Methionine uptake and cytopathogenicity of viable but nonculturable Shigella dysenteriae type 1.

A pathogenic strain of Shigella dysenteriae type 1 was selected for study to elucidate the physiology and potential pathogenicity of organisms in the viable but nonculturable (VBNC) state in the environment. Studies in our laboratory have shown that S. dysenteriae type 1 survives in laboratory microcosms in the VBNC state for long periods of time, i.e., more than 6 months. VBNC cells of S. dysenteriae type 1 were found to retain cytopathogenicity for cultured HeLa cells. To determine whether VBNC S. dysenteriae type 1 expressed protein after loss of culturability, 35S-labelled methionine was added to suspensions of VBNC cells. Total cellular proteins were extracted and examined by autoradiography. Results indicate that VBNC S. dysenteriae type 1 is capable of both active uptake of methionine and incorporation of methionine into protein. Amino acid uptake and protein synthesis substantiate the viability of cells of S. dysenteriae type 1 in the VBNC state, i.e., although the cells are unable to be cultured on laboratory media by standard bacteriological methods, the cells remain metabolically active. Furthermore, VBNC cells of S. dysenteriae type 1 may pose a potential public health hazard that has not yet been recognized.     

Use of the polymerase chain reaction and fluorescent-antibody methods for detecting viable but nonculturable Shigella dysenteriae type 1 in laboratory microcosms.

Epidemiological studies of shigellosis in Bangladesh have demonstrated that surface-water sources can act as foci of infection. Studies of laboratory microcosms have shown that shigellae become nonculturable but remain viable when exposed to environmental samples of water. The present study was carried out to detect viable but nonculturable Shigella dysenteriae 1 from laboratory microcosms by the polymerase chain reaction and the fluorescent-antibody techniques. S. dysenteriae 1 was inoculated into laboratory microcosms consisting of water samples collected from ponds, lakes, rivers, and drains in Bangladesh. The survival of S. dysenteriae in microcosms was assessed by viable counting on MacConkey agar. After 2 to 3 weeks, S. dysenteriae 1 became nonculturable but remained viable. After 6 weeks, this nonculturable but viable S. dysenteriae 1 was detected by both the polymerase chain reaction and the fluorescent-antibody methods. The viable but nonculturable state of S. dysenteriae 1 demonstrated in this study may be important for understanding the epidemiology of shigellosis.     

Retention of virulence in a viable but nonculturable Edwardsiella tarda isolate

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 4 degrees C. 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. 10(9) 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.     

Membrane fatty acid and virulence changes in the viable but nonculturable state of Vibrio vulnificus.

The nonculturable state of Vibrio vulnificus and, for comparison, that of Escherichia coli were studied in artificial-seawater microcosms at 5 degrees C. Total cell counts were monitored by acridine orange epifluorescence, metabolic activity by direct viable counts, and culturability by plate counts on selective and nonselective media. Whereas total counts remained constant, plate counts of V. vulnificus suggested nonculturability by day 24. In contrast, direct viable counts indicated significant cell viability throughout 32 days of incubation. As an indication of the metabolic changes that occurred as cells entered the state of nonrecoverability, membrane fatty acid analyses were performed. At the point of nonculturability of V. vulnificus, the major fatty acid species (C16 and C16:1) had decreased 57% from the T0 level, concomitant with the appearance of several short-chain acids. Although the bacteria were still recoverable, a similar trend was observed with E. coli. Electron microscopy of nonculturable V. vulnificus showed that the cells were rounded and reduced in size and contained fewer ribosomes. Mouse infectivity studies conducted with these cells suggested loss of virulence.     

Membrane fatty acid and virulence changes in the viable but nonculturable state of Vibrio vulnificus

The nonculturable state of Vibrio vulnificus and, for comparison, that of Escherichia coli were studied in artificial-seawater microcosms at 5 degrees C. Total cell counts were monitored by acridine orange epifluorescence, metabolic activity by direct viable counts, and culturability by plate counts on selective and nonselective media. Whereas total counts remained constant, plate counts of V. vulnificus suggested nonculturability by day 24. In contrast, direct viable counts indicated significant cell viability throughout 32 days of incubation. As an indication of the metabolic changes that occurred as cells entered the state of nonrecoverability, membrane fatty acid analyses were performed. At the point of nonculturability of V. vulnificus, the major fatty acid species (C16 and C16:1) had decreased 57% from the T0 level, concomitant with the appearance of several short-chain acids. Although the bacteria were still recoverable, a similar trend was observed with E. coli. Electron microscopy of nonculturable V. vulnificus showed that the cells were rounded and reduced in size and contained fewer ribosomes. Mouse infectivity studies conducted with these cells suggested loss of virulence.     

