Epidemiological evidence of lesser role of thermostable direct hemolysin (TDH)-related hemolysin (TRH) than TDH on Vibrio parahaemolyticus pathogenicity.

Vibrio parahaemolyticus carrying the tdh gene, encoding the thermostable direct hemolysin (TDH), or the trh gene, encoding the TDH-related hemolysin (TRH), are both considered virulent strains. There are, however, disproportionally fewer reports of infections caused by seafood contaminated with trh-positive strains than by seafood contaminated with tdh-positive strains. Bivalves such as clams and oysters are the major seafood varieties associated with the infections. In this study, the prevalence of strains possessing the tdh and trh genes was investigated in Japan in 74 samples collected in 2007-2008 and in 177 samples collected in 2010 of domestic bivalves, bloody clams, hen clams, short-neck clams, and rock oysters. The tdh-positive and trh-negative, tdh-negative and trh-positive, and tdh-positive and trh-positive samples represented 5.4%, 12.2%, and 4.1% of all samples collected in 2007-2008, and 5.1%, 18.6%, and 5.6% of all samples collected in 2010, respectively. As determined by polymerase chain reaction, the prevalence of tdh negative and trh positive in all samples was two to four times higher than that of tdh positive and trh negative. In the samples collected in 2010, the tdh-negative and trh-positive V. parahaemolyticus (20 samples) was more often isolated than tdh-positive and trh-negative V. parahaemolyticus (7 samples). The most common serotype of tdh-positive isolates (22 of 24 strains) was pandemic O3:K6. The trh-positive isolates (61 strains) were various serotypes including OUT:KUT. In 330 V. parahaemolyticus outbreaks and sporadic infections in Japan, most outbreaks and sporadic infections were caused by tdh-positive and trh-negative strains (89.4%). The frequencies of infections caused by tdh-negative and trh-positive, and both tdh- and trh-positive strains were 1.2% and 3.0%, respectively. This finding suggests that the virulence of trh might be less than that of tdh, although trh-positive V. parahaemolyticus frequently contaminated bivalves.

[Molecular Detection Methods for Vibrio parahaemolyticus in Seafood].

To detect Vibrio parahaemolyticus in seafood, we evaluated efficient combinations of molecular methods with DNA extraction methods using heat extraction and alkaline heat extraction, and PCR, real-time PCR and loop-mediated isothermal amplification (LAMP) assays were performed targeting V parahaemolyticus species-specific genes (tlh and rpoD) and pathogenic factors genes (tdh and trh). The species-specific genes were detected in all combinations of two strains (a tdh * trh1-positive strain and a trh2-positive strain), two kinds of shellfish (oyster and bloody clams) and molecular methods with tlh-real time PCR or rpoD-LAMP assays with DNA of alkaline heat extraction at 85-145cfu/test level. tdh was detected in both seafoods with real time PCR assay with DNA of heat extraction at 85cfu/test level, and detected with the LAMP and real time PCR assays with DNA of alkaline heat extraction at 85cfu/test level. Detection of both trh1 and trh2 with the PCR assay with DNA of alkaline heat extraction was comparatively high though trh2 was detected with the LAMP assay with DNA of alkaline heat extraction at 145cfu/test level. It, however, is necessary to investigate more sensitive trh-detection methods. In this study, the results indicated that tlh-real time PCR or rpoD-LAMP, tdh-real time PCR and tdh-LAMP assays with DNA of alkaline heat extraction are relatively-sensitive methods to detect V. parahaemolyticus in seafood.

Characteristics of bacterial and fungal growth in plastic bottled beverages under a consuming condition model.

Microbial contamination in unfinished beverages can occur when drinking directly from the bottle. Various microorganisms, including foodborne pathogens, are able to grow in these beverages at room temperature or in a refrigerator. In this study, we elucidated the characteristics of microorganism growth in bottled beverages under consuming condition models. Furthermore, we provide insight into the safety of partially consumed bottled beverages with respect to food hygiene. We inoculated microorganisms, including foodborne pathogens, into various plastic bottled beverages and analysed the dynamic growth of microorganisms as well as bacterial toxin production in the beverages. Eight bottled beverage types were tested in this study, namely green tea, apple juice drink, tomato juice, carbonated drink, sport drink, coffee with milk, isotonic water and mineral water, and in these beverages several microorganism types were used: nine bacteria including three toxin producers, three yeasts, and five moulds. Following inoculation, the bottles were incubated at 35°C for 48 h for bacteria, 25°C for 48 h for yeasts, and 25°C for 28 days for moulds. During the incubation period, the number of bacteria and yeasts and visible changes in mould-growth were determined over time. Our results indicated that combinations of the beverage types and microorganism species correlated with the degree of growth. Regarding factors that affect the growth and toxin-productivity of microorganisms in beverages, it is speculated that the pH, static/shaking culture, temperature, additives, or ingredients, such as carbon dioxide or organic matter (especially of plant origin), may be important for microorganism growth in beverages. Our results suggest that various types of unfinished beverages have microorganism growth and can include food borne pathogens and bacterial toxins. Therefore, our results indicate that in terms of food hygiene it is necessary to consume beverages immediately after opening the bottle.

