Hospital Water Distribution System Research Articles

Antibiotic resistance pattern of waterborne causative agents of healthcare-associated infections: A call for biofilm control in hospital water systems

BackgroundIn recent years, the global spread of antimicrobial resistance has become a concerning issue, often referred to as a “silent pandemic”. Healthcare-associated infections (HAIs) caused by antibiotic-resistant bacteria (ARB) are a recurring problem, with some originating from waterborne route. The study aimed to investigate the presence of clinically relevant opportunistic bacteria and antibiotic resistance genes (ARGs) in hospital water distribution systems (WDSs). MethodsWater and biofilm samples (n = 192) were collected from nine hospitals in Isfahan and Kashan, located in central Iran, between May 2022 and June 2023. The samples were analyzed to determine the presence and quantities of opportunistic bacteria and ARGs using cultural and molecular methods. ResultsStaphylococcus spp. were highly detected in WDS samples (90 isolates), with 33 % of them harboring mecA gene. However, the occurrences of E. coli (1 isolate), Acinetobacter baumannii (3 isolates), and Pseudomonas aeruginosa (14 isolates) were low. Moreover, several Gram-negative bacteria containing ARGs were identified in the samples, mainly belonging to Stenotrophomonas, Sphingomonas and Brevundimonas genera. Various ARGs, as well as intI1, were found in hospital WDSs (ranging from 14 % to 60 %), with higher occurrences in the biofilm samples. ConclusionOur results underscore the importance of biofilms in water taps as hotspots for the dissemination of opportunistic bacteria and ARG within hospital environments. The identification of multiple opportunistic bacteria and ARGs raises concerns about the potential exposure and acquisition of HAIs, emphasizing the need for proactive measures, particularly in controlling biofilms, to mitigate infection risks in healthcare settings.

Fungal contamination of the water distribution system of a tertiary hospital water supply system in a resource-limited setting.

Fungal contamination of hospital water distribution systems has been implicated in outbreaks of healthcare-associated infections. To evaluate the prevalence of fungi in the water distribution system of a tertiary hospital in Nigeria. This was a descriptive cross-sectional study. Swabs and water samples were collected from taps and faucets in the hospital categorized into low (Accidents and Emergency Unit, Children Emergency Unit, Acute Stroke Unit and the 24 in-patient hospital wards) and high-risk (Renal Dialysis Unit, Central Sterile Services Department, Theatres and Intensive Care Units (ICUs)) units based on the vulnerability of patients being managed there. The membrane filtration method for water analysis was used. Where possible, isolates cultured were identified to species level. In total, 105 water and 49 swab samples were collected for analysis. All analysed water samples grew fungi. A total of 289 (high-risk; n = 178; low-risk; n = 111) and 76 fungi isolates were recorded from water and swab samples, respectively, with 31 different species identified. Aspergillus was the most predominant genus with five different species: Aspergillus niger (9.9%), terreus (4.4%), flavus (3.3%), fumigatus (8.8%) and versicolor (2.20%) isolated. Twenty-five and 18 species of fungi were identified in the low and high-risk units, respectively. The labour ward (n = 46; 25.8%) and modular theatre (n = 47; 42.3%) were the most contaminated units. Cladosporium spp. and Paecilomyces spp. were the most frequently isolated fungi in the low and high-risk units, respectively. The dialysis centre (n = 9; 8.1%) and renal transplant theatre (n = 7; 6.31%) had the lowest contamination rates in the high-risk units. Aspergillus niger, Cephalosporium curtipes, Penicillium chrysogenum and Penicillium glabrum were each identified in 4/6 units from which swabs were taken. The facility had no documented protocol for its water safety and quality. Our data reveal a high rate of contamination of hospital water sources by fungi, some of which are known to cause life-threatening infections. For better water treatment and water tank cleaning and disinfection, a standard protocol is advised. Ensuring that the water distribution systems in hospital settings are free of fungal contaminants is important to prevent the possibility of waterborne mycosis outbreaks.

The Detection Limits of Legionella According to the EU Directive 2020/2184. Could That Be Too Permissive?

