scholarly journals Survival ofErwinia amylovorain mature apple fruit calyces through the viable but nonculturable (VBNC) state

2009 ◽  
Vol 107 (1) ◽  
pp. 106-116 ◽  
Author(s):  
M. Ordax ◽  
E.G. Biosca ◽  
S.C. Wimalajeewa ◽  
M.M. López ◽  
E. Marco-Noales
Keyword(s):  
Apple Fruit ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Mature Apple

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.

scholarly journals Viable-but-NonculturableListeria monocytogenesandSalmonella entericaSerovar Thompson Induced by Chlorine Stress Remain Infectious

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
pp. e00540-18 ◽  
Author(s):  
Callum J. Highmore ◽  
Jennifer C. Warner ◽  
Steve D. Rothwell ◽  
Sandra A. Wilks ◽  
C. William Keevil
Keyword(s):  
Listeria Monocytogenes ◽  
Caenorhabditis Elegans ◽  
Life Span ◽  
Fresh Produce ◽  
Environmental Stresses ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Content Type ◽  
Food Borne Pathogens ◽  
Food Borne

ABSTRACTThe microbiological safety of fresh produce is monitored almost exclusively by culture-based detection methods. However, bacterial food-borne pathogens are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses such as chlorine, which is commonly used for fresh produce decontamination. Here, complete VBNC induction of green fluorescent protein-taggedListeria monocytogenesandSalmonella entericaserovar Thompson was achieved by exposure to 12 and 3 ppm chlorine, respectively. The pathogens were subjected to chlorine washing following incubation on spinach leaves. Culture data revealed that total viableL. monocytogenesandSalmonellaThompson populations became VBNC by 50 and 100 ppm chlorine, respectively, while enumeration by direct viable counting found that chlorine caused a <1-log reduction in viability. The pathogenicity of chlorine-induced VBNCL. monocytogenesandSalmonellaThompson was assessed by usingCaenorhabditis elegans. Ingestion of VBNC pathogens byC. elegansresulted in a significant life span reduction (P= 0.0064 andP< 0.0001), and no significant difference between the life span reductions caused by the VBNC and culturableL. monocytogenestreatments was observed.L. monocytogeneswas visualized beyond the nematode intestinal lumen, indicating resuscitation and cell invasion. These data emphasize the risk that VBNC food-borne pathogens could pose to public health should they continue to go undetected.IMPORTANCEMany bacteria are known to enter a viable-but-nonculturable (VBNC) state in response to environmental stresses. VBNC cells cannot be detected by standard laboratory culture techniques, presenting a problem for the food industry, which uses these techniques to detect pathogen contaminants. This study found that chlorine, a sanitizer commonly used for fresh produce, induces a VBNC state in the food-borne pathogensListeria monocytogenesandSalmonella enterica. It was also found that chlorine is ineffective at killing total populations of the pathogens. A life span reduction was observed inCaenorhabditis elegansthat ingested these VBNC pathogens, with VBNCL. monocytogenesas infectious as its culturable counterpart. These data show that VBNC food-borne pathogens can both be generated and avoid detection by industrial practices while potentially retaining the ability to cause disease.

Infection frequency of mature apple fruit with Erwinia amylovora deposited on pedicels and its survival in the fruit stored at low temperature

2005 ◽  
Vol 71 (4) ◽  
pp. 296-301 ◽  
Author(s):  
Takanori Tsukamoto ◽  
Koji Azegami ◽  
Takayuki Matsuura ◽  
Tatsuji Ohara ◽  
Yasuhiro Inoue ◽  
...  
Keyword(s):  
Low Temperature ◽  
Erwinia Amylovora ◽  
Apple Fruit ◽  
Infection Frequency ◽  
Mature Apple

scholarly journals mRNA Detection by Reverse Transcription-PCR for Monitoring Viability over Time in an Enterococcus faecalis Viable but Nonculturable Population Maintained in a Laboratory Microcosm

2000 ◽  
Vol 66 (10) ◽  
pp. 4564-4567 ◽  
Author(s):  
Maria del Mar Lleò ◽  
Sabrina Pierobon ◽  
Maria Carla Tafi ◽  
Caterina Signoretto ◽  
Pietro Canepari
Keyword(s):  
Enterococcus Faecalis ◽  
Reverse Transcription ◽  
Microbiological Quality ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Reverse Transcription Pcr ◽  
Pathogenic Factors ◽  
Laboratory Microcosm ◽  
Or Genes

ABSTRACT The viable but nonculturable (VBNC) state is a survival strategy adopted by bacteria when they are exposed to hostile environmental conditions. It has been shown that VBNC forms of bacteria are no longer capable of growing on conventional bacteriological media but conserve pathogenic factors and/or genes. It is thus necessary to develop methods capable of detecting nonculturable bacteria and of establishing their viability when the microbiological quality of environments is monitored. In this study we demonstrated that a gene was expressed during the VBNC state in a low-nutrient-concentration microcosm through detection of Enterococcus faecalis pbp5 mRNA by reverse transcription-PCR over a 3-month period. The presence of mRNA correlated with metabolic activity and resuscitation capability, indicating the viability of the VBNC cells.

scholarly journals A Proteomic Signature of Dormancy in the Actinobacterium Micrococcus luteus

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
Sujina Mali ◽  
Morgan Mitchell ◽  
Spencer Havis ◽  
Abiodun Bodunrin ◽  
Jonathan Rangel ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
High Performance ◽  
Micrococcus Luteus ◽  
Treponema Pallidum ◽  
Gc Content ◽  
Antibiotic Tolerance ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Content Type ◽  
Regulatory Processes

