Nonculturable Forms Research Articles

Effect of Previous Incubation of Aeromonas hydrophila in Wastewater Prior to Its Transfer Into Marine Water Microcosms

The occurrence of the mesophilic motile Gram negative non enterobacterial species A. hydrophila in the wild is a major problem that deserves to be resolved since it is a potentially pathogen able to enter into a non-culturable state on routine bacteriological plating media. These non-culturable forms can be detected by several direct or indirect visualization methods. This species has frequently been isolated from pathological forms in fish farming marine areas, especially near wastewater discharges. Consequently, we studied A. hydrophila in marine water microcosms placed during a 24 hour period in treated waste waters and compared with the homologous strain not placed in the same conditions. Thus, two kinds of microcosms were prepared using filtered and autoclaved marine water or waste water, inoculated by A. hydrophila and maintained at 25°C in darkness. The results obtained indicated that A. hydrophila population incubated at 25°C in marine water declined rapidly (3.21 log units in plate count number) during the first three days. Additionally, we noted that A. hydrophila incubated in marine water after a previous treatment in waste water declined progressively to 2.74 log units (in plate count number). Nevertheless, we showed no significant variations of the number of total bacterial cells for A. hydrophila developed in marine water after prior treatment in waste water, despite the appearance of the VBNC form. During this state, rods of normal size, elongated cells and cocci were obtained. Concomitantly, we determined several changes in biochemical and antimicrobial patterns of stressed A. hydrophila, notably the acquisition of adipate metabolization and an increase of resistance to antimicrobial compounds, especially for A. hydrophila incubated in marine water after treatment in waste water.

Alpha-ketoglutarate oxidoreductase, an essential salvage enzyme of energy metabolism, in coccoid form of Helicobacter pylori

In the Krebs cycle of Helicobacter pylori, the absence of alpha-ketoglutarate dehydrogenase and succinyl CoA synthetase are shown. Instead, alpha-ketoglutarate is converted to succinyl CoA and succinate by alpha-ketoglutarate oxidoreductase (KOR) and CoA transferase (CoAT). In the present study, when H. pylori transformed to the coccoid form, a viable but non-culturable form of H. pylori with reduced metabolic activity, the KOR activity was enhanced while the CoAT activity was reduced. Direct inactivation of KOR could potently kill the bacteria without allowing conversion to the coccoid form, suggesting a novel treatment strategy for the eradication of H. pylori, especially in cases infected with multiple antibiotic-resistant strains.

Phytoplankton-linked viable non-culturable Vibrio cholerae O1 (VNC) from rivers in Tucuman, Argentina

Plankton-linked Vibrio cholerae has been detected in aquatic environments since the first decades of the 20th century. Several mechanisms have been proposed to explain the survival of V. cholerae during inter-epidemic periods, one of which is through existence in its viable non-culturable (VNC) form in biological reservoirs. This latent form of V. cholerae (VNC) would explain the seasonal outbreaks and the way the pathogen survives in the environment. The objectives of this study were to assay V. cholerae O1 in its viable non-culturable form in two rivers in Tucuman, Argentina, as well as its possible association to phytoplankton and its relation to four environmental variables (pH, temperature, conductivity and dissolved oxygen), using direct immunofluorescence. Analysis of phytoplankton revealed that diatoms were predominant with percentages between 85 and 100%. Nitzschia palea was the only species found at all three sample sites with percentages between 0 and 38%. Of the 54 samples obtained during the 18 sampling periods, V. cholerae VNC was detected through direct immunofluorescence in 39% of the cases and at all three sampling sites. Positive samples were analysed for association of VNC with phytoplankton and between 1 and 10 bacteria were found adhered to a single algal cell. This confirms for the first time in northwestern Argentina adherence of this microorganism to the genera Stigeoclonium and Nitzschia as environmental reservoirs. No correlation could be found between the latent form of Vibrio and the environmental variables assayed.

Aeromonas–Acanthamoeba interaction and early shift to a viable but nonculturable state of Aeromonas by Acanthamoeba

