Assessment of antibiotic susceptibility of Legionella pneumophila isolated from water systems in Poland.

INTRODUCTION AND OBJECTIVE: Several studies have reported therapy failures in patients with legionnaires'disease; however, antimicrobial resistance of clinical and environmental isolates of Legionella spp. has not yet been documented. Routine susceptibility testing of Legionella spp. is not recommended because of difficulties in determining standard minimal inhibitory concentration values. The purpose of this study was to analyze the antimicrobial susceptibility of Legionella pneumophila. strains isolated from a water supply system. MATERIALS AND METHOD: Twenty-eight isolates of L. pneumophila (16 - L. pneumophila SG 1, 12 - L. pneumophila SG 2-14) obtained from water systems in public buildings in Poland were tested. Susceptibility testing was performed using the E-test method. The tested antibiotic were azithromycin, ciprofloxacin, and rifampicin. The medium used for the susceptibility testing was BCYE-, a special medium for Legionella cultivation. RESULTS: Among the tested strains, L. pneumophila was the only one resistant to azithromycin. It was a strain of L. pneumophila SG 2-14 isolated from the water system in a sanitorium. All isolates were found to be sensitive to ciprofloxacin and rifampicin. However, the azithromycin-resistant strain exhibited higher ciprofloxacin and rifampicin MIC (1.5 µg/ml, and 0.19 µg/ml, respectively). The MIC50 for azithromycin, ciprofloxacin, and rifampicin were 0,032, 0,125, and 0,003 µg/ml, respectively. The MIC90 for azithromycin, ciprofloxacin, and rifampicin were 0,032, 0,125, and 0,003 µg/ml, respectively. CONCLUSIONS: Azithromycin resistance was found in one strain of L. pneumophila SG 2-14, but the resistance mechanism is unknown and needs further study. It is possible that therapeutic failures in Legionnaires' disease may be associated with bacterial resistance which should be taken into account. The antibiotic sensitivity testing described in this study could be helpful in detecting the resistance of clinical L. pneumophila isolates. Ciprofloxacin and rifampicin have good in vitro activity against environmental L. pneumophila SG 1 and SG 2-14 in Poland.

[Occurrence of Legionella in technological water and studies of the total number of bacteria and fungi in indoor air at workplaces where water aerosol is generated]. / Wystepowanie bakterii z rodzaju Legionella w wodzie technologicznej oraz badania ogólnej liczby bakterii i grzybów powietrzu na stanowiskach pracy, na których generowany jest aerozol wodny.

BACKGROUND: The aim of the study was to confirm the fact that technological water is a significant carrier of Legionella, a potential serious threat to the health of operators of mechanical devices generating contaminated water aerosol. MATERIAL AND METHODS: Microbiological analyses of water and indoor air were conducted in 8 different production facilities involved in mechanical processing of glass. The study covered 81 samples of water collected from technical water systems. Isolation of Legionella and the determination of total number of microorganisms were processed according to PN-EN ISO 11731-2:2008E and PN-EN ISO 6222:2004P, respectively. Air samples were collected using air samplers and total numbers of bacteria and fungi were determined. RESULTS: The studies of process water, indicated the presence of Legionella in 27.2% of collected samples. These bacteria were present in both closed and open process water reservoirs at 10 cfu/100 ml to 2.9 x 10(4) cfu/100 ml. The count of other associated bacteria exceeded 10(3) cfu/ml. All strains isolated from Legionella-positive samples were identified as L. pneumophila SG 2-14. In 5 of 8 studied production facilities an increased total number of aerial bacteria and fungi was observed in samples collected in close vicinity of aerosol source. CONCLUSIONS: To reduce the number of microorganisms in water it is required to introduce technological water quality monitoring and procedures for the cleaning and disinfecting of mechanical devices generating water aerosol.

Persistent colonization of 2 hospital water supplies by L. pneumophila strains through 7 years--sequence-based typing and serotyping as useful tools for a complex risk analysis.

