Molecular vs culture methods for the detection of bacterial faecal indicators in groundwater for human use

AIMS: The current standard culture methods are unable to detect nongrowing bacteria and, thus, might not be sufficient for precise monitoring of the microbiological quality of waters. The use of a molecular method such as PCR could be a valid alternative to detect bacterial faecal contamination indicators such as Escherichia coli and Enterococcus faecalis and reveal the presence of culturable and nonculturable bacterial forms. METHODS AND RESULTS: The presence of E. coli and Ent. faecalis cells in 30 groundwater samples was evaluated with the standard culture method and compared with a specific PCR protocol. A substantial percentage (50%) of the samples not containing culturable cells proved positive in the search for Ent. faecalis DNA by PCR. Quantification by competitive PCR (cPCR) of the DNA detected allowed us to calculate the number of nonculturable cells present in water samples: the number varied from 2 to 120 cells ml(-1). Only four samples were positive for E. coli DNA and the corresponding nonculturable cells varied from 24 to 70 ml(-1). CONCLUSIONS: This study demonstrates that the standard culture methods in use are unable to detect a substantial proportion of the bacterial population which is nonculturable but, as previously demonstrated, potentially still viable and able to express those pathogenic factors needed for causing infections in humans. SIGNIFICANCE AND IMPACT OF THE STUDY: To protect human health it is necessary to develop and use methods which detect the nonculturable as well as culturable bacteria present in water.     

Recovery of hydrogen peroxide-sensitive culturable cells of Vibrio vulnificus gives the appearance of resuscitation from a viable but nonculturable state

The viabilities of five strains of Vibrio vulnificus were evaluated during the storage of the organisms in sterile seawater at 5 degrees C. The number of CFU was measured by plate count methods on rich media. The total cell numbers were determined by direct microscopic count methods. The titer of CFU declined logarithmically to undetectable levels over a period of 2 to 3 weeks, while the total cell numbers were unchanged. Midway through each study, higher culturable cell counts began to be observed on plates containing catalase or sodium pyruvate; during the latter stages of the study, the plate counts on such media were up to 1,000-fold higher than those on unsupplemented plates. Because autoclaving is known to generate hydrogen peroxide in rich media, and because catalase and sodium pyruvate are known to eliminate hydrogen peroxide, it appears that the conditions of the experiments led to the selection of a hydrogen peroxide-sensitive culturable cell subpopulation. At the time of the final stage of the decline in viability of each culture, hydrogen peroxide-sensitive cells were the only culturable cells present. Warming samples of the cultures to room temperature led to the growth of these residual culturable cells, utilizing nutrients provided by the nonculturable cells. The cells that grew recovered hydrogen peroxide resistance. When mixtures of culturable and nonculturable cells were diluted to the point where only nonculturable cells were present, or when the hydrogen peroxide-sensitive culturable cells had declined to undetectable levels, warming had no effect; no culturable cells were recovered. Warming has been reported to "resuscitate" nonculturable cells. Recognition of the existence of hydrogen peroxide-sensitive culturable cell populations, as well as their ability to grow to high levels in the warmed seawater microcosms, leads instead to the conclusion that while warming permits culturable cells to grow, it has no effect on nonculturable cells.     

