Exploration of genes associated with induction of the viable but non-culturable state of Campylobacter jejuni.

Campylobacter jejuni is known to enter a viable but non-culturable (VBNC) state when exposed to environmental stresses. Microarray and quantitative real-time polymerase chain reaction (qPCR) analyses were performed to elucidate the genes related to the induction of the VBNC state. The C. jejuni NCTC11168 strain was cultured under low-temperature or high-osmotic stress conditions to induce the VBNC state. mRNA expression in the VBNC state was investigated using microarray analysis, and the gene encoding peptidoglycan-associated lipoprotein, Pal, was selected as the internal control gene using qPCR analysis and software. The three genes showing particularly large increases in mRNA expression, cj1500, cj1254, and cj1040, were involved in respiration, DNA repair, and transporters, respectively. However, formate dehydrogenase encoded by cj1500 showed decreased activity in the VBNC state. Taken together, C. jejuni actively changed its mRNA expression during induction of the VBNC state, and protein activities did not always match the mRNA expression levels.

Quantitative Proteomic Analysis on the Slightly Acidic Electrolyzed Water Triggered Viable but Non-Culturable Listeria monocytogenes.

This study undertakes a comprehensive exploration of the impact of slightly acidic electrolyzed water (SAEW) on Listeria monocytogenes, a common foodborne pathogen, with a particular focus on understanding the molecular mechanisms leading to the viable but nonculturable (VBNC) state. Given the widespread application of SAEW as an effective disinfectant in the food industry, uncovering these molecular pathways is crucial for improving food safety measures. We employed tandem mass tags (TMT), labeling proteomic techniques and LC-MS/MS to identify differentially expressed proteins under two doses of SAEW conditions. We indicated 203 differential expressed proteins (DEPs), including 78 up-regulated and 125 down-regulated DEPs. The functional enrichment analysis of these proteins indicated that ribosomes, biosynthesis of secondary metabolites, and aminoacyl-tRNA biosynthesis were enriched functions affected by SAEW. Further, we delved into the role of protein chlorination, a potential consequence of reactive chlorine species generated during the SAEW production process, by identifying 31 chlorinated peptides from 22 proteins, with a dominant sequence motif of Rxxxxx[cY] and functionally enriched in translation. Our findings suggest that SAEW might prompt alterations in the protein translation process and trigger compensatory ribosome biosynthesis. However, an imbalance in the levels of elongation factors and AARSs could hinder recovery, leading to the VBNC state. This research carries substantial implications for food safety and sanitation, as it adds to our understanding of the SAEW-induced VBNC state in L. monocytogenes and offers potential strategies for its control.

Intensified inactivation of model and environmental bacteria by an atmospheric-pressure air-liquid discharge plasma compared with chlorination.

Water-borne pathogenic bacteria are always the top priority to be removed through disinfection process in water treatment due to their threat to human health. It was necessary to develop novel disinfection methods since the conventional chlorine disinfection was inefficient in inactivating chlorine-resistant bacteria, inducing the viable but non-culturable (VBNC) bacteria and forming disinfection by-products (DBPs). In this study, the inactivation of four model strains including Gram-negative (G-), Gram-positive (G+) and environmental samples by atmospheric-pressure air-liquid discharge plasma (ALDP) was assessed systematically. The results showed that ALDP was superior in inactivating all of the samples compared with chlorination. During 10 min ALDP treatment, the G- bacteria were completely inactivated, and the G+ one was inactivated by more than 4.61 logs. The inactivation of bacteria from a campus lake and a wastewater treatment plant effluent exceeded 99.82% and 97.78%, respectively. For G- bacteria, ALDP resulted in a much lower (102∼103 times) levels of VBNC cells than chlorination. ALDP could effectively remove the chlorine-resistant bacteria. More than 96.41% of the intracellular DNA and 99.99% of the extracellular DNA were removed, whereas it was only 56.35% and 12.82% for chlorination. ALDP had a stronger ability to destroy cell structure than chlorination, presumably due to the existence of ROS (·OH, 1O2 and O2-). GC-MS analysis showed that ALDP produced less DBPs than chlorination. These findings provided new insights for the application of discharge plasma in water disinfection, which could be complemental or alternative to the conventional disinfection methods.