To investigate the hypothesis that amoeba may comprise a significant environmental reservoir for Aeromonas, Acanthamoeba-Aeromonas interaction experiments were performed. Acanthamoeba were grown in monoculture and co-cultures with three different species of Aeromonas. Survival, invasion and viable but nonculturable state experiments were performed. We showed that at a low initial bacterial cell density, growth of Aeromonas spp. was inhibited by Acanthamoeba castellanii, while A. castellanii growth was unaffected. In contrast, a high initial bacterial cell density, Aeromonas hydrophila AEW44 and Aeromonas veronii biovar sobria AEW104 suppressed the growth of A. castellanii. Fluorescent and phase-contrast microscopic observations of GFP tagged Aer. hydrophila AEW44 demonstrated that the bacterial cells aggregated on A. castellanii cells after 15 min of incubation and internalized. Aeromonas hydrophila AEW44 cells were found to be actively moving. Interestingly, Aer. hydrophila AEW44 cells shifted more rapidly to a viable but nonculturable form when co-cultured with A. castellanii than in monoculture. We demonstrated that Aeromonas spp. are able to interact with and to infect the protozoan A. castellanii under laboratory conditions. Free-living amoeba might play a role as reservoir for Aeromonas, and thus may increase the transmission of Aeromonas by acting as a vehicle.

Survival and viability of Helicobacter pylori after inoculation into chlorinated drinking water

The aim of this work was to assess the effect of chlorine water treatment on Helicobacter pylori and to study the succession of cellular alterations in response to chlorine exposure. H. pylori NCTC 11637 reference strain was used for inoculating water samples. The culturability, substrate responsiveness combined with fluorescent in situ hybridization detection (DVC–FISH assay), RNA content, DNA content, and mRNA changes of H. pylori cells were analyzed. Culturability was lost at 5 min in water with 0.96 mg/l of free chlorine. Viable cells were detected by DVC–FISH after 3 h of exposure to chlorine but not after 24 h. The percentage of coccoid forms was higher than spiral forms after 40 s of chlorine exposure, but even after 24 h, FISH detection revealed the presence of spiral cells. After 24 h, amplification of the specific H. pylori 16S rDNA gene was achieved. Expression of the vacA gene was detected with the same intensity at all time points tested, demonstrating that these genes are expressed in non-culturable H. pylori cells. Levels of 16S rRNA were constant during the chlorine treatment, so killing of bacteria with chlorine probably does not involve ribosome degradation. According to our results, H. pylori could survive to disinfection practices normally used in drinking water treatment in the viable but non-culturable form, which would allow them to reach final consumption points and, at the same time, enable them to be undetectable by culture methods.

A Rapid Molecular-Based Assay for Direct Quantification of Viable Bacteria in Slaughterhouses

A rapid test for microbial quantification in carcass and environmental swabs that does not require enrichment and provides results in less than 4 h is described here. Steps in the assay include the rapid concentration of bacteria on sponge swabs by vacuum filtration followed by real-time PCR detection. The assay has been applied for the detection of coliforms, Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes on carcass swabs and environmental samples in a slaughterhouse-processing line. Comparison of this rapid method with standard culture techniques for coliform counts on beef and pork carcass swabs revealed higher numbers of bacteria (2- to 50-fold) by the rapid test compared with the plate counts. This was due to the detection of all bacteria (live, dead, and non-culturable forms) in the rapid assay. To allow detection of only viable bacteria, concentrated samples were treated with ethidium monoazide (EMA) prior to DNA extraction and real-time PCR detection, thereby preventing the amplification of DNA from bacteria with damaged cell walls and allowing only the DNA from bacteria with intact membranes to be detected. EMA treatment resulted in a significant reduction (P <0.001) in the number of coliforms detected compared to real-time PCR without EMA treatment. In beef swabs, the counts obtained in EMA real-time PCR were not significantly different (P <0.08) from the culture counts and the correlation coefficient between the two assays was 0.7385. A lower correlation coefficient (0.402) was obtained with pork swabs. The assay described herein has the potential to be applied on a routine basis to slaughterhouse lines for the detection of indicator organisms or specific pathogens.

Salinity-induced survival strategy of Vibrio cholerae associated with copepods in Cochin backwaters

The occurrence of Vibrio cholerae in water, sediment and copepods was studied over a wide range of salinity using conventional and polymerase chain reaction (PCR) techniques in the Cochin backwaters. V. cholerae occurred either as culturable or non-culturable form in this tropical estuary. During the pre monsoon period when high salinity prevailed (35.5 psu) V. cholerae was associated with copepods only in non-culturable form, but with the onset of monsoon when lower salinity prevailed, copepod-associated- V. cholerae reverted back to culturable form. The optimum salinity range for culturability was found to be 15–20 psu. This points to the fact that salinity is a major factor that which influences V. cholerae’s culturability and its association with copepods in Cochin backwaters.

