Isolation of Legionella species from drinking water

Three different species of Legionella were recovered from samples of water taken from chlorinated public water supplies where no coliform bacteria were simultaneously detected. Five of 856 samples yielded Legionella isolates. Three isolates were identified as Legionella pneumophila serogroup 1, the fourth was identified as Legionella dumoffii, and the fifth was identified as Legionella jordanis. Studies to determine the survival of L. pneumophila Flint 1 serogroup 1 in tap water at various temperatures and in tap water with added sodium hypochlorite were done. These organisms were found to survive for 299 days in tap water at 24 and 5 degrees C but not at 35 degrees C. A concentration of at least 0.2 mg of residual chlorine per ml was required to eliminate at least 90% of L. pneumophila and Escherichia coli inocula in 2 h.     

Isolation of Legionella pneumophila from water systems: methods and preliminary results.

A preliminary survey of water systems in hospitals and hotels showed that Legionella pneumophila may be found in water storage and distribution systems as well as in the recirculating cooling water of air-conditioning plants. Altogether 42 isolates of L pneumophila were made from 31 establishments, six of which were associated with cases of legionnaires'' disease but in 25 of which there was no known association with disease. In the six establishments implicated epidemiologically as the source of legionnaires'' disease, these organisms were found in each of their water-distribution systems and also in the cooling water from each of the three with cooling towers. In establishments not associated with cases, water from three out of nine cooling towers, four out of 24 taps or showers, and one out of 15 storage tanks was found to contain legionellae. The organisms were isolated by guinea-pig inoculation and subsequent culture of their peritoneal fluid, liver, and spleen. Finding L pneumophila in water systems in the absence of cases of legionnaires'' disease should not at present be an indication for attempts at eradication.     

Isolation of Legionella species from drinking water.

Three different species of Legionella were recovered from samples of water taken from chlorinated public water supplies where no coliform bacteria were simultaneously detected. Five of 856 samples yielded Legionella isolates. Three isolates were identified as Legionella pneumophila serogroup 1, the fourth was identified as Legionella dumoffii, and the fifth was identified as Legionella jordanis. Studies to determine the survival of L. pneumophila Flint 1 serogroup 1 in tap water at various temperatures and in tap water with added sodium hypochlorite were done. These organisms were found to survive for 299 days in tap water at 24 and 5 degrees C but not at 35 degrees C. A concentration of at least 0.2 mg of residual chlorine per ml was required to eliminate at least 90% of L. pneumophila and Escherichia coli inocula in 2 h.     

Isolation of mycobacteria from acidic forest soil samples: comparison of culture methods

To evaluate which combination of decontamination method and medium is most reliable when examining acidic, organic forest soils for mycobacteria, three decontamination methods and five media supplemented with cycloheximide were compared. Before decontamination, the samples were incubated at 37°C for 5 h to allow germination of microbial spores. The recovery of mycobacteria was significantly influenced both by the method and by medium. Decontamination with NaOH or H₂SO₄ both combined with malachite green and cycloheximide yielded higher viable counts of mycobacteria than decontamination with NaOH followed by oxalic acid. Egg media at pH 5·5 resulted in lower mycobacterial counts than egg media at pH 6·5 or Mycobacteria 7H11 agar. The numbers of slopes totally free of contaminants revealed Mycobacteria 7H11 agar medium to be more prone to contamination than the four egg media tested. The highest counts of mycobacteria and a low rate of contamination were obtained when decontamination with NaOH-malachite green–cycloheximide was combined with culture on glycerol and cycloheximide supplemented egg medium at pH 6·5.     

Detection and quantification of Legionella pneumophila in water samples using competitive PCR

The presence of high levels of Legionella pneumophila in man-made aquatic systems correlates with the incidence of nosocomial Legionnaires' disease. This requires a rapid, reliable, and sensitive quantification of L. pneumophila concentrations in suspected water systems. In this research, a homologous competitor was developed and evaluated in a L. pneumophila competitive polymerase chain reaction (cPCR) to quantify this human pathogen in a quick, cost-effective, and reliable way. Accuracy of cPCR was evaluated by analyzing cooling tower and tap water samples spiked with known concentrations of L. pneumophila bacteria, in parallel with the standard culture method. Legionella pneumophila amounts detected and calculated from cPCR and culture correlated very well: r = 0.998, P = 0.002 for tap water and r = 0.990, P = 0.009 for cooling tower water. Nevertheless, for both kinds of water samples, mean numbers of L. pneumophila calculated from cPCR results were always higher than those obtained by culture. This study makes it clear that the rapid, sensitive, and cost-effective L. pneumophila cPCR is a promising alternative to the standard time-consuming culture method and expensive real-time PCR to enumerate L. pneumophila bacteria in environmental water samples.     