Purification of a cell-associated hemagglutinin from Shigella dysenteriae type 1

Abstract A cell-associated hemagglutinin (HA) was isolated and purified from a clinical isolate of Shigella dysenteriae type 1 by affinity chromatography on a fetuin-agarose column. The purified hemagglutinin produced a single-stained protein band of around 66 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). In an immunodiffusion test, HA-antisera produced a single precipitin band against the purified HA without exhibiting any reactivity towards lipopolysaccharide (LPS) of S. dysenteriae type 1 strain. Inhibition of the hemagglutination by the glycoproteins fetuin, asialofetuin and a sugar derivative N -acetyl-neuraminic acid but not by simple sugars, suggested the specific requirement of complex carbohydrate for binding. Electron micrographs of the purified HA revealed a morphology typical of globular protein.     

Resuscitation of Vibrio vulnificus from the viable but nonculturable state.

Stationary-phase-grown cells of the estuarine bacterium Vibrio vulnificus became nonculturable in nutrient-limited artificial seawater microcosms after 27 days at 5 degrees C. When the nonculturable cells were subjected to temperature upshift by being placed at room temperature, the original bacterial numbers were detectable by plate counts after 3 days, with a corresponding increase in the direct viable counts from 3% to over 80% of the total cell count. No increase in the total cell count was observed during resuscitation, indicating that the plate count increases were not due to growth of a few culturable cells. Chloramphenicol and ampicillin totally inhibited resuscitation of the nonculturable cells when added to samples that had been at room temperature for up to 24 h. After 72 h of resuscitation, the inhibitors had an easily detectable but reduced effect on the resuscitated cells, indicating that protein and peptidoglycan synthesis were still ongoing. Major changes in the morphology of the cells were discovered. Nonculturable cells of V. vulnificus were small cocci (approximately 1.0 micron in diameter). Upon resuscitation, the cells became large rods with a size of mid-log-phase cells (3.0 microns in length). Four days after the cells had become fully resuscitated, the cell size had decreased to approximately 1.5 micron in length and 0.7 micron in width. The cells were able to go through at least two cycles of nonculturability and subsequent resuscitation without changes in the total cell count. This is the first report of resuscitation, without the addition of nutrient, of nonculturable cells, and it is suggested that temperature may be the determining factor in the resuscitation from this survival, or adaptation, state of certain species in estuarine environments.     

A case of shigellosis caused by Shigella dysenteriae type 1 and Shigella flexneri type 5B

Shigella dysenteriae type 1 and Shigella flexneri type 5b strains were isolated as causative agents of bacterial dysentery in a patient having visited South-East Asia. Both strains are a rare finding for Bulgaria. S. dysenteriae 1 strains have not been isolated since 1962, and there were only single isolates of S. flexneri 5b. The strains were of the same antibiotic resistance patterns. Conjugation experiments showed that resistance is determined by transferrable R-plasmids having identical characteristics. It is assumed that in the patient's gut transfer of an R-plasmid occurs from E. coli of the normal flora to the pathogenic shigellae.     

