C. Cells were sampled for subsequent analyses before VBNC induction, during it, and up to 220 days afterwards. We found that, after cold incubation for 220 days, the viable plate count was zero, but active cells could still be observed (as green fluorescent cells) under a fluorescence microscope, indicating that Lacticaseibacillus paracasei Zhang entered the VBNC state under these conditions. Scanning electron microscopy revealed the altered ultra-morphology of the VBNC cells, characterized by a shortened cell length and a wrinkled cell surface. Principal component analysis of the Raman spectra profiles revealed obvious differences in the intracellular biochemical constituents between normal and VBNC cells. Comparative analysis of the Raman spectra identified 12 main differential peaks between normal and VBNC cells, corresponding to carbohydrates, lipids, nucleic acids, and proteins. Our results suggested that there were obvious cellular structural intracellular macromolecular differences between normal and VBNC cells. During the induction of the VBNC state, the relative contents of carbohydrates (such as fructose), saturated fatty acids (such as palmitic acid), nucleic acid constituents, and some amino acids changed obviously, which could constitute a bacterial adaptive mechanism against adverse environmental conditions. Our study provides a theoretical basis for revealing the formation mechanism of a VBNC state in lactic acid bacteria.

Viable but nonculturable (VBNC) state, an underestimated and controversial microbial survival strategy.

As a unique microbial response to adverse circumstances, the viable but nonculturable (VBNC) state is characterized by the loss of culturability of microbial cells on/in nutrient media that normally support their growth, while maintaining metabolic activity. These cells can resuscitate to a culturable state under suitable conditions. Given the intrinsic importance of the VBNC state and recent debates surrounding it, there is a need to redefine and standardize the term, and to address essential questions such as 'How to differentiate VBNC from other similar terms?' and 'How can VBNC cells be standardly and accurately determined?'. This opinion piece aims at contributing to an improved understanding of the VBNC state and promoting its proper handling as an underestimated and controversial microbial survival strategy.

Transcriptional profiling of Xanthomonas campestris pv. campestris in viable but nonculturable state.

BACKGROUND: Xanthomonas campestris pv. campestris (Xcc) is an important seed-borne plant pathogenic bacteria that can cause a serious threat to cruciferous crops. Bacteria can enter into the viable but non-culturable (VBNC) state under stress conditions, and cause potential risks to agricultural production because the VBNC bacterial cells will evade culture-based detection. However, little is known about the mechanism of VBNC. Our previous study showed that Xcc could be induced into VBNC state by copper ion (Cu2+). RESULTS: Here, RNA-seq was performed to explore the mechanism of VBNC state. The results indicated that expression profiling was changed dramatically in the different VBNC stages (0 d, 1 d, 2 d and 10 d). Moreover, metabolism related pathways were enriched according to COG, GO and KEGG analysis of differentially expressed genes (DEGs). The DEGs associated with cell motility were down-regulated, whereas pathogenicity related genes were up-regulated. This study revealed that the high expression of genes related to stress response could trigger the active cells to VBNC state, while the genes involved in transcription and translation category, as well as transport and metabolism category, were ascribed to maintaining the VBNC state. CONCLUSION: This study summarized not only the related pathways that might trigger and maintain VBNC state, but also the expression profiling of genes in different survival state of bacteria under stress. It provided a new kind of gene expression profile and new ideas for studying VBNC state mechanism in X. campestris pv. campestris.

The Viable but Non-Culturable (VBNC) State, a Poorly Explored Aspect of Beneficial Bacteria.

Many bacteria have the ability to survive in challenging environments; however, they cannot all grow on standard culture media, a phenomenon known as the viable but non-culturable (VBNC) state. Bacteria commonly enter the VBNC state under nutrient-poor environments or under stressful conditions. This review explores the concept of the VBNC state, providing insights into the beneficial bacteria known to employ this strategy. The investigation covers different chemical and physical factors that can induce the latency state, cell features, and gene expression observed in cells in the VBNC state. The review also covers the significance and applications of beneficial bacteria, methods of evaluating bacterial viability, the ability of bacteria to persist in environments associated with higher organisms, and the factors that facilitate the return to the culturable state. Knowledge about beneficial bacteria capable of entering the VBNC state remains limited; however, beneficial bacteria in this state could face adverse environmental conditions and return to a culturable state when the conditions become suitable and continue to exert their beneficial effects. Likewise, this unique feature positions them as potential candidates for healthcare applications, such as the use of probiotic bacteria to enhance human health, applications in industrial microbiology for the production of prebiotics and functional foods, and in the beer and wine industry. Moreover, their use in formulations to increase crop yields and for bacterial bioremediation offers an alternative pathway to harness their beneficial attributes.