Bacteriostasis of nisin against planktonic and biofilm bacteria: Its mechanism and application.

Food safety incidents caused by bacterial contamination have always been one of the public safety issues of social concern. Planktonic cells, viable but non-culturable (VBNC) cells, and biofilm cells of bacteria can coexist in food or food processing, posing more serious challenges to public health and safety by increasing bacterial survival and difficulty in detection. As a non-toxic, no side effect, and highly effective bacteriostatic substance, nisin has received wide attention from researchers. In this review, we summarized the species and biosynthesis of nisin, the effects of nisin alone or in combination with other treatments on planktonic and biofilm cells, and its applications in the fields of food, feed, and medicine by consulting numerous studies. Meanwhile, the mechanism of nisin on planktonic and biofilm cells was proposed based on existing researches. Nisin not only has antibacterial activity against most G+ bacteria but also exhibits a bacteriostatic effect on G- bacteria when combined with other antibacterial treatments. In addition to planktonic cells, nisin also has significant effects on bacterial cells in biofilms by changing the thickness, density, and composition of biofilms. Based on the three action processes of nisin on biofilms, we summarized the changes of bacteria in biofilms, including the causes of bacterial death and the formation of the VBNC state. We consider that research on the relationship between nisin and VBNC state should be strengthened.

Effect of RpoS on the survival, induction, resuscitation, morphology, and gene expression of viable but non-culturable Salmonella Enteritidis in powdered infant formula.

Involvement of the transcriptional regulator RpoS in the persistence of viable but non-culturable (VBNC) state has been demonstrated in several species of bacteria. This study investigated the role of the RpoS in the formation and resuscitation of VBNC state in Salmonella enterica serovar Enteritidis CICC 21482 by measuring bacterial survival, morphology, physiological characteristics, and gene expression in wild-type (WT) and rpoS-deletion (ΔrpoS) strains during long-term storage in powdered infant formula (PIF). The ΔrpoS strain was produced by allelic exchange using a suicide plasmid. Bacteria were inoculated into PIF for 635-day storage. Survival, morphology, intracellular reactive oxygen species (ROS) levels and intercellular quorum sensing autoinducer-2 (AI-2) contents were regularly measured. Resuscitation assays were conducted after obtaining VBNC cells. Gene expression was measured using real-time quantitative polymerase chain reaction (qPCR). The results showed that RpoS and low temperature conditions were associated with enhanced culturability and recoverability of Salmonella Enteritidis after desiccation storage in low water activity (aw) PIF. In addition, the synthesis of intracellular ROS and intercellular quorum sensing AI-2 was regulated by RpoS, inducing the formation and resuscitation of VBNC cells. Gene expression of soxS, katG and relA was found strongly associated with RpoS. Due to the lack of RpoS factor, the ΔrpoS strain could not normally synthesize SoxS, catalase and (p)ppGpp, resulting in its early shift to the VBNC state. This study elucidates the role of rpoS in desiccation stress and the formation and resuscitation mechanism of VBNC cells under desiccation stress. It serves as the basis for preventing and controlling the recovery of pathogenic bacteria in VBNC state in low aw foods.

Persistence and viable but non-culturable state induced by streptomycin in Erwinia amylovora.

Persister cell and viable but non-culturable (VBNC) state of bacteria are survival strategies against antibiotics and various environmental stresses, respectively, but they tend to be ignored in agriculture fields, even though bacteria can regain their abilities to survive and produce disease once those stresses disappear. This study was carried out to determine whether persister cell and VBNC state in Erwinia amylovora are present after exposures to streptomycin, the length of their persistence, and the steps needed to decrease the inoculum. Persister cells were observed using biphasic killed growth curve for 4-8 h when the late stationary phase cells of E. amylovora were cultured in liquid medium containing streptomycin. This state was maintained for up to 12 h based on the colony forming units (CFUs) of the colonies that grew on the mannitol glutamate yeast extract (MGY) medium after streptomycin was removed. The CFUs on the MGY medium were lower than the total count determined using the LIVE/DEAD Kit, suggesting that persister cells and VBNC state might co-exist for up to 12 h after exposure to streptomycin. However, after 12 h, E. amylovora cells did not continue to grow on the medium for 9 days, suggesting that they entered a VBNC state at that time and remained in a persistent state. In addition, based on the Redox Sensor Green staining method, the presence of both states was confirmed for up to 12 h, and only then did the VBNC state became apparent. Furthermore, persister cells were observed for up to 24 h, and damaged cells reduced when E. amylovora cells were culture in distilled water with streptomycin, indicating that the uptake of lower nutrients in E. amylovora led to prolonged persister cells and VBNC state, which are more likely to survive after streptomycin treatments. The addition of sucrose and oxytetracycline to distilled water containing streptomycin reduced persister cells than other sources did. Thus, to inhibit the spread of fire blight, management techniques must consider the hazards of using streptomycin treatments that induce dormancy, such as persister cells and VBNC state, beyond the development of resistant strain.

