Viable But Nonculturable State Research Articles

Development of a Direct and Rapid Detection Method for Viable but Non-culturable State of Pediococcus acidilactici.

Pediococcus acidilactici may significantly reduce the pH-value, and thus has different influence, including serving as a probiotic in human microbiota but a spoilage in human food as it could change the flavor. Pediococcus acidilactici is also capable of entering into the viable but non-culturable (VBNC) state causing false negative results of standard culture-based detection method. Thus, development of detection method for VBNC state P. acidilactici is of great significance. In this study, propidium monoazide (PMA) combined with cross priming amplification (CPA) was developed to detect the VBNC cells of P. acidilactici and applied on the detection in different systems. With detection limit of 104 cells/ml, high sensitivity, and 100% specificity, PMA-CPA can successfully detect VBNC cells of P. acidilactici and be applied in with high robustness.

Evaluation of antibacterial activities of silver nanoparticles on culturability and cell viability of Escherichia coli

Nanoparticles released into the environment are attracting increasing concern because of their potential toxic effects. Conventional methods for assessing the toxicity of nanoparticles are usually confined to cultivable cells, but not applicable to viable but non-culturable (VBNC) cells. However, it remains unknown whether silver nanoparticles (AgNPs), a typical antimicrobial agent, could induce bacteria into a VBNC state in natural environments. In this work, the viability of E. coli, an indicator bacterium widely used for assessing the antibacterial activity of AgNPs, was examined through coupling plate counting, fluorescence staining and adenosine triphosphate (ATP) production. AgNPs were found to have a considerable antibacterial ability, which resulted in less than 0.0004% of culturable cells on plates. However, more than 80% of the cells still maintained their cell membrane integrity under the stress of 80 mg/L AgNPs. Meanwhile, the residue of ATP production (0.6%) was 1500 times higher than that of the culturable cells (< 0.0004%). These results clearly demonstrate that when exposed to AgNPs, most of cells fell into a VBNC state, instead of dying. Environmental factors, e.g., Cl− and illumination, which could change the dissolution, hydrophilicity and zeta potential of AgNPs, eventually influenced the culturability of E. coli. Inhibition of dissolved Ag+ and reactive oxygen species was found to facilitate the mitigation of the strain into a VBNC state. Our findings suggest the necessity of re-evaluating the environmental effects and antibacterial activities of AgNPs.

Induction of a Viable but Nonculturable State, Thermal and Sanitizer Tolerance, and Gene Expression Correlation with Desiccation-Adapted Biofilm and Planktonic Salmonella in Powdered Infant Formula

This study was conducted to investigate the effects of the physiological state, desiccation adaptation, and storage of powdered infant formula on Salmonella cell survival and expression of desiccation stress-related genes. Powdered infant formula was inoculated with Salmonella Typhimurium in the biofilm state on beads and in the planktonic state on nitrocellulose filters and stored at 25°C for up to 270 days. Both 5-cyano-2,3-ditoyl tetrazolium chloride flow cytometry and xylose lysine deoxycholate agar plate counts revealed that biofilm-forming Salmonella cells tended to enter the viable but nonculturable (VBNC) state (P < 0.05). The population reduction of all desiccation-adapted Salmonella Typhimurium decreased significantly in both physiological states after exposure to mild heat (60°C) compared with nonadapted control cells (P < 0.05). Salmonella cells were cross-protected from heat in both physiological states, but cross-protection against hydrogen peroxide was induced for only planktonic Salmonella cells. The reverse transcription quantitative PCR results revealed that the rpoS gene in biofilm Salmonella cells on all desiccation adaption days and in planktonic Salmonella cells on day 7 of dry storage was significantly upregulated (P < 0.05). The rpoE, grpE, and invA genes in Salmonella cells in both physiological states were significantly down-regulated (P < 0.05). Physiological state and storage time might affect expression of these genes. Prior exposure to adverse conditions, including low water activity, and the physiological state impacted Salmonella survival, and its ability to enter the VBNC state and gene expression.

