scholarly journals Analysis of the Resuscitation-Availability of Viable-But-Nonculturable Cells ofVibrio parahaemolyticusupon Exposure to the Refrigerator Temperature

2018 ◽  
Author(s):  
Jae-Hyun Yoon ◽  
Young-Min Bae ◽  
Buom-Young Rye ◽  
Chang-Sun Choi ◽  
Sung-Gwon Moon ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Sea Water ◽  
Ethylenediaminetetraacetic Acid ◽  
Bacterial Cells ◽  
Vbnc State ◽  
Sodium Pyruvate ◽  
Viable But Nonculturable ◽  
Nonculturable Cells ◽  
Pure Cultures ◽  
Refrigerator Temperature

ABSTRACTMajor pathogenic strains ofVibrio parahaemolyticuscan enter into the viable-but-nonculturable (VBNC) state when subjected to environmental conditions commonly encountered during food processing. Especially, VBNC cells can be recovered to the culturable state reversibly by removing the causative stress, expressing higher levels of virulence factors. Therefore, the aim of this study was to determine if VBNCV. parahaemolyticusstrains retain the resuscitation-availability upon eliminating the adverse condition, followed by the enrichment in developed resuscitation-facilitating buffers. Bacterial cells were shown to enter into the VBNC state in artificial sea water (ASW, pH 6) microcosms at 4°C within 70 days. VBNC cells were harvested, inoculated in formulated resuscitation-buffers, and then incubated at 25°C for several days. TSB (pH 8) supplemented with 3% NaCl (TSBA) exhibited the higher resuscitation-availability of VBNC cells. It was also shown that TSBAcontaining 10,000 U/mg/protein catalase, 2% sodium pyruvate, 20 mM MgSO4, 5 mM ethylenediaminetetraacetic acid (EDTA), and cell free supernatants extracted from the pure cultures ofV. parahaemolyticuswas more effective in resuscitating VBNC cells ofV. parahaemolyticus, showing by 7.69-8.91 log10CFU/ml.IMPORTANCEGenerally, higher concentrations (≤40%) of NaCl are used for preserving different sorts of food products from bacterial contaminations. However, it was shown from the present study that strains ofV. parahaemolyticuswere able to persist in maintaining the cellular viability, thereby entering into the VBNC state upon exposure to the refrigerator temperature for 80 days. Hence, the ability of VBNCV. parahaemolyticusto re-enter into the culturable state was examined, using various resuscitation buffers that were formulated in this study. VBNC cells re-gained the culturability successfully when transferred onto the resuscitation-buffer D, and then incubated at 25°C for several days. Resuscitation-facilitating agent D is consisting of antioxidizing agents, mineral, an emulsifier, and cell free supernatants from the actively growing cells ofV. parahaemolyticus. It appeared that such a reversible conversion of VBNC cells to the culturable state would depend on multiple resuscitation-related channels.

scholarly journals Characteristics of viable but nonculturable Vibrio parahaemolyticus induced under extended periods of cold-starvation with various NaCl concentrations

2020 ◽  
Author(s):  
Jae-Hyun Yoon ◽  
Jeong-Eun Hyun ◽  
Sung-Kwon Moon ◽  
Sun-Young Lee
Keyword(s):  
Membrane Potential ◽  
Vibrio Parahaemolyticus ◽  
Sea Water ◽  
Animal Cell ◽  
Hexadecenoic Acid ◽  
Bacterial Cells ◽  
Cellular Composition ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Cell Functions

AbstractThis study was undertaken to examine the induction of VBNC states of Vibrio parahaemolyticus under prolonged cold-starvation with various NaCl concentrations and their responsive characteristics to maintain cell viability. V. parahaemolyticus entered the viable but nonculturable (VBNC) state in artificial sea water at 4°C within 80 day and persisted in the VBNC state for 150 days. During cold-starvation, bacterial cells were used to estimate their cell functions, including cytotoxicity, fatty acid composition, membrane potential, and morphology. Cytotoxic effect of V. parahaemolyticus cells against animal cell lines was decreased to below 50% after 80 days. VBNC V. parahaemolyticus cells showed decreasing levels of palmitic, vaccenic, and hexadecenoic acid on membrane, concomitantly with the formation of empty gaps between the cytoplasmic and outer membrane, in comparison with those of the pure cultures. Starvation at 4°C for 30 days resulted in a high increase in N-phenyl-1-napthylamine intensity within V. parahaemolyticus cells. Membrane potential and cellular composition were strongly affected by increasing NaCl contents of the microcosms after its evolution into the VBNC state. VBNC V. parahaemolyticus cells may undergo selected physiological changes such as the modulation of membrane potential and re-arrangement of cellular composition.