Evidence for viable but nonculturable yeasts in botrytis-affected wine

AIMS: In Botrytis-affected wine, high concentrations of SO2 are added to stop the alcoholic fermentation and to stabilize the wine. During maturing in barrels or bottle-ageing, an unwanted refermentation can sometimes occur. However, results of the usual plate count of wine samples at the beginning of maturing suggest wine microbial stability. The aim of this study was to investigate the mode of yeasts survival after the addition of SO2 and to identify surviving yeasts. METHODS AND RESULTS: Using direct epifluorescence technique, we observed the behaviour of cells after SO2 addition and compared the cell number determined by this method with the result of plate counts. The persistent yeast species were identified using two methods: polymerase chain reaction (PCR)-restriction fragment length polymorphism and PCR-denaturing gradient gel electrophoresis. They were identified as Saccharomyces cerevisiae and Candida stellata, and after few months of maturing, other spoiling yeasts appeared, like Rhodotorula mucilaginosa or Zygosaccharomyces bailii. CONCLUSIONS: All characteristics of the cells lead to the conclusion that yeast persisted in wine in a viable but nonculturable-like state (VBNC). Suppression of the effect of free-SO2 did not lead to the resuscitation of the cells; however, another method proved the capacity of the cells to exit from the VBNC-like state. SIGNIFICANCE AND IMPACT OF THE STUDY: This study permits the characterization of the presence of VBNC-like yeasts in wine. The 'refermentation' phenomenon is probably due to the exit of the VBNC state.     

Shigella dysenteriae type 1-specific bacteriophage from environmental waters in Bangladesh

Shigella dysenteriae type 1 is the causative agent of the most severe form of bacillary dysentery, which occurs as epidemics in many developing countries. We isolated a bacteriophage from surface water samples from Bangladesh that specifically lyses strains of S. dysenteriae type 1. This phage, designated SF-9, belongs to the Podoviridae family and has a 41-kb double-stranded DNA genome. Further screening of water samples for the prevalence of the phage revealed 9 of 71 (12.6%) water samples which were positive for the phage. These water samples were also positive in PCR assays for one or more S. dysenteriae type 1-specific genes, including ipaBCD and stx1, and live S. dysenteriae type 1 was isolated from three phage-positive samples. The results of this study suggest that phage SF-9 may have epidemiological applications in tracing the presence of S. dysenteriae type 1 in environmental waters.     

In planta recovery of Erwinia amylovora viable but nonculturable cells

Little is known about the survival mechanisms of Erwinia amylovora outside its hosts. It has been demonstrated that it enters the viable but nonculturable state (VBNC) when exposed to different types of stress. In the VBNC state, bacterial cells remain viable but unable to grow on the solid general media where they usually do, and are thus undetectable by conventional culture-dependent methods. In this work, we have evaluated the recovery of E. amylovora VBNC cells by passage through pear plantlets, in comparison with other recovery methods commonly used for this pathogen: incubation in KB broth and inoculation of immature fruits. VBNC cells were obtained by exposure of bacterial cells to different types of stress (oligotrophy, nutrient deprivation and chlorine), and recovery assays were performed at 26°C. In all cases, the recovery of VBNC cells was more effective in plantlets than in liquid KB or immature fruits. In fact, when cells were exposed to chlorine for more than 30 min, only passage through host plant gave positive result, enabling recovery of E. amylovora cells few days after inoculation of plants. These results suggest a higher effectiveness of in planta recovery than those performed with liquid KB or detached fruits. Our results support the hypothesis of the VBNC state being part of the E. amylovora life cycle. The potential existence of this physiological state in nature should be taken in consideration in epidemiological studies of fire blight, with the aim to optimize the management and control of this disease.     

Starvation survival and viable but nonculturable states in Aeromonas hydrophila

The behavior of Aeromonas hydrophila stored at 4 degrees C and 25 degrees C in nutrient-poor filtered sterilized distilled water was investigated. At 4 degrees C, the A. hydrophila population declined below the detection level (0.1 cell mL(-1)) after 7 weeks, whereas the number of cells with intact membrane as determined by the LIVE/DEAD method decreased only by 1 log unit. Although, this response is reminiscent of the so-called VBNC state, the cells could not be resuscitated by an upshift to 25 degrees C. A mixture of rods with normal size and elongated cells was observed in this state. At 25 degrees C, viable cells and cells with intact membrane declined only by 0.8 log unit over the 10-week storage period, and thus A. hydrophila entered the classical starvation survival state. During this state, a mixture of rods and cocci was observed. Prestarvation at 25 degrees C for 24 h and especially 49 days delayed significantly the rate of entry into the VBNC state. However, stationary phase cells were not significantly more tolerant than exponential phase cells. No significant improvements in recovery yield were obtained on LB agar plates amended with catalase or sodium pyruvate. During cold incubation, high variability in responses was observed. Intermittent cryptic regrowth might be responsible for this variability in responses.     