Formation of nonculturable Escherichia coli in drinking water

AIMS: To examine whether incubation of Escherichia coli in nondisinfected drinking water result in development of cells that are not detectable using standard procedures but maintain a potential for metabolic activity and cell division. METHODS AND RESULTS: Survival and detectability of four different E. coli strains were studied using drinking water microcosms and samples from contaminated drinking water wells. Recovery of E. coli was compared using different cultivation-dependent methods, fluorescence in situ hybridization (FISH) using specific oligonucleotide probes, direct viable counts (DVC), and by enumeration of gfp-tagged E. coli (green fluorescent protein, GFP). Two levels of stress responses were observed after incubation of E. coli in nondisinfected drinking water: (i) the presence of cells that were not detected using standard cultivation methods but could be cultivated after gentle resuscitation on nonselective nutrient-rich media, and (ii) the presence of cells that responded to nutrient addition but could only be detected by cultivation-independent methods (DVC, FISH and GFP). Collectively, the experiments demonstrated that incubation for 20-60 days in nondisinfected drinking water resulted in detection of only 0.7-5% of the initial E. coli population using standard cultivation methods, whereas 1-20% could be resuscitated to a culturable state, and 17-49% could be clearly detected using cultivation-independent methods. CONCLUSIONS: Resuscitation of stressed E. coli on nonselective nutrient-rich media increased cell counts in drinking water using both traditional (CFU), and cultivation-independent methods (DVC, FISH and GFP). The cultivation-independent methods resulted in detection of 10-20 times more E. coli than the traditional methods. The results indicate that a subpopulation of substrate-responsive but apparent nonculturable E. coli may develop in drinking water during long-term starvation survival. SIGNIFICANCE AND IMPACT OF THE STUDY: The existence of substrate-responsive but nonculturable cells should be considered when evaluating the survival potential of E. coli in nondisinfected drinking water.     

An investigation of the presence of ultramicrocells in natural mineral water

The presence of 'ultramicrocells' in natural mineral water, capable of passing through a 0.2 micron filter, has been demonstrated. Filters allowing the greatest proportion of viable (culturable) cells to pass ranked in the order, 0.4 micron polycarbonate (5.02%) > 0.2 micron polycarbonate (0.02%) > or = 0.45 micron cellulose nitrate (0.02%) > 0.2 micron cellulose acetate (< 0.002%). Following incubation for 4 d at 22 degrees C, viable counts in filtered mineral water increased from < 2-8.7 x 10(2) cfu ml-1(-2).8 x 10(4)-1.9 x 10(6) cfu ml-1. Successive filtration/incubation cycles of mineral water increased the proportion of cells passing through a 0.2 micron cellulose acetate filter from < 0.003% to 0.11% and 0.69%, suggesting selection for 'ultramicrocells'. Cells isolated from this process and grown on liquid R2A medium were thin, Gram-negative rods, of 0.15-0.40 micron wide and 0.50-6.20 microns long. Membrane filtration techniques used for pathogen detection in mineral waters will not retain all the cells present. If pathogens are able to form ultramicrocells, these may go undetected.     

Examination of recovery in vitro and in vivo of nonculturable Escherichia coli O157:H7

Escherichia coli O157:H7 (strains ATCC 43895 and FO46) became nonculturable in sterile, distilled, deionized water or after exposure to chlorine. Recovery of nonculturable E. coli O157:H7 was examined by in vitro and in vivo methods. The decline in culturability of starved E. coli O157:H7 was measured by plate count on rich medium. Recovery in vitro of nonculturable cells was conducted with media amended with catalase or sodium pyruvate; however, there was no apparent increase over culturable cell counts on amended versus nonamended media. Although nonculturable E. coli O157:H7 did not recover under in vitro conditions, a mouse model was used to determine if in vivo conditions would provide sufficient conditions for recovery of nonculturable E. coli O157:H7. In separate studies, mice were orally challenged with starvation-induced nonculturable cells (FO46) or chlorine-induced nonculturable cells (43895 and FO46). Passage through the mouse gastrointestinal tract had no effect on recovery of nonculturable (starvation or chlorine induced) E. coli O157:H7 (43895 or FO46), based on analysis of fecal samples. Mouse kidneys were assayed for the presence of Shiga toxin using the Vero cell assay. Differences in cytotoxicity towards Vero cells from kidney samples of mice receiving nonculturable cells and control mice were not significant, suggesting a loss of virulence.     

