Efficacy of the detection of Legionella in hot and cold water samples by culture and PCR. I. Standardization of methods.

The aim of the present study was: - to compare methods for concentration and isolation of Legionella DNA from water; - to examine the efficacy of various modifications of PCR test (PCR, semi-nested PCR, and real-time PCR) for the detection of known numbers of Legionella pneumophila in water samples artificially contaminated with the strain of this bacterium and in randomly selected samples of environmental water, in parallel with examination by culture. It was found that filtration is much more effective than centrifugation for the concentration of DNA in water samples, and that the Qiamp DNA Mini-Kit is the most efficient for isolation of Legionella DNA from water. The semi-nested PCR and real-time PCR proved to be the most sensitive methods for detection of Legionella DNA in water samples. Both PCR modifications showed a high correlation with recovery of Legionella by culture (p<0.01), while no correlation occurred between the results of one-stage PCR and culture (p>0.1).

Efficacy of the detection of Legionella in hot and cold water samples by culture and PCR. II. Examination of native samples from various sources.

A total of 123 water samples were examined in parallel by culture and semi-nested PCR for the presence of Legionella. They comprised: 35 samples of hot water distributed by the urban municipal water supply system (MWSS) taken in institutions, 45 samples of hot water distributed by urban MWSS taken in dwellings, 27 samples of cold water distributed by rural MWSS taken in dwellings, and 16 samples of cold well water taken in rural areas. The greatest frequency of the isolation of Legionella by culture (88.6%) was recorded in the samples of hot water from the urban institutions, having been greater compared to all other sources (p<0.001). The frequency of Legionella isolation from hot water in urban dwellings (28.9%) was significantly greater compared to the combined value (2.3%) for cold water from rural MWSS and wells (p<0.001). Strains belonging to Legionella pneumophila serogroups 2-14 predominated in the examined samples, while strains of L. pneumophila serogroup 1 and strains of Legionella spp. (other than L. pneumophila) were 3-fold less numerous. The rates of positive findings in the semi-nested PCR (stage 2) were greater than culture isolations in all kinds of samples, except for urban institutions. The correlation between the culture and PCR results was positive for samples of hot water from urban MWSS (p<0.01), but not for samples of cold water from rural MWSS and wells (p>0.5). A significant correlation was found between rates of PCR-positive results and numbers of Legionella pneumophila serogroups 2-14 strains, but not for other Legionella serogroups or species. In conclusion, our results support the opinion that though PCR cannot be a substitute for the isolation of Legionella by culture, it could be regarded as an useful complementary method.

Co-existence of Legionella and other Gram-negative bacteria in potable water from various rural and urban sources.

A total of 320 potable water samples were collected from various rural and urban sources located in the Lublin region of eastern Poland. They comprised: 55 samples of treated (chlorinated) tap water from rural dwellings distributed by the municipal water supply system (MWSS), 111 samples of treated tap water from urban dwellings distributed by the MWSS, 45 samples of untreated well water from household wells and 109 samples from private water supply systems (PWSS) distributing untreated well water. Water samples were examined for the presence and species composition of Legionella, Yersinia, Gram-negative bacteria belonging to family Enterobacteriaceae (GNB-E) and Gram-negative bacteria not belonging to the family Enterobacteriaceae (GNB-NE), by filtering through cellulose filters and culture on respectively GVPC, CIN, EMB and tryptic soya agar media. The occurrence of Legionella in the samples taken from the outlets of the urban MWSS was high (77.5%), and significantly greater compared to frequencies noted in rural MWSS (7.3%), and samples of well water from household wells (28.9%) and PWSS (13.8%) (p<0.001). Strains L. pneumophila serogroups 2-14, L. pneumophila serogroup 1 and Legionella spp. (species other than L. pneumophila) formed respectively 64.3%, 17.5%, and 18.2% of total isolates from urban MWSS, 100%, 0, and 0 of those from rural MWSS, 69.2%, 7.7%, and 23.1% of those from household wells, and 66.7%, 0, and 33.3% of those from PWSS. The concentration of Legionella strains in the positive samples from urban MWSS exceeded the threshold limit value of 100 cfu/100 ml in 86.1%, while in the other sources this value was not exceeded. No Yersinia strains were isolated from the examined water samples. Altogether 8 species or genera of Gram-negative bacteria belonging to Enterobacteriaceae family (GNB-E) and 10 species or genera of Gram-negative bacteria not belonging to the Enterobacteriaceae family (GNB-NE) were found in the examined samples. In the MWSS samples, an inverse relationship was found between Legionella and GNB-E and the numbers of Enterobacter spp. and Serratia spp. strains were significantly more common in the samples without Legionella. By contrast, in the PWSS samples, the numbers of Enterobacter spp., Klebsiella spp. and Salmonella spp. were distinctly and significantly greater (p<0.01-p<0.001) in the samples containing Legionella. Among GNB-NE, Pseudomonas aeruginosa strains occurred significantly more frequently in samples containing Legionella (for MWSS and well water separately p<0.05, for total samples p<0.001). Similarly, strains of Flavobacterium breve and Xanthomonas spp. occurred significantly more often in the samples with Legionella, while the numbers of Aeromonas spp. and Vibrio spp. strains were significantly greater in the samples not containing Legionella. In conclusion, a health risk could be associated with exposure to the water from urban MWSS because of the high prevalence and concentration of Legionella, and with exposure to well water from PWSS because of the correlation of occurrence of Legionella and potentially pathogenic Enterobacteriaceae strains, and the possibility of synergistic eff ects. The adverse eff ects could be also due to the significant correlation of Legionella and Pseudomonas aeruginosa that occured in water from various sources.