Microbial contamination associated with consumption and the growth in plastic bottled beverage.

Plastic bottles enable the storage of unfinished beverages, and most of microbial contamination has occurred in the unfinished beverage that was left. Therefore, we investigated microorganisms in various beverages contaminated by pouring and drinking directly by mouth from the bottle, and analyzed the growth of microorganisms in the beverages at room temperature. In the pouring test, microbial growth was detected in 60 of 320 samples, and 13 bacterial strains, 49 mold strains, and 8 yeast strains were isolated. Molds including Cladosporium spp., Tramets spp., Bjerkandera spp., and Penicillium spp. accounted for the majority of isolated microorganisms. In the drinking test, microbial growth was detected in 181 of 352 samples, and 225 bacterial strains, 27 mold strains and 77 yeast strains were isolated. Bacteria including Streptococcus spp. such as S. salivarius and Staphylococcus spp. such as S. aureus accounted for the majority of isolated microorganisms. Enterotoxin-producing S. aureus and Bacillus cereus were also isolated. The pH of the beverage influenced the growth of bacteria. The Brix values of the beverage did not correlate with the growth of microorganisms. These results revealed that various microorganisms including foodborne pathogens were able to grow in numerous types of beverages and that the storage of unfinished beverage in inappropriate condition, such as the storage at room temperature led microorganism to grow easily in beverage. Therefore, it is necessary to consume beverages as soon as possible after opening the bottle.

Specific detection of viable Legionella cells by combined use of photoactivated ethidium monoazide and PCR/real-time PCR.

Legionella organisms are prevalent in manmade water systems and cause legionellosis in humans. A rapid detection method for viable Legionella cells combining ethidium monoazide (EMA) and PCR/real-time PCR was assessed. EMA could specifically intercalate and cleave the genomic DNA of heat- and chlorine-treated dead Legionella cells. The EMA-PCR assay clearly showed an amplified fragment specific for Legionella DNA from viable cells, but it could not do so for DNA from dead cells. The number of EMA-treated dead Legionella cells estimated by real-time PCR exhibited a 10(4)- to 10(5)-fold decrease compared to the number of dead Legionella cells without EMA treatment. Conversely, no significant difference in the numbers of EMA-treated and untreated viable Legionella cells was detected by the real-time PCR assay. The combined assay was also confirmed to be useful for specific detection of culturable Legionella cells from water samples obtained from spas. Therefore, the combined use of EMA and PCR/real-time PCR detects viable Legionella cells rapidly and specifically and may be useful in environmental surveillance for Legionella.

[Legionella contamination risk factors in non-circulating hot spring water].

We examined water from 182 non-circulating hot spring bathing facilities in Japan for possible Legionella occurrence from June 2005 to December 2006, finding Legionella-positive cultures in 119 (29.5%) of 403 samples. Legionellae occurrence was most prevalent in bathtub water (39.4%), followed by storage tank water (23.8%), water from faucets at the bathtub edge (22.3%), and source-spring water (8.3%), indicating no statistically significant difference, in the number of legionellae, having an overall mean of 66 CFU/100mL. The maximum number of legionellae in water increased as water was sampled downstream:180 CFU/100 mL from source spring, 670 from storage tanks, 4,000 from inlet faucets, and 6,800 from bathtubs. The majority--85.7%--of isolated species were identified as L. pneumophila : L. pneumophila serogroup (SG) 1 in 22%, SG 5 in 21%, and SG 6 in 22% of positive samples. Multivariate logistic regression models used to determine the characteristics of facilities and sanitary management associated with Legionella contamination indicated that legionellae was prevalent in bathtub water under conditions where it was isolated from inlet faucet/pouring gate water (odds ratio [OR] = 6.98, 95% confidence interval [CI] = 2.14 to 22.8). Risk of occurrence was also high when the bathtub volume exceeded 5 m3 (OR = 2.74, 95% CI = 1.28 to 5.89). Legionellae occurrence was significantly reduced when the bathing water pH was lower than 6.0 (OR = 0.12, 95% CI = 0.02 to 0.63). Similarly, occurrence was rare in inlet faucet water or the upper part of the plumbing system for which pH was lower than 6.0 (OR = 0.06, 95% CI = 0.01 to 0.48), and when the water temperature was maintained at 55 degrees C or more (OR = 0.10, 95% CI = 0.01 to 0.77). We also examined the occurrence of amoeba, Mycobacterium spp., Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus in water samples.