Development of a Gram-negative selective agar (GNSA) for the detection of Gram-negative microflora in sputa in patients with cystic fibrosis

AIMS: To develop a selective agar medium to help detect and quantify Gram-negative flora in the sputum of patients with cystic fibrosis (CF). METHODS AND RESULTS: A novel Gram-negative Selective Agar (GNSA) medium was developed consisting of tryptone soya broth (30 g), bacteriological agar no.1 (10 g), yeast extract (5 g), crystal violet (2 mg), nisin (48 mg), novobiocin (5 mg), cycloheximide (100 mg), amphotericin (2 mg) and double distilled water (1 l), for the selective culture of all Gram-negative flora from the sputum of patients with CF. GNSA was able to support the proliferation of all 34 Gram-negative organisms examined, including 23 species most commonly associated with CF, but was unable to support the growth of the 12 Gram-positive or seven fungal organisms examined. Sensitivity studies demonstrated that the GNSA medium was able to detect not less than 1.50 x 102 CFU ml-1 sputum Pseudomonas aeruginosa, 2.38 x 102 CFU ml-1 sputum Burkholderia cepacia genomovar IIIb and 6.70 x 103 CFU ml-1 sputum Stenotrophomonas maltophilia. A comparison of the microbial flora detected in the sputa of 12 adult CF patients by employment of routine bacteriological agar media and GNSA, demonstrated that GNSA was able to detect all Gram-negative organisms cultured by routine media, but had the advantage of detecting Alcaligenes xylosoxidans in two CF patients, whom had no previous history of Gram-negative infection. CONCLUSIONS: GNSA was unable to support the proliferation of any Gram-positive organism or yeast/fungi, but was successful in supporting the growth of all Gram-negative organisms challenged. SIGNIFICANCE AND IMPACT OF THE STUDY: Employment of this medium coupled with semi-automated technology may aid in helping to efficiently determine Gram-negative loading of respiratory secretions, particularly in response to antibiotic intervention.     

Maintenance of pathogenicity during entry into and resuscitation from viable but nonculturable state in Aeromonas hydrophila exposed to natural seawater at low temperature

AIMS: To investigate the fate of Aeromonas hydrophila pathogenicity when cells switch, in nutrient-poor filtered sterilized seawater, between the culturable and nonculturable state. METHODS AND RESULTS: Aeromonas hydrophila ATCC 7966, rendered non culturable within 50-55 days of exposure to marine stress conditions, was tested for its ability to maintain haemolysin and to adhere to McCoy cells. Results showed that pathogenicity was lost concomitantly with culturability, whereas cell viability remained undamaged, as determined by the Kogure cell elongation test. However, this loss is only temporary because, following temperature shift from 5 to 23 degrees C, multiple biological activities of recovered Aer. hydrophila cells, which include their ability to lyse human erythrocytes and to attach and destroy McCoy cells were regained. During the temperature-induced resuscitation, constant total cell counts were observed. Moreover, no significant improvement in recovery yield was obtained on brain-heart infusion (BHI) agar plates amended with catalase. We suggest that in addition to the growth of the few undetected culturable cells, there is repair and growth of some mildly injured viable but nonculturable cells. CONCLUSIONS: The possibility that nonculturable cells of normally culturable Aer. hydrophila in natural marine environment may constitute a source of infectious diseases posing a public health problem was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments may mimic what happens when Aer. hydrophila cells are released in natural seawater with careful attention to the conditions in which surrounding waters gradually become warmer in late summer/early autumn.     

Is Urografin density gradient centrifugation suitable to separate nonculturable cells from <Emphasis Type="Italic">Escherichia coli</Emphasis> populations?

The ability of Urografin or Percoll density gradient centrifugations to separate nonculturable subpopulations from heterogeneous Escherichia coli populations was analysed. Bacterial counts (total, active and culturable cells) and flow cytometric analyses were carried out in all recovered bands. After Urografin centrifugation, and despite the different origin of E. coli populations, a common pattern was obtained. High-density bands were formed mainly by nonculturable cells. However, the increase in cell density would not be common to all nonculturable cells, since part of this subpopulations banded in low-density zones, mixed with culturable cells. Bands obtained after Percoll centrifugation were heterogeneous and culturable and nonculturable cells were recovered along the gradient. Thus, fractionation in Urografin cannot be only attributed to changes in buoyant densities during the transition from culturable to nonculturable state. Urografin density gradients allow us to obtain enriched fractions in nonculturable subpopulations from a heterogeneous population, but working conditions should be carefully chosen to avoid Urografin toxicity.     