Survival of viable but nonculturable Cronobacter sakazakii in macrophages contributes to infections

Cronobacter sakazakii (C. sakazakii), a pathogen that exists in dry and low-moisture environments, such as powder infant formula (PIF), can enter a viable but nonculturable (VBNC) state under harsh conditions, which enables it to escape traditional detection methods and thus poses a potential public health risk. This study aimed at assessing the virulent nature of VBNC C. sakazakii. Our results showed that VBNC C. sakazakii induced intestinal inflammation in neonatal rats. However, the degree of inflammation was significantly lower than that of culturable bacteria due to decreasing endotoxin production, motility, adhesion, and invasion ability in the VBNC state. From the perspective of bacterial translocation, the numbers of C. sakazakii in the blood, liver, and spleen of rats treated with VBNC cells were in the same order of magnitude as those treated with its culturable counterpart and may lead to the same degree of bacteremia. According to the macrophage survival assays, the survival rate of VBNC C. sakazakii within macrophages was 4.7 times higher than that of culturable cells. Based on these findings, we hypothesize that VBNC C. sakazakii evaded the host immune defense system, penetrated the tissue barrier, and translocated to the bloodstream, liver, and spleen through macrophages. Thus, our study reveals that VBNC C. sakazakii could be a potential risk for infants’ health.

Coupled DNA-labeling and sequencing approach enables the detection of viable-but-non-culturable Vibrio spp. in irrigation water sources in the Chesapeake Bay watershed

Nontraditional irrigation water sources (e.g., recycled water, brackish water) may harbor human pathogens, including Vibrio spp., that could be present in a viable-but-nonculturable (VBNC) state, stymieing current culture-based detection methods. To overcome this challenge, we coupled 5-bromo-2′-deoxyuridine (BrdU) labeling, enrichment techniques, and 16S rRNA sequencing to identify metabolically-active Vibrio spp. in nontraditional irrigation water (recycled water, pond water, non-tidal freshwater, and tidal brackish water). Our coupled BrdU-labeling and sequencing approach revealed the presence of metabolically-active Vibrio spp. at all sampling sites. Whereas, the culture-based method only detected vibrios at three of the four sites. We observed the presence of V. cholerae, V. vulnificus, and V. parahaemolyticus using both methods, while V. aesturianus and V. shilonii were detected only through our labeling/sequencing approach. Multiple other pathogens of concern to human health were also identified through our labeling/sequencing approach including P. shigelloides, B. cereus and E. cloacae. Most importantly, 16S rRNA sequencing of BrdU-labeled samples resulted in Vibrio spp. detection even when our culture-based methods resulted in negative detection. This suggests that our novel approach can effectively detect metabolically-active Vibrio spp. that may have been present in a VBNC state, refining our understanding of the prevalence of vibrios in nontraditional irrigation waters.

Rapid determination of viable but non-culturable Campylobacter jejuni in food products by loop-mediated isothermal amplification coupling propidium monoazide treatment

Campylobacter is the leading cause of foodborne human diarrhea worldwide. This microbe in the viable but non-culturable (VBNC) state can evade detection by routinely used culture-based methods and remain viable for extended periods of time. Bacteria in this dormancy state can resume their metabolic activity and virulence by resuscitation under favorable conditions, and subsequently cause infections. In this study, an assay combining loop-mediated isothermal amplification (LAMP) and propidium monoazide (PMA) treatment was developed for the detection and quantification of VBNC C. jejuni in agri-foods. PMA-qLAMP targeting the hipO gene demonstrated 100% high specificity to C. jejuni. A linear detection of C. jejuni was achieved between 8.77 × 102 and 8.77 × 07 CFU/mL with a coefficient of determination (R2) of 0.9956, indicating a good quantitative capacity. C. jejuni was effectively induced into the VBNC state by osmotic stress (i.e., 7% NaCl, w/v) over 48 h. VBNC C. jejuni cells were spiked into three representative food products and determined by PMA-qLAMP coupled with plating assay. The detection limits of PMA-qLAMP were 1.58 × 102 CFU/mL in milk, 3.78 × 102 CFU/g in chicken breast meat, and 4.33 × 102 CFU/g in romaine lettuce. PMA-qLAMP demonstrated rapid (25–40 min), specific (100% inclusivity and 100% exclusivity) and sensitive (~102 CFU/mL) determination of VBNC C. jejuni. This method can be applied in the agri-food industry to decrease the risks related to the consumption of contaminated agri-foods with pathogenic bacteria in the VBNC state and reduce the burden of C. jejuni infections to public health.