BD Vacutainer™ Urine Culture & Sensitivity Preservative PLUS Plastic Tubes Minimize the Harmful Impact of Stressors Dependent on Temperature and Time Storage in Uropathogenic Bacteria.

Background: Urinary tract infection is a worldwide health problem. According to the Clinical Laboratory Improvement Amendments and the European Urinalysis Guideline, urine samples should be tested within 2 h of collection. Thus, using chemical preservatives that guarantee the pre-analytical conditions is a practical tool. However, the effects of temperature and storage time as uropathogenic bacteria stressors are unclear. Methods: Gram-negative and -positive ATTC strains, E. coli, P. mirabilis, E. faecalis, and S. aureus, were used in this study. Strains in liquid media were stored at 4, 25, and 37 °C for 0, 2, 12, 24, and 48 h in tubes with and without preservatives. Then, reactive oxygen species (ROS) levels, viable but non-culturable bacteria (VBNC), and bacteria growth were analyzed. Results: A high ROS level was associated with the presence of VBNC and dead bacteria with low CFU counts, but a low ROS level increased the CFU number, depending on temperature and storage time in tubes without preservatives (boric acid, sodium borate, and formate). The BD Vacutainer™ Urine Culture & Sensitivity Preservative PLUS Plastic Tubes (C&S-PP) prevent this ROS increase, maintaining the CFU number for longer. Conclusions: C&S-PP tubes minimize the stressor effects (temperature and time storage) on uropathogenic bacteria when stored, improving the pre-analytical conditions of cultures realized by the clinical laboratory.

Quantification and cultivation of Helicobacter pylori (H. pylori) from various urban water environments: A comprehensive analysis of precondition methods and sample characteristics.

Substantial evidence suggests that all types of water, such as drinking water, wastewater, surface water, and groundwater, can be potential sources of Helicobacter pylori (H. pylori) infection. Thus, it is critical to thoroughly investigate all possible preconditioning methods to enhance the recovery of H. pylori, improve the reproducibility of subsequent detection, and optimize the suitability for various water types and different detection purposes. In this study, we proposed and evaluated five distinct preconditioning methods for treating water samples collected from multiple urban water environments, aiming to maximize the quantitative qPCR readouts and achieve effective selective cultivation. According to the experimental results, when using the qPCR technique to examine WWTP influent, effluent, septic tank, and wetland water samples, the significance of having a preliminary cleaning step becomes more evident as it can profoundly influence qPCR detection results. In contrast, the simple, straightforward membrane filtration method could perform best when isolating and culturing H. pylori from all water samples. Upon examining the cultivation and qPCR results obtained from groundwater samples, the presence of infectious H. pylori (potentially other pathogens) in aquifers must represent a pressing environmental emergency demanding immediate attention. Furthermore, we believe groundwater can be used as a medium to reflect the H. pylori prevalence in a highly populated community due to its straightforward analytical matrix, consistent detection performance, and minimal interferences from human activities, temperature, precipitation, and other environmental fluctuations.

Removal of bacterial pathogens and antibiotic resistance bacteria by anaerobic sludge digestion with thermal hydrolysis pre-treatment and alkaline stabilization post-treatment.

