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

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.

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Evaluation of Propidium Monoazide and Quantitative PCR To Quantify Viable Campylobacter jejuni Biofilm and Planktonic Cells in Log Phase and in a Viable but Nonculturable State

Journal of Food Protection ◽

10.4315/0362-028x.jfp-14-583 ◽

2015 ◽

Vol 78 (7) ◽

pp. 1303-1311 ◽

Cited By ~ 9

Author(s):

BRENDA MAGAJNA ◽

HEIDI SCHRAFT

Keyword(s):

Quantitative Pcr ◽

Campylobacter Jejuni ◽

Viable Cell ◽

Propidium Monoazide ◽

Vbnc State ◽

Planktonic Cells ◽

Viable But Nonculturable ◽

Cell Counts ◽

Routine Monitoring ◽

Novel Method

Despite being considered fragile and fastidious, Campylobacter jejuni remains the leading cause of bacterial gastroenteritis in the developed world. C. jejuni survives stresses by forming biofilms or entering a viable but nonculturable (VBNC) state. To investigate the number of viable cells in samples exposed to low nutrient and temperature stress, a novel method, propidium monoazide quantitative PCR (PMAqPCR), was compared with BacLight biovolume analysis and conventional plate counting for the enumeration of C. jejuni–removed biofilm cells and separately grown planktonic cells in late log phase (24 h). There were no significant differences between viable cell counts obtained using PMAqPCR and those from plate counts or BacLight biovolume analyses for each sample, confirming that this method provides results consistent with those from accepted enumeration methods (P > 0.05). To induce a VBNC state, C. jejuni planktonic cells and dislodged and washed biofilm cells were separately incubated in phosphate-buffered saline at 4°C for up to 60 days. Even when cells exposed to stress were provided with enrichment in Bolton broth before plating, treated biofilm cells lost culturability by day 10, whereas their planktonic counterparts remained culturable to day 60. The nonculturable biofilm cells remained viable in high numbers to day 60, and viable cell counts from the PMAqPCR (6.15 log cells per ml) were not significantly different from those obtained using the BacLight assay (6.98 log cells per ml) (P > 0.05), confirming that this novel method is also reliable for cells exposed to stress for extended periods. PMAqPCR shows promise for analysis where C. jejuni exists in biofilms or in the VBNC state. Adopting PMAqPCR in routine monitoring, in conjunction with improved biofilm cell collection methods, will allow for more accurate enumeration of viable and potentially virulent cells, leading to improved sanitation and reduced incidence of infection.

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Induction and Resuscitation of Viable but Nonculturable Corynebacterium diphtheriae

Microorganisms ◽

10.3390/microorganisms9050927 ◽

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.

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Vancomycin Resistance Is Maintained in Enterococci in the Viable but Nonculturable State and after Division Is Resumed

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.3.1154-1156.2003 ◽

2003 ◽

Vol 47 (3) ◽

pp. 1154-1156 ◽

Cited By ~ 38

Author(s):

Maria del Mar Lleò ◽

Barbara Bonato ◽

Caterina Signoretto ◽

Pietro Canepari

Keyword(s):

Vancomycin Resistance ◽

Survival Strategy ◽

Vbnc State ◽

Additional Risk ◽

Viable But Nonculturable ◽

Vancomycin Resistant Enterococci ◽

Nonculturable State ◽

Resistance Trait ◽

Vancomycin Resistant ◽

Viable But Nonculturable State

ABSTRACT Stressed vancomycin-resistant enterococci (VRE) can activate a survival strategy known as the viable but nonculturable (VBNC) state and are able to maintain vancomycin resistance. During restoration of division they continue to express the vancomycin resistance trait. We suggest that VBNC enterococci may constitute further reservoirs of VRE and therefore represent an additional risk for human health.