The problem of detecting legionella after a case of legionellosis from the source of environmental contamination has been known since a long time ago. Legionella is a bacterium present in various natural and artificial habitats and especially in surface fresh waters. It is found in greater concentration in warm waters, at temperatures between 20°C and 42°C. The greatest risk factor for humans is represented by the presence of Legionella in water distribution systems in hospitals, medical equipment (e.g. respirators, dialyzers, inhalers, humidifiers, water, massage equipment used in balneotherapy) and turbines used in dental practices, especially for hospitalized individuals. In the EU directive 2020/2184, issued by the European parliament on 16/12/2020, the concentration of Legionella was added to the parameters to be determined in assessing the quality of drinking water intended for human consumption. The objectives were to improve the quality standard of drinking water, reduce the consumption of bottled water and consequently reduce plastic waste. The WHO notes that Legionella causes the greatest burden from a health point of view and it is included among the parameters that require careful monitoring with a limit of less than 1000 CFU/L. The aim of this report was to evaluate the new EU directive 2020/2184 on the light of our laboratory experience. A total of 459 samples were processed at our Hygiene of food Laboratory - Department of Medical Sciences and Public Health. All statistical analyses were conducted using the SPSS statistical package (version 23 for Windows. SPSS, Inc. Chicago, Ill). Of the 67 structures examined where the cases occurred, 35 showed samples with at least one over-threshold value considering the reference value of 100 CFU/L, whereas using the new limit of 1000 CFU/L, only 25 structures resulted as having at least one sample above the threshold. In our experience as a regional reference laboratory for Legionella research, the increase from 100 CFU/L to 1,000 CFU/L could lead to a lower alert level. In fact, in the period between October 2017 and October 2021, the median value of CFU/L in presence of a case was 0 (0-100). Despite the large amount of studies on Legionella only a few relate the withdrawals and the consequent CFU/L with the confirmed cases of legionellosis, as in our analysis. The 75° percentile values of the Legionella concentration equal to 100 CFU/L in all samples associated with cases and clusters leads us to hypothesize that the limit equal to 1000 CFU/L that will be introduced for environmental monitoring as per recent European regulations may not be sufficiently protective for minimizing risk in the population, especially in healthcare facilities where fragile patients are assisted.

Detection of Legionella pneumophila in the Water Samples of Food Industries and Hospitals in Bangladesh

Background: The human pathogen Legionella pneumophila causes a serious pneumonia-like respiratory disease called Legionnaires' disease, mainly in elderly and immunocompromised individuals. This pathogen can be found in the water distribution systems of large constructions with cooling towers which is a common phenomenon at present in Bangladesh due to its rapid economic growth. But there is a dearth of information on the incidence of Legionella in Bangladesh. Therefore, the current study aimed to investigate the presence of Legionella pneumophila in hospital and industrial water distribution systems in Dhaka, Bangladesh. Methods: A total of 114 water samples collected from two hospitals and five food industries were inoculated on the Legionella-specific medium Buffer Charcoal Yeast Extract (BCYE) agar medium before and after the treatment with acid, heat, or a combination of both. Samples producing Legionella-like colonies on BCYE agar medium were screened by Legionella Latex Test Kit, and the metagenomic DNAs obtained from these samples were analyzed by PCR using L. pneumophila-specific 16S rRNA primers. Results: Among 114 samples, Legionella-like colonies were observed in 30 water samples which demonstrated no agglutination in the Latex agglutination test. PCR analysis showed the presence of L. pneumophila in seven water samples, four in the potable water, chiller water, and cooling tower water of two different food industries, and three in ICU tap water, cooling tower water, and Fan Coil Units of two different hospitals. Sequence analysis of amplicons revealed that all seven sequences had 100% similarity with L. pneumophila. Conclusion: The presence of L. pneumophila in the water samples of local hospitals and food industries indicates that these habitats might serve as a potential site for Legionnaires' infection in Bangladesh. The results also showed that PCR, contrary to the conventional culture methods, could be more efficient and rapid in the identification of L. pneumophila.