Contamination with Legionella spp. of hot water system (HWS) in hospitals is a considerable problem and elimination of bacteria poses difficulties. Obligatory control of Legionella spp. in hospital HWS was implemented in Poland in 2008y. After that, Legionella spp. has been isolated repeatedly from HWS of the majority of hospitals. The aim of our study was to confirm the permanent colonization with Legionella spp. of 2 hospital HWSs based on the antigenic (serogroup/subgroups) and genetic properties (SBT, rtxA) of L.pneumophila strains isolated in 2004-2011. The dynamic of L.pneumophila population was also examined due to methods of disinfections applied during 7 years. Totally, 134 environmental samples were collected from two hospitals in 2004-2011 (118 from HWSs). During the study disinfection by chlorine dioxide was implemented in both hospitals, while thermal shock was added in the hospital A. Isolated L.pneumophila were serogrouped (105 strains) using Dresden MAb Panel, genotyped by sequence based typing (53) and by harboring of rtxA gene (58 isolates). Legionella spp. were still presented in both systems after 7 years. Exactly the same strains (ST1, ST87, ST114, ST992) were found in the hospital B. While changes of L.pneumophila population were observed in the hospital A: strains still occurred after 7 years (ST835 Sg6, ST114 Sg6); modified antigenic properties (ST835 - Sg12 vs. Sg6); eliminated or maybe not detected (ST81, ST838, ST959). Moreover, the majority of examined strains ST1 (Sg1, OLDA) harboured rtxA gene (hospital B). Our results and data in the EWGLI SBT base indicated higher risk of Legionella infection in the hospital B than A--because of heavy colonization with L.pneumophila ST1. The risk assessment of Legionella infection based only on technical parameters, extent of colonization/contamination level may be not completed. It should be supplemented with the additional examination: serotyping, genotyping and virulence testing of isolated strains.

[Respiratory symptoms among industrial workers exposed to water aerosol. A pilot study of process water and air microbial quality]. / Analiza wystepowania dolegliwosci i objawów ze strony ukladu oddechowego u osób zatrudnionych w przemysle na stanowiskach pracy generujacych aerozol wodny. Badania pilotazowe jakosci mikrobiologicznej powietrza i wody technologicznej.

BACKGROUND: The frequency of respiratory symptoms in workers exposed to water aerosol was evaluated along with the preliminary assessment of microbiological contamination of air and water used in glass processing plants. MATERIAL AND METHODS: A questionnaire survey was conducted in 131 workers from 9 glass processing plants. Questions focused on working conditions, respiratory symptoms and smoking habits. A pilot study of air and water microbiological contamination in one glass processing plant was performed. Water samples were tested for Legionella in accordance with EN ISO 11731-2:2008 and for total colony count according to PN-EN ISO 6222:2004. Air samples were tested for total numbers of molds and mildews. RESULTS: During the year preceding the survey acute respiratory symptoms occurred in 28.2% of participants, while chronic symptoms were reported by 29% of respondents. Increased risks of cough and acute symptoms suggestive of pneumonia were found among the respondents working at a distance up to 20 m from the source of water aerosol compared to other workers (OR = 2.7), with no difference in the frequency of other symptoms. A microbiological analysis of water samples from selected glass plant revealed the presence of L. pneumophila, exceeding 1000 cfu/100 ml. The number of bacteria and fungi detected in air samples (above 1000 cfu/m3) suggested that water aerosol at workplaces can be one of the sources of the air microbial contamination. CONCLUSIONS: The questionnaire survey revealed an increased risk of cough and acute symptoms suggestive of pneumonia in the group working at a shortest distance form the source of water aerosol.

Assessment of cytokine release after in vitro stimulation of whole blood with legionella pneumophila in immunocompromised patients.

This study we examined ex vivo potential of the immune response after stimulation of whole blood with L. pneumophila SG 1, SG 2-14 and L. pneumophila standard strain ATCC 33152 in immunocompromised patients, such as: hemodialysis patients and patients after renal transplantation. The levels of TNF-α and IFN-γ in supernatants were measured with the use of commercial ELISA kits. The synthesis of TNF-α and IFN-γ after stimulation with L. pneumophila were analyzed in two aspects: differentiated stimulatory activity in relation to SG 1, SG 2-14 and ATCC 33152 L. pneumophila and differentiated response of the hemodialysis patients and patients after renal transplantation in relation to the control group. The positive and negative results of anti-L. pneumophila antibodies of two groups of our patients were found for the analysis of the stimulatory activity of L.pneumophila as a primary or secondary response. In patients with immunosuppression the response in the secretion of cytokines (TNF-α and IFN-γ) was reduced after stimulation of L. pneumophila SG 1 but in varying degrees after stimulation of L. pneumophila SG 2-14, which indicates that the risk of the infection is varied.

[Optimization of Cryptosporidium and Giardia detection in water environment using automatic elution station Filta-Max xpress]. / Optymalizacja metody wykrywania pierwotniaków pasozytniczych Cryptosporidium i Giardia w srodowisku wodnym przy zastosowaniu automatycznej stacji pluczacej Filta-Max xpress.

