Improved Direct Viable Count Procedure for Quantitative Estimation of Bacterial Viability in Freshwater Environments

A direct viable count (DVC) procedure was developed which clearly and easily discriminates the viability of bacterial cells. In this quantitative DVC (qDVC) procedure, viable cells are selectively lysed by spheroplast formation caused by incubation with antibiotics and glycine. This glycine effect leads to swollen cells with a very loose cell wall. The viable cells then are lysed easily by a single freeze-thaw treatment. The number of viable cells was obtained by subtracting the number of remaining cells after the qDVC procedure from the total cell number before the qDVC incubation. This improved procedure should provide useful information about the metabolic potential of natural bacterial communities.     

Improved direct viable count procedure for quantitative estimation of bacterial viability in freshwater environments

A direct viable count (DVC) procedure was developed which clearly and easily discriminates the viability of bacterial cells. In this quantitative DVC (qDVC) procedure, viable cells are selectively lysed by spheroplast formation caused by incubation with antibiotics and glycine. This glycine effect leads to swollen cells with a very loose cell wall. The viable cells then are lysed easily by a single freeze-thaw treatment. The number of viable cells was obtained by subtracting the number of remaining cells after the qDVC procedure from the total cell number before the qDVC incubation. This improved procedure should provide useful information about the metabolic potential of natural bacterial communities.     

Enumeration and characterization of bacteria in mineral water by improved direct viable count method

Fifteen strains from two emergent mineral waters were isolated and tentatively identified with API 20NE and BIOLOG GN systems. These strains were screened for their sensitivities to seven replication-inhibiting antibiotics of the (fluoro)quinolone group (nalidixic and pipemidic acid, flumequine, norfloxacin, ofloxacin, pefloxacin and ciprofloxacin). It was shown that the direct viable count (DVC) procedure could be improved by using certain antibiotic cocktails, which were active against the isolates. Geometric bacterial features were successfully determined with image analysis and adapted software (ICONIX, Perfect Image). Elongations were significant and allowed rapid discrimination of antibiotic inhibited and non-inhibited strains. Particular isolates in a mixed culture were characterized and enumerated after only 14 h exposure with the appropriate antibiotic cocktail. This method can also be applied to other communities, such as mixed cultures in bio-fermentors or in food with known microflora.     

Ecological Application of Antibiotics as Respiratory Inhibitors of Bacterial Populations

Two terregenous and four marine bacterial isolates were treated with six antibiotics and antibiotic combinations. Comparisons made between responses of cells in early and late logarithmic and stationary growth phases indicated variable sensitivity to the agents. Bacteria in stationary and late log-phase cultures exhibited the greatest resistance, whereas the early log-phase cells exhibited greatest antibiotic susceptibility. We conclude that the tested antibiotics cannot be used for ecological purposes to delineate bacterial respiration in mixed microbial communities.     

The application of viable count procedures for measuring viable cells in the various marine environments

Aims: To investigate whether the use of direct viable count (DVC), quantitative viable count (qDVC), colony‐forming units and the contribution of capsule‐bearing bacteria to the total number of bacteria and esterase‐active bacteria could be used to clearly differentiate viable cells in various trophic status of seawater. Methods and Results: Hundred and four marine isolates from various marine environments in Turkey (Western Black Sea, northern part of the Sea of Marmara, Northern Aegean Sea and eastern part of the Sea of Marmara) were screened. Seawater samples were taken from the surface (the upper 0–30 cm) and deeper layers (from 5 to 500 m) of the sea at different time periods between February 2002 and June 2007. For the assessment of cell elongation, minor modifications were made on DVC procedure in order to optimize the concentration of yeast extract and incubation time for enumeration of bacteria in response to nutrient addition. The best results were obtained when the yeast extract was used at a final concentration of 250 mg l^?1 (at 35°C 24 h incubation) for bacteria isolated from eutrophic areas and a final concentration of 50 mg l^?1 for those selected from oligotrophic areas. A positive correlation was found between the trophic level and the level of metabolically active bacteria. Among these methods, the bacterial number obtained by qDVC is higher than those gained by other methods. Conclusions: The results indicate that the qDVC procedure could easily differentiate between viable cells and dormant or dead cells. We suggest that this method may be applicable to detecting the level of metabolic potential of bacterial communities in marine environments. Significance and Impact of the Study: The study resulted in increased knowledge on the applicability of the qDVC method that arranges the substrate amount and incubation time as well as on the comparison of various viable bacteria count procedures related to trophic situation of seawater samples.     