Monitoring the Starvation-Survival Response of Edwardsiella piscicida and E. tarda in Freshwater Microcosms, at Various Temperatures.

Edwardsiella piscicida is an important fish pathogen responsible for economic losses in global aquaculture, and E. tarda is also a human zoonotic pathogen. In this study, the survival of E. piscicida and E. tarda strains kept in filtered and sterilized lake water microcosms was investigated during a 20-week period at 7 °C, 15 °C and 25 °C, as well as its pathogenicity retention during a starvation period. E. tarda V43.2 stayed culturable for 6 weeks at 7 °C, 9 weeks at 25 °C and 12 weeks at 15 °C. Both E. piscicida strains (V12.1 and V57.2) stayed culturable even longer, for at least 12 weeks at 7 °C, 15 °C and 25 °C under the same starvation conditions. After Edwardsiella cells entered into the VBNC state, some became shorter and "rounded up," but others aggregated and retained a short rod shape. Aggregates of Edwardsiella cells were common throughout the VBNC period, and a well-formed biofilm was observed for all tested strains at the end of the experiment. The growth capacity of VBNC cells was restored by cultivating microcosm water samples in LB broth at 28 °C. Resuscitated E. piscicida cells were as virulent for the European eel as the controls. Natural waters can be a reservoir for Edwardsiella, and its underestimation in environmental samples poses a risk to public health and aquaculture.

Listeria monocytogenes Sublethal Injury and Viable-but-Nonculturable State Induced by Acidic Conditions and Disinfectants.

The dormancy continuum hypothesis states that in response to stress, cells enter different stages of dormancy ranging from unstressed living cells to cell death, in order to ensure their long-term survival under adverse conditions. Exposure of Listeria monocytogenes cells to sublethal stressors related to food processing may induce sublethal injury and the viable-but-nonculturable (VBNC) state. In this study, exposure to acetic acid (AA), hydrochloric acid (HCl), and two disinfectants, peracetic acid (PAA) and sodium hypochlorite (SH), at 20°C and 4°C was used to evaluate the potential induction of L. monocytogenes strain Scott A into different stages of dormancy. To differentiate the noninjured subpopulation from the total population, tryptic soy agar with 0.6% yeast extract (TSAYE), supplemented or not with 5% NaCl, was used. Sublethally injured and VBNC cells were detected by comparing plate counts obtained with fluorescence microscopy and by using combinations of carboxyfluorescein and propidium iodide (viable/dead cells). Induction of sublethal injury was more intense after PAA treatment. Two subpopulations were detected, with phenotypes of untreated cells and small colony variants (SCVs). SCVs appeared as smaller colonies of various sizes and were first observed after 5 min of exposure to 5 ppm PAA at 20°C. Increasing the stress intensity from 5 to 40 ppm PAA led to earlier detection of SCVs. L. monocytogenes remained culturable after exposure to 20 and 30 ppm PAA for 3 h. At 40 ppm, after 3 h of exposure, the whole population was considered nonculturable, while cells remained metabolically active. These results corroborate the induction of the VBNC state. IMPORTANCE Sublethally injured and VBNC cells may evade detection, resulting in underestimation of a food product's microbial load. Under favorable conditions, cells may regain their growth capacity and acquire new resistant characteristics, posing a major threat for public health. Induction of the VBNC state is crucial for foodborne pathogens, such as L. monocytogenes, the detection of which relies almost exclusively on the use of culture recovery techniques. In the present study, we confirmed that sublethal injury is an initial stage of dormancy in L. monocytogenes that is followed by the VBNC state. Our results showed that PAA induced SCVs (a phenomenon potentially triggered by external factors) and the VBNC state in L. monocytogenes, indicating that tests of lethality based only on culturability may provide false-positive results regarding the effectiveness of an inactivation treatment.

Quantitative detection of trace VBNC Cronobacter sakazakii by immunomagnetic separation in combination with PMAxx-ddPCR in dairy products.