scholarly journals Physiological Characteristics of Viable-but-nonculturableVibrio parahaemolyticusupon Prolonged Exposure to the Refrigerator Temperature

2018 ◽  
Author(s):  
Jae-Hyun Yoon ◽  
Sun-Young Lee
Keyword(s):  
Prolonged Exposure ◽  
Sea Water ◽  
Morphological Changes ◽  
Saturated Fatty Acids ◽  
Cellular Membrane ◽  
Physiological Characteristics ◽  
Time Interval ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Refrigerator Temperature

ABSTRACTAlthough it has been reported thatviable-but-nonculturable (VBNC) cells ofVibrio parahaemolyticuscan be developed by a prolonged duration of cold-starvation there are restricted cellular characteristics available on understanding the exact mechanisms governing the entry of pathogens into the VBNC state. Therefore, this research was aimed at determining the cellular profile of VBNC cells ofV. parahaemolyticusupon exposure to the refrigerator temperature. Strains ofV. parahaemolyticuswere incubated in artificial sea water (ASW) microcosms (pH 6) added with different amounts of NaCl at 4°C until these pathogens entered into such a dormant state. At a regular time-interval, both culturability and viability of these bacteria were enumerated, and then cellular profiling were carried out in terms of cellular membrane permeability, enzymatic activity, hydrophobicity, fatty acid composition, and morphological changes after cells ofV. parahaemolyticusbecame the VBNC state. Three strains ofV. parahaemolyticusused in this study showed that VBNC cells retained the strong virulent properties to Vero and CACO-2 cell lines, re-gained the cytotoxicity even after resuscitation, became permeabilized in terms of the outer membrane, showed lower levels of enzymatic (catalase and glutathione-S-transferase) activities, exerted the increasing hydrophobicity, and then exhibited increasing amounts of saturated fatty acids.IMPORTANCETo the current best knowledge, there are restricted information available on understanding the physiological characterization of viable-but-nonculturable cells. Most previous studies are still making a degree of efforts in discovering the causative effector causing microorganisms to be induced into the VBNC state. Herein, the present study showed that pathogenicV. parahaemolyticuscan enter into the VBNC state when challenged by a certain environmental stress where higher amounts of NaCl combined with acidic pHs was artificially controlled. Importantly, it was indicated that VBNCV. parahaemolyticusmaintained peculiarly different physiological characteristics. Furthermore, this study proposed a novel approach on the transient/stepwise conversion of the bacteria into the VBNC state. Specific alternative tools for measuring and controlling the incidence of VBNC pathogens on food are not established until now. In this aspect, results obtained from this study will used to provide an effective insight in determining physiological properties of viable-but-nonculturableV. parahaemolyticus.

scholarly journals Real-Time Reverse Transcription-PCR for Transcriptional Expression Analysis of Virulence and Housekeeping Genes in Viable but Nonculturable Vibrio parahaemolyticus after Recovery of Culturability

2007 ◽  
Vol 73 (16) ◽  
pp. 5183-5189 ◽  
Author(s):  
François Coutard ◽  
Solen Lozach ◽  
Monique Pommepuy ◽  
Dominique Hervio-Heath
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Vibrio Parahaemolyticus ◽  
Virulence Genes ◽  
Housekeeping Genes ◽  
Clinical Strain ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Reverse Transcription Pcr ◽  
Study Results

ABSTRACT A real-time reverse transcription-PCR method was developed to determine whether the recovery of culturability of viable but nonculturable (VBNC) Vibrio parahaemolyticus induced the expression of virulence genes coding for the thermostable direct hemolysin and for type III secretion system 2 (TTSS2). The culturability of clinical strain Vp5 of V. parahaemolyticus in artificial seawater at 4°C was monitored, and the VBNC state was obtained. One day after entry into the VBNC state, temperature upshifts to 20 and 37°C allowed the recovery of culturability. Standard curves for the relative quantification of expression of the housekeeping genes rpoS, pvsA, fur, and pvuA; the tdh2 gene; and the TTSS2 genes (VPA1354, VPA1346, and VPA1342) were established. The levels of expression of the pvsA and pvuA genes were stable and were used to normalize the levels of expression of the other genes. No transcriptional induction of the selected virulence genes under the temperature conditions used to recover the culturability of the VBNC bacteria was observed. The study results demonstrate that the recovery of culturability of VBNC cells of pathogenic V. parahaemolyticus is restricted to regrowth, without correlation with the induction of virulence gene expression. Disease induction would depend mainly on host-pathogen interactions that allow the expression of the virulence genes. This is the first time that the use of mRNA to detect viable cells was evaluated by computing the half-lives of multiple mRNA species under conditions inducing the VBNC state.