Comparison of conventional culture and PCR methods for the detection of Legionella pneumophila in water

A comparative assessment of conventional culture and nucleic acid techniques in the detection of Legionella pneumophila in seeded tap water samples was performed, using bacterial concentrations ranging from 994 to 0·015 cfu ml⁻¹. Different filtration and centrifugation protocols were evaluated. The results permitted the development of a tentative algorithm for the detection of legionellae in tap water. Samples should first be analysed using PCR methods. In the event of quantitative data and bacterial strains for epidemiologic typing being required, the same sample, or a greater volume of the sample, if positive with PCR, can be re-tested by filtration through polycarbonate membranes followed by plating a homogenate of the filter. If samples are found to be negative with PCR, they can be re-analysed in greater volumes by filtration through polycarbonate membranes followed by direct placing of the filter on culture media, to allow detection of very low numbers of bacteria. This protocol should be validated in the field before it can be routinely implemented.     

Postantibiotic effect of various antibiotics on Legionella pneumophila strains isolated from water systems

The postantibiotic effects (PAE) of azithromycin, clarithromycin, ciprofloxacin, and levofloxacin were investigated against Legionella pneumophila (L. pneumophila) strains isolated from several hot water systems of different buildings in Istanbul. Each strain in logarithmic phase of growth was exposed to concentrations of antibiotics equal to minimum inhibitory concentration (MIC) and 4× MIC for 1?h. Recovery periods of test cultures were evaluated after centrifugation using the viable counting method. The mean values of PAEs for the strains of L. pneumophila, azithromycin at a concentration equal to and 4 times of MIC values were found 1.75?±?0.28 h and 4.06?±?0.44?h, for clarithromycin 2.98?±?0.70?h and 4.18?±?0.95?h, for ciprofloxacin 2.97?±?0.63?h and 4.70?±?0.63?h, for levofloxacin 2.05?±?0.33?h and 3.78?±?0.46?h, respectively. All of the antibiotics showed increased PAE values in a concentration-dependent manner. The findings of our study may play useful role in selecting the appropriate timing of doses during therapy with antimicrobials to treat patients infected with L. pneumophila.     

Evaluation of the methods for enumerating coliform bacteria from water samples using precise reference standards

AIMS: To use BioBall cultures as a precise reference standard to evaluate methods for enumeration of Escherichia coli and other coliform bacteria in water samples. METHODS AND RESULTS: Eight methods were evaluated including membrane filtration, standard plate count (pour and spread plate methods), defined substrate technology methods (Colilert and Colisure), the most probable number method and the Petrifilm disposable plate method. Escherichia coli and Enterobacter aerogenes BioBall cultures containing 30 organisms each were used. All tests were performed using 10 replicates. The mean recovery of both bacteria varied with the different methods employed. CONCLUSIONS: The best and most consistent results were obtained with Petrifilm and the pour plate method. Other methods either yielded a low recovery or showed significantly high variability between replicates. SIGNIFICANCE AND IMPACT OF THE STUDY: The BioBall is a very suitable quality control tool for evaluating the efficiency of methods for bacterial enumeration in water samples.     

Species detection using environmental DNA from water samples

The assessment of species distribution is a first critical phase of biodiversity studies and is necessary to many disciplines such as biogeography, conservation biology and ecology. However, several species are difficult to detect, especially during particular time periods or developmental stages, potentially biasing study outcomes. Here we present a novel approach, based on the limited persistence of DNA in the environment, to detect the presence of a species in fresh water. We used specific primers that amplify short mitochondrial DNA sequences to track the presence of a frog (Rana catesbeiana) in controlled environments and natural wetlands. A multi-sampling approach allowed for species detection in all environments where it was present, even at low densities. The reliability of the results was demonstrated by the identification of amplified DNA fragments, using traditional sequencing and parallel pyrosequencing techniques. As the environment can retain the molecular imprint of inhabiting species, our approach allows the reliable detection of secretive organisms in wetlands without direct observation. Combined with massive sequencing and the development of DNA barcodes that enable species identification, this approach opens new perspectives for the assessment of current biodiversity from environmental samples.     