Demonstration of viable but nonculturable Vibrio cholerae O1 in fresh water environment of India using ciprofloxacin DFA-DVC method

Aim: To demonstrate the presence of culturable and nonculturable viable pathogenic Vibrio cholerae O1 in fresh water environments of a cholera‐endemic region in India. Methods and Results: Conventional culture and ciprofloxacin DFA–DVC were utilized to investigate the existence of V. cholerae O1. We isolated pathogenic culturable V. cholerae O1 from water samples collected from cholera‐affected areas. No culturable V. cholerae O1 was isolated from water and plankton samples from natural fresh water bodies. Ciprofloxacin was used for DFA–DVC as V. cholerae O1 are 100% resistant to nalidixic acid in our region. The viable but nonculturable O1 cells were demonstrated in 2·21 and 40·69% samples from natural water bodies and cholera‐affected areas, respectively. Conclusion: Vibrio cholerae O1 VBNC could be demonstrated using modified DFA–DVC technique. Ciprofloxacin is preferable to nalidixic acid for DVC in view of existing high‐level resistance to nalidixic acid in cholera‐endemic areas. Significance and Impact of the study: We endorse that for public health surveillance, cholera outbreak investigation and disease control water samples in addition to culture should be tested for V. cholerae using DFA–DVC.     

Detection of culturable and nonculturable Legionella species from hot water systems of public buildings in Japan

Aims: To investigate the prevalence of culturable and nonculturable Legionella species in hot water systems of public buildings in Japan and assess the risk factors associated with Legionella contamination in hot water systems. Methods and Results: Legionella species were detected by conventional culture and molecular methods in 130 water samples collected from 40 buildings. A total of 26 (20·0%) water samples from 17 (42·5%) buildings were positive by culture, qualitative PCR or both methods: Legionella pneumophila and Leg. anisa were detected in four samples by a culture method, whereas 23 samples were positive by qualitative PCR, with the presence of various Legionella species confirmed by sequencing. Of these 23 samples, bacterial counts were quantifiable in 21 by real‐time PCR (from 1·7 × 10⁵ to 2·6 × 10¹¹ cells per litre). Phylogenetic analysis of amplified partial 16S rRNA gene showed close relations to various species of Legionella, including Leg. anisa and Leg. micdadei, all of which have been associated with respiratory diseases or increased antibody titres in human sera. Assessment of risk factors showed that turbidity, free chlorine concentration, iron concentration and heterotrophic plate count (HPC) were significantly associated with Legionella contamination (P < 0·05). Conclusions: Contamination of hot water systems of public buildings with culturable and nonculturable Legionella species may be a potential risk factor for Legionella infection in Japan. Adequate levels of chlorine, low levels of iron and HPC are important maintenance measures in the reduction of Legionella contamination in hot water systems. Significance and Impact of the Study: More than 40% of hot water systems in the Japanese public buildings examined were contaminated by not only culturable Leg. pneumophila and Leg. anisa but also by nonculturable pathogenic species. To our knowledge, this is the first report of both culturable and nonculturable Legionella contamination in hot water systems of public buildings in Japan.     

Examination of Recovery In Vitro and In Vivo of Nonculturable Escherichia coli O157:H7

Escherichia coli O157:H7 (strains ATCC 43895 and FO46) became nonculturable in sterile, distilled, deionized water or after exposure to chlorine. Recovery of nonculturable E. coli O157:H7 was examined by in vitro and in vivo methods. The decline in culturability of starved E. coli O157:H7 was measured by plate count on rich medium. Recovery in vitro of nonculturable cells was conducted with media amended with catalase or sodium pyruvate; however, there was no apparent increase over culturable cell counts on amended versus nonamended media. Although nonculturable E. coli O157:H7 did not recover under in vitro conditions, a mouse model was used to determine if in vivo conditions would provide sufficient conditions for recovery of nonculturable E. coli O157:H7. In separate studies, mice were orally challenged with starvation-induced nonculturable cells (FO46) or chlorine-induced nonculturable cells (43895 and FO46). Passage through the mouse gastrointestinal tract had no effect on recovery of nonculturable (starvation or chlorine induced) E. coli O157:H7 (43895 or FO46), based on analysis of fecal samples. Mouse kidneys were assayed for the presence of Shiga toxin using the Vero cell assay. Differences in cytotoxicity towards Vero cells from kidney samples of mice receiving nonculturable cells and control mice were not significant, suggesting a loss of virulence.     