scholarly journals Induction of Escherichia coli and Salmonella typhimurium into the viable but nonculturable state following chlorination of wastewater

2005 ◽  
Vol 3 (3) ◽  
pp. 249-257 ◽  
Author(s):  
James D. Oliver ◽  
Maya Dagher ◽  
Karl Linden
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Stationary Phases ◽  
Health Hazard ◽  
Physiological State ◽  
Treated Wastewater ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Nonculturable Cells ◽  
Nonculturable State

We examined the effects of chlorine disinfection on Escherichia coli and Salmonella typhimurium in secondary-treated wastewater to determine whether such treatment might induce these bacteria into the viable but nonculturable (VBNC) state. In this state, cells lose culturability but retain viability and the potential to revert to the metabolically active and infectious state. To examine the effects of chlorination on cells in different physiological states, cells from the logarithmic and stationary phases, or nutrient starved, or grown in natural wastewater, were studied. Isogenic cells with and without plasmids were also examined. Whereas a mixture of free and combined chlorine, as occurs under typical wastewater disinfection, was found to be rapidly lethal to most cells, regardless of their physiological state or plasmid content, c. 104 of the original 106 cells ml−1 did survive in the VBNC state. While we were not successful in resuscitating these cells to the culturable state, the presence of such nonculturable cells in treated wastewater offers a potential public health hazard.

scholarly journals Inactivation Kinetics ofVibrio parahaemolyticuson Sand Shrimp(Metapenaeus ensis)by Cinnamaldehyde at 4°C

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
DongLai Zhang ◽  
QingLi Dong ◽  
Thomas Ross
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Bile Salts ◽  
Inactivation Kinetics ◽  
Vbnc State ◽  
Minimum Level ◽  
Viable But Nonculturable ◽  
Metapenaeus Ensis ◽  
Cold Environments ◽  
Shrimp Shell ◽  
Inactivation Process

Sand shrimp(Metapenaeus ensis), shrimp shell, and shrimp meat were inoculated with a three-strain cocktail ofVibrio parahaemolyticuswith or without the natural antimicrobial cinnamaldehyde (2.5 mg/ml) and were, then, stored at 4°C for up to 25 days and 18 inactivation curves were obtained.V. parahaemolyticuswere inactivated down to the minimum level of detection (2.48 log CFU/g) on thiosulfate citrate bile salts sucrose agar (TCBS) plates within 7 and 10 days with low and high densities ofV. parahaemolyticusinoculation, 4.5 log CFU/g and 8.2 log CFU/g, respectively. With adding cinnamaldehyde, the inactivation process ofV. parahaemolyticuswith low populations, 4.5 log CFU/g, lasted for only 4 days. Therefore, cinnamaldehyde inactivated cells faster as expected. However, unexpectedly, in shrimp meat cases, cells have much more persistence of over even 25 days before entering the minimum level of detection both with and without cinnamaldehyde treatment. Therefore, a hypothesis was formed that when cells kept in cold environments (4°C) after several days recovered to up to 103–104CFU/g towards the end of the experiments and with starvation (shell and shrimp studies), cells might render a viable but nonculturable (VBNC) state.

scholarly journals Roles of Alkyl Hydroperoxide Reductase Subunit C (AhpC) in Viable but Nonculturable Vibrio parahaemolyticus

2013 ◽  
Vol 79 (12) ◽  
pp. 3734-3743 ◽  
Author(s):  
Hen-Wei Wang ◽  
Chun-Hui Chung ◽  
Tsung-Yong Ma ◽  
Hin-chung Wong
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Parent Strain ◽  
Vbnc State ◽  
Alkyl Hydroperoxide Reductase ◽  
Subunit C ◽  
Viable But Nonculturable ◽  
Content Type ◽  
Alkyl Hydroperoxide ◽  
The Times