Molecular typing of Japanese Escherichia coli O157 : H7 isolates from clinical specimens by multilocus variable-number tandem repeat analysis and PFGE.

The multilocus variable-number tandem repeat analysis (MLVA) method to target eight variable-number tandem repeat loci, based on agarose gel electrophoresis separation of multiplexed PCR products, and the PFGE method were applied to clinical isolates of Escherichia coli O157 : H7 with the aim of comparing their performance as methods of typing this bacterium. Using MLVA, a total of 57 isolates from patients in Shizuoka prefecture, Japan, were divided into 20 types and classified into 23 PFGE types. Twenty-four isolates from four sporadic infections, four household contact infections and one outbreak that occurred in central parts of Shizuoka prefecture during August to November in 2005 were shown to be the same MLVA type, and most of the isolates had identical PFGE banding patterns, suggesting the diffuse outbreak in these parts of Japan. Thus, there was a good correlation between MLVA types and PFGE types, with both methods displaying broadly similar discriminatory powers. However, the MLVA typing proved to be a much easier and more rapid method for the analysis of E. coli O157 : H7 strain relatedness to identify transmission routes. Hence, our MLVA method would be a suitable technique for routine typing in many laboratories, including public health agencies, and even in hospitals.

Expression of heat shock protein 27 with the transition from proliferation to differentiation of acinar precursor cell in regenerating submandibular gland of rats.

Heat shock protein 27 (Hsp27) has been suggested to participate in the cell proliferation and differentiation during tissue development. In fact, we have demonstrated the transient occurrence of Hsp27 during the differentiation of salivary gland acinar cells in postnatal rats. The purpose of the present study is to explore the potential role of Hsp27 in the proliferation and differentiation of the acinar cells during regeneration of the salivary gland. Using the experimental regeneration model of the rat submandibular gland after the release of duct ligation, the spatio-temporal localization of Hsp27 was investigated in immunohistochemistry in regenerating acini. No epithelial cells were immunoreactive for Hsp27 immediately after unligation, but Hsp27-immunoreactive cells were observed in regenerating acini located at the end portion of survived ductal tissues on the third day after unligation. The number of Hsp27-immunoreactive cells in regenerating acini reached its peak on the 5th day after unligation, and started to decline on the 7th day. They were undetectable on the 14th day. Importantly, the increase in the number of Hsp27-immunoreactive cells was preceded by the decline in the cell proliferative activity, and Hsp27-immunoreactivity declined and disappeared in conjunction with the progression of acinar cell differentiation, as judged by the double-immunostaining for Hsp27 and proliferating cell nuclear antigen, a cell proliferation marker, or glycine-rich protein-alpha, a specific marker of differentiated acinar cells. All the findings suggest that Hsp27 is expressed with the transition from the cell proliferation to differentiation of the acinar precursor cells during the regenerating process.

Draft Genome Sequence of Mycobacterium sp. Strain shizuoka-1, a Novel Mycobacterium Isolated from Groundwater of a Bathing Facility in Shizuoka, Japan.

Mycobacterium sp. strain shizuoka-1 is a rapidly growing scotochromogenic mycobacterium and was isolated from well water for a bathing facility in Shizuoka Prefecture in Japan. Here, we report the draft sequence of its genome, comprising a 6.5-Mb chromosome. This mycobacterium has 83.1% identity with Mycobacterium rhodesiae, a human pathogen.

Induction of apoptosis by beta-diketones in human tumor cells.