BACKGROUND: The presence of parasitic protozoa in drinking water is mostly a result of improperly maintened the water treatment process. Currently, in Poland the testing of Cryptosporidium and Giardia in water as a part of routine monitoring of water is not perform. OBJECTIVE: The aim of this study was the optimization of the method of Cryptosporidium and Giardia detection in water according to the main principles of standard ISO 15553:2006 and using Filta-Max xpress automatic elution station. MATERIAL AND METHOD: Preliminary tests were performed on the samples contaminated with oocysts and cysts of reference strains of both parasitic protozoa. Further studies were carried out on environmental samples of surface water sampled directly from the intakes of water (21 samples from Vistula River and 8 samples from Zegrzynski Lake). Filtration process and samples volume reducing were performed using an automatic elution system Filta-Max xpress. Next, samples were purified during immunomagnetic separation process (IMS). Isolated cysts and oocysts were stained with FITC and DAPI and than the microscopic observation using an epifluorescence microscope was carried out. RESULTS: Recovery of parasite protozoa in all contaminated water samples after 9-cycles elution process applied was mean 60.6% for Cryptosporidium oocysts and 36.1% for Giardia cysts. Studies on the environmental surface water samples showed the presence of both parasitic protozoa. Number of detected Giardia cysts ranged from 1.0/10 L up to 4.5/10 L in samples from Zegrzynski Lake and from 1.0/10 L up to 38.9/10 L in samples from Vistula River. Cryptosporidium oocysts were present in 50% of samples from the Zegrzynski Lake and in 47.6% of samples from the Vistula River, and their number in both cases was similar and ranged from 0.5 up to 2.5 oocyst/10 L. The results show that applied procedure is appropriate for detection the presence of parasitic protosoan in water, but when water contains much amount of inorganic matter and suspended solids test method have to be modified like subsamples preparation and filtration process speed reduction. CONCLUSIONS: The applied method with the modification using Filta-Max xpress system can be useful for the routine monitoring of water. Detection of Cryptosporidium and Giardia in all samples of water taken from the intakes of surface water shows the possibility oftransfering of the protozoan cysts into the water intended for the consumption, therefore the testing of Cryptosporidium and Giardia should be included into the monitoring of water.

Development of a miniaturized DNA microarray for identification of 66 virulence genes of Legionella pneumophila.

INTRODUCTION: For the last five years, Legionella sp. infections and legionnaire's disease in Poland have been receiving a lot of attention, because of the new regulations concerning microbiological quality of drinking water. This was the inspiration to search for and develop a new assay to identify many virulence genes of Legionella pneumophila to better understand their distribution in environmental and clinical strains. The method might be an invaluable help in infection risk assessment and in epidemiological investigations. MATERIAL/METHODS: The microarray is based on Array Tube technology. It contains 3 positive and 1 negative control. Target genes encode structural elements of T4SS, effector proteins and factors not related to T4SS. Probes were designed using OligoWiz software and data analyzed using IconoClust software. To isolate environmental and clinical strains, BAL samples and samples of hot water from different and independent hot water distribution systems of public utility buildings were collected. RESULTS: We have developed a miniaturized DNA microarray for identification of 66 virulence genes of L. pneumophila. The assay is specific to L. pneumophila sg 1 with sensitivity sufficient to perform the assay using DNA isolated from a single L. pneumophila colony. Seven environmental strains were analyzed. Two exhibited a hybridization pattern distinct from the reference strain. DISCUSSION: The method is time- and cost-effective. Initial studies have shown that genes encoding effector proteins may vary among environmental strains. Further studies might help to identify set of genes increasing the risk of clinical disease and to determine the pathogenic potential of environmental strains.

[Investigation of susceptibility of materials contacting with drinking water for microbial growth]. / Badania podatnosci materialów kontaktujacych sie z woda przeznaczona do spozycia na powstawanie obrostów mikrobiologicznych.

Main purpose of this paper was to confirm of results obtained by new method based on bioluminescence technique for determination of susceptibility of materials contacting with drinking water were repeatable. Investigation was conducted during 8 weeks in dynamic conditions using continuous flow reactor UPE. Every week swabbes from tested materials were collected and level of ATP was examined. The results obtained from the surface of tested materials were repeatable and clearly approximate to those obtained from the surface of negative control material (stainless steel, low susceptibility for microbial growth). The level of the ATP on the surface of positive control material (paraffin, large susceptibility for microbial growth) was fifty times higher than that observed on other materials. Presented investigation was the main part of validation process, which in short time will serve to initiate system of complete assessment for materials contacting with drinking water according to Council Directives 89/106/EEC and 98/83/EC requirements.

[Microbiological evaluation of risk of legionellosis and practical methods applied for elimination of Legionella pneumophila from hospital water systems]. / Mikrobiologiczna ocena zagrozenia legioneloza oraz zastosowane metody eliminacji Legionella pneumophila z sieci wodnych budynków szpitalnych.