Exchange of chromosomal markers by natural transformation between the soil isolate,Pseudomonas stutzeri JM300, and the marine isolate,Pseudomonas stutzeri strain ZoBell

Both the soil isolate,Pseudomonas stutzeri JM300, and the marine isolate,Pseudomonas stutzeri strain ZoBell, have been shown previously to be naturally transformable. This study reports the detection of genetic exchange by natural transformation between these two isolates. Transformation frequency was determined by filter transformation procedures. Three independent antibiotic resistance loci were used as chromosomal markers to monitor this exchange event: resistance to rifampicin, streptomycin, and nalidixic acid. The maximum frequencies of transformation were on the order of 3.1 to 3.8×10^-6 transformants per recipient; frequencies over an order of magnitude greater than those for spontaneous antibiotic resistance, although they are lower than those observed for soil: soil or marine: marine strain crosses. This exchange was inhibited by DNase I. Transformation was observed between soil and marine strains, both by filter transformation using purified DNA solutions and when transforming DNA was added in the form of viable donor cells. The results from this study support the close genetic relationship betweenP. stutzeri JM300 andP. stutzeri strain ZoBell. These results also further validate the utility ofP. stutzeri as a benchmark organism for modeling gene transfer by natural transformation in both soil and marine habitats.     

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.     

Development of a direct viable count procedure for some Gram-positive bacteria

The direct viable count (DVC) is a procedure for enumerating viable-nonculturable cells. It should be noted, however, that bacteria demonstrating the viable but nonculturable phase have to date included only Gram-negative species, mainly because the DVC procedure does not lend itself to the analysis of Gram-positive bacteria since the DVC procedure is dependent on the bacterium being sensitive to nalidixic acid. The authors report here concerning studies on an analogous procedure for the direct enumeration of viable-nonculturable Gram-positive bacteria.
To facilitate a differential DVC for Gram-positive bacteria, ciprofloxacin, enoxacin, norfloxacin or isopropyl cinodine were substituted for nalidixic acid. These antibiotics were chosen because, like nalidixic acid, they are DNA gyrase inhibitors. The concentrations used for each antibiotic were 1000 μg ml^-1, 100 μg ml^-1 and 10 mg ml^-1. Pure cultures of Staphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae, Listeria monocytogenes and Bacillus subtilis were obtained from the culture collection at the University of Wyoming and a faecal streptococcus was isolated from the Laramie wastewater treatment plant. An antibiotic and optimal concentration thereof was found which gave enlarged cells for all the organisms except the faecal streptococcus isolated from the wastewater plant for which no enlarged cells were ever seen. The antibiotic and concentration thereof which gave the optimal percent enlarged cells in the DVC procedure varied between organisms.     

Impact of an urban effluent on antibiotic resistance of riverine Enterobacteriaceae and Aeromonas spp

In order to evaluate the impact of an urban effluent on antibiotic resistance of freshwater bacterial populations, water samples were collected from the Arga river (Spain), upstream and downstream from the wastewater discharge of the city of Pamplona. Strains of Enterobacteriaceae (representative of the human and animal commensal flora) (110 isolates) and Aeromonas (typically waterborne bacteria) (118 isolates) were selected for antibiotic susceptibility testing. Most of the Aeromonas strains (72%) and many of the Enterobacteriaceae (20%) were resistant to nalidixic acid. Singly nalidixic acid-resistant strains were frequent regardless of the sampling site for Aeromonas, whereas they were more common upstream from the discharge for enterobacteria. The most common resistances to antibiotics other than quinolones were to tetracycline (24.3%) and beta-lactams (20.5%) for Enterobacteriaceae and to tetracycline (27.5%) and co-trimoxazole (26.6%) for Aeromonas. The rates of these antibiotic resistances increased downstream from the discharge at similar degrees for the two bacterial groups; it remained at high levels for enterobacteria but decreased along the 30-km study zone for Aeromonas. Genetic analysis of representative strains demonstrated that these resistances were mostly (enterobacteria) or exclusively (Aeromonas) chromosomally mediated. Moreover, a reference strain of Aeromonas caviae (CIP 7616) could not be transformed with conjugative R plasmids of enterobacteria. Thus, the urban effluent resulted in an increase of the rates of resistance to antibiotics other than quinolones in the riverine bacterial populations, despite limited genetic exchanges between enterobacteria and Aeromonas. Quinolone resistance probably was selected by heavy antibiotic discharges of unknown origin upstream from the urban effluent.     

Determination of viability within serotypes of a soil population of Rhizobium leguminosarum bv. trifolii.