Assessment of the risk of Legionella pneumophila in water distribution systems in hospitals of Tehran city.

When a sensitive host inhales aerosols containing these bacteria, Legionella infection occurs. Therefore, monitoring and assessing Legionella in the environment and water distribution systems of such places are critical due to the prone population in hospitals. However, the health risks of Legionella bacteria in the environment are not adequately evaluated. In this study, for hospitalized patients, we performed a quantitative health risk assessment of Legionella in selected hospitals in Tehran city using two scenarios of shower and toilet faucet exposure. This study identified Legionella in 38 cases (38%) out of 100 samples collected from toilet faucets and showers in 8 hospitals. The information gathered was used for quantitative microbial risk assessment (QMRA). The microbial load transmitted by inhalation was calculated using the concentration of Legionella in water. Other exposure parameters (inhalation rate and exposure time) were obtained using information from other studies and the median length of hospital stay (3.6days). The exponential model was used to estimate the risk of infection (γ = 0.06) due to Legionella pneumophila (L. pneumophila) inhalation for each exposure event. For the mean concentration obtained for Legionella (103CFU/L), the risk of infection for toilet faucets and showers was in the range of 0.23-2.3 and 3.5-21.9, respectively, per 10,000 hospitalized patients. The results were compared with the tolerable risk level of infection determined by the US EPA and WHO. The risk values exceeded the WHO values for waterborne pathogens in hospitals in both exposure scenarios. As a result, our QMRA results based on monitoring data showed that despite using treated water (from distribution networks in the urban areas) by hospitals, 38% of the samples were contaminated with Legionella, and faucets and showers can be sources of Legionella transmission. Hence, to protect the health of hospitalized patients, the risk of Legionella infection should be considered.

Environmental Surveillance of Legionella spp. in an Italian University Hospital Results of 10 Years of Analysis

The occurrence of Legionella spp. in the water distribution systems of large hospitals and other healthcare facilities is considered particularly dangerous, due to the critical nature of the hospitalized patients. The aim of this study is to present a pluri-annual environmental surveillance in a large university hospital assessing the prevalence of Legionella spp. and underlining its variability over the years. The samples of water were collected in accordance with the Italian National Guidelines and the sampling sites considered in this study were selected favoring wards with very high-risk patients and with patients at increased risk. The laboratory analyzed a total of 305 water samples deriving from 24 different sampling points. Legionella spp. were detected in 39.4% of samples, the majority of which were contaminated by Legionella pneumophila serogroups 2–14 (68.7%). Statistically significant differences were found among different seasons with a linear trend in positive proportion from summer to spring. Several experimental interventions to prevent and reduce Legionella colonization were attempted, but there is no a definitive method for the complete eradication of this microorganism. The permanent monitoring of hospital water distribution systems is fundamental to preventing the potential risk of nosocomial Legionellosis and to implementing procedures to minimize the risk of Legionella spp. colonization.

Identification and isolation of homoserine lactones (HSLs) produced by Pseudomonas aeruginosa and the effects on Legionella pneumophila growth

Risk for infections from Legionella pneumophila represents a critical issue for immunocompromised individuals, especially when this species is present within the biofilm of the water distribution systems of hospitals or other health facilities where the complex ancient water network creates environmental conditions that are suitable for Legionella persistence and multiplication. The shock hyperchlorination and subsequent systemic continuous chlorination is a disinfection treatment reported in the Italian Guideline for prevention and control of nosocomial legionellosis as an effective procedure to reduce L. pneumophila colonization of hospital water sources. Although this disinfection strategy has been proven to be effective in the short-term, eradication of Legionella from water networks is usually not achieved. The objective of this study is the identification and purification of P. aeruginosa bacteriocins, such as homoserine lactones (HSLs), by the development of an experimental method by High performance-liquid chromatography (HPLC), coupled to diode array detection (DAD) and the evaluation of the effectiveness of these molecules in the managing of the persistence of L. pneumophila in experimental cocultures.

Occurrence of Fungi in the Potable Water of Hospitals: A Public Health Threat.