ABSTRACT Dormancy is a protective state in which diverse bacteria, including Mycobacterium tuberculosis, Staphylococcus aureus, Treponema pallidum (syphilis), and Borrelia burgdorferi (Lyme disease), curtail metabolic activity to survive external stresses, including antibiotics. Evidence suggests dormancy consists of a continuum of interrelated states, including viable but nonculturable (VBNC) and persistence states. VBNC and persistence contribute to antibiotic tolerance, reemergence from latent infections, and even quorum sensing and biofilm formation. Previous studies indicate that the protein mechanisms regulating persistence and VBNC states are not well understood. We have queried the VBNC state of Micrococcus luteus NCTC 2665 (MI-2665) by quantitative proteomics combining gel electrophoresis, high-performance liquid chromatography, and tandem mass spectrometry to elucidate some of these mechanisms. MI-2665 is a nonpathogenic actinobacterium containing a small (2.5-Mb), high-GC-content genome which exhibits a well-defined VBNC state induced by nutrient deprivation. The MI-2665 VBNC state demonstrated a loss of protein diversity accompanied by increased levels of 18 proteins that are conserved across actinobacteria, 14 of which have not been previously identified in VNBC. These proteins implicate an anaplerotic strategy in the transition to VBNC, including changes in the glyoxylate shunt, redox and amino acid metabolism, and ribosomal regulatory processes. Our data suggest that MI-2665 is a viable model for dissecting the protein mechanisms underlying the VBNC stress response and provide the first protein-level signature of this state. We expect that this protein signature will enable future studies deciphering the protein mechanisms of dormancy and identify novel therapeutic strategies effective against antibiotic-tolerant bacterial infections. IMPORTANCE Dormancy is a protective state enabling bacteria to survive antibiotics, starvation, and the immune system. Dormancy is comprised of different states, including persistent and viable but nonculturable (VBNC) states that contribute to the spread of bacterial infections. Therefore, it is imperative to identify how bacteria utilize these different dormancy states to survive antibiotic treatment. The objective of our research is to eliminate dormancy as a route to antibiotic tolerance by understanding the proteins that control dormancy in Micrococcus luteus NCTC 2665. This bacterium has unique advantages for studying dormancy, including a small genome and a well-defined and reproducible VBNC state. Our experiments implicate four previously identified and 14 novel proteins upregulated in VBNC that may regulate this critical survival mechanism.

scholarly journals New Insights into the Formation of Viable but Nonculturable Escherichia coli O157:H7 Induced by High-Pressure CO 2

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Feng Zhao ◽  
Yongtao Wang ◽  
Haoran An ◽  
Yanling Hao ◽  
Xiaosong Hu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Pressure ◽  
Cell Division ◽  
State Formation ◽  
Metabolic Activity ◽  
Rna Seq ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
E Coli ◽  
State Entry

ABSTRACT The formation of viable but nonculturable (VBNC) Escherichia coli O157:H7 induced by high-pressure CO 2 (HPCD) was investigated using RNA sequencing (RNA-Seq) transcriptomics and isobaric tag for relative and absolute quantitation (iTRAQ) proteomic methods. The analyses revealed that 97 genes and 56 proteins were significantly changed upon VBNC state entry. Genes and proteins related to membrane transport, central metabolisms, DNA replication, and cell division were mainly downregulated in the VBNC cells. This caused low metabolic activity concurrently with a division arrest in cells, which may be related to VBNC state formation. Cell division repression and outer membrane overexpression were confirmed to be involved in VBNC state formation by homologous expression of z2046 coding for transcriptional repressor and ompF encoding outer membrane protein F. Upon VBNC state entry, pyruvate catabolism in the cells shifted from the tricarboxylic acid (TCA) cycle toward the fermentative route; this led to a low level of ATP. Combating the low energy supply, ATP production in the VBNC cells was compensated by the degradation of l -serine and l -threonine, the increased AMP generation, and the enhanced electron transfer. Furthermore, tolerance of the cells with respect to HPCD-induced acid, oxidation, and high CO 2 stresses was enhanced by promoting the production of ammonia and NADPH and by reducing CO 2 production during VBNC state formation. Most genes and proteins related to pathogenicity were downregulated in the VBNC cells. This would decrease the cell pathogenicity, which was confirmed by adhesion assays. In conclusion, the decreased metabolic activity, repressed cell division, and enhanced survival ability in E. coli O157:H7 might cause HPCD-induced VBNC state formation. IMPORTANCE Escherichia coli O157:H7 has been implicated in large foodborne outbreaks worldwide. It has been reported that the presence of as few as 10 cells in food could cause illness. However, the presence of only 0.73 to 1.5 culturable E. coli O157:H7 cells in salted salmon roe caused infection in Japan. Investigators found that E. coli O157:H7 in the viable but nonculturable (VBNC) state was the source of the outbreak. So far, formation mechanisms of VBNC state are not well known. In a previous study, we demonstrated that high-pressure CO 2 (HPCD) could induce the transition of E. coli O157:H7 into the VBNC state. In this study, we used RNA-Seq transcriptomic analysis combined with the iTRAQ proteomic method to investigate the formation of VBNC E. coli O157:H7 induced by HPCD treatment. Finally, we proposed a putative formation mechanism of the VBNC cells induced by HPCD, which may provide a theoretical foundation for controlling the VBNC state entry induced by HPCD treatment.

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