ABSTRACTAlkyl hydroperoxide reductase subunit C (AhpC) is the catalytic subunit responsible for the detoxification of reactive oxygen species that form in bacterial cells or are derived from the host; thus, AhpC facilitates the survival of pathogenic bacteria under environmental stresses or during infection. This study investigates the role of AhpC in the induction and maintenance of a viable but nonculturable (VBNC) state inVibrio parahaemolyticus. In this investigation,ahpC1(VPA1683) andahpC2(VP0580) were identified in chromosomes II and I of this pathogen, respectively. Mutants with deletions of these twoahpCgenes and their complementary strains were constructed from the parent strain KX-V231. The growth of these strains was monitored on tryptic soy agar–3% NaCl in the presence of the extrinsic peroxides H2O2andtert-butyl hydroperoxide (t-BOOH) at different incubation temperatures. The results revealed that bothahpCgenes were protective againstt-BOOH, whileahpC1was protective against H2O2. The protective function ofahpC2at 4°C was higher than that ofahpC1. The times required to induce the VBNC state (4.7 weeks) at 4°C in a modified Morita mineral salt solution with 0.5% NaCl and then to maintain the VBNC state (4.7 weeks) in anahpC2mutant and anahpC1 ahpC2double mutant were significantly shorter than those for the parent strain (for induction, 6.2 weeks; for maintenance, 7.8 weeks) and theahpC1mutant (for induction, 6.0 weeks; for maintenance, 8.0 weeks) (P< 0.03). Complementation with anahpC2gene reversed the effects of theahpC2mutation in shortening the times for induction and maintenance of the VBNC state. This investigation identified the different functions of the twoahpCgenes and confirmed the particular role ofahpC2in the VBNC state ofV. parahaemolyticus.

A Mixed Culture Recovery Method Indicates that Enteric Bacteria Do Not Enter the Viable but Nonculturable State

1998 ◽  
Vol 64 (5) ◽  
pp. 1736-1742 ◽  
Author(s):  
Gregg Bogosian ◽  
Patricia J. L. Morris ◽  
Julia P. O’Neil
Keyword(s):  
Mixed Culture ◽  
Enterobacter Aerogenes ◽  
Cell Types ◽  
Enteric Bacteria ◽  
Bacterial Cells ◽  
Recovery Method ◽  
Viable But Nonculturable ◽  
Nonculturable Cells ◽  
Nonculturable State ◽  
Viable But Nonculturable State

ABSTRACT A new method, called the mixed culture recovery (MCR) method, has been developed to determine whether recovery of culturable bacterial cells from a population of largely nonculturable cells is due to resuscitation of the nonculturable cells from a viable but nonculturable state or simply to growth of residual culturable cells. The MCR method addresses this issue in that it involves the mixing of two easily distinguishable strains (e.g., lactose positive and negative) in such a way that large numbers of nonculturable cells of both strains are present together with a small number of culturable cells of only one strain, performing a nutrient addition resuscitation procedure, and then plating the cells to determine whether both cell types are recoverable. In repeated experiments with strains ofEscherichia coli, Klebsiella pneumoniae,Enterococcus faecalis, Enterobacter aerogenes, and Salmonella choleraesuis, only cells of the culturable strain were recovered after application of various resuscitation techniques. These results suggest that the nonculturable cells were dead and that the apparent resuscitation was merely due to the growth of the remaining culturable cells.

Biochemical and Virulence Characterization of Viable but Nonculturable Cells of Vibrio parahaemolyticus

2004 ◽  
Vol 67 (11) ◽  
pp. 2430-2435 ◽  
Author(s):  
HIN-CHUNG WONG ◽  
CHI-TSUNG SHEN ◽  
CHIA-NI CHANG ◽  
YEONG-SHENG LEE ◽  
JAMES D. OLIVER
Keyword(s):  
Fatty Acid ◽  
Vibrio Parahaemolyticus ◽  
Foodborne Pathogen ◽  
Major Change ◽  
Exponential Phase ◽  
Activity Level ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Protective Enzyme

Vibrio parahaemolyticus is a common foodborne pathogen frequently causing outbreaks in summer. Maintenance of virulence by the viable but nonculturable (VBNC) state of this pathogen would allow its threat to human health to persist. This study reports on the change in virulence and concomitant changes in activity of two enzymes and fatty acid profiles when V. parahaemolyticus ST550 entered the VBNC state in the modified Morita mineral salt–0.5% NaCl medium incubated at 4°C. The major change in fatty acid composition occurred in the first week, with a rapid increase in C15:0 fatty acid and saturated/unsaturated ratio while a rapid decrease in C16:1 was observed. The activity level of the inducible protective enzyme superoxide dismutase became undetectable in the VBNC state, whereas that of constitutive glucose-6-phosphate dehydrogenase did not change in either the exponential phase or the VBNC state. Cytotoxicity against HEp-2 cells and a suckling mouse assay showed that virulence was lowered in the VBNC state compared with exponential-phase cells. Longer incubation times were required by the VBNC cells to achieve the same level of virulence as seen in exponential-phase cells. Culturable cells were recovered on selective agar medium from the VBNC cultures injected into suckling mice, probably as the result of in vivo resuscitation. Results of this study add to our understanding of the biochemical and physiological changes that have not been reported when V. parahaemolyticus enters into the VBNC state.