Soil microbial counts and identification of culturable bacteria in an extreme by arid zone

Sixteen samples of two soil cores (about 550 and 180 cm in depth) were drilled at intervals in the lower reach of Heihe river basin (northwest of China) in order to illustrate soil microbial characteristics and diversity of culturable bacteria in an extreme by arid environment. Soil water content, organic matter, total nitrogen, pH, direct cell counts, and culturable microorganism counts were evaluated. The total cell concentration was 19-1120/microg (i.e. 0.19-11.2 x 10(8) per g) soil, the culturable bacteria count being 0.2-10.9 per microg (i.e. 2 x 10(5)-10.9 x 10(6) CFU/g) soil. The number of direct cell counts obtained by 4',6-diamidino-2-phenylindole-staining or the cound of culturable microbes after enrichment with different media were statistically significantly correlated with soil organic matters, total nitrogen content, soil water content and surface vegetation; this partly explained the larger number in the deeper first core than in the shallower one. As part of identification of 228 colonies isolated from the two cores, thirty-two were selected for 16S rDNA amplification, sequencing and molecular identification. These 32 isolates were affiliated to 5 major groups of bacteria: alpha-Proteobacteria, 5-Proteobacteria, gamma-Proteobacteria, the high-G+C G+-bacteria, the low-G+C G- -bacteria, and the Cytophaga-Flexibacter-Bacteroides group. Twenty-eight were rod- or short-rod shaped, which accounted for >87.5% of all species; only 4 of 32 species were cocci (<12.5%).     

Phylogenetic analysis and in situ identification of the intestinal microbial community of rainbow trout (Oncorhynchus mykiss, Walbaum)

AIMS: To identify the dominant culturable and nonculturable microbiota of rainbow trout intestine. METHODS AND RESULTS: Microbial density of rainbow trout intestine was estimated by direct microscopic counts (4',6-diamidino-2-phenylindole, DAPI) and by culturing on tryptone soya agar (TSA). Differential gradient gel electrophoresis analysis of bacterial DNA from intestinal samples, re-amplification of bands and sequence analysis was used to identify the bacteria that dominated samples where aerobic counts were < or =2% of the DAPI counts. 16S rDNA gene sequences of 146 bacterial isolates and three sequences of uncultured bacteria were identified. A set of oligonucleotide probes was constructed and used to detect and enumerate the bacterial community structure of the gastrointestinal tract of rainbow trout by fluorescence in situ hybridization (FISH). Members of the gamma subclass of Proteobacteria (mainly Aeromonas and Enterobacteriaceae) dominated the bacterial population structure. Acinetobacter, Pseudomonas, Shewanella, Plesiomonas and Proteus were also identified together with isolates belonging to the beta subclass of Proteobacteria and Gram-positive bacteria with high and low DNA G + C content. In most samples, the aerobic count (on TSA) was 50-90% of the direct (DAPI) count. A bacterium representing a previously unknown phylogenetic lineage with only 89% 16S rRNA gene sequence similarity to Anaerofilum pentosovorans was detected in intestinal samples where aerobic counts were < or =2% of direct (DAPI) counts. Ten to 75% of the microbial population in samples with low aerobic counts hybridized (FISH) with a probe constructed against this not-yet cultured bacterium. CONCLUSIONS: Proteobacteria belonging to the gamma subclass dominated the intestinal microbiota of rainbow trout. However, in some samples the microflora was dominated by uncultivated, presumed anaerobic, micro-organisms. The bacterial population structure of rainbow trout intestine, as well as total bacterial counts, varied from fish to fish. SIGNIFICANCE AND IMPACT OF THE STUDY: Good correlation was seen between cultivation results and in situ analysis, however, a molecular approach was crucial for the identification of organisms uncultivated on TSA.     