Concern has been raised about the percentage of viable cells within soil rhizobia populations measured by the immunofluorescence direct count method. The purpose of this study was to evaluate a direct viable count technique which is based on the fact that viable bacteria in natural populations undergo cell elongation when they are exposed to a combination of substrate and the inhibitor of DNA gyrase, nalidixic acid. A soil extraction procedure was developed to recover a high proportion of soil bacteria (ca. 10(9)/g of soil) in suspensions with an optical clarity suitable for accurate microscopic enumeration. After incubation for 16 to 20 h at 27 degrees C in the presence of yeast extract (200 mg/liter) and nalidixic acid (10 mg/liter), between 65 and 74% of the bacteria in soil suspension became significantly elongated (greater than or equal to 4.2 microns). In contrast, less than or equal to 0.5% of the same population could be cultured, regardless of the medium composition, nutrient concentration, or incubation conditions. The direct viable count method was combined with immunofluorescence to compare the percent viability and kinetics of appearance of elongated cells within serotypes of a soil population of Rhizobium leguminosarum bv. trifolii. Although the majority of these organisms were viable, as observed by immunofluorescence, we obtained evidence that subpopulations within the soil rhizobia community were in different states of competence to respond to substrate. A consistently low percentage (less than or equal to 30%) of the population of serotype 23 was elongated even after 24 h of incubation and regardless of when the soil was sampled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of viability within serotypes of a soil population of Rhizobium leguminosarum bv. trifolii

Concern has been raised about the percentage of viable cells within soil rhizobia populations measured by the immunofluorescence direct count method. The purpose of this study was to evaluate a direct viable count technique which is based on the fact that viable bacteria in natural populations undergo cell elongation when they are exposed to a combination of substrate and the inhibitor of DNA gyrase, nalidixic acid. A soil extraction procedure was developed to recover a high proportion of soil bacteria (ca. 10(9)/g of soil) in suspensions with an optical clarity suitable for accurate microscopic enumeration. After incubation for 16 to 20 h at 27 degrees C in the presence of yeast extract (200 mg/liter) and nalidixic acid (10 mg/liter), between 65 and 74% of the bacteria in soil suspension became significantly elongated (greater than or equal to 4.2 microns). In contrast, less than or equal to 0.5% of the same population could be cultured, regardless of the medium composition, nutrient concentration, or incubation conditions. The direct viable count method was combined with immunofluorescence to compare the percent viability and kinetics of appearance of elongated cells within serotypes of a soil population of Rhizobium leguminosarum bv. trifolii. Although the majority of these organisms were viable, as observed by immunofluorescence, we obtained evidence that subpopulations within the soil rhizobia community were in different states of competence to respond to substrate. A consistently low percentage (less than or equal to 30%) of the population of serotype 23 was elongated even after 24 h of incubation and regardless of when the soil was sampled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microbial antagonism: a neglected avenue of natural products research

Competition amongst microbes for space and nutrients in the marine environment is a powerful selective force which has led to the evolution of a variety of effective strategies for colonising and growing on surfaces. We are particularly interested in the chemical ecology of marine epibiotic bacteria which live on the surfaces of marine algae or invertebrates. Over 400 strains of surface-associated bacteria from various species of seaweed and invertebrate from Scottish coastal waters were isolated and 35% of them shown to produce antimicrobial compounds. This is a much higher proportion than free living marine isolates or soil bacteria. In addition, many strains which did not normally produce antibiotics could be induced to do so by exposing them to small amounts of live cells, supernatants from other bacterial cultures or other chemicals. Thus the number of strains able to produce antibiotics appears to be much higher than previously thought. Induction of antibiotic production was elicited by other marine epibionts and also by terrestrial human pathogens such as Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. An understanding of this type of chemical induction and the factors regulating non-constitutive secretion of antimicrobial compounds will allow more effective strategies for searching for new chemotherapeutic antibiotics to be designed.     

Metal tolerance and antibiotic resistance patterns of a bacterial population isolated from sea water

The total aerobic heterotrophic and metal-resistant bacterial communities were studied in marine water. The resistance patterns, expressed as MICs, for 81 bacterial isolates to eight heavy metals were surveyed by using the agar dilution method. A great proportion of the isolates were sensitive to cadmium (99%), mercury (91%), zinc (84%) and cobalt (83%). On the other hand, 94%, 40%, 35% and 22% were resistant to lead, nickel, arsenate and copper, respectively. The majority of the tested strains (95.06%) were multiple metal-resistant, with pentametal resistance as the major pattern (25.9%). The response of the isolates to 11 tested antibiotics was tested and ranged from complete resistance to total sensitivity and multiple antibiotic resistance was exhibited by 70.38% of the total isolated population. The highest incidence of metal-antibiotic double resistance existed between lead and all antibiotics (100%), copper and penicillin (95%) and nickel and ampicillin (83.3%).     