A variety of beta-diketones were evaluated for their cytotoxic profiles against oral human normal and tumor cells. Among 22 compounds (BD1-22) tested, the cytotoxicity of 3-formylchromone (BD17) (CC50=7.8 microg/mL) against human oral squamous cell carcinoma (HSC-2) cells was higher than that of curcumin (CC50=23.6 microg/mL). Tumor cell-specific cytotoxicity was also detected in BD17 which exhibited little cytotoxic activity against a normal human cell, gingival fibroblast (HGF). (-)-3- (BD13) (CC50=21.7 microg/mL) and (+)-3-(Trifluoroacetyl)camphor (BD12) (CC50=29.7 microg/mL) are enantiomers and showed cytotoxicity comparable to curcumin and dibenzoylmethane (BD2) (CC50=22.5 microg/mL). BD13 did not induce DNA fragmentation in HL-60 cells nor activate caspase 3, 8 and 9 in both HL-60 and HSC-2 cells, regardless of the presence or absence of FeCl3. On the other hand, BD17 was found to induce apoptosis in HSC-2 and HL-60 cells, as judged by internucleosomal DNA fragmentation, caspase 3, 8 and 9 activation and dysfunction of mitochondrial membrane potential. The cytotoxic activity of BD13, BD17 and curcumin was significantly reduced by chelation with FeCl3. The tumor-specific cytotoxicity and apoptosis-inducing activity of BD17 against human tumor cells undoubtedly warrant further studies of its efficacy as a cancer chemotherapeutic agent.

Serotype, Shiga toxin (Stx) type, and antimicrobial resistance of Stx-producing Escherichia coli isolated from humans in Shizuoka Prefecture, Japan (2003-2007).

The serotype, Shiga toxin (Stx) type, and antimicrobial resistance patterns of 138 Stx-producing Escherichia coli (STEC) strains isolated from humans between 2003 and 2007 in Shizuoka Prefecture, Japan were characterized. The predominant O serogroups of the STEC isolates were O157, O26, and O111. Antimicrobial susceptibility testing of the STEC isolates showed that 31 of the 138 isolates (22.5%) were resistant to antibiotics. Compared to the results reported in the previous studies, a higher rate of STEC O157 isolates were susceptible to all the antimicrobial agents used in this study. However, antimicrobial susceptibility data from this study showed that antimicrobial resistance patterns have increased by 6 compared to the survey performed by Masuda et al. between 1987 and 2002 (Jpn. J. Food Microbiol., 21, 44-51, 2004). This indicates that STEC isolates have evolved to show a variety of antimicrobial resistance patterns. It is important to consider the population of isolates showing decreased susceptibility to clinically relevant drugs such as ciprofloxacin (CPFX) and fosfomycin (FOM). All the 3 STEC isolates resistant to nalidixic acid showed low susceptibility to CPFX (MIC, 0.25-0.5 µg/ml). In addition, a decreased susceptibility to FOM was clearly observed in the E. coli O26 isolates. Our findings also showed that 1 STEC O26 strain could possibly be a chromosomal AmpC ß-lactamase hyperproducer. These results suggest that antimicrobial therapy may be less effective in patients with non-O157 STEC infections than in those with STEC O157 infections.

Factors for occurrence of extended-spectrum ß-lactamase-producing Escherichia coli in broilers.

To clarify the factors for occurrence of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in broilers, two flocks (1 day of age) fed a diet with or without antibiotics were kept in a broiler house sanitized with disinfectants. ESBL-producing E. coli, however, was detected at a concentration of over 10(6) CFU/g of feces at 9 days of age to 49 days of age in both broiler flocks. Therefore, this indicated that the antibiotics other than cephalosporins used in this study had no effect due to co-selection on the numbers of ESBL-producing E. coli in broiler feces during this period. When a flock was kept with diet containing antibiotics for 49 days in a laboratory animal room, no ESBL-producing E. coli was detected in the flock. These results suggest that the occurrence of ESBL-producing E. coli may not be related to feeding with antibiotics and that the contamination of broiler houses with ESBL-producing E. coli might be an important factor.

Antibiotic resistance in bacterial pathogens from retail raw meats and food-producing animals in Japan.