Temperature-Dependent Genome Degradation in the Coccoid Form of Campylobacter jejuni

Campylobacter jejuni undergoes a dramatic morphological transformation from a corkscrew-shaped rod to a coccoid form in response to unfavorable conditions. It has been speculated that the coccoid plays an important role in the survival and dissemination of C. jejuni but questions still remain regarding the viability of coccoid cells. Characterization of the genome of coccoid cells found that newly formed coccoid cells (i.e., 1-3 days) had a SmaI-digestion profile identical to that of spiral-shaped cells; however, there was a progressive degradation of the DNA with continued incubation at 37 degrees C. Concomitant with genome degradation was the detection of DNA in supernatants of coccoid cells. In contrast, cells incubated at 4 degrees C retained a spiral shape and their SmaI-digestion profile for 8 weeks and released little DNA into the medium. Thus, low temperature inhibited both coccoid formation and genome degradation. Collectively, these data support the theory that the coccoid form of C. jejuni is a manifestation of cellular degradation and spiral-shaped cells, or possibly coccoid cells formed at low temperature, are the most probable candidates for a viable but nonculturable form of this pathogen.

Viable but nonculturable Vibrio cholerae O1 in the aquatic environment of Argentina.

In Argentina, as in other countries of Latin America, cholera has occurred in an epidemic pattern. Vibrio cholerae O1 is native to the aquatic environment, and it occurs in both culturable and viable but nonculturable (VNC) forms, the latter during interepidemic periods. This is the first report of the presence of VNC V. cholerae O1 in the estuarine and marine waters of the Rio de la Plata and the Argentine shelf of the Atlantic Ocean, respectively. Employing immunofluorescence and PCR methods, we were able to detect reservoirs of V. cholerae O1 carrying the virulence-associated genes ctxA and tcpA. The VNC forms of V. cholerae O1 were identified in samples of water, phytoplankton, and zooplankton; the latter organisms were mainly the copepods Acartia tonsa, Diaptomus sp., Paracalanus crassirostris, and Paracalanus parvus. We found that under favorable conditions, the VNC form of V. cholerae can revert to the pathogenic, transmissible state. We concluded that V. cholerae O1 is a resident of Argentinean waters, as has been shown to be the case in other geographic regions of the world.

Viable but non-culturable forms of food and waterborne bacteria: Quo Vadis?

There is increasing evidence for a viable but non-culturable (VBNC) state in microbes, particularly in the stressing environment presented by modern foods with their varied pre-treatment and packaging strategies. This is a cause for concern because of evidence that microbial pathogens in such a state may retain their capacity to cause infections after ingestion by the consumer despite their inability to grow under the conditions employed in procedures for determining their presence in foods. Heavily stressed pathogenic species of bacteria in a VBNC or not immediately culturable state are potentially dangerous public health problems, particularly as stressed cells may be more virulent than well-fed bacteria. In this viewpoint article, I wish to focus on possible procedures for detecting such organisms and assessing their physiological state.

Electrochemical investigation of the dynamics of Mycobacterium smegmatis cells' transformation to dormant, nonculturable form

Dynamics of transformation of Mycobacterium smegmatis cells by cultivation under nonoptimal conditions (partial starvation) to dormant, nonculturable form has been studied. For this aim, an electrochemical method was developed to detect both viable and ‘viable but nonculturable’ (VBNC) cells. The current produced by bacteria placed at the electrode surface was measured in the presence of 2,6-dichlorophenol indophenol (DCIP) at the applied potential of 350 mV. It has been established that electrochemical activity changes parallel with the growth of biomass. The transition of M. smegmatis to a dormant, nonculturable state goes along with the decrease of the detection current up to 20% of the maximum level. This means that nonculturable cells have rather high rest metabolic activity. The course of the CFU values has a complicated character during bacterial growth. The placement of the bacterial culture on the solid medium appears to cause a new stress that stops proliferation and stimulates aggregation. Both processes distort CFU measurement results. The quantitative estimation of the viable but nonculturable cells by counting colonies, measuring optical density and current produced by bacteria has been discussed.

Detection of Arcobacter spp. in the coastal environment of the Mediterranean Sea.

The occurrence of Arcobacter spp. was studied in seawater and plankton samples collected from the Straits of Messina, Italy, during an annual period of observation by using cultural and molecular techniques. A PCR assay with three pairs of primers targeting the 16S and 23S rRNA genes was used for detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii in cultures and environmental samples. Only one of the Arcobacter species, A. butzleri, was isolated from seawater and plankton samples. With some samples the A. butzleri PCR assay gave amplified products when cultures were negative. A. cryaerophilus and A. skirrowii were never detected by culture on selective agar plates; they were detected only by PCR performed directly with environmental samples. Collectively, our data suggest that culturable and nonculturable forms of Arcobacter are present in marine environments. The assay was useful for detecting Arcobacter spp. both as free forms and intimately associated with plankton. This is the first report showing both direct isolation of A. butzleri and the presence of nonculturable Arcobacter spp. in the coastal environment of the Mediterranean Sea.