One immunomagnetic separation (IMS) assay based on immunomagnetic beads (IMBs) has been evaluated as a potential pretreatment tool for the separation and enrichment of target bacteria. In this study, we successfully immobilized antibodies onto magnetic bead surfaces to form IMBs through biotin and a streptavidin (SA) system to capture viable but nonculturable (VBNC) Cronobacter sakazakii (C. sakazakii) from dairy products. Various parameters that affected the capture efficiency (CE) of IMS, including the number of antibodies, IMBs dose, incubation time, magnetic separation time, and immunoreaction temperature, were systematically investigated. We further determined the optimal enrichment conditions for different dairy substrates to ensure maximum enrichment of target pathogens in the system. An IMS technique combining improved propidium monoazide (PMAxx) and droplet digital PCR (ddPCR) was established to detect the pathogenic VBNC C. sakazakii. The IMS-PMAxx-ddPCR method after IMBs enrichment showed higher accuracy when the VBNC C. sakazakii was under 1 Log10 copies/g. The detection limit for this method in a background of powdered infant formula (PIF) was 5.6 copies/g. In summary, the developed IMS-PMAxx-ddPCR method has great potential for the analysis and detection of VBNC bacteria in food.

Study on the Viable but Non-culturable (VBNC) State Formation of Staphylococcus aureus and Its Control in Food System.

A Viable but non-culturable (VBNC) state is a bacterial survival strategy under reverse conditions. It poses a significant challenge for public health and food safety. In this study, the effect of external environmental conditions including acid, nutrition, and salt concentrations on the formation of S. aureus VBNC states at low temperatures were investigated. Different acidity and nutritional conditions were then applied to food products to control the VBNC state formation. Four different concentration levels of each factor (acid, nutrition, and salt) were selected in a total of 16 experimental groups. Nutrition showed the highest influence on the VBNC state formation S. aureus, followed by acid and salt. The addition of 1% acetic acid could directly kill S. aureus cells and inhibit the formation of the VBNC state with a nutrition concentration of 25, 50, and 100%. A propidium monoazide-polymerase chain reaction (PMA-PCR) assay was applied and considered as a rapid and sensitive method to detect S. aureus in VBNC state with the detection limit of 104 CFU/mL.

Effect of Thermosonication and Physicochemical Properties of Wine on Culturability, Viability, and Metabolic Activity of Brettanomyces bruxellensis Yeast in Red Wines.

The aim of this research was to investigate the short- and long-term effects of thermosonication and different physicochemical properties of wine on culturability, viability, and metabolic activity of Brettanomyces bruxellensis yeast. Thermosonication was conducted at 43 °C during 1, 2, and 3 min, while wine variations included several pH, alcohol, and sugar levels. Cell culturability and viability were determined immediately after treatment and during 90 days of storage, while metabolic activity was determined after 90 days of storage. Results showed that, although culturability was not confirmed in dry wines immediately after 3 min of treatment, thermosonication did not result in complete inactivation of the B. bruxellensis population. Herein, the first evidence of a viable but not culturable (VBNC) state of B. bruxellensis after thermosonication exposure was observed. Moreover, thermosonication reduced the production of volatile phenols. Obtained results suggest application of thermosonication for reduction of the B. bruxellensis population only in early stages of wine contamination.

Induction and Recovery of the Viable but Nonculturable State of Hop-Resistance Lactobacillus brevis.

Lactobacillus brevis is a major hop-resistance bacterium which poses significant challenge for the brewing industry, mainly due to the difficulty or incapability in detection by routine culturing methodology and its beer spoilage ability.This study aimed at investigating the VBNC state of a hop-resistance strain, L. brevis BM-LB13908. The culturable, total and viable numbers of L. brevis cells were calculated by MRS agar plate counting, acridine orange direct count (AODC) method and Live/Dead BacLight bacterial viability kit with fluorescence microscope. VBNC formation was induced by 189 ± 5.7 days under low-temperature storage or 27 ± 1.2 subcultures by continuous passage in beer, and VBNC cells induced by both strategies were recovered by adding catalase. In addition, insignificant difference in beer-spoilage ability was found in 3 states of L. brevis, including logarithmic growing, VBNC and recovered cells. This is the first study on the formation of VBNC state for L. brevis and beer-spoilage ability of both VBNC and recovered cells, which indicate L. brevis strain could cause beer spoilage without being detected by routine methodologies. The results derived from this study may support further study on L. brevis and other hop-resistance bacteria, and guidance on beer spoilage prevention and control, such as improvement for brewers on the microbiological quality control by using the improved culture method with catalase supplementation.

