Legionella contamination of dental-unit waters

Volume 61, no. 4, p. 1211, column 2, line 40: this sentence should read as follows. "The viable-culture methods for Legionella sp. detection, however, often fail, and the Centers for Disease Control and Prevention has turned, when necessary, to PCR for epidemiological investigations of Legionnaires' disease and Pontiac fever (19)." Line 46: "(6)" should read "(11)." [This corrects the article on p. 1208 in vol. 61.].     

Microbial biofilm formation and contamination of dental-unit water systems in general dental practice

Dental-unit water systems (DUWS) harbor bacterial biofilms, which may serve as a haven for pathogens. The aim of this study was to investigate the microbial load of water from DUWS in general dental practices and the biofouling of DUWS tubing. Water and tube samples were taken from 55 dental surgeries in southwestern England. Contamination was determined by viable counts on environmentally selective, clinically selective, and pathogen-selective media, and biofouling was determined by using microscopic and image analysis techniques. Microbial loading ranged from 500 to 10(5) CFU. ml(-1); in 95% of DUWS water samples, it exceeded European Union drinking water guidelines and in 83% it exceeded American Dental Association DUWS standards. Among visible bacteria, 68% were viable by BacLight staining, but only 5% of this "viable by BacLight" fraction produced colonies on agar plates. Legionella pneumophila, Mycobacterium spp., Candida spp., and Pseudomonas spp. were detected in one, five, two, and nine different surgeries, respectively. Presumptive oral streptococci and Fusobacterium spp. were detected in four and one surgeries, respectively, suggesting back siphonage and failure of antiretraction devices. Hepatitis B virus was never detected. Decontamination strategies (5 of 55 surgeries) significantly reduced biofilm coverage but significantly increased microbial numbers in the water phase (in both cases, P < 0.05). Microbial loads were not significantly different in DUWS fed with soft, hard, deionized, or distilled water or in different DUWS (main, tank, or bottle fed). Microbiologically, no DUWS can be considered "cleaner" than others. DUWS deliver water to patients with microbial levels exceeding those considered safe for drinking water.     

Legionella anisa: a new species of Legionella isolated from potable waters and a cooling tower.

Between March 1980 and June 1981, five strains of Legionella-like organisms were isolated from water. Four were recovered from potable water collected from hospitals in Chicago, Ill., and Los Angeles, Calif., during outbreaks of nosocomial legionellosis. The fifth strain was isolated from water collected from an industrial cooling tower in Jamestown, N.Y. The strains exhibited biochemical reactions typical of Legionella species and were gram-negative motile rods which grew on buffered charcoal-yeast extract agar but not on blood agar, required cysteine, and were catalase positive, urease negative, nitrate negative, hippurate negative, and nonfermentative. All strains were positive for oxidase and beta-lactamase and produced a brown, diffusible pigment. Of the five strains, four exhibited blue-white autofluorescence under long-wavelength UV light. The fatty-acid composition and ubiquinone content of these strains were consistent with those of other Legionella species. Direct fluorescent-antibody examination of the five strains with conjugates to previously described Legionella species demonstrated no cross-reactions except with the conjugates to L. longbeachae serogroup 2 and L. bozemanii serogroup 2. Four strains gave a 4+ reaction to the L. longbeachae serogroup 2 conjugate and the fifth strain gave a 1+ reaction. Each of the five strains gave a 4+ reaction with the conjugate to L. bozemanii serogroup 2. DNAs from the five strains were highly related (84 to 99%) and showed 5 to 57% relatedness to other Legionella species. These strains constitute a new species in the genus Legionella, and the name Legionella anisa sp. nov. is proposed. The type strain of L. anisa is WA-316-C3 (ATCC 35292).     

Sensitivity of Legionella pneumophila to sunlight in fresh and marine waters.

Studies were carried out to assess the sunlight sensitivity of Legionella pneumophila suspended in fresh and marine waters. Comparison studies on sunlight sensitivity of lake water bacteria, Pseudomonas aeruginosa, Escherichia coli and Streptococcus faecalis, were also undertaken. The effects of full sunlight and polyacrylic-screened sunlight were monitored in the study. Results indicate that L. pneumophila cells are slightly more sensitive to sunlight in seawater than in fresh water. Enumeration of sunlight-stressed bacteria in fresh water was found to be dependent on the medium used, and the following order of sensitivity to sunlight, from least to most sensitive, was noted: natural lake water bacteria, L. pneumophila, P. aeruginosa, E. coli, and S. faecalis.     