Induction of Viable but Nonculturable Salmonella in Exponentially Grown Cells by Exposure to a Low-Humidity Environment and Their Resuscitation by Catalase

2017 ◽  
Vol 80 (2) ◽  
pp. 288-294 ◽  
Author(s):  
Yuta Morishige ◽  
Atsushi Koike ◽  
Ai Tamura-Ueyama ◽  
Fumio Amano
Keyword(s):  
Salmonella Enteritidis ◽  
Room Temperature ◽  
Vbnc State ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Sodium Pyruvate ◽  
Viable But Nonculturable ◽  
Nutritional Conditions ◽  
Low Humidity ◽  
Humidity Environment ◽  
Subsequent Resuscitation

ABSTRACTSalmonella is a major cause of foodborne disease that sometimes occurs in massive outbreaks around the world. This pathogen is tolerant of low-humidity conditions. We previously described a method for induction of viable but nonculturable (VBNC) Salmonella enterica serovar Enteritidis by treatment with hydrogen peroxide (H2O2) and subsequent resuscitation with 0.3 mM sodium pyruvate. Here, we report a new method for the induction of the VBNC state in Salmonella Enteritidis cells, one involving dehydration. Exposure of Salmonella Enteritidis cells to dehydration stress under poor nutritional conditions (0.9% [wt/vol] NaCl) and 10 to 20% relative humidity at room temperature decreased the presence of culturable population to 0.0067%, but respiratory and glucose uptake active populations were maintained at 0.46 and 1.12%, respectively, meaning that approximately 1% may have entered the VBNC state. Furthermore, these VBNC cells could be resuscitated to acquire culturability by incubation with catalase in M9 minimal medium without glucose in a manner dependent on the dose of catalase but not sodium pyruvate. These results suggest that a low-humidity environment could cause Salmonella Enteritidis cells to enter the VBNC state and the cells could then be resuscitated for growth by treatment with catalase, suggesting a potential risk of Salmonella Enteritidis to survive in low water activity foods in the VBNC state and to start regrowth for foodborne illness.

scholarly journals Association of a d-Alanyl-d-Alanine Carboxypeptidase Gene with the Formation of Aberrantly Shaped Cells during the Induction of Viable but Nonculturable Vibrio parahaemolyticus

2013 ◽  
Vol 79 (23) ◽  
pp. 7305-7312 ◽  
Author(s):  
Wei-cheng Hung ◽  
Wann-Neng Jane ◽  
Hin-chung Wong
Keyword(s):  
Cell Wall ◽  
Vibrio Parahaemolyticus ◽  
Culture Medium ◽  
Culture Media ◽  
Cell Wall Synthesis ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Content Type ◽  
Initial Stage ◽  
Enhanced Expression

ABSTRACTVibrio parahaemolyticusis a halophilic Gram-negative bacterium that causes human gastroenteritis. When the viable but nonculturable (VBNC) state of this bacterium was induced by incubation at 4°C in Morita minimal salt solution containing 0.5% NaCl, the rod-shaped cells became coccoid, and various aberrantly shaped intermediates were formed in the initial stage. This study examined the factors that influence the formation of these aberrantly shaped cells. The proportion of aberrantly shaped cells was not affected in a medium containingd-cycloserine (50 μg/ml) but was lower in a medium containing cephalosporin C (10 μg/ml) than in the control medium without antibiotics. The proportion of aberrantly shaped cells was higher in a culture medium that contained 0.5% NaCl than in culture media containing 1.0 or 1.5% NaCl. The expression of 15 of 17 selected genes associated with cell wall synthesis was enhanced, and the expression of VP2468 (dacB), which encodesd-alanyl-d-alanine carboxypeptidase, was enhanced the most. The proportion of aberrantly shaped cells was significantly lower in thedacBmutant strain than in the parent strain, but the proportion was restored in the presence of the complementarydacBgene. This study suggests that disturbance of the dynamics of cell wall synthesis by enhanced expression of the VP2468 gene is associated with the formation of aberrantly shaped cells in the initial stage of induction of VBNCV. parahaemolyticuscells under specific conditions.

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