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 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 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 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.

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.

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.

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 Induction and Resuscitation of Viable but Nonculturable Corynebacterium diphtheriae

2021 ◽  
Vol 9 (5) ◽  
pp. 927
Author(s):  
Takashi Hamabata ◽  
Mitsutoshi Senoh ◽  
Masaaki Iwaki ◽  
Ayae Nishiyama ◽  
Akihiko Yamamoto ◽  
...  
Keyword(s):  
Low Temperatures ◽  
Diphtheria Toxin ◽  
Pathogenic Bacteria ◽  
Corynebacterium Diphtheriae ◽  
Human Pathogen ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Environmental Survival ◽  
Reverse Transcription Quantitative Pcr ◽  
Morphological Differences

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.

scholarly journals Survival Strategy of Erwinia amylovora against Copper: Induction of the Viable-but-Nonculturable State

2006 ◽  
Vol 72 (5) ◽  
pp. 3482-3488 ◽  
Author(s):  
M�nica Ordax ◽  
Ester Marco-Noales ◽  
Mar�a M. L�pez ◽  
Elena G. Biosca
Keyword(s):  
Pathogenic Bacteria ◽  
Erwinia Amylovora ◽  
Control Plant ◽  
Viable Cell ◽  
Epifluorescence Microscopy ◽  
Survival Strategy ◽  
Vbnc State ◽  
Tetrazolium Chloride ◽  
Viable But Nonculturable ◽  
Cell Counts

ABSTRACT Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu2+ was inoculated with 107 CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper.

scholarly journals Induction of Viable but Nonculturable Escherichia coli O157:H7 in the Phyllosphere of Lettuce: a Food Safety Risk Factor

2011 ◽  
Vol 77 (23) ◽  
pp. 8295-8302 ◽  
Author(s):  
Laura-Dorina Dinu ◽  
Susan Bach
Keyword(s):  
Escherichia Coli ◽  
Food Safety ◽  
Low Temperature ◽  
Cell Density ◽  
Prolonged Storage ◽  
Potential Hazard ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Content Type ◽  
E Coli

ABSTRACTEscherichia coliO157:H7 continues to be an important human pathogen and has been increasingly linked to food-borne illness associated with fresh produce, particularly leafy greens. The aim of this work was to investigate the fate ofE. coliO157:H7 on the phyllosphere of lettuce under low temperature and to evaluate the potential hazard of viable but nonculturable (VBNC) cells induced under such stressful conditions. First, we studied the survival of six bacterial strains following prolonged storage in water at low temperature (4°C) and selected two strains with different nonculturable responses for the construction ofE. coliO157:H7 Tn7gfptransformants in order to quantitatively assess the occurrence of human pathogens on the plant surface. Under a suboptimal growth temperature (16°C), bothE. coliO157:H7 strains maintained culturability on lettuce leaves, but under more stressful conditions (8°C), the bacterial populations evolved toward the VBNC state. The strain-dependent nonculturable response was more evident in the experiments with different inoculum doses (109and 106E. coliO157:H7 bacteria per g of leaf) when strain BRMSID 188 lost culturability after 15 days and strain ATCC 43895 lost culturability within 7 days, regardless of the inoculum dose. However, the number of cells entering the VBNC state in high-cell-density inoculum (approximately 55%) was lower than in low-cell-density inoculum (approximately 70%). We recorded the presence of verotoxin for 3 days in samples that contained a VBNC population of 4 to 5 log10cells but did not detect culturable cells. These findings indicate thatE. coliO157:H7 VBNC cells are induced on lettuce plants, and this may have implications regarding food safety.

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