Since the last decade, attention towards the occurrence of fungi in potable water has increased. Commensal and saprophytic microorganisms widely distributed in nature are also responsible for causing public health problems. Fungi can contaminate hospital environments, surviving and proliferating in moist and unsterile conditions. According to Italian regulations, the absence of fungi is not a mandatory parameter to define potable water, as a threshold value for the fungal occurrence has not been defined. This study evaluated the occurrence of fungi in potable water distribution systems in hospitals. The frequency of samples positive for the presence of fungi was 56.9%; among them, filamentous fungi and yeasts were isolated from 94.2% and 9.2% of the samples, respectively. The intensive care unit (87.1%) had the highest frequency of positive samples. Multivariable model (p < 0.0001), the variables of the period of the year (p < 0.0001) and type of department (p = 0.0002) were found to be statistically significant, suggesting a high distribution of filamentous fungi in the potable water of hospitals. Further studies are necessary to validate these results and identify the threshold values of fungi levels for different types of water used for various purposes to ensure the water is safe for consumption and protect public health.

The role of water distribution system in HCAIs

Abstract Introduction The water distribution system in hospitals may constitute a source of healthcare-associated infections. Understanding the means of acquisition, sources and reservoirs of nosocomial pathogens is crucial for developing methods to reduce the incidence of nosocomial infections. The aim of our study was to evaluate the microbiological contamination by Pseudomonas aeruginosa in the water system of the University Hospital “G. Martino” in Messina over a period of nine years, in order to a) clarify possible relationship between the presence of aerators and contamination, b) to highlight the presence of a yearly variation and c) to verify the statistical difference between microbiological contamination of taps with and without filters. Materials and Methods We carried out a retrospective study for nine year through the collection of water samples that were analysed for total bacteria count and Pseudomonas aeruginosa. The differences between the results recorded over the nine-year study period were evaluated using variance analysis. P values of &amp;lt; 0.05 were considered statistically significant. Software R was used for statistical assessment. Results We analysed a total of 6168 samples with a positive rate of 9.31% and a decreasing trend over the nine years to a value of 2.44% (p &amp;lt; 0.001), due to the elimination of the aerators of taps without filters and also to the introduction of filters in taps of high-risk wards and surgical rooms. We found statistical difference between taps with or without filter (p &amp;lt; 0.001) and a higher positive rate during the summer season compared to the other months. Also, an increase of the positivity rate was detected in some year due to the lack of monthly replacement of the filter. Conclusions These results reveal a high level of contamination of taps by opportunistic bacteria with severe implications especially for high-risk settings and therefore, the need to improve the management of these devices. Key messages Particular attention must be reserved to the water distribution systems in hospitals in order to prevent the spread of healthcare associated infections. The continued application and strengthening of guidelines and procedures is essential to limit the spread of multidrug resistant pathogens.

Legionella pneumophila and Protozoan Hosts: Implications for the Control of Hospital and Potable Water Systems.

Legionella pneumophila is an opportunistic waterborne pathogen of public health concern. It is the causative agent of Legionnaires’ disease (LD) and Pontiac fever and is ubiquitous in manufactured water systems, where protozoan hosts and complex microbial communities provide protection from disinfection procedures. This review collates the literature describing interactions between L. pneumophila and protozoan hosts in hospital and municipal potable water distribution systems. The effectiveness of currently available water disinfection protocols to control L. pneumophila and its protozoan hosts is explored. The studies identified in this systematic literature review demonstrated the failure of common disinfection procedures to achieve long term elimination of L. pneumophila and protozoan hosts from potable water. It has been demonstrated that protozoan hosts facilitate the intracellular replication and packaging of viable L. pneumophila in infectious vesicles; whereas, cyst-forming protozoans provide protection from prolonged environmental stress. Disinfection procedures and protozoan hosts also facilitate biogenesis of viable but non-culturable (VBNC) L. pneumophila which have been shown to be highly resistant to many water disinfection protocols. In conclusion, a better understanding of L. pneumophila-protozoan interactions and the structure of complex microbial biofilms is required for the improved management of L. pneumophila and the prevention of LD.