To determine the prevalence and antimicrobial susceptibility profiles of Campylobacter, Salmonella, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and vancomycin-resistant enterococci (VRE) in food-producing animals and retail raw meats in Japan, raw meat samples as well as food-producing animal feces, cutaneous swabs, and nasal swabs collected from 2004 to 2006 were analyzed. Isolation rates of Campylobacter jejuni and Campylobacter coli, Salmonella, and S. aureus were 34.6% (363 of 1,050), 2.7% (28 of 1,050), and 32.8% (238 of 725), respectively. MRSA was isolated from 3% (9 of 300) of meat samples. No VRE were isolated in this study. Antibiotic resistance in C. coli was higher than that in C. jejuni. Three C. jejuni isolates from a patient with diarrhea in a hospital of Shizuoka Prefecture and two chicken samples that exhibited resistance to ciprofloxacin had identical pulsed-field gel electrophoresis patterns, suggesting that ciprofloxacin-resistant C. jejuni could have been distributed in meat. S. aureus isolates showed the highest level of resistance to ampicillin and tetracycline. Resistance to tetracycline in S. aureus isolates from beef was lower than that seen in isolates from chicken and pork (P < 0.01). This study revealed that the prevalence of MRSA and VRE were low in food-producing animals and retail domestic meats in Japan, although Campylobacter isolates resistant to fluoroquinolone and erythromycin were detected. The occurrence of antimicrobial-resistant pathogens should be monitored continuously to improve the management of the risks associated with antimicrobial drug resistance transferred from food-producing animals to humans.

Detection and characterization of p44/msp2 transcript variants of Anaplasma phagocytophilum from naturally infected ticks and wild deer in Japan.

Anaplasma phagocytophilum is an obligate intracellular bacterium and causes a febrile illness in humans and livestock. In nature, this bacterium is sustained in a tick-mammal cycle. Several p44/msp2-related genes are expressed from a single expression locus by gene conversion. In this study, we obtained 119 cDNA sequences of p44/msp2 transcripts from A. phagocytophilum in 6 Haemaphysalis ticks and 3 wild sika deer (Cervus nippon) in Japan. These 119 sequences were classified into 36 different variant sequences based on their similarities. The 36 cDNA sequences were phylogenetically grouped into 2 major clusters--tick- and deer-associated. The tick-associated sequences were further classified into 4 distinct subclusters, suggesting that A. phagocytophilum in ticks seems to selectively express specific p44/msp2 transcripts, such as the transcripts in the 4 subclusters that were closely related to previously identified p44/msp2 genes. The deer-associated sequences were also grouped into 4 subclusters, but these transcripts were probably more diverse than the transcripts derived from ticks. This might be due to the relatively nonselective expression of p44/msp2 in deer or the strain differences in A. phagocytophilum from ticks and deer in separate geographic regions or both. Thus, this study may contribute to the understanding of A. phagocytophilum p44/msp2 expression in nature in Japan.

Prevalence of extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae in food-producing animals.

To evaluate the diversity of extended-spectrum ß-lactamases (ESBL) genes among food-producing animals, 48 isolates of ESBL-producing Escherichia coli isolates were obtained from rectal samples of broilers, layers, beef cattle and pigs, at the slaughterhouse level. ESBL-carrying E. coli were isolated from 60.0% of individual broiler rectal samples, 5.9% of layers, 12.5% of beef cattle and 3% of pigs. One ESBL-producing Klebsiella pneumoniae was isolated from a broiler. The ESBL-positive E. coli isolates from broilers harbored various ESBL genes: bla (SHV-12), bla(CTX-M-2), bla(CTX-M-14), bla(CTX-M-15) and bla(CTX-M-44). The plasmid DNAs were analyzed by restriction patterns. Homogeneous band patterns were yielded in those of K. pneumoniae and E. coli isolates harboring the bla(CTX-M-2) gene from different farms. No genetic relation between the 2 CTX-M-14 ESBL-producing strains was found by pulsed-field gel electrophoresis, although 2 plasmids in these strains, obtained from different broiler farms, were similar to each other. This study provides evidence that the proliferation of CTX-M-producing E. coli is due to the growth of indigenous CTX-M-producing strains and the possible emergence of strains that acquired CTX-M genes by horizontal transfer in different broiler farms. CTX-M-producing coliforms in broilers should be controlled due to the critical importance of cephalosporins and the zoonotic potential of ESBL-producing bacteria.

Characteristics of a sharp decrease in Vibrio parahaemolyticus infections and seafood contamination in Japan.