Gram-negative bacteria in water distribution systems of hospitals.

A total of 67 samples of tap water were collected from faucets and showerheads in 6 hospitals located in the Lublin province (eastern Poland). The samples were examined for the presence and species composition of Legionella, Gram-negative bacteria belonging to family Enterobacteriaceae (GNB-E) and Gram-negative bacteria not belonging to family Enterobacteriaceae (GNB-NE), by filtering through cellulose filters and culture on respectively GVPC, EMB and tryptic soya agar media. On average, Legionella was isolated from 65.7% of the water samples taken in hospitals. Strains of the Legionella pneumophila types 2-14 predominated, forming 74.6% of total Legionella isolates. Legionella pneumophila type 1 strains constituted 13.5% of the total count, while other species of Legionella (referred to as Legionella spp.) formed 11.9% of the total. The concentrations of Legionella in positive water samples ranged from 3-350 cfu/100 ml. GNB-E were not found in the examined water samples. GNB-NE were isolated from 79.1% of the water samples taken in hospitals in the concentrations 11-300 cfu/100 ml. Species of the family Pseudomonadaceae predominated among GNB-NE strains isolated from the examined water samples, forming on average 71.5% of the total count. Altogether, 20 GNB-NE species were identified in the examined samples, out of which 12 were potentially pathogenic. In conclusion, Gram-negative flora of water samples taken in the examined hospitals complies with potable water sanitary standards by the lack of Enterobacteriaceae species, but creates a moderate health risk because of mediocre concentrations of Legionella and the presence of potentially pathogenic non-enterobacterial species.

Antinuclear antibodies are not increased in the early phase of Borrelia infection.

In the literature, there are case reports suggesting that Borrelia burgdorferi infection may induce autoimmune diseases dependent on antinuclear antibodies (ANA). The present study was undertaken in order to verify this possibility in a prospective manner. The study group comprised 78 consecutive patients (51 women and 27 men, median age 41.5 years) referred to our Department for the serologic diagnosis of Borrelia infection. The patients' sera were tested for Borrelia-specific IgM and IgG (Recombinant Antigen Enzyme Immunoassays, Biomedica). Antibodies against Borrelia were detected in 31 (39.7 %) persons. 15 persons (19.2 %) had positive IgM, another 15 (19.2 %)--positive IgG, and 1 person (3.2 %)--both IgM and IgG. Frequent positivity of IgM antibodies suggests that persons in the early phase of infection prevailed in the group. Tests for anti-dsDNA, anti-RNP, anti-Sm antibodies, and a screening test for systemic rheumatic diseases (ANA Rheuma Screen) were carried out using Varelisa Enzyme Immunoassays (Pharmacia and Upjohn). The spectrum of autoimmune diseases covered by these tests included SLE, MCTD, Sjogren's syndrome, scleroderma, polymyositis, and dermatomyositis. ANA were detected in 15 persons (19.2 %): anti-dsDNA in 7 (9.0 %), anti-RNP in 1 (1.3 %), anti-Sm in 2 (2.6 %), and ANA Rheuma Screen was positive in 6 persons (7.7 %). Statistical analysis of differences in the ANA frequency between Borrelia-positive and -negative groups was carried out using Fisher's exact chi-square test (both without and with gender and age matching). No significant differences were found between the groups. Based on the above results, we conclude that there is no increase in the frequency of antinuclear antibodies in the early phase of Borrelia infection.