Heterotrophic plate counts of surface water samples by using impedance methods.

Membrane filtration, spread plating, and pour plating are conventional methods used to determine the heterotrophic plate counts of water samples. Impedance methods were investigated as an alternative to conventional methods, since sample dilution is not required and the bacterial count can be estimated within 24 h. Comparisons of impedance signals obtained with different water samples revealed that capacitance produced faster detection times than conductance. Moreover, the correlation between heterotrophic plate count and detection time was highest (r = 0.966) when capacitance was used. Linear and quadratic regressions of heterotrophic plate count and impedance detection time were affected by incubation temperatures. Regressions between heterotrophic plate counts based on conventional methods and detection times of water samples incubated at less than or equal to 25 degrees C had R2 values of 0.878 to 0.933. However, regressions using detection times of water samples incubated at greater than or equal to 30 degrees C had lower R2 values, even though water samples produced faster detection times. Comparisons between broth-based versions of R2A medium and plate count agar revealed that the latter correlated highly with heterotrophic plate count, provided that water samples were incubated at 25 degrees C and impedance measurements were conducted with the capacitance signal (r = 0.966). When the linear regression of this relationship was tested with 100 water samples, the correlation between predicted and actual log10 CFU milliliter-1 was 0.869. These results indicate that impedance methods provide a suitable alternative to conventional methods.     

Detection of Legionella spp. and Legionella pneumophila in water samples of Spain by specific real-time PCR

Legionella pneumophila is the primary cause of the legionellosis diseases (90 %) (Yu et al. in J Infect Dis 186:127–128, 2002; Doleans et al. in J Clin Microbiol 42:458–460, 2004; Den Boer et al. in Clin Microbiol Infect 14:459–466, 2008). In this study, methodologies based on molecular biology were developed in order to provide a quick diagnosis of the bacterial presence in water samples of Spain. Multiplex real-time polymerase chain reaction assays were realized to target the 16S rRNA and macrophage infectivity potentiator (mip) genes of, respectively, Legionella spp. and L. pneumophila including in the design of an internal control. The results obtained by the culture and the gene amplification methods agreed in 94.44 % for the 16S rRNA gene, and a concordance of 66.67 % of the cases was obtained for the mip gene.     

Isolation of Salmonella from environmental samples collected in the reptile department of Antwerp Zoo using different selective methods

AIMS: To evaluate the environmental spread of Salmonella strains in the reptile department of Antwerp Zoo and to compare different isolation methods for Salmonella. METHODS AND RESULTS: One hundred environmental samples were collected in the service sections and public spaces of the reptile department. After pre-enrichment in buffered peptone water (BPW), selective enrichment was performed in Rappaport Vassiliadis Single Component Enrichment Broth (RVS), Selenite Cystine Broth (SEL) and Mueller Kauffman Tetrathionate Broth (MKTTn). Subculturing on Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium, and the combined use of immunomagnetic separation (IMS) and RVS was evaluated. The isolation media used were Hektoen Enteric Agar (HE), Phenol Red Brilliant Green Agar (BG) and Xylose Lysine Decarboxylase Agar (XLD). Salmonella strains were found in 47 samples (47.0%). Most isolations were made on HE after combined IMS/RVS enrichment. Sixty-six Salmonella strains were serotyped, 29 belonged to Salmonella enterica ssp. enterica (I), 3 to ssp. salamae (II), 29 to ssp. arizonae or diarizonae (IIIa/b), 4 to ssp. houtenae (IV) and 1 strain showed autoagglutination. In addition, a 10-year survey (1995-2004) of Salmonella serovars isolated from reptiles at Antwerp Zoo is presented. CONCLUSIONS: A high prevalence of Salmonella strains was noted in the service sections of the reptile department. Only a few isolations were made in the public spaces. Selective enrichment in RVS was the most efficient. In combination with IMS, this method gave an even higher isolation rate than the International Standard method (ISO 6579:2002). SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms the importance of reptiles as spreaders of Salmonella in their surroundings. The possible infectious risks for zoo personnel and visitors are evaluated. Improved laboratory protocols for the isolation of Salmonella from the environment are suggested.     

Heterotrophic plate counts of surface water samples by using impedance methods.