Legionella contamination in hot water of Italian hotels

A cross-sectional multicenter survey of Italian hotels was conducted to investigate Legionella spp. contamination of hot water. Chemical parameters (hardness, free chlorine concentration, and trace element concentrations), water systems, and building characteristics were evaluated to study risk factors for colonization. The hot water systems of Italian hotels were strongly colonized by Legionella; 75% of the buildings examined and 60% of the water samples were contaminated, mainly at levels of > or =10(3) CFU liter(-1), and Legionella pneumophila was the most frequently isolated species (87%). L. pneumophila serogroup 1 was isolated from 45.8% of the contaminated sites and from 32.5% of the hotels examined. When a multivariate logistic model was used, only hotel age was associated with contamination, but the risk factors differed depending on the contaminating species and serogroup. Soft water with higher chlorine levels and higher temperatures were associated with L. pneumophila serogroup 1 colonization, whereas the opposite was observed for serogroups 2 to 14. In conclusion, Italian hotels, particularly those located in old buildings, represent a major source of risk for Legionnaires' disease due to the high frequency of Legionella contamination, high germ concentration, and major L. pneumophila serogroup 1 colonization. The possible role of chlorine in favoring the survival of Legionella species is discussed.     

Comamonas acidovorans contamination of dental unit waters

A study was carried out to evaluate the extent of the colonization of dental water systems by Comamonas acidovorans and to investigate how the occurrence of these bacteria is related to certain water characteristics. The 152 water samples were collected from the oral rinsing cup, air-water syringe, turbine and supply lines to dental units. Comamonas acidovorans was found most frequently and in greatest quantities in samples taken from water entering the units and in samples with a lower total bacterial count at 22 degrees C, higher temperature, lower content of organic matter and, in general, higher concentrations of residual chlorine.     

Legionella anisa: a new species of Legionella isolated from potable waters and a cooling tower

Between March 1980 and June 1981, five strains of Legionella-like organisms were isolated from water. Four were recovered from potable water collected from hospitals in Chicago, Ill., and Los Angeles, Calif., during outbreaks of nosocomial legionellosis. The fifth strain was isolated from water collected from an industrial cooling tower in Jamestown, N.Y. The strains exhibited biochemical reactions typical of Legionella species and were gram-negative motile rods which grew on buffered charcoal-yeast extract agar but not on blood agar, required cysteine, and were catalase positive, urease negative, nitrate negative, hippurate negative, and nonfermentative. All strains were positive for oxidase and beta-lactamase and produced a brown, diffusible pigment. Of the five strains, four exhibited blue-white autofluorescence under long-wavelength UV light. The fatty-acid composition and ubiquinone content of these strains were consistent with those of other Legionella species. Direct fluorescent-antibody examination of the five strains with conjugates to previously described Legionella species demonstrated no cross-reactions except with the conjugates to L. longbeachae serogroup 2 and L. bozemanii serogroup 2. Four strains gave a 4+ reaction to the L. longbeachae serogroup 2 conjugate and the fifth strain gave a 1+ reaction. Each of the five strains gave a 4+ reaction with the conjugate to L. bozemanii serogroup 2. DNAs from the five strains were highly related (84 to 99%) and showed 5 to 57% relatedness to other Legionella species. These strains constitute a new species in the genus Legionella, and the name Legionella anisa sp. nov. is proposed. The type strain of L. anisa is WA-316-C3 (ATCC 35292).     

Risk factors for contamination of hotel water distribution systems by Legionella species

The Legionella colonization frequency at 385 Greek hotel hot and cold water distribution systems was 20.8%. Legionella contamination was associated with the presence of an oil heater (odds ratio [OR]=2.04, 95% confidence interval [CI]=1.12 to 3.70), with the sample temperature (OR=0.26, 95% CI=0.1 to 0.5), with seasonal operation (OR=3.23, 95% CI=1.52 to 6.87), and with the presence of an independent disinfection system (OR=0.30, 95% CI=0.15 to 0.62). The same water temperatures, free-chlorine levels, and pHs differently affect the survival of various Legionella spp.     

应用双重PCR检测环境水体中的军团菌

建立双重PCR方法以检出环境水体中的军团菌。设计两对引物,分别扩增军团菌的16S rRNA和M ip基因,扩增片段长各为375bp和996bp。该方法检测军团菌的灵敏度为5.8×102cfu/m l,6株嗜肺标准军团菌均扩增出996bp和375bp两条带,4株非嗜肺军团菌扩增出375bp条带,4株非军团菌无条带;检测71份环境水样,5份出现两条条带,2份可见375bp条带,阳性率为7.0%。该方法快速、灵敏、特异,为水体中的嗜肺军团菌检测提供了有效方法。     

Developing an ecologically relevant heterogeneous biofilm model for dental-unit waterlines

This study monitored the biodiversity of microbes cultured from a heterogeneous biofilm which had formed on the lumen of a section of dental waterline tubing over a period of 910 days. By day 2 bacterial counts on the outlet-water showed that contamination of the system had occurred. After 14 days, a biofilm comparable to that of clinical waterlines, consisting of bacteria, fungi and amoebae had formed. This showed that the proprietary silver coating applied to the luminal surface of the commercial waterline tubing failed to prevent biofilm formation. Molecular barcoding of isolated culturable microorganisms showed some degree of the diversity of taxa in the biofilm, including the opportunistic pathogen Legionella pneumophila. Whilst the system used for isolation and identification of contaminating microorganisms may underestimate the diversity of organisms in the biofilm, their similarity to those found in the clinical environment makes this a promising test-bed for future biocide testing.     