Expression of rpoS, ompA and hfq genes of Cronobacter sakazakii strain Yrt2a during stress and viable but nonculturable state.

Cronobacter spp. in powdered infant formula has been etiologically linked to meningitis and necrotizing enterocolitis in certain groups of infants. This study aimed to determine whether C. sakazakii Yrt2a strain experiencing desiccation stress could enter viable but nonculturable (VBNC) state as well as to examine the expression of genes associated with stress and virulence during the above states. Stress and VBNC conditions were determined based on viability and culturability assays. Expression of genes related to stress (rpoS) and virulence (hfq and ompA) was evaluated by real-time PCR. The results showed that C. sakazakii Yrt2a entered VBNC 24 days post exposure to 2 h of desiccation treatment. The expression of rpoS, hfq and ompA genes was up-regulated during stress conditions, suggesting that Cronobacter successfully managed stress to maintain its culturability while maintaining its virulence. The expression of the target genes decreased at VBNC state but remained higher than that of a normal state. These findings reinforce the assumption that C. sakazakii undergoing VBNC state maintains its pathogenicity.

The viable but nonculturable state induction and genomic analyses of Lactobacillus casei BM-LC14617, a beer-spoilage bacterium.

This study aimed to investigate the viable but nonculturable (VBNC) state and genomic features of a beer-spoilage strain, Lactobacillus caseiBM-LC14617. Induction on the VBNC state of L. casei strain BM-LC14617 was conducted by both low-temperature storage and continuous passage in beer, and formation of VBNC state was detected after 196 ± 3.3 days and 32 ± 1.6 subcultures, respectively. Resuscitation of VBNC cells was successfully induced by addition of catalase, and culturable, VBNC, and resuscitated cells shared similar beer-spoilage capability. Whole genome sequencing was performed, and out of a total of 3,964 predicted genes, several potential VBNC and beer-spoilage-associated genes were identified. L. casei is capable of entering into and resuscitating from the VBNC state and possesses beer-spoilage capability. The genomic characterization yield insightful elucidation of VBNC state for L. casei. This study represents the first evidence on VBNC state induction of L. casei and beer-spoilage capability of VBNC and resuscitated cells. Also, this is the first genomic characterization of L. casei as a beer-spoilage bacterium. The current study may aid in further study on L. casei and other beer-spoilage bacteria, and guide the prevention and control of beer spoilage.

Towards understanding clinical campylobacter infection and its transmission: time for a different approach?

Campylobacter spp. are among the most commonly diagnosed causes of human infection. Methods for detection of the 29 campylobacter species have mainly focused on cultivation of the thermophilic species. More than 99% of clinical campylobacter isolates notified in the UK in the recent past have been from faecal samples and associated with gastroenteritis. Campylobacter enteritis notifications in temperate zones show a seasonal increase during the summer months with a sharp decrease in the winter months, a pattern which remains incompletely understood. The striking seasonality in the expression of many human genes, some concerned with inflammation and immunity, suggests a need for further study of the host regarding the temporal distribution of many human infections, including campylobacteriosis. A tendency for campylobacter to enter a non-cultivable state under adverse conditions effects a reduction in the number of isolations. A Polymerase Chain Reaction (PCR)-based screening approach for the presence of the Campylobacter genus and followed by speciation has provided some insight into the limitations of cultivation for campylobacter, also allowing the discovery of new species. The increased sensitivity of the PCR-based approach over culture-based methods may make it difficult for the laboratory to differentiate asymptomatic campylobacter carriage from clinical campylobacter infection in non-sterile body sites. Campylobacter infection depends on a combination of host factors, and on acquisition of a suitably virulent strain with a tropism for human epithelium. The possibility of persistence of campylobacter in a viable but non-culturable latent form in the human body may also require further investigation. The scope of this review includes a discussion of current methods for diagnosing acute campylobacter infection and for detecting campylobacter in water and foodstuffs. The review also questions the prevailing view that poultry is the most common source of campylobacteriosis.