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Prewashing with Acidified Sodium Chlorite Reduces Pathogenic Bacteria in Lightly Fermented Chinese Cabbage

Journal of Food Protection ◽

10.4315/0362-028x-68.5.999 ◽

2005 ◽

Vol 68 (5) ◽

pp. 999-1004 ◽

Cited By ~ 17

Author(s):

YASUHIRO INATSU ◽

YUTAKA MAEDA ◽

M. L. BARI ◽

SUSUMU KAWASAKI ◽

SHINICHI KAWAMOTO

Keyword(s):

Chinese Cabbage ◽

Salmonella Enteritidis ◽

Pathogenic Bacteria ◽

Control Experiment ◽

Viable Cell ◽

Sodium Chlorite ◽

Distilled Water ◽

Escherichia Coli O157 ◽

Cell Counts ◽

Natural Microflora

Efficacy of prewashing with acidified sodium chlorite (ASC) for the sanitation of lightly fermented Chinese cabbage was evaluated. The population of the natural microflora on the cabbage leaves was reduced about 2.0 log CFU/g just after washing with ASC, a significant reduction compared with the control distilled water wash (P ≤ 0.05). In the control experiment, viable aerobic bacteria increased gradually when incubated at 10°C; however, ASC-washed cabbage maintained a lower microbial concentration. The treatment of Chinese cabbage with ASC reduced the population of artificially inoculated Escherichia coli O157:H7, Salmonella Enteritidis, Staphylococcus aureus, and Listeria monocytogenes by 2.4 log CFU/g. The sanitation efficacy of ASC was 1.6 log CFU/g higher than that of distilled water washing. The viable cell counts of all pathogenic bacteria tested remained constant during 8 days of storage at 10°C for both washing treatments, with the exception of L. monocytogenes, whose viable cell counts increased gradually with time for both treatments. No significant differences in color, odor, taste, and texture in raw leaves were observed after the ASC wash compared with after the distilled water wash. These results indicate that prewashing with ASC could control bacterial growth in lightly fermented Chinese cabbage without changing the product quality.

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Recovery in Embryonated Eggs of Viable but Nonculturable Campylobacter jejuni Cells and Maintenance of Ability To Adhere to HeLa Cells after Resuscitation

Applied and Environmental Microbiology ◽

10.1128/aem.65.11.5154-5157.1999 ◽

1999 ◽

Vol 65 (11) ◽

pp. 5154-5157 ◽

Cited By ~ 63

Author(s):

J. M. Cappelier ◽

J. Minet ◽

C. Magras ◽

R. R. Colwell ◽

M. Federighi

Keyword(s):

Surface Water ◽

Campylobacter Jejuni ◽

Hela Cells ◽

Pathogenic Bacteria ◽

Vbnc State ◽

Adhesion Properties ◽

Viable But Nonculturable

ABSTRACT The existence of a viable but nonculturable (VBNC) state has been described for Campylobacter jejuni as it had been for a number pathogenic bacteria. Three C. jejuni human isolates were suspended in surface water and subsequently entered the VBNC state. After starvation for 30 days, VBNC cells were inoculated in the yolk sacs of embryonated eggs. Culturable cells were detected in a large proportion of the embryonated eggs inoculated with VBNC C. jejuni cells. Recovered cells kept their adhesion properties.

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Development of a Quantitative Competitive PCR Assay for Detection and Quantification of Escherichia coliO157:H7 Cells

Applied and Environmental Microbiology ◽

10.1128/aem.67.7.3291-3294.2001 ◽

2001 ◽

Vol 67 (7) ◽

pp. 3291-3294 ◽

Cited By ~ 26

Author(s):

Wenli Li ◽

Mary Anne Drake

Keyword(s):

Pathogenic Bacteria ◽

Skim Milk ◽

Viable Cell ◽

Quantitative Detection ◽

Competitive Pcr ◽

Pcr Assay ◽

E Coli ◽

Cell Counts ◽

Cell Densities ◽

Detection And Quantification

ABSTRACT A quantitative competitive PCR (QC-PCR) assay was developed to detect and quantify Escherichia coli O157:H7 cells. From 103 to 108 CFU of E. coli O157:H7 cells/ml was quantified in broth or skim milk, and cell densities predicted by QC-PCR were highly related to viable cell counts (r 2 = 0.99 and 0.93, respectively). QC-PCR has potential for quantitative detection of pathogenic bacteria in foods.

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Retention of Virulence in a Viable but Nonculturable Edwardsiella tarda Isolate

Applied and Environmental Microbiology ◽

10.1128/aem.02243-06 ◽

2006 ◽

Vol 73 (4) ◽

pp. 1349-1354 ◽

Cited By ~ 64

Author(s):

Meng Du ◽

Jixiang Chen ◽

Xiaohua Zhang ◽

Aijuan Li ◽

Yun Li ◽

...