Primary sludge (PS) is associated with public health and environmental risks, so regulations focus on reducing the pathogenic and heavy metal contents of the treated material (biosolids), intended for soil amendments and land reclamation. The regulations set limits for Escherichia coli (or fecal coliforms), Salmonella spp., helminth eggs and enterovirus. However, the potential risk due to antibiotic resistant bacteria (ARB) and other human potential pathogenic bacteria (HPB) are not considered. In this work, three sludge treatment processes, having in common an anaerobic digestion step, were applied to assess the removal of regulated bacteria (fecal coliforms, Salmonella spp), ARB and HPB. The treatment arrangements, fed with PS from a full-scale wastewater treatment plant were: 1) Mesophilic anaerobic digestion followed by alkaline stabilization post-treatment (MAD-CaO); 2) Thermophilic anaerobic digestion (TAD) and, 3) Pre-treatment (mild thermo-hydrolysis) followed by TAD (PT-TAD). The results address the identification, quantification (colony forming units) and taxonomic characterization of ARB resistant to ß-lactams and vancomycin, as well as the taxonomic characterization of HPB by sequencing with PacBio. In addition, quantification based on culture media of fecal coliforms and Salmonella spp. is presented. The capabilities and limitations of microbiological and metataxonomomic analyses based on PacBio sequencing are discussed, emphasizing that they complement each other. Genus Aeromonas, Acinetobacter, Citrobacter, Enterobacter, Escherichia, Klebsiella, Ochrobactrum, Pseudomonas and Raoultella, among others, were found in the PS, which are of clinical or environmental importance, being either HPB, HPB-ARB, or non-pathogenic ARB with the potentiality of horizontal gene transfer. Based on the analysis of fecal coliforms and Salmonella spp., the three processes produced class A (highest) biosolids, suitable for unrestricted agriculture applications. Mild thermo-hydrolisis was effective in decreasing ARB cultivability, but it reappeared after the following TAD. O. intermedium (HPB-ARB) was enriched in MAD and TAD while Laribacter hongkongensis (HPB) did persist after the applied treatments.

Transcriptome Analysis of Viable but Non-Culturable Brettanomyces bruxellensis Induced by Hop Bitter Acids.

The viable but non-culturable (VBNC) state has been studied in detail in bacteria. However, it has received much less attention in eukaryotic cells. The induction of a VBNC beer-spoilage yeast (Brettanomyces bruxellensis) by hop bitter acids with different concentrations and its recovery were studied in this work. B. bruxellensis cells were completely induced into the VBNC state by treatment of 250 mg/L hop bitter acids for 2 h. The addition of catalase at a concentration of 2,000 U/plate on YPD agars enabled these VBNC cells to recover their culturability within 2 days. Moreover, the transcriptome profiling revealed that 267 and 197 genes were significantly changed upon VBNC state entry and resuscitation, respectively. The differentially expressed genes involved in the peroxisome activities, ABC transporter, organic acid metabolism, and TCA cycle were mainly downregulated in the VBNC cells. In contrast, the amino acid and carbohydrate metabolism, cell division, and DNA replication were promoted. This study supplies a theoretical basis for microbial risk assessment in the brewing industry.

Recovery of microbiological quality of long-term stagnant tap water in university buildings during the COVID-19 pandemic.

Stagnant water can cause water quality deterioration and, in particular, microbiological contaminations, posing potential health risks to occupants. University buildings were unoccupied with little water usage during the COVID-19 pandemic. It's an opportunity to study microbiological quality of long-term stagnant water (LTSW) in university buildings. The tap water samples were collected for three months from four types of campus buildings to monitor water quality and microbial risks after long-term stagnation. Specifically, the residual chlorine, turbidity, and iron/zinc were disqualified, and the heterotrophic plate counts (HPC) exceeded the Chinese national standard above 100 times. It took 4-54 days for these parameters to recover to the routine levels. Six species of pathogens were detected with high frequency and levels (101-105 copies/100 mL). Remarkably, L. pneumophilia occurred in 91% of samples with turbidity > 1 NTU. The absence of the culturable cells for these bacteria possibly implied their occurrence in a viable but non-culturable (VBNC) status. The bacterial community of the stagnant tap water differed significantly and reached a steady state in more than 50 days. Furthermore, a high concentration of endotoxin (>10 EU/mL) was found in LTSW, which was in accordance with the high proportion of dead bacteria. The results suggested that the increased microbiological risks require more attention and the countermeasures before the building reopens should be taken.