Nucleic acids from the host bacterium as a major source of nucleotides for three marine bacteriophages

Abstract The incorporation of ³²P-phosphorus into marine bacteriophage nucleic acid was studied in culture experiments to investigate the source of nucleotides used by the phage. We consistently found that the ³²P-specific activity in the phage genome increased during the 11 h incubation and was low relative to the specific activity in the medium, averaging 21% (±SD 5.9) for the three phage isolates. This was in accordance with a mathematical model where most of the nucleotides for phage DNA synthesis were derived from the host cell nucleic acid rather than de novo synthesis. We propose that this metabolic strategy may be common among marine phages, as an adaptation to a nutrient poor environment. Consequently, the contribution of free DNA to the dissolved fraction through phage lysis of bacteria, may be less that previously thought. Also during radiolabelling of bacteriophages in natural water samples, isotope dilution may be dependent on the specific growth rate of the bacterial host.     

Rapid detection of chlorine-induced bacterial injury by the direct viable count method using image analysis.

A modified direct viable count method to detect living bacteria was used with image analysis for the rapid enumeration of chlorine-injured cells in an Escherichia coli culture. The method was also used for determining chlorine-induced injury in coliform isolates and enteric pathogenic bacteria. Cultures were incubated in phosphate-buffered saline, containing 0.3% Casamino Acids (Difco Laboratories, Detroit, Mich.), 0.03% yeast extract, and optimal concentrations of nalidixic acid. Samples were withdrawn before and after incubation and stained with acridine orange, and cell lengths and breadths were measured by computerized image analysis. After incubation, cells which exceeded the mean preincubation length (viable cells) were enumerated and the results were compared with those obtained by the plate count method. Injury in the chlorine-exposed cell population was determined from the difference in viable count obtained with a nonselective Casamino Acids-yeast extract-nalidixic acid medium and a selective Casamino Acids-yeast extract-nalidixic acid medium containing sodium deoxycholate or sodium lauryl sulfate. The levels of injury determined by the direct viable count technique by using image analysis were comparable to those determined by the plate count method. The results showed that image analysis, under optimal conditions, enumerated significantly higher numbers of stressed E. coli than the plate count method did and detected injury in various cultures in 4 to 6 h.     

Development of a direct viable count procedure for the investigation of VBNC state in Listeria monocytogenes

A viable but non-culturable (VBNC) bacterial state was originally detected in studies in environmental microbiology. In particular, this state has been demonstrated for a number of human pathogens (Escherichia coli, Salmonella enteritidis, Vibrio cholerae, Legionella pneumophila and Campylobacter jejuni). The presence of VBNC cells poses a major public health problem since they cannot be detected by traditional culturing methods and the cells remain potentially pathogenic under favourable conditions. But, as far as we know, the VBNC state has not been yet described in Listeria monocytogenes. In most studies, this has been assessed by the Kogure procedure based on cellular elongation in the presence of DNA gyrase inhibitors. The antibiotic used was nalidixic acid in order to prevent DNA replication, only efficient in Gram-negative bacteria studies. In this study, we describe a new DVC procedure to detect and count viable of L. monocytogenes suspended in filtered, sterilized distilled water. We used different concentrations of ciprofloxacin, efficient both in Gram-negative and Gram-positive bacteria. Bacteria cells were removed and resuspended in BHI broth, with yeast extract and ciprofloxacin. The mixture was incubated at different incubation times at 37 degrees C. After different incubation times, cells were filtered through an isopore polycarbonate black membrane filter and covered with a DAPI solution or orange acridine. The filters were prepared and examined by epifluorescence microscopy. Elongated cells were counted as viable cells, whereas normal size was regarded as nonactive ones. This method allows determination of ciprofloxacin concentration and incubation time optimal to detect maximum viable cells percentage in L. monocytogenes.     

Intergeneric natural plasmid transformation between E. coli and a marine Vibrio species

Natural transformation is the mechanism of procaryotic gene transfer that involves the uptake and expression of genetic information encoded in extracellular DNA. This process has been regarded as a mechanism to transfer genes (primarily chromosomal markers) between closely related strains or species. Here we demonstrate the cell-contact-dependent transfer of a non-conjugative plasmid from a laboratory E. coli strain to a marine Vibrio species, the first report of intergeneric natural plasmid transformation involving a marine bacterium. The nucleic acid synthesis inhibitors nalidixic acid and rifampicin inhibited the ability of the E. coli to function as a donor. However, dead cells also served as efficient donors. There was an obligate requirement for cell contact. No transfer occurred in the presence of DNase I, when donors and recipients were separated by a 0.2-micron filter, or when spent medium alone was used as a source of transforming DNA. These results indicate that contact-mediated intergeneric plasmid exchange can occur in the absence of detectable viable donor cells and that small non-conjugative plasmids can be spread through heterogeneous microbial communities by a process previously not recognized, natural plasmid transformation. These findings are important in the assessment of genetic risk to the environment, particularly from wastewater treatment systems and the use of genetically engineered organisms in the environment.     