Keyword(s):

Morphological Changes ◽

Large Population ◽

Edwardsiella Tarda ◽

Vbnc State ◽

Viable But Nonculturable ◽

Cell Counts ◽

Nonculturable State ◽

Plate Counts ◽

Temperature Upshift ◽

Viable But Nonculturable State

ABSTRACT Edwardsiella tarda is pathogen of fish and other animals. The aim of this study was to investigate the viable but nonculturable (VBNC) state and virulence retention of this bacterium. Edwardsiella tarda CW7 was cultured in sterilized aged seawater at 4ï¿½C. Total cell counts remained constant throughout the 28-day period by acridine orange direct counting, while plate counts declined to undetectable levels (<0.1 CFU/ml) within 28 days by plate counting. The direct viable counts, on the other hand, declined to ca. 109 CFU/ml active cells and remained fairly constant at this level by direct viable counting. These results indicated that a large population of cells existed in a viable but nonculturable state. VBNC E. tarda CW7 could resuscitate in experimental chick embryos and in the presence of nutrition with a temperature upshift. The resuscitative times were 6 days and 8 days, respectively. The morphological changes of VBNC, normal, and resuscitative E. tarda CW7 cells were studied with a scanning electron microscope. The results showed that when the cells entered into the VBNC state, they gradually changed in shape from short rods to coccoid and decreased in size, but the resuscitative cells did not show any obvious differences from the normal cells. The VBNC and the resuscitative E. tarda CW7 cells were intraperitoneally inoculated into turbot separately, and the fish inoculated with the resuscitative cells died within 7 days, which suggested that VBNC E. tarda CW7 might retain pathogenicity.

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Effects of the Essential Oil from Origanum vulgare L. on Survival of Pathogenic Bacteria and Starter Lactic Acid Bacteria in Semihard Cheese Broth and Slurry

Journal of Food Protection ◽

10.4315/0362-028x.jfp-15-172 ◽

2016 ◽

Vol 79 (2) ◽

pp. 246-252 ◽

Cited By ~ 16

Author(s):

GEANY TARGINO de SOUZA ◽

RAYSSA JULLIANE de CARVALHO ◽

JOSSANA PEREIRA de SOUSA ◽

JOSEAN FECHINE TAVARES ◽

DONALD SCHAFFNER ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Lactic Acid ◽

Lactic Acid Bacteria ◽

Essential Oil ◽

Pathogenic Bacteria ◽

Viable Cell ◽

Inhibitory Effects ◽

Origanum Vulgare ◽

Cell Counts ◽

Cheese Slurry

ABSTRACT This study assessed the inhibitory effects of the essential oil from Origanum vulgare L. (OVEO) on Staphylococcus aureus, Listeria monocytogenes, and a mesophilic starter coculture composed of lactic acid bacteria (Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris) in Brazilian coalho cheese systems. The MIC of OVEO was 2.5 μl/ml against both S. aureus and L. monocytogenes and 0.6 μl/ml against the tested starter coculture. In cheese broth containing OVEO at 0.6 μl/ml, no decrease in viable cell counts (VCC) of both pathogenic bacteria was observed, whereas the initial VCC of the starter coculture decreased approximately 1.0 log CFU/ml after 24 h of exposure at 10°C. OVEO at 1.25 and 2.5 μl/ml caused reductions of up to 2.0 and 2.5 log CFU/ml in S. aureus and L. monocytogenes ,respectively, after 24 h of exposure in cheese broth. At these same concentrations, OVEO caused a greater decrease of initial VCC of the starter coculture following 4 h of exposure. Higher concentrations of OVEO were required to decrease the VCC of all target bacteria in semisolid coalho cheese slurry compared with cheese broth. The VCC of Lactococcus spp. in coalho cheese slurry containing OVEO were always lower than those of pathogenic bacteria under the same conditions. These results suggest that the concentrations of OVEO used to control pathogenic bacteria in semihard cheese should be carefully evaluated because of its inhibitory effects on the growth of starter lactic acid cultures used during the production of the product.

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