Exploring the Potential of Micrococcus luteus Culture Supernatant With Resuscitation-Promoting Factor for Enhancing the Culturability of Soil Bacteria.

A bacterial species is best characterized after its isolation in a pure culture. This is an arduous endeavor for many soil microorganisms, but it can be simplified by several techniques for improving culturability: for example, by using growth-promoting factors. We investigated the potential of a Micrococcus luteus culture supernatant containing resuscitation-promoting factor (SRpf) to increase the number and diversity of cultured bacterial taxa from a nutrient-rich compost soil. Phosphate-buffered saline and inactivated SRpf were included as controls. After agitation with SRpf at 28°C for 1 day, the soil suspension was diluted and plated on two different solid, oligotrophic media: tenfold diluted Reasoner's 2A agar (R2A) and soil extract-based agar (SA). Colonies were collected from the plates to assess the differences in diversity between different treatments and cultivation media. The diversity on both R2A and SA was higher in the SRpf-amended extracts than the controls, but the differences on R2A were higher. Importantly, 51 potentially novel bacterial species were isolated on R2A and SA after SRpf treatment. Diversity in the soil extracts was also determined by high-throughput 16S rRNA amplicon sequencing, which showed an increase in the abundance of specific taxa before their successful cultivation. Conclusively, SRpf can effectively enhance the growth of soil bacterial species, including those hitherto uncultured.

Optimization of a propidium monoazide-qPCR method for Escherichia coli quantification in raw seafood.

The present study compared different concentrations of propidium monoazide (PMA), time of exposure to light and different light intensities to determine the optimal conditions for the quantification of viable Escherichia coli in cell suspension and in food matrix. The influence of cell density and the effectiveness of PMA in viable but non-culturable (VBNC) E. coli cells were evaluated and also applied in food matrix. For that purpose, different concentrations of PMA (20 µM, 40 µM, 50 µM, 60 µM and 80 µM) under different times of exposure (5 min, 10 min, 15 min, 20 min and 30 min) to lights of different intensities (500 W and 650 W) were evaluated. After determining the optimal conditions, the PMA-qPCR methods were applied to different compositions of live and heat-killed E. coli suspensions (v:v; 0:1; 1:0; 1:1) in concentrations ranging from 3 Log to 7 Log CFU/mL. The same dilutions were prepared with E. coli in VBNC state and applied in food matrix. The results obtained from qPCR, PMA-qPCR and plate counts were compared. The results suggested that a PMA treatment of 50 µM PMA for 15 min under 650 W light intensity was optimal under our conditions. For E. coli cell suspensions, the amplification of heat-killed cells was inhibited greatly by PMA when concentrations were ≤ 5 Log CFU/mL. For the samples of oyster inoculated with heat-killed cells, E. coli was not detected by PMA-qPCR in concentrations ≤4 Log CFU/g. Regarding the results with VBNC state, we considered the PMA-qPCR method to be applicable for enumerating E. coli VBNC cells in oyster samples. Based on our findings, we further recommend the use of PMA-qPCR with the aim of reducing the amplification of dead cells for improving its performance, since false-positives could still occur depending on the level of E. coli in the sample. The application of the PMA-qPCR for quantification of bacteria, compared to the use of culture-dependent methods, is quite promising. However, further studies are recommended, especially using different food matrices.

Alterations in the Cell Wall of Rhodococcus biphenylivorans Under Norfloxacin Stress.

Many microorganisms can enter a viable but non-culturable (VBNC) state under various environmental stresses, while they can also resuscitate when the surroundings turn to suitable conditions. Cell walls play a vital role in maintaining cellular integrity and protecting cells from ambient threats. Here, we investigated the alterations in the cell wall of Rhodococcus biphenylivorans TG9 at VBNC state under norfloxacin stress and then at resuscitated state in fresh lysogeny broth medium. Electron microscopy analyses presented that TG9 in the VBNC state had a thicker and rougher cell wall than that in exponential phase or resuscitated state. Meanwhile, the results from infrared spectroscopy also showed that its VBNC state has different peptidoglycan structures in the cell wall. Moreover, in the VBNC cells the gene expressions related to cell wall synthesis and remodeling maintain a relatively high level. It indicates that the morphological variations of TG9 at the VBNC state might result from kinetic changes in the cell wall synthesis and remodeling. As a consequence, the alterations in the cell wall of VBNC TG9 may somewhat account for its tolerance mechanisms to antibiotic treatment.