Rapid detection of chlorine-induced bacterial injury by the direct viable count method using image analysis

A modified direct viable count method to detect living bacteria was used with image analysis for the rapid enumeration of chlorine-injured cells in an Escherichia coli culture. The method was also used for determining chlorine-induced injury in coliform isolates and enteric pathogenic bacteria. Cultures were incubated in phosphate-buffered saline, containing 0.3% Casamino Acids (Difco Laboratories, Detroit, Mich.), 0.03% yeast extract, and optimal concentrations of nalidixic acid. Samples were withdrawn before and after incubation and stained with acridine orange, and cell lengths and breadths were measured by computerized image analysis. After incubation, cells which exceeded the mean preincubation length (viable cells) were enumerated and the results were compared with those obtained by the plate count method. Injury in the chlorine-exposed cell population was determined from the difference in viable count obtained with a nonselective Casamino Acids-yeast extract-nalidixic acid medium and a selective Casamino Acids-yeast extract-nalidixic acid medium containing sodium deoxycholate or sodium lauryl sulfate. The levels of injury determined by the direct viable count technique by using image analysis were comparable to those determined by the plate count method. The results showed that image analysis, under optimal conditions, enumerated significantly higher numbers of stressed E. coli than the plate count method did and detected injury in various cultures in 4 to 6 h.     

In-situ enumeration and probing of pyrene-degrading soil bacteria

Inferences about which microorganisms degrade polycyclic aromatic hydrocarbons in contaminated soils have largely been obtained using culture-based techniques, despite the low percentage of microorganisms in soil that are believed to be culturable. We used a substrate-responsive direct viable count method to identify and quantify potential polycyclic aromatic hydrocarbon-degrading bacteria in a soil containing petroleum wastes. Bacteria were extracted and their response to substrates determined in the presence of DNA gyrase inhibitors, which cause viable and active cells to elongate. When yeast extract, a widely used carbon source, was added as a growth substrate, together with nalidixic acid, piromidic acid and ciprofloxacin, a significant increase in elongated cells to 47%, 37% and 22%, respectively, was observed within 24 h. With pyrene as the main substrate, 10 mg L(-1) of nalidixic acid or piromidic acid caused 18-22% and 8-12%, respectively, of the cells to elongate within 24 h; whereas the effect of 0.5 mg L(-1) ciprofloxacin was not significant until 53 h later. Enlarged cells were identified and enumerated by fluorescent in situ hybridization, using Alpha-, Beta- and Gammaproteobacteria, and domain Bacteria-specific probes. The Bacteria-specific probe detected 35-71% of the total microorganisms detected by the DNA-binding dye 4,6-diamidino-2-phenylindole. Initially, 44%, 13% and 5% of the total bacteria in the soil extract were Alpha-, Beta- and Gammaproteobacteria, respectively. Without pyrene or a gyrase inhibitor, these subgroups decreased to 30% of the total population but were predominant with piromidic acid or unchanged with ciprofloxacin when pyrene was the main substrate. The proportion of elongated Alpha- and Betaproteobacteria (potential pyrene degraders) increased significantly (P<0.05). This approach links phylogenetic information with physiological function in situ without the conventional cultivation of bacteria and can be used to probe and enumerate degradative groups at even a finer level of discrimination.     

Resistance to copper ions and antibiotics in marine heterotrophic bacteria in the coastal waters of Vietnam

Copper and antibiotic resistance was experimentally studied for the first time in marine heterotrophic bacteria that were isolated from microfoulings of copper and copper-alloy test plates in coastal waters of Vietnam. Resistance to copper ions and to at least one of the antibiotics tested was detected in 78.7% of the isolates. A total of 28 models of antibiotic resistance were found in the bacteria. All strains isolated from the foulings of the copper plates were resistant to seven or more antimicrobials. The microfouling communities of copper and copper-alloy plates were dominated by Bacillus spp. and Staphylococcus spp. Copper and antibiotic resistance was statistically independent of the taxon of the tested bacteria.