Induction and Resuscitation of Viable but Nonculturable Corynebacterium diphtheriae.

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.

Pathogenic and Virulence Factor Detection on Viable but Non-culturable Methicillin-Resistant Staphylococcus aureus.

Food safety and foodborne infections and diseases have been a leading hotspot in public health, and methicillin-resistant Staphylococcus aureus (MRSA) has been recently documented to be an important foodborne pathogen, in addition to its recognition to be a leading clinical pathogen for some decades. Standard identification for MRSA has been commonly performed in both clinical settings and food routine detection; however, most of such so-called “standards,” “guidelines,” or “gold standards” are incapable of detecting viable but non-culturable (VBNC) cells. In this study, two major types of staphylococcal food poisoning (SFP), staphylococcal enterotoxins A (sea) and staphylococcal enterotoxins B (seb), as well as the panton-valentine leucocidin (pvl) genes, were selected to develop a cross-priming amplification (CPA) method. Limit of detection (LOD) of CPA for sea, seb, and pvl was 75, 107.5, and 85 ng/μl, indicating that the analytical sensitivity of CPA is significantly higher than that of conventional PCR. In addition, a rapid VBNC cells detection method, designated as PMA-CPA, was developed and further applied. PMA-CPA showed significant advantages when compared with PCR assays, in terms of rapidity, sensitivity, specificity, and accuracy. Compared with conventional VBNC confirmation methods, the PMA-CPA showed 100% accordance, which had demonstrated that the PMA-CPA assays were capable of detecting different toxins in MRSA in VBNC state. In conclusion, three CPA assays were developed on three important toxins for MRSA, and in combination with PMA, the PMA-CPA assay was capable of detecting virulent gene expression in MRSA in the VBNC state. Also, the above assays were further applied to real samples. As concluded, the PMA-CPA assay developed in this study was capable of detecting MRSA toxins in the VBNC state, representing first time the detection of toxins in the VBNC state.

High concentration of sodium chloride could induce the viable and culturable states of Escherichia coli O157:H7 and Salmonella enterica serovar Enteritidis.

In the present study, Escherichia coli O157:H7 and Salmonella enterica serovar Enteritidis were transferred into Luria-Bertani medium without NaCl (LBWS) and adjusted to various pHs (4, 5, 6 and 7) with lactic acid containing 0·75, 5, 10 and 30% NaCl, and stored at 25°C until the bacterial populations reached below detectable levels on tryptic soy agar (TSA). Although E.coli O157:H7 and S.Enteritidis did not grow on TSA when incubated in LBWS with 30% NaCl for 35 and 7days, more than 60 and 70% of the bacterial cells were shown to be viable via fluorescent staining with SYTO9 and propidium iodide (PI), respectively, suggesting that a number of cells could be induced into the viable but nonculturable (VBNC) state. These bacteria that were induced into a VBNC state were transferred to a newly prepared tryptic soy broth (TSB) and then incubated at 37°C for several days. After more than 7days, E.coli O157:H7 and S.Enteritidis regained their culturability. We, therefore, suggest that E.coli O157:H7 and S.Enteritidis entered the VBNC state under the adverse condition of higher salt concentrations and were revived when these conditions were reversed.

Survival of escherichia coli in Water Microcosm Study and Rethinking its Use as Indicator

We investigated the fate of Escherichia coli in natural waters, addressing survival, viable but non-culturable (VBNC) state, changes in phenotype and genomic diversification under laboratory microcosm environment. Five E. coli isolates (three marine, one clinical and one laboratory strain) were inoculated in microcosms of sterile distilled water, as well as, water collected from river, pond, sea, and estuary. A viable count was measured every week for up to 33 weeks. Strains which undergone VBNC state in microcosms were resuscitated. The revived isolates were tested to determine changes in antibiotic susceptibility, thermotolerance and genetic fingerprinting. We found extended persistence of five E. coli strains in water microcosms at room temperature for durations ranging from four to 33 weeks. Even in distilled water, some isolates persisted up to seven weeks; proving E. coli have excellent tenacity to survive. At VBNC state in microcosms, bacteria were small coccoid in shape and formed biofilms in most cases. Resuscitated laboratory strains showed changes in antibiotic susceptibility and thermotolerance compared to the original counterpart. However, the revived marine isolates showed very little to no difference in those tests. Resuscitation media could not revived the pathogenic isolates. The REP- and BOX-PCR genomic fingerprinting also showed some genetic diversification among resuscitated strains during the extended survival and VBNC state. The overall observation supports for the E. coli to survive in natural waters and to enter into VBNC state. It ultimately proves that their presence in water does not necessarily indicate faecal contamination but still beyond acceptance.