Improved Culturing Techniques for Campylobacter

ABSTRACT Improved techniques for culturing and enumerating Campylobacter jejuni were developed. Parameters included (1) growth vessel type, (2) growth atmosphere, (3) incubation time, and (4) frequency of subcul‐turing. Improved growth and consistent morphology were obtained using a tissue culture flask compared with an Erlenmeyer flask or a test tube as well as exposure to modified atmosphere (10% CO2, 85% N2, and 5% O2). When cultures of C. jejuni were incubated more than 24 h, transformation to the coccoid increased, and fewer colony‐forming units were detected. Excessive subculturing (&gt;2 times) resulted in increased formation of coc‐coid forms. Decreased concentrations of colony‐forming units of C. jejuni commonly attributed to the production of a viable, nonculturable form also may be due to cellular clumping. These techniques should alleviate difficulty and reduce variation when working with Campylobacter.

Retention of virulence in viable but non-culturable halophilic Vibrio spp.

The viable but non-culturable (VBNC) forms of two environmental strains of Vibrio alginolyticus 1 and Vibrio parahaemolyticus 66 and one strain of V. parahaemolyticus ATCC 43996 showing virulence characteristics (hemolysin production, adhesive and/or cytotoxic ability, in vivo enteropathogenicity) were obtained by culturing bacteria in a microcosm consisting of artificial sea water (ASW) and incubating at 5 °C with shaking. Every 2 days, culturability of the cells in the microcosm was monitored by spread plates on BHI agar and total count and the percentage of viable cells were determined by double staining with DAPI and CTC. When cell growth was not detectable (<0.1 CFU/ml), the population was considered non-culturable and, then, the VBNC forms were resuscitated in a murine model. For each strain, eight male Balb/C mice were intragastrically inoculated with 0.1 ml of concentrated ASW bacterial culture. Two mice from each group were sacrificed at 2, 4, 8, and 12 days after challenge for autopsy and re-isolation of the microorganisms from the intestinal tissue cultures. Isolation was obtained in 25% of the animals challenged with the VBNC V. alginolyticus strain, in 37.5% of those challenged with the VBNC V. parahaemolyticus strain of environmental origin and in 50% of the animals infected with VBNC V. parahaemolyticus ATCC 43996. The strains thus isolated were again subjected to biological assays to determine the retention of pathogenicity. The virulence characteristics that seemed to disappear after resuscitation in the mouse were subsequently reactivated by means of two consecutive passages of the strains in the rat ileal loop model. The results obtained indicate that VBNC forms of the strains examined can be resuscitated and retain their virulence properties.

Survival and resuscitation of ten strains of Campylobacter jejuni and Campylobacter coli under acid conditions.

The culturability of 10 strains of Campylobacter jejuni and Campylobacter coli was studied after the bacteria were exposed to acid conditions for various periods of time. Campylobacter cells could not survive 2 h under acid conditions (formic acid at pH 4). The 10 Campylobacter strains could not be recovered, even when enrichment media were used. Viable cells, however, could be detected by a double-staining (5-cyano-2,3-ditolyl tetrazolium chloride [CTC]-4',6'-diamidino-2-phenylindole [DAPI]) technique, demonstrating that the treated bacteria changed into a viable but nonculturable (VBNC) form; the number of VBNC forms decreased over time. Moreover, some VBNC forms of Campylobacter could be successfully resuscitated in specific-free-pathogen fertilized eggs via two routes, amniotic and yolk sac injecting.

An investigation into the changed physiological state of Vibrio bacteria as a survival mechanism in response to cold temperatures and studies on their sensitivity to heating and freezing.