Vibrio parahaemolyticus has been one of the most important foodborne pathogens in Japan since the 1960s, and a large epidemic was caused by the pandemic serotype O3:K6 from 1997 to 2001. V. parahaemolyticus infections, however, have sharply declined since that time. Data on serotypes isolated from 977 outbreaks were collected and analysed. Total and pathogenic, thermostable direct hemolysin (TDH) gene-positive V. parahaemolyticus were qualitatively and quantitatively detected in 842 seafood samples from wholesale markets in 2007-2009. Strains isolated from patients and seafood were analysed by serotyping, tdh-PCR, group-specific PCR for pandemic strains, and pulsed-field gel electrophoresis (PFGE). The sharp decrease in the infections from 1999 onwards was noted not only for O3:K6 infections but also for other serotypes. The change in the seafood contamination situation from 2001 to 2007-2009 was characterised by a decrease to three-fourths in the frequency of tdh-positive samples, although that decrease was small compared to the 18-fold decrease in the cases of V. parahaemolyticus outbreaks. PFGE detected the pandemic O3:K6 serotype in the same profile in seafood and patients from 1998 to the present. Because of no large decrease in seafood contamination by V. parahaemolyticus from the production to distribution stages and the presence of pandemic O3:K6 serotype in seafood to the present, it was suggested that the change of seafood contamination was unrelated to the sharp decrease in V. parahaemolyticus infections. V. parahaemolyticus infections might be prevented at the stages after the distribution stage.

A survey of ß-lactamase-producing Escherichia coli in farm animals and raw retail meat in Shizuoka Prefecture, Japan.

We surveyed ß-lactamase-producing Escherichia coli from farm animals (chickens, pigs, and cattle) and raw retail meat in Shizuoka Prefecture, Japan. In total 305 E. coli isolates, 15 isolates collected from broilers, beef cattle, chicken meat, and pork meat, were found to have ß-lactamase genes encoding CTX-M-2, CTX-M-14, CMY-2, SHV-2, and/or TEM-1, whereas 7 possessed mutations in the ampC promoter region. The findings suggest that broilers are more important than other farm animals with regards to the surveillance of ß-lactamase-producing E. coli in this region.

A rapid detection method using flow cytometry to monitor the risk of Legionella in bath water.

Legionella species are the causative agents of human legionellosis, and bathing facilities have been identified as the sources of infection in several outbreaks in Japan. Researchers in Japan have recently reported evidence of significant associations between bacterial counts and the occurrence of Legionella in bathing facilities and in a hot tub model. A convenient and quantitative bacterial enumeration method is therefore required as an indicator of Legionella contamination or disinfection to replace existing methods such as time-consuming Legionella culture and expensive Legionella-DNA amplification. In this study, we developed a rapid detection method (RDM) to monitor the risk of Legionella using an automated microbial analyzing device based on flow cytometry techniques to measure the total number of bacteria in water samples within two minutes, by detecting typical patterns of scattered light and fluorescence. We first compared the results of our RDM with plate counting results for five filtered hot spring water samples spiked with three species of bacteria, including Legionella. Inactivation of these samples by chlorine was also assessed by the RDM, a live/dead bacterial fluorescence assay and plate counting. Using the RDM, the lower limit of quantitative bacterial counts in the spiked samples was determined as 3.0×10(3)(3.48log)counts mL(-1). We then used a laboratory model of a hot tub and found that the RDM could monitor the growth curve of naturally occurring heterotrophic bacteria with 1 and 2 days' delayed growth of amoeba and Legionella, respectively, and could also determine the killing curve of these bacteria by chlorination. Finally, samples with ≥3.48 or <3.48log total bacterial counts mL(-1) were tested using the RDM from 149 different hot tubs, and were found to be significantly associated with the positive or negative detection of Legionella with 95% sensitivity and 84% specificity. These findings indicated that the RDM can be used for Legionella control at bathing facilities, especially those where the effectiveness of chlorine is reduced by the presence of Fe(2+), Mn(2+), NH(4)(+), skin debris, and/or biofilms in the water.

Comparison of ethidium monoazide and propidium monoazide for the selective detection of viable Legionella cells.

Ethidium monoazide (EMA) and propidium monoazide (PMA) have been utilized for selective PCR amplification of DNA from viable bacterial cells. In this study, we compared the abilities of EMA and PMA, together with real-time PCR, to specifically distinguish dead Legionella cells from viable cells. Several experiments showed that PMA or EMA treatment could specifically prevent the PCR amplification of DNA from dead Legionella cells in water samples. However, a 4-fold higher concentration of PMA than EMA was required to achieve this effect. EMA may therefore be more useful for practical environmental investigations of Legionella.