Fate of Enterobacter cloacae JP120 and Alcaligenes eutrophus AEO106(pRO101) in soil during water stress: effects on culturability and viability.

A sandy loam soil near field capacity moisture content (psi = -0.050 MPa) or air dried (psi = -300 MPa) was inoculated with about 3 x 10(7) CFU of Enterobacter cloacae JP120 and Alcaligenes eutrophus AEO106(pRO101) per g and incubated in 40-g portions at 17 degrees C in closed or open Erlenmeyer flasks. In the field-moist soil, selective plating, direct viable counts, and DNA hybridization showed only minor changes in the numbers of E. cloacae and A. eutrophus cells with time (14 days), and the results obtained with the three detection methods generally agreed. In the air-dried soil, the majority of both bacteria were found as intact DNA-carrying cells that were neither culturable nor viable by the methods employed in this study. The numbers of culturable E. cloacae and A. eutrophus cells dropped to 10(5) and 10(2) CFU/g, respectively, 2 h after inoculation. Direct viable counts showed that only about 1% of the cells detected by immunofluorescence microscopy were viable, but a fraction of viable nonculturable cells of both bacteria was present. A. eutrophus did not tolerate desiccation as well as E. cloacae. Only a minor fraction of the two test organisms regained their culturability or viability after rewetting of the air-dried soil; the number of total heterotrophic culturable bacteria, however, increased more than 10-fold and reached 73% of the level found in the field-moist soil at day 14.     

Survival and gene expression of enterotoxigenic Escherichia coli during long‐term incubation in sea water and freshwater

Aims: In this study, the main objective was to verify the hypothesis of induction of ‘viable but non‐culturable’ (VBNC) forms of enterotoxigenic Escherichia coli (ETEC) during incubation in water. Methods and Results: Six clinically isolated ETEC strains were studied. Viable counts showed culturable ETEC bacteria for up to 3 months in freshwater but only two out of six strains were culturable in seawater at this time point. Although the bacterial cells remained intact, no production or secretion of heat‐labile (LT) or heat‐stable (ST) enterotoxins was observed using GM1‐ELISA methods. However, genes encoding ETEC toxins (STh and LT), colonization factors (CS7 and CS17), gapA and 16S RNA were expressed during 3 months in both sea water and freshwater microcosms as determined by real‐time RT‐PCR on cDNA derived from the bacteria. Conclusions: Clinically isolated ETEC strains can survive for long periods in both sea water and freshwater. The bacterial cells remain intact, and the gene expression of virulence genes and genes involved in metabolic pathways are detected after 3 months. Significance and Impact of the Study: These results indicate that ETEC bacteria can enter a VBNC state during stressful conditions and suggest that ETEC has the potential to be infectious after long‐term incubation in water.     

Predominance of Nonculturable Cells of the Biocontrol Strain Pseudomonas fluorescens CHA0 in the Surface Horizon of Large Outdoor Lysimeters

The persistence of the biocontrol agent Pseudomonas fluorescens CHA0 in the surface horizon of 12 large outdoor lysimeters planted with winter wheat, Phacelia tanacetifolia followed by spring wheat, or maize was monitored for 1 year. Soil was inoculated with a spontaneous rifampin-resistant mutant (CHA0-Rif) of CHA0, and the strain was studied by using colony counts, Kogure's direct viable counts, and total counts (immunofluorescence). The number of culturable cells of the inoculant decreased progressively from 8 to 2 log CFU/g of soil or lower. However, culturable cells of CHA0-Rif accounted for less than 1% of the total cells of the inoculant 8 months after release in autumn. Since viable but nonculturable cells represented less than a quarter of the latter, most cells of CHA0-Rif in soil were thus inactive-dormant or dead at that time. Nonculturable cells of the inoculant were predominant also in the surface horizon of the lysimeters inoculated in the spring, and a significant fraction of them were viable. Results suggest that the occurrence of nonculturable cells of CHA0-Rif was influenced by climatic factors (water availability and soil temperature) and the abundance of roots in soil. The fact that the inoculant persisted as mixed populations of cells of different physiological states, in which nonculturable cells were predominant, needs to be taken into account when assessing the autecology of wild-type or genetically modified pseudomonads released into the soil ecosystem.     