Microbiological evaluation of risk of legionellosis was based on the results of water samples collected from hospital water systems examinations. The percentage of positive water samples, number of detected Legionella spp. cfu/100ml, determined serogroup and subgroup of isolated strains as well as genetic virulence markers (rtxA, mompS) were analyzed by multivariable regression tests. The ability to adhere to cells A549 and proliferate in THP-1 cells were also examined. Strong correlation was found between the detected number of Legionella spp. cells (cfu/100ml) and percentage of positive samples, presence of gene depending ability bacteria to move, adhere to A549 cells and presence of mompS gene for special protein building capsule. From other side, correlation between identified cases or infections due to L. pneumophila and presence of gene: rtxA, mompS, and epitop MAb3/l was determined. In conclusion it was indicated that evaluation of risk of legionellosis should be based on ability the legionellae strains to prolify, and grow in water systems and also on virulence properties of isolated strains, especially those producing RTX toxin, flagella and presenting virulence epitop MAb3/l. Two examples of practical application methods for reduction of risk of nosocomial infection were described also. It was shown that cooperation of engineers and microbiologist is essential for effective elimination of Legionella from water systems and reduction of the risk of Legionella infection in hospital.

[Elaboration of the method for assessment of susceptibility to microbial growth of materials contacting with drinking water]. / Opracowanie metody oceny podatnosci materialów kontaktujacych sie z woda przeznaczona do spozycia na powstawanie obrostów mikrobiologicznych.

Initial methodological study was conducted to determine the susceptibility to microbial growth of different materials applied in contact with drinking water. The purpose of this assay was to elaborate the method for determining the ability of different materials contacting with drinking water to promote microbial growth and to determine possible correlation of results obtained by two analytic methods: inoculation of microorganisms swabbed from materials surface into a medium and ATP level examination. The assay was conducted during 16 weeks in dynamic conditions using continuous flow reactor. Every two weeks swabbes from examinated materials were collected and the total number of microorganisms was determined after incubation in 22 degrees C for 72 hours and in 37 degrees C for 48 hours respectively. Determination of microorganisms number and ATP level were both examinated in water inlet. Difference in susceptibility to microbial growth of different materials used in contact with drinking water was observed. This was confirmed by both analytic methods. Microbial growth on the surface of negative control materials (glass, poliethylene and stainless steel) was several time less intensive than on positive control material (floor finish not destined for contact with drinking water). Correlation between the quantity of microorganisms and ATP level on the surface of the same kind of materials was confirmed. Usefulness of continous flow reactor was confirmed.

[Occurrence of bacteria from the Legionella genus in cooling water systems]. / Wystepowanie bakterii z rodzaju Legionella w systemach wody chlodniczej.

Results of Legionella bacteria study in water samples from industrial cooling water systems and air-conditioner systems were presented. Legionella pneumophila was detected in 32,4%. of 518 examined water samples collected from 1998 to 2006. The most dangerous Legionella pneumophila sg 1 was detected in 11.3% of all positive samples. The high number (more than 10(4) jtk/l) of isolated Legionella bacteria was determinated in 60% of water samples taken from cooling waters systems and 33% of samples of waters related with air-conditioning that is alarming. High pollution requires immediate intervention (installations cleaning and disinfection). Data indicated real health risk connected with Legionella pollution of cooling water. Creation of low regulations which will put the obligation of cooling water systems monitoring of Legionella bacteria occurrence and taking preventive measures is necessary.

[Outbreak of hospital acquired Legionnaires' disease in patients of ophthalmic ward. Nosocomial Legionella infections for the first time observed in Poland]. / Ognisko choroby legionistów na oddziale okulistycznym. Szpitalne zakazenie Legionella pneumophila po raz pierwszy obserwowane w Polsce.

The aim of the paper is to describe the criteria used for identification of first and so far the only one outbreak of hospital bacterial infections due to L. pneumophila. The infected persons were patients hospitalized at ophthalmic ward for more than 10 days. Four patients were found ill among 27 hospitalized (15%) at ophthalmic ward and 3 of them died (75%) in spite treatment in intensive care unit. The source of infection was found in the hospital hot water system. It was shown that L. pneumophila sg 1 and sg 2-14 were settled in the tanks and pipelines of hot water installations. The high number of L. pneumophila sg I and sg 2-14 colony forming units (> 10 000 cfu /100 ml) were found in the water specimens taken from the hospital water system, showing the high risk of Legionella infection for patients. Cleaning and disinfection of hot water system was repeated three times using composition every time modified as stronger mechanical, thermal and chemical methods. Complete elimination of Legionella from hot water system was achieved after cutting off deadlegs of water and replacement of both old hot water reservoirs with new ones. Collected experience served for preparation of guidelines for control and prevention of Legionella infections in hospital buildings, published on National Institute of Hygiene web site A month later Polish Ministry of Health published the Directives concerning the quality of drinking water to which the control of Legionella infection has been included.