Studies on the occurrence of Gram-negative bacteria in ticks: Ixodes ricinus as a potential vector of Pasteurella.

A total of 372 Ixodes ricinus ticks (101 females, 122 males, and 149 nymphs) collected by flagging in 6 mixed woodlands of eastern Poland were examined by culture for the presence of internal Gram-negative bacteria other than Borrelia burgdorferi. Adult ticks were examined in pools of 2 specimens each and nymphs were examined in pools of 3-5 specimens each. Ticks were disinfected in 70 % ethanol and homogenized in 0.85% NaCl. The diluted homogenate was inoculated onto 3 kinds of agar media: buffered charcoal yeast extract (BCYE-alpha) for isolation of fastidious Gram-negative bacteria, eosin methylene blue agar (EMB) for isolation of enterobacteria, and tryptic soya agar for isolation of all other non-fastidious Gram-negative bacteria. The Gram-negative isolates were identified with the API Systems 20E and NE microtests. A total of 9 species of Gram-negative bacteria were identified, of which the commonest were strains determined as Pasteurella pneumotropica/haemolytica, which were isolated on BCYE-alpha agar from ticks collected in all 6 examined woodlands. The total number of these strains (49) exceeded the total number of all other strains of Gram-negative bacteria recovered from ticks (30). Of the total number of examined ticks, the minimum infection rate with Pasteurella pneumotropica/haemolytica was highest in females (18.8%), and slightly lower in males (12.3%) and nymphs (10%). Besides Pasteurella pneumotropica/haemolytica, the following species of Gram-negative bacteria were isolated from examined ticks: Pantoea agglomerans, Serratia marcescens, Serratia plymuthica on EMB agar and Aeromonas hydrophila, Burkholderia cepacia, Chromobacterium violaceum, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia on tryptic soya agar. Minimal infection rates with these bacteria were low, ranging from 0.7-5.9%. Of the isolated bacteria, Chromobacterium violaceum, Pasteurella pneumotropica/haemolytica, Pseudomonas aeruginosa, and Serratia marcescens are potentially pathogenic for man and/or animals. In particular, the common occurrence of Pasteurella pneumotropica/haemolytica in Ixodes ricinus ticks poses a potential risk of pasteurellosis for humans and animals exposed to tick bites.

Microbial quality of water in dental unit reservoirs.

Microbial quality of water in a dental unit is of considerable importance since patients and dental staff are regularly exposed to water and aerosol generated by the unit. Water delivered to a dental unit by the so-called independent water system is the water coming from a reservoir which, at the same time, is an initial part of dental unit waterlines (DUWL). Thus, microbiological quality of this water is extremely important for the quality of water flowing from dental handpieces. The aim of the study was to assess microbiologically the water contained in dental unit reservoirs. Water samples were collected aseptically from the water reservoirs of 19 dental units. Results concerning microbial contamination: potable water quality indices, and detection and isolation of Legionella species bacteria, were presented. Over a half of the samples did not comply with the norms for potable water. In 63.1% of the cases, the number of colony forming units (cfu/ml) and of coliform organisms significantly exceeded acceptable values. Enterococcus was not detected in the samples of examined water. Similarly, no Legionella were found in the samples of dental unit reservoirs water. Reservoirs as water supplies and initial segment of DUWL should be subject to protocol to eliminate microbial contamination and routine monitoring to guarantee an appropriate quality of water used in dental treatment.

Legionella in sprinkling water as a potential occupational risk factor for gardeners.

To study a possibility of infection with Legionella at gardening by inhaling of water aerosolized at sprinkling of plants, samples of tap water used for sprinkling of plants grown in outdoor gardens and in greenhouses in the Lublin province (eastern Poland) were examined for the presence of Legionella, along with the samples of soil, artificial medium and air collected in modern greenhouses. The strains of Legionella were isolated from 8 out of 36 samples of water (22.2%) collected from outdoor taps used for sprinkling plants cultivated in outdoor gardens, and from 5 out of 20 samples of water (25.0%) collected from indoor taps used for sprinkling of plants cultivated in traditional greenhouses or foil tunnels. Both in the samples collected from outdoor and indoor taps Legionella pneumophila 2-14 was more common than Legionella spp. (respectively 13.9% vs. 8.3%, and 15.0% vs. 10.0%). No legionellae were found in 18 samples of tap water, 14 samples of soil, 14 samples of artificial medium or 6 samples of air collected in modern greenhouses. The results of this preliminary study suggest that water aerosolized at sprinkling of plants represents a potential source of Legionella infection among gardeners.