Extracellular organic matter from Micrococcus luteus containing resuscitation-promoting factor in sequencing batch reactor for effective nutrient and phenol removal.

Culture supernatant containing resuscitation-promoting factor (SRpf) from Micrococcus luteus was added to the sequencing batch reactor (SBR) for effective treatment of phenol-containing wastewater. SRpf acclimation significantly improved combined removal of phenol and nutrients. Moreover, the Illumina high-throughput sequencing analysis revealed that the SRpf boosted bacteria diversity, which enhanced the stability of the system under phenol stress. Addition of SRpf increased the abundances of Actinobacteria and Proteobacteria phyla which are involved in nutrient and phenol removal. Specifically, SRpf promoted Nitrosomonas and Nitrospira which participate in nitrification, family Comamonadaceae, genera Dechloromonas and Pseudomonas involved in denitrification, and Acinetobacter, Pseudomonas and Rhodocyclus which remove phosphorus elements. Moreover, the abundances of Bacillus and Klebsiella responsible for phenol removal as well as Pseudomonas and Acinetobacter were significantly increased after SRpf acclimation. These results show that SBR combined with SRpf acclimation provide optimal nutrient and phenol removal.

Resuscitation, isolation and immobilization of bacterial species for efficient textile wastewater treatment: A critical review and update.

Given highly complex and recalcitrant nature of synthetic dyes, textile wastewater poses a serious challenge on surrounding environments. Until now, biological treatment of textile wastewater using efficient bacterial species is still considered as an environmentally friendly and cost-effective approach. The advances in resuscitating viable but non-culturable (VBNC) bacteria via signaling compounds such as resuscitation-promoting factors (Rpfs) and quorum sensing (QS) autoinducers, provide a vast majority of potent microbial resources for biological wastewater treatment. So far, textile wastewater treatment from resuscitating and isolating VBNC state bacteria has not been critically reviewed. Thus, this review aims to provide a comprehensive picture of resuscitation, isolation and application of bacterial species with this new strategy, while the recent advances in synthetic dye decolorization were also elaborated together with the mechanisms involved. Discussion was further extended to immobilization methods to tackle its application. We concluded that the resuscitation of VBNC bacteria via signaling compounds, together with biochar-based immobilization technologies, may lead to an appealing biological treatment of textile wastewater. However, further development and optimization of the integrated process are still required for their wide applications.

Development and Application of a Simple "Easy To Operate" Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coli.

O157 Escherichia coli is one of the most important foodborne pathogens causing disease even at low cellular numbers. Thus, the early and accurate detection of this pathogen is important. However, due to the formation of viable but non-culturable (VBNC) status, the golden standard culturing methodology fails to identify O157 E. coli once it enters VBNC status. Crossing priming amplification (CPA) is a novel, simple, easy-to-operate detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to firstly develop and apply a CPA assay with propidium monoazide (PMA) for the rapid detection of the foodborne E. coli O157:H7 in VBNC state. Five primers (2a/1s, 2a, 3a, 4s, and 5a) were specially designed for recognizing three targets, which were rfbE, stx1, and stx2, and evaluated for its effectiveness in detecting VBNC cell of E. coli O157:H7 with detection limits of pure VBNC culture at 103, 105, and 105 colony-forming units (CFUs)/ml for rfbE, stx1, and stx2, respectively, whereas those of food samples (frozen pastry and steamed bread) were 103, 105, and 105 CFUs/ml. The application of the PMA-CPA assay was successfully used on detecting E. coli O157:H7 in VBNC state from food samples. In conclusion, this is the first development of PMA-CPA assay on the detection of VBNC cell, which was found to be useful and a powerful tool for the rapid detection of E. coli O157:H7 in VBNC state. Undoubtedly, the PMA-CPA method can be of high value to the food industry owing to its various advantages such as speed, specificity, sensitivity, and cost-effectiveness.

Quantification of Viable but Non-Culturable Cells of Legionella pneumophila.