Photoreactivation of UV-irradiated Legionella pneumophila and other Legionella species.

Shortwave UV light was assessed as a feasible modality for the control of Legionnaires disease bacterium in water. The results of this study show that Legionella pneumophila and six other Legionella species are very sensitive to low doses of UV. However, all Legionella species tested effectively countered the germicidal effect of UV when subsequently exposed to photoreactiving light.     

Karelian larids under the conditions of global contamination of waters with organochlorines

The influence of chlorinated pesticides (DDE and lindane) on Karelian larids has been investigated. Data on the content of pollutants in eggs of the investigated bird species have been obtained by gas chromatography. These data show that the level of anthropogenic contamination is statistically significant, but in general low. This is related with the restriction of these pesticides in modern agriculture and the total decrease in agriculture activity in the northern parts of the country.     

PCB contamination in surface sediments in the coastal waters of Costa Rica

This paper describes the initial investigation of PCB concentrations in four geographical regions (three on the Pacific and one in the Caribbean) of coastal Costa Rica: Bahia Culebra, Golfo Dulce, Golfo de Nicoya, and Limón. Overall total concentrations of PCB were low in all areas except around the port of Golfito (Golfo Dulce). Overall average concentration is 2.80 ng/g dw, with a standard deviation of 2.75. The low concentration could be due to lack of contamination or the subsequent degradation in the warm climate, or the low sorptive capacity of the sediment. Further investigation is ongoing.     

A second serogroup of Legionella erythra serologically indistinguishable from Legionella rubrilucens.

Twenty-two red-autofluorescent Legionella strains were identified serologically as either Legionella rubrilucens or L. erythra. A rRNA probe was used for restriction fragment length polymorphism (RFLP) analysis of the strains and the patterns generated were used as an additional method of identifying the strains to species level. In two instances strains which were identified as L. rubrilucens by serology appeared to belong to the species L. erythra by RFLP analysis. This apparent contradiction was resolved by measurements of DNA/DNA homology which confirmed the existence of a second serogroup of L. erythra serologically indistinguishable from L. rubrilucens.     

LAMP-based method for a rapid identification of Legionella spp. and Legionella pneumophila

Legionella pneumophila is accounted for more than 80% of Legionella infection. However it is difficult to discriminate between the L. pneumophila and non-L. pneumophila species rapidly. In order to detect the Legionella spp. and distinguish L. pneumophila from Legionella spp., a real-time loop-mediated isothermal amplification (LAMP) platform that targets a specific sequence of the 16S rRNA gene was developed. LS-LAMP amplifies the fragment of the 16S rRNA gene to detect all species of Legionella genus. A specific sequence appears at the 16S rRNA gene of L. pneumophila, while non-L. pneumophila strains have a variable sequence in this site, which can be recognized by the primer of LP-LAMP. In the present study, 61 reference strains were used for the method verification. We found that the specificity was 100% for both LS-LAMP and LP-LAMP, and the sensitivity of LAMP assay for L. pneumophila detection was between 52 and 5.2 copies per reaction. In the environmental water samples detection, a total of 107 water samples were identified by the method. The culture and serological test were used as reference methods. The specificity of LS-LAMP and LP-LAMP for the samples detection were 91.59% (98/107) and 93.33% (56/60), respectively. The sensitivity of LS-LAMP and LP-LAMP were 100% (51/51) and 100% (18/18). The results suggest that real-time LAMP, as a new assay, provides a specific and sensitive method for rapid detection and differentiation of Legionella spp. and L. pneumophila and should be utilized to test environmental water samples for increased rates of detection.     

Ecological distribution of Legionella pneumophila.

Bacteria were concentrated 500-fold from 20-liter water samples collected from 67 different lakes and rivers in the United States. The data suggest that Legionella pneumophila is part of the natural aquatic environment and that the bacterium is capable of surviving extreme ranges of environmental conditions. The data further demonstrate the effectiveness of the direct fluorescent-antibody technique for detecting L. pneumophila in natural aquatic systems. Smears of the concentrated samples were screened microscopically for serogroups of L. pneumophila by the direct fluorescent-antibody technique. Virtually all of the 793 samples were found to be positive by this method. The 318 samples containing the largest numbers of positive bacteria which were morphologically consistent with L. pneumophila were injected into guinea pigs for attempted isolations. Isolates were obtained from habitats with a wide range of physical, chemical, and biological parameters. Samples collected monthly from a thermally altered lake and injected into guinea pigs demonstrated a seasonality of infection, with the highest frequency of infection occurring during the summer months.     