Membrane filtration, spread plating, and pour plating are conventional methods used to determine the heterotrophic plate counts of water samples. Impedance methods were investigated as an alternative to conventional methods, since sample dilution is not required and the bacterial count can be estimated within 24 h. Comparisons of impedance signals obtained with different water samples revealed that capacitance produced faster detection times than conductance. Moreover, the correlation between heterotrophic plate count and detection time was highest (r = 0.966) when capacitance was used. Linear and quadratic regressions of heterotrophic plate count and impedance detection time were affected by incubation temperatures. Regressions between heterotrophic plate counts based on conventional methods and detection times of water samples incubated at less than or equal to 25 degrees C had R2 values of 0.878 to 0.933. However, regressions using detection times of water samples incubated at greater than or equal to 30 degrees C had lower R2 values, even though water samples produced faster detection times. Comparisons between broth-based versions of R2A medium and plate count agar revealed that the latter correlated highly with heterotrophic plate count, provided that water samples were incubated at 25 degrees C and impedance measurements were conducted with the capacitance signal (r = 0.966). When the linear regression of this relationship was tested with 100 water samples, the correlation between predicted and actual log10 CFU milliliter-1 was 0.869. These results indicate that impedance methods provide a suitable alternative to conventional methods.     

Quantitative real-time Legionella PCR for environmental water samples: data interpretation

Quantitative Legionella PCRs targeting the 16S rRNA gene (specific for the genus Legionella) and the mip gene (specific for the species Legionella pneumophila) were applied to a total of 223 hot water system samples (131 in one laboratory and 92 in another laboratory) and 37 cooling tower samples (all in the same laboratory). The PCR results were compared with those of conventional culture. 16S rRNA gene PCR results were nonquantifiable for 2.8% of cooling tower samples and up to 39.1% of hot water system samples, and this was highly predictive of Legionella CFU counts below 250/liter. PCR cutoff values for identifying hot water system samples containing >10(3) CFU/liter legionellae were determined separately in each laboratory. The cutoffs differed widely between the laboratories and had sensitivities from 87.7 to 92.9% and specificities from 77.3 to 96.5%. The best specificity was obtained with mip PCR. PCR cutoffs could not be determined for cooling tower samples, as the results were highly variable and often high for culture-negative samples. Thus, quantitative Legionella PCR appears to be applicable to samples from hot water systems, but the positivity cutoff has to be determined in each laboratory.     

Isolation of Legionella longbeachae serogroup 1 from potting mixes

Following a statewide outbreak of legionellosis due to Legionella longbeachae serogroup 1 in South Australia in 1988 and 1989, studies were performed to find a source of the organism. A number of water and soil samples with and without acid decontamination were examined for L. longbeachae by using a selective medium containing vancomycin, aztreonam, and pimafucin. There were no isolations of L. longbeachae from water samples. Organisms resembling L. longbeachae were isolated from a number of samples of potting mixes and from soil surrounding plants in pots collected from the homes of four patients. The organisms were found to persist for 7 months in two potting mixes stored at room temperature. Legionellae were isolated with difficulty from potting mixes which were allowed to dry out. Identification of isolates as L. longbeachae serogroup 1 was confirmed by quantitative DNA hybridization and serological tests. Restriction-fragment-length-polymorphism studies showed minor differences between patient and environmental isolates but differentiated these readily from L. longbeachae serogroup 2 and other antigenically related legionellae. The isolation of L. longbeachae from some potting mixes and the prolonged survival of the organisms in this medium suggest that soil rather than water is the natural habitat of this species and may be the source of human infections.     

Isolation of Legionella pneumophila from nonepidemic-related aquatic habitats.

Continuous centrifugation of large volumes of water from natural southeastern lakes allowed quantitative detection of Legionella pneumophila by direct immunofluorescent staining. Positive samples were injected intraperitoneally into guinea pigs, and the L. pneumophila were isolated and identified by their morphological, cultural, physiological, and serological characteristics.     

Isolation of Legionella pneumophila from nonepidemic-related aquatic habitats.

Continuous centrifugation of large volumes of water from natural southeastern lakes allowed quantitative detection of Legionella pneumophila by direct immunofluorescent staining. Positive samples were injected intraperitoneally into guinea pigs, and the L. pneumophila were isolated and identified by their morphological, cultural, physiological, and serological characteristics.