Direct Detection of Viable but Non-culturable (VBNC) Salmonella in Real Food System by a Rapid and Accurate PMA-CPA Technique.

Salmonella enterica is a typical foodborne pathogen with multiple toxic effects, including invasiveness, endotoxins, and enterotoxins. Viable but nonculturable (VBNC) is a type of dormant form preserving the vitality of microorganisms, but it cannot be cultured by traditional laboratory techniques. The aim of this study is to develop a propidium monoazide-crossing priming amplification (PMA-CPA) method that can successfully detect S. enterica rapidly with high sensitivity and can identify VBNC cells in food samples. Five primers (4s, 5a, 2a/1s, 2a, and 3a) were specially designed for recognizing the specific invA gene. The specificity of the CPA assay was tested by 20 different bacterial strains, including 2 standard S. enterica and 18 non-S. enterica bacteria strains covering Gram-negative and Gram-positive isolates. Except for the two standard S. enterica ATCC14028 and ATCC29629, all strains showed negative results. Moreover, PMA-CPA can detect the VBNC cells both in pure culture and three types of food samples with significant color change. In conclusion, the PMA-CPA assay was successfully applied on detecting S. enterica in VBNC state from food samples.

High pressure processing combined with selected hurdles: Enhancement in the inactivation of vegetative microorganisms.

High pressure processing (HPP) as a nonthermal processing (NTP) technology can ensure microbial safety to some extent without compromising food quality. However, for vegetative microorganisms, the existence of pressure-resistant subpopulations, the revival of sublethal injury (SLI) state cells, and the resuscitation of viable but nonculturable (VBNC) state cells may constitute potential food safety risks and pose challenges for the further development of HPP application. HPP combined with selected hurdles, such as moderately elevated or low temperature, low pH, natural antimicrobials (bacteriocin, lactate, reuterin, endolysin, lactoferrin, lactoperoxidase system, chitosan, essential oils), or other NTP (CO2 , UV-TiO2 photocatalysis, ultrasound, pulsed electric field, ultrafiltration), have been highlighted as feasible alternatives to enhance microbial inactivation (synergistic or additive effect). These combinations can effectively eliminate the pressure-resistant subpopulation, reduce the population of SLI or VBNC state cells and inhibit their revival or resuscitation. This review provides an updated overview of the microbial inactivation by the combination of HPP and selected hurdles and restructures the possible inactivation mechanisms.

How to Evaluate Non-Growing Cells-Current Strategies for Determining Antimicrobial Resistance of VBNC Bacteria.

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.

The diagnostic tools for viable but nonculturable pathogens in the food industry: Current status and future prospects.

Viable but nonculturable (VBNC) microorganisms have been recognized as pathogenic contaminants in foods and environments. The failure of VBNC cells to form the visible colonies hinders the ability to use conventional media for their detection. Efficient and rapid detection of pathogens in the VBNC state is a prerequisite to ensure the food safety and public health. Despite their nonculturability, VBNC cells have distinct characteristics, such as morphology, metabolism, chemical composition, and gene and protein expression, that have been used as the basis for the development of abundant diagnostic tools. This review covers the current status and advances in various approaches for examining microorganisms in the VBNC state, including but not limited to the methodological aspects, advantages, and drawbacks of each technique. Existing methods, such as direct viable count, SYTO/PI dual staining, and propidium monoazide quantitative polymerase chain reaction (PCR), as well as some techniques with potential to be applied in the future, such as digital PCR, enhanced-surface Raman spectroscopy, and impedance-based techniques, are summarized in depth. Finally, future prospects for the one-step detection of VBNC bacteria are proposed and discussed. We believe that this review can provide more optional methods for researchers and promote the development of rapid, accurate detecting methods, and for inspectors, the diagnostic tools can provide data to undertake risk analysis of VBNC cells.

Detection and Potential Virulence of Viable but Non-Culturable (VBNC) Listeria monocytogenes: A Review.