Use of multiparameter analysis for Vibrio alginolyticus viable but nonculturable state determination.

BACKGROUND: Vibrio alginolyticus is known to enter into a viable but nonculturable (VBNC) state in response to environmental conditions unfavorable to the growth. Cells in VBNC condition pose a public health threat because they are potentially pathogenic. METHODS: We constructed a pathway for the identification of the most significant variables and the characterization of those variables able to discriminate the groups under investigation. Different parameters measured by the image processing software were chosen as the most representative of V. alginolyticus cell morphology (length index for dimension) and metabolic activity (density profile indexes). To detect relationships between the groups of treatment performed, we carried out a principal components analysis (PCA). RESULTS: The PCA analysis indicated that increasing coccoid shape transformation was related to both metabolic and dimension variations, delineating a well defined graph profile. Indeed, we discovered that specific morphological variations occur when cells in the culturable state pass into VBNC condition, namely comma-shaped culturable bacteria are converted into coccoid-shaped VBNC cells. The results were also supported by scanning electron microscopy analysis. CONCLUSIONS: This technique allows the analysis of a large number of vibrio samples in a short period of time. The obtained multiparameter information may complement genetic/molecular analyses facilitating, in an automatic fashion, further studies to evaluate the potential risk of this pathogen in the environment. It may also be a useful tool for large-scale cell biology studies and high content screening.     

Localization of Shiga toxin gene in the region of Shigella dysenteriae 1 chromosome specifying virulence functions

Abstract R' plasmids have been generated that contain a determinant of Shigella dysenteriae 1 which directs the synthesis of Shiga toxin in Escherichia coli K-12. This gene is closely linked to the argE locus and thus is located in a region of the Shigella chromosome known to contain determinants of virulence factors.     

Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii.

Legionella pneumophila is an aquatic bacterium and is responsible for Legionnaires' disease in humans. Free-living amoebae are parasitized by legionellae and provide the intracellular environment required for the replication of this bacterium. In low-nutrient environments, however, L. pneumophila is able to enter a non-replicative viable but nonculturable (VBNC) state. In this study, L. pneumophila Philadelphia I JR 32 was suspended in sterilized tap water at 10(4) cells/ml. The decreasing number of bacteria was monitored by CFU measurements, acridine orange direct count (AODC), and hybridization with 16S rRNA-targeted oligonucleotide probes. After 125 days of incubation in water, the cells were no longer culturable on routine plating media; however, they were still detectable by AODC and by in situ hybridization. The addition of Acanthamoeba castellanii to the dormant bacteria resulted in the resuscitation of L. pneumophila JR 32 to a culturable state. A comparison of plate-grown legionellae and reactivated cells showed that the capacity for intracellular survival in human monocytes and intraperitoneally infected guinea pigs, which is considered a parameter for virulence, was not reduced in the reactivated cells. However, reactivation of dormant legionellae was not observed in the animal model.     

Fluorescent-antibody method useful for detecting viable but nonculturable Salmonella spp. in chlorinated wastewater

An indirect fluorescent-antibody (IFA) technique, which employed adsorbed Behring polyvalent I O antiserum, was used to detect Salmonella spp. in environmental water systems. The IFA method used in this study detected 95% of Salmonella serotypes encountered in human infections in France, with a sensitivity threshold of 7.5 x 10(3) bacteria per ml of wastewater. Specificity was assessed by testing IFA against Salmonella-free seawater and a variety of bacteria other than Salmonella spp. When used to examine raw and chlorinated wastewater over a 2-month period, the IFA method was successful in detecting Salmonella spp. in all 12 of the samples examined, with total numbers determined to be 4.5 x 10(5) to 3.3 x 10(7) salmonellae per 100 ml. In comparison, for the same samples, enumeration by culture, using the most-probable-number technique, was effective in detecting Salmonella spp. in only four of eight raw-water samples and one of four chlorinated water samples tested. Three samples were further tested by using the direct viable count procedure combined with IFA and results showed that 5 to 31.5% of the Salmonella spp. enumerated by this method in chlorinated water were substrate responsive.