To induce pathogenic Vibrio bacteria into a changed physiological state, in response to cold temperatures in sea water, and assess their sensitivity to heating and freezing, as compared with normal cells. Cells of exponential phase Vibrio vulnificus, V. cholerae and V. parahaemolyticus were washed and inoculated into flasks of sea water, which were stored at 20 and 4 degrees C. Cells stored at 20 degrees C could be recovered after 60 d on non-selective agar (heart infusion agar; HIA) and on the selective agar (thiosulphate citrate bile salts agar) which is used in most Vibrio detection methodology. At 4 degrees C cells became non-culturable on both agars over time. The non-culturable cells appeared to be metabolically active and maintained their membrane integrity, whilst undergoing a change in morphology from rod-shaped to coccoid cells. Resuscitation was possible, in some cases, by an upshift in temperature before plating and the addition of catalase to HIA plates was found to increase recovery. Studies were carried out to assess the sensitivity of the non-culturable cells to heating and freezing compared with the normal cells. Vibrio organisms, whether culturable or in the non-culturable form, were not inactivated by freezing to -20 degrees C. Heating studies showed that V. parahaemolyticus was very heat resistant at low temperatures. However, a pasteurization regime of 2 min at 70 degrees C was found to be effective against all three strains. Experiments showed that the non-culturable cells of all three strains were similar in their heat resistance or, in some cases, were more heat sensitive than cells in the normal form. Cells in the changed physiological form would not be detected in fish or seafood products by the current Vibrio detection methods. Freezing had no effect in reducing cell numbers. Vibrio parahaemolyticus was very heat resistant in the low temperature pasteurization studies. The higher pasteurization regime of 70 degrees C for 2 min was effective against all three pathogens. Non-culturable cells had similar heat sensitivity or were more heat sensitive than cells in the normal state. SIGNIFICANCE OF IMPACT OF THE STUDY: The study has highlighted a need for the development of better Vibrio detection methods. The low temperature pasteurization of oysters, which has been recommended in the USA, would not be adequate against the strain of V. parahaemolyticus used in this study. Heating regimes which were found to control cells in the normal form will also be effective for the control of the cells with changed physiology.

Meeting on the Microbiology of Soils, Autumn 2001Estimation of protozoan diversity in soil

Different methods of estimating protozoan diversity in soil are discussed in this paper, with the major emphasis on heterotrophic flagellates. Although many species of ciliates and testate amoebae seem to be unique to the soil environment, the communities of heterotrophic flagellates and naked amoebae are probably best considered as restricted versions of their aquatic counterparts. Soil protozoa are difficult to observe directly, hence culture techniques are usually used. These techniques enable us to explore certain functional aspects, but their major drawback is that some soil protozoa cannot be cultured. Molecular methods, however, have the potential to detect such non-culturable forms.

Cold-adaptation of Antarctic microorganisms - possible involvement of viable but nonculturable state

Occurrence of the viable but nonculturable (VBNC) form of microorganisms in nature is well known. Evidence available in the literature indicates that low temperature may induce microorganisms to form VBNC cells. The VBNC state of some bacteria, collected from Antarctic lakes, has been recently reported. Hence it is postulated that formation of VBNC cells may be one of the strategies adopted by Antarctic microorganisms to survive in the extreme cold climate.

Competitive polymerase chain reaction for quantification of nonculturable Enterococcus faecalis cells in lake water.

Among the survival strategies developed by bacteria when faced with adverse environmental conditions, the viable but nonculturable (VNC) state has been described. In this state, bacteria are unable to form colonies but are still alive and capable of metabolic activity. The VNC state has been described in numerous Gram-negative species, but recently also in Enterococcus faecalis, a Gram-positive species which can be found in the environment. In this study we describe a competitive PCR (cPCR) protocol to detect and quantify a specific sequence of DNA from culturable and nonculturable E. faecalis cells present in water samples. The protocol was found to be specific and capable of detecting amounts of DNA up to 0.1 pg corresponding to approximately 2 cells ml(-1). Moreover, it allows an internal standard to be used to quantify the amount of specific DNA present in samples from different environments. The application of this cPCR method to water samples from Lake Garda enabled us to demonstrate the presence of nonculturable forms of E. faecalis in lake water and to quantify their DNA and the corresponding concentration of nonculturable cells.

Competitive polymerase chain reaction for quantification of nonculturable Enterococcus faecalis cells in lake water

Among the survival strategies developed by bacteria when faced with adverse environmental conditions, the viable but nonculturable (VNC) state has been described. In this state, bacteria are unable to form colonies but are still alive and capable of metabolic activity. The VNC state has been described in numerous Gram-negative species, but recently also in Enterococcus faecalis, a Gram-positive species which can be found in the environment. In this study we describe a competitive PCR (cPCR) protocol to detect and quantify a specific sequence of DNA from culturable and nonculturable E. faecalis cells present in water samples. The protocol was found to be specific and capable of detecting amounts of DNA up to 0.1 pg corresponding to approximately 2 cells ml −1. Moreover, it allows an internal standard to be used to quantify the amount of specific DNA present in samples from different environments. The application of this cPCR method to water samples from Lake Garda enabled us to demonstrate the presence of nonculturable forms of E. faecalis in lake water and to quantify their DNA and the corresponding concentration of nonculturable cells.