Survival of Aeromonas salmonicida in lake water

The survival of Aeromonas salmonicida subsp. salmonicida in lake water was investigated by using a variety of techniques. They included acridine orange epifluorescence, respiration, cell culture, cell revival, flow cytometry, plasmid maintenance, and membrane fatty acid analysis. During a 21-day study, A. salmonicida became nonculturable in sterile lake water samples. Flow cytometry and direct microscopy indicated that cells were present. Although the nonculturable cells could not be revived, the recovery method did indicate that the presence of low numbers of culturable cells within samples could produce misleading results. Plasmid DNA, genomic DNA, and RNA were maintained in the nonculturable cells; in addition, changes in the fatty acid profiles were also detected. Although viability could not be proven, it was shown that the morphological integrity of nonculturable cells was maintained.     

Survival of Aeromonas salmonicida in lake water.

The survival of Aeromonas salmonicida subsp. salmonicida in lake water was investigated by using a variety of techniques. They included acridine orange epifluorescence, respiration, cell culture, cell revival, flow cytometry, plasmid maintenance, and membrane fatty acid analysis. During a 21-day study, A. salmonicida became nonculturable in sterile lake water samples. Flow cytometry and direct microscopy indicated that cells were present. Although the nonculturable cells could not be revived, the recovery method did indicate that the presence of low numbers of culturable cells within samples could produce misleading results. Plasmid DNA, genomic DNA, and RNA were maintained in the nonculturable cells; in addition, changes in the fatty acid profiles were also detected. Although viability could not be proven, it was shown that the morphological integrity of nonculturable cells was maintained.     

Distribution of viruses in the Chesapeake Bay.

High virus counts were found in water samples collected from the Chesapeake Bay. Viruses were enumerated by ultracentrifugation of water samples onto grids which were visualized by transmission electron microscopy. Virus counts in September 1990, April 1991, June 1991, August 1991, and October 1991 ranged between 2.6 x 10(6) and 1.4 x 10(8) viruses ml-1 with a mean of 2.5 x 10(7) viruses ml-1. Virus counts were usually at least three times higher than direct bacterial counts in corresponding samples. Virus counts in August and October were significantly higher than at the other sampling times, whereas bacterial counts were significantly lower at that time, yielding mean virus-to-bacterium ratios of 12.6 and 25.6, respectively. From analysis of morphology of the virus particles, it is concluded that a large proportion of the viruses are bacteriophages. The high virus counts obtained in this study suggest that viruses may be an important factor affecting bacterial populations in the Chesapeake Bay, with implications for gene transfer in natural aquatic bacterial populations and release of genetically engineered microorganisms to estuarine and coastal environments.     

Distribution of viruses in the Chesapeake Bay.

High virus counts were found in water samples collected from the Chesapeake Bay. Viruses were enumerated by ultracentrifugation of water samples onto grids which were visualized by transmission electron microscopy. Virus counts in September 1990, April 1991, June 1991, August 1991, and October 1991 ranged between 2.6 x 10(6) and 1.4 x 10(8) viruses ml-1 with a mean of 2.5 x 10(7) viruses ml-1. Virus counts were usually at least three times higher than direct bacterial counts in corresponding samples. Virus counts in August and October were significantly higher than at the other sampling times, whereas bacterial counts were significantly lower at that time, yielding mean virus-to-bacterium ratios of 12.6 and 25.6, respectively. From analysis of morphology of the virus particles, it is concluded that a large proportion of the viruses are bacteriophages. The high virus counts obtained in this study suggest that viruses may be an important factor affecting bacterial populations in the Chesapeake Bay, with implications for gene transfer in natural aquatic bacterial populations and release of genetically engineered microorganisms to estuarine and coastal environments.