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

Bacterial dormancy: A subpopulation of viable but non-culturable cells demonstrates better fitness for revival.

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.

First Report on the Rapid Detection and Identification of Methicillin-Resistant Staphylococcus aureus (MRSA) in Viable but Non-culturable (VBNC) Under Food Storage Conditions.

Formation of viable but non-culturable (VBNC) status in methicillin-resistant Staphylococcus aureus (MRSA) has never been reported, and it poses a significant concern for food safety. Thus, this study aimed to firstly develop a rapid, cost-effective, and efficient testing method to detect and differentiate MRSA strains in the VBNC state and further apply this in real food samples. Two targets were selected for detection of MRSA and toxin, and rapid isothermal amplification detection assays were developed based on cross-priming amplification methodology. VBNC formation was performed for MRSA strain in both pure culture and in artificially contaminated samples, then propidium monoazide (PMA) treatment was further conducted. Development, optimization, and evaluation of PMA-crossing priming amplification (CPA) were further performed on detection of MRSA in the VBNC state. Finally, application of PMA-CPA was further applied for detection on MRSA in the VBNC state in contaminated food samples. As concluded in this study, formation of the VBNC state in MRSA strains has been verified, then two PMA-CPA assays have been developed and applied to detect MRSA in the VBNC state from pure culture and food samples.

Detection of Viable but Non-Culturable (Vbnc) Escherichia Coli from Beef Liver Using Pma-PCR Assay

Escherichia coli, the most important foodborne pathogen worldwide, can enter a dormant state called viable but nonculturable (VBNC) state under various stress conditions. Hence, it escapes detection by traditional culturing methods. The present study used Propidium monoazide (PMA)-Polymerase chain reaction (PCR) assay in order to verify whether E. coli, especially O157 strains, converted to the VBNC state under the influence of cool-warm and freeze-thaw stresses. 120 beef liver samples were collected from local markets. The samples that were positive to pathogenic E. coli were treated with cooling (4 °C)-warm and freezing (-18 °C)-thaw cycles to induce the VBNC state. Samples that demonstrated the inability to form colonies on EMB agar were tested by PMA-PCR assays to confirm the existence of VBNC E. coli. 9 samples (7.5 %) of 120 beef liver samples were positive to pathogenic E. coli by microbiological tests. Cool-warm and freeze-thaw stresses on the 9 samples led to the inability of 3 of them to grow on EMB agar plates. By using PMA-PCR assay, we found that 4 °C cool-warm cycle induced E. coli into VBNC state at a higher percentage ratio than -18 °C freeze-thaw cycle. Even though further experiments are still required, in rare cases, at -18 °C, E. coli VBNC may lose expression of genes responsible for viability and metabolic activity. Additionally, our study evaluated the PMA-PCR assay as a rapid and accurate method to verify the existence of VBNC-associated foodborne E. coli O157.

The effects of the recombinant YeaZ of Vibrio harveyi on the resuscitation and growth of soil bacteria in extreme soil environment.

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.

Laboratory Protocols for Isolation and Identification of Toxigenic Strains of Vibrio cholerae: A Review

Vibrio cholerae is a pathogenic bacteria that accounts for diarrheal illness which can be life-threatening in lack of treatment and accounts for great morbidity and mortality. The common mode of transmission includes fecal-oral route transmission mediated through contaminated food and water. The strains 01 and 0139 have been associated with epidemic outbreak of cholera with severe clinical manifestation whereas the infection caused by non O1 and non O139 strains are recognized as non-epidemic cholera characterized by mild-severe diarrheal syndrome without epidemic potential. Laboratory diagnosis through Culture and determining Antimicrobial susceptibility tests are recommended for diagnosis of cholera in all clinical laboratory settings. The simple conventional laboratory diagnosis through culture should be effective for diagnosis of cholera in the resource (PCR) limited laboratories. However, Vibrio cholerae in VBNC (Viable but non-culturable) state can only be detected through molecular methods. Therefore, molecular methods need to be extended in all epidemiological laboratories for preventing misidentification of Vibrio cholerae especially from environmental samples. Epidemic outbreak of cholera has explained the need of immediate preparedness with preventive, diagnosis and curative improvement in all health sectors. Therefore, immediate health and sanitation related awareness are necessary for prompt control of the disease before it leads community to global burden.