Viral contamination of some drinking waters in Romania: a twenty years survey

A synthesis is made on the 20 years virological survey of the drinking water from some towns of Romania. The sampling of the water was made by the gauze-pad method. The virus concentration method by adsorption-elution with the yeast cells was applied concomitantly, at first with the PE60 method, then, during the last years, either with aluminum hydroxide or with the polymer PV methods, and the concentrates were inoculated both into suckling mice and into cell cultures. Various types of coxsackievirus A and B, poliovirus and adenovirus were detected. The proportions of positive samples varied between 0 and 25% annually, in diverse towns, the mean proportion being 2.1%. These proportions are relative low, although two concentrating and two detecting methods were applied concomitantly.     

Ulva lactuca as bioindicator of metal contamination in intertidal waters in Hong Kong

The use of Ulva lactuca L. as an indicator of metal contamination was assessed by analysing the levels of Mn, Fe, Ni, Cu, Zn, Cd and Pb in the alga collected from 24 intertidal sites around the Island of Hong Kong. Twelve of the sites are in the rural southern parts of the Island where the coastal waters are relatively clean. The remaining 12 sites are located in the north and within Victoria Harbour which receives, apart from industrial effluents, untreated domestic sewage from a population of some 3.5 million. The mean levels of Mn, Fe, Ni, Cu, Zn and Pb in Ulva from the urban sites were respectively 4.0, 4.6, 1.8, 2.3, 2.4 and 4.6 folds those from the rural sites. However, similar levels of Cd were found in the alga amongst all the sites. Locations of high levels of metal contamination, particularly to the eastern end of the Harbour, have been identified. Preliminary results indicate that Ulva is a good indicator of Mn, Fe, Cu, Zn and Pb contaminations.     

Serospecific antigens of Legionella pneumophila.

Serospecific antigens isolated by EDTA extraction from four serogroups of Legionella pneumophila were analyzed for their chemical composition, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The antigens were shown to be lipopolysaccharides and to differ from the lipopolysaccharides of other gram-negative bacteria. The serospecific antigens contained rhamnose, mannose, glucosamine, and two unidentified sugars together with 2-keto-3-deoxyoctonate, phosphate, and fatty acids. The fatty acid composition was predominantly branched-chain acids with smaller amounts of 3-hydroxymyristic acid. The antigens contain periodate-sensitive groups; mannosyl residues were completely cleaved by periodate oxidation. Hydrolysis of the total lipopolysaccharide by acetic acid resulted in the separation of a lipid A-like material that cross-reacted with the antiserum to lipid A from Salmonella minnesota but did not comigrate with it on sodium dodecyl sulfate gels. None of the four antigens contained heptose. All of the antigen preparations showed endotoxicity when tested by the Limulus amebocyte lysate assay. The results of this study indicate that the serogroup-specific antigens of L. pneumophila are lipopolysaccharides containing an unusual lipid A and core structure and different from those of other gram-negative bacteria.     

Legionella sainthelensi: a new species of Legionella isolated from water near Mt. St. Helens.

Six strains of a new species, Legionella sainthelensi, were isolated from freshwater in areas affected by the volcanic eruptions of Mt. St. Helens in the state of Washington. Strains of L. sainthelensi are culturally and biochemically similar to other legionellae. They grow on buffered charcoal yeast agar but not on media that lack cysteine. They are gram-negative, nonsporeforming, motile rods that are positive in reactions for catalase, oxidase, gelatin liquefaction, and beta-lactamase. They are negative in reactions for urease, hydrolysis of hippurate, reduction of nitrates, fermentation of glucose, and blue-white autofluorescence. Their cell wall fatty acid composition is qualitatively similar to those of other legionellae, with 50 to 62% branched-chain fatty acids. They contain the isobranched-chain 14- and 16-carbon acids and anteisobranched-chain 15- and 17-carbon acids and relatively large amounts of straight-chain 16-carbon acid. All strains of L. sainthelensi contain approximately equal amounts of ubiquinones Q9, Q10, Q11, and Q12, a pattern similar to those of Legionella bozemanii, Legionella dumoffi, and Legionella longbeachae. Serological cross-reactions were observed between L. sainthelensi, both serogroups of L. longbeachae, and Legionella oakridgensis. Three strains of L. sainthelensi were greater than 90% related by DNA hybridization. The type strain of L. sainthelensi, Mt. St. Helens 4, was 36% related to the type strain of L. longbeachae and 3 to 14% related to the other nine described Legionella species.