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

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.02776-06 ◽

2007 ◽

Vol 73 (16) ◽

pp. 5183-5189 ◽

Cited By ~ 34

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.

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Determination of Bovine Rotavirus G and P Serotypes in Italy by PCR

Journal of Clinical Microbiology ◽

10.1128/jcm.37.12.3879-3882.1999 ◽

1999 ◽

Vol 37 (12) ◽

pp. 3879-3882 ◽

Cited By ~ 37

Author(s):

E. Falcone ◽

M. Tarantino ◽

L. Di Trani ◽

P. Cordioli ◽

A. Lavazza ◽

...

Keyword(s):

Reverse Transcription ◽

Clinical Signs ◽

Specific Primers ◽

Bovine Rotavirus ◽

Reverse Transcription Pcr ◽

Rotavirus Vaccines ◽

Intestinal Contents ◽

Group A

Determination of the G and P serotypes of group A bovine rotaviruses from 149 samples of feces or intestinal contents collected from calves showing clinical signs of neonatal diarrhea was performed by a nested reverse transcription-PCR typing assay. The G6 serotype was the most prevalent, accounting for viruses in 55.7% of the samples; viruses of the G10 and G8 serotypes were found in 34.9 and 4.7% of the samples, respectively. The virus in one sample (0.7%) was not classified due to concomitant infection with G6 and G8 strains, whereas viruses in six samples (4.0%) could not be characterized with any of the three G serotype-specific primers selected for the present study. When examined for their P-serotype specificities, viruses in 55 and 42.3% of the samples were characterized as P[11] and P[5], respectively, no P[1] serotype was identified, and viruses in 2.7% of the samples could not be classified due to multiple reactivity with both P[5]- and P[11]-specific primers. Various combinations of G and P serotypes were observed, the most frequent being G6,P[5] (38.3%), G10,P[11] (31.5%), and G6,P[11] (15.4%). The results of the present study, while contributing to a better understanding of the epidemiology of bovine rotaviruses in Italy, address the relevance of serotype specificity with regard to the constancy of the quality of bovine rotavirus vaccines under different field conditions.

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Persistence of Enterococcus faecalis in Aquatic Environments via Surface Interactions with Copepods

Applied and Environmental Microbiology ◽

10.1128/aem.71.5.2756-2761.2005 ◽

2005 ◽

Vol 71 (5) ◽

pp. 2756-2761 ◽

Cited By ~ 31

Author(s):

Caterina Signoretto ◽

Gloria Burlacchini ◽

Carla Pruzzo ◽

Pietro Canepari

Keyword(s):

Enterococcus Faecalis ◽

Microbiological Quality ◽

Lipoteichoic Acid ◽

Survival Strategies ◽

Natural Environments ◽

Human Pathogens ◽

Vbnc State ◽

Adhesive Properties ◽

Main Component

ABSTRACT Several human pathogens and fecal-pollution indicators may persist as viable organisms in natural environments, owing to their ability to activate different types of survival strategies. These strategies include adhesion on both abiotic and biotic surfaces and the entrance to the so-called viable but nonculturable (VBNC) state. In an 18-month survey for the detection of enterococci in both lake water and seawater, C. Signoretto et al. (Appl. Environ. Microbiol. 70:6892-6896, 2004) have shown that Enterococcus faecalis was detected mostly bound to plankton and in the VBNC state. In the present study, we show that in vitro adhesion of E. faecalis to copepods accelerated the entry of cells into the VBNC state relative to that of planktonic bacteria. VBNC E. faecalis cells maintained their adhesive properties to copepods and chitin (the main component of the copepod carapace), though to a reduced extent in comparison with growing cells. Sugar competition experiments showed interference with adhesion to both copepods and chitin by GlcNAc and only to copepods by d-mannose. Four enterococcal cell wall proteins present in both growing and VBNC cells and lipoteichoic acid were shown to be capable of binding chitin. The results indicate that copepods may represent an additional environmental reservoir of enterococci, thus suggesting the advisability of redesigning the protocols currently used for microbial detection during the evaluation of the microbiological quality of environmental samples.

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The Viable but Nonculturable State and Starvation Are Different Stress Responses of Enterococcus faecalis, as Determined by Proteome Analysis

Journal of Bacteriology ◽

10.1128/jb.184.23.6739-6745.2002 ◽

2002 ◽

Vol 184 (23) ◽

pp. 6739-6745 ◽

Cited By ~ 132

Author(s):

Sabina Heim ◽

Maria Del Mar Lleo ◽

Barbara Bonato ◽

Carlos A. Guzman ◽

Pietro Canepari

Keyword(s):

Life Cycle ◽

Enterococcus Faecalis ◽

Stress Responses ◽

Protein Profile ◽

Expression Patterns ◽

Proteome Analysis ◽

Vbnc State ◽

Viable But Nonculturable ◽

Nonculturable State ◽

Viable But Nonculturable State

ABSTRACT The protein expression patterns of exponentially growing, starved, and viable but nonculturable (VBNC) Enterococcus faecalis cells were analyzed to establish whether differences exist between the VBNC state and other stress responses. The results indicate that the protein profile of VBNC cells differs from that of either starved or exponentially growing bacteria. This demonstrates that the VBNC state is a distinct physiological phase within the life cycle of E. faecalis, which is activated in response to multiple environmental stresses.

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