Legionella pneumophila, among other bacteria, may enter a viable but non-culturable state as a means for survival in stressful conditions. Bacterial cells in the viable but non-culturable state cannot grow on standard medium; however, they continue to exhibit characteristics that are associated with live cells, such as respiration, transcription, and cell wall integrity. The present paper outlines a detailed protocol for the detection of viable but non-culturable L. pneumophila cells via Syto® 9 and propidium iodide staining coupled with flow cytometry.

Global Proteomic Analysis of the Resuscitation State of Vibrio parahaemolyticus Compared With the Normal and Viable but Non-culturable State.

Vibrio parahaemolyticus is a common pathogen which has become a major concern of seafood products. The bacteria in the viable but non-culturable (VBNC) state are unable to form colonies on growth media, but under appropriate conditions they can regain culturability. In this study, V. parahaemolyticus was induced into VBNC state at low temperature and oligotrophic condition, and was resuscitated to culturable state. The aim of this study is to explore the comparative proteomic profiles of the resuscitation state compared with the VBNC state and the exponential phase of V. parahaemolyticus using isobaric tags for relative and absolute quantitation (iTRAQ) technique. The differentially expressed proteins (DEPs) were subjected to GO functional annotations and KEGG pathway analysis. The results indicated that a total of 429 proteins were identified as the significant DEPs in the resuscitation cells compared with the VBNC cells, including 330 up-regulated and 99 down-regulated DEPs. Meanwhile, the resuscitation cells displayed 25 up-regulated and 36 down-regulated DEPs (total of 61 DEPs) in comparison with the exponential phase cells. The remarkable DEPs including ribosomal proteins, ABC transporters, outer membrane proteins and flagellar proteins. GO annotation showed that the 429 DEPs were classified into 37 GO terms, of which 17 biological process (BP) terms, 9 cellular component (CC) terms and 11 molecular function (MF) terms. The up-regulated proteins presented in all GO terms except two terms of developmental process and reproduction. The 61 DEPs were assigned to 23 GO terms, the up- and down-regulated DEPs were both mainly involved in cellular process, establishment of localization, metabolic process and so on. KEGG pathway analysis revealed that the 429 DEPs were assigned to 35 KEGG pathways, and the pathways of ribosome, glyoxylate and dicarboxylate metabolism were significantly enriched. Moreover, the 61 DEPs located in 26 KEGG pathways, including the significantly enriched KEGG pathways of ABC transporters and two-component system. This study would contribute to a better understanding of the molecular mechanism underlying the resuscitation of the VBNC state of V. parahaemolyticus.

Induction of Viable but Nonculturable Escherichia coli O157:H7 by Low Temperature and Its Resuscitation.

Viable but non-culturable (VBNC) cells are alive bacteria cells, but lose their culturability in conventional culture media, usually escape detection by the plate count method and pose a serious threat to food safety and public health. Therefore, it is urgent to study the VBNC status, and to provide theoretical basis and scientific basis for food processing and safety control caused by pathogenic microorganisms. In this study, Escherichia coli O157:H7 was induced to the VBNC state at two different temperatures (-20°C and 4°C) and its resuscitation and morphological changes under different nutritional conditions were studied. The initial inoculum of 2.1 × 107 CFU/mL E. coli O157:H7 cells were induced into the VBNC state in normal saline, distilled water, LB broth at -20 °C after 176, 160, 80 days, respectively. The results showed that E. coli O157:H7 reserved at -20°C, and LB culture medium were easier to enter VBNC state than others conditions, the cells still had metabolic activity and the cell morphology changed from the typical rod shape to short rod and the cell size decreased. The resuscitate ways including the direct warming resuscitation, gradual warming resuscitation, adding chemical substance resuscitation, and adding nutrients resuscitation were studied. The results showed that the optimal conditions of 5% Tween 80 and 3% Tween 80 acculated the resuscitation of E. coli O157:H7 VBNC state cells induced by low temperature LB medium and low temperature saline. E. coli O157:H7 VBNC state failed from resuscitation when incubating in LB broth, respectively using direct warming and adding nutrients substance. This study provides new insights into induction and resuscitation of VBNC E. coli O157:H7 and offers an approach for investigating the formation mechanism of VBNC foodborne pathogens in food safety.