Legionella control in a tertiary care hospital in Italy: adapting solutions to changing scenarios

Abstract Background Legionella contamination of hospital water distribution system still remains a critical issue in healthcare settings, despite the disputed relationship between environmental legionella and hospital-acquired legionellosis. We report the experience of our 1200-beds hospital, where a new water safety plan (WSP) has been applied since 2017, and the results obtained with the application of different methods to control Legionella water contamination, especially in critical areas. Methods Policlinico San Martino is the referral tertiary acute-care university hospital in Liguria region, North-West Italy. It’s made of 21 buildings of different dimensions, 7 of which host high risk patients, according to Italian Guidelines for legionellosis prevention and control. A sampling plan for Legionella detection was implemented in 2017, focusing on the water network critical points. Different secondary disinfection procedures were used, including systemic (chlorine dioxide, monochloramine), focal (point-of-use filtration) and short-term methods (hyperchlorination), the last in case of contamination higher than 10^4 CFU/l. Results Respectively, during 2017 and 2018 a total of 201 and 119 hot water samples were collected, distributed among the 7 critical buildings of the hospital. Negative samples raised from 69.7% to 74.8%, while positive samples &amp;gt;10^3 CFU/l (recommended cut-off for acute disinfection according to Italian guidelines) lowered from 21.9% to 9.2% of total samples. Out of 55 positive samples, Legionella sg 3 accounted for 98.2% of the total. Conclusions The new WSP determined a significant reduction of Legionella contamination of water distribution networks in our hospital critical buildings. The continuous surveillance of critical water system points provided evidence to elaborate an effective protocol for routine and extraordinary disinfection interventions, as result of a close collaboration between hospital hygiene unit and engineering and technical services. Key messages The implementation of an effective water safety plan and different disinfection procedures in hospital critical areas allow us to reduce and control the risk of hospital-acquired Legionella infection. Continuous surveillance cultures of drinking water to detect Legionella are necessary to adapt disinfection methods to local results.

Real-Time Continuous Surveillance of Temperature and Flow Events Presents a Novel Monitoring Approach for Hospital and Healthcare Water Distribution Systems.

Within hospitals and healthcare facilities opportunistic premise plumbing pathogens (OPPPs) are a major and preventable cause of healthcare-acquired infections. This study presents a novel approach for monitoring building water quality using real-time surveillance of parameters measured at thermostatic mixing valves (TMVs) across a hospital water distribution system. Temperature was measured continuously in real-time at the outlet of 220 TMVs located across a hospital over a three-year period and analysis of this temperature data was used to identify flow events. This real-time temperature and flow information was then compared with microbial water quality. Water samples were collected randomly from faucets over the three-year period. These were tested for total heterotrophic bacteria, Legionella spp. and L. pneumophila. A statistically significant association with total heterotrophic bacteria concentrations and the number of flow events seven days prior (rs[865] = −0.188, p < 0.01) and three days prior to sampling (rs[865] = −0.151, p < 0.01) was observed, with decreased heterotrophic bacteria linked to increased flushing events. Only four samples were positive for Legionella and statistical associations could not be determined; however, the environmental conditions for these four samples were associated with higher heterotrophic counts. This study validated a simple and effective remote monitoring approach to identifying changes in water quality and flagging high risk situations in real-time. This provides a complementary surveillance strategy that overcomes the time delay associated with microbial culture results. Future research is needed to explore the use of this monitoring approach as an indicator for different opportunistic pathogens.

Prevalence of Legionella spp. in hot water samples from Polish hospitals in 2009-2013

Background: Microbiological threat from Legionella spp. is associated with the current widespread use of air-condition systems and frequent colonization of hot water distribution systems in public facilities (hospitals, hotels, nursing homes). This poses a particularly high risk in hospitalized patients, as many of them are immunocompromised. More than 50 species and 72 serogroups of Legionella spp. have been described thus far, including more than 20 species being pathogenic to humans. Material/Methods: Microbiological threat posed by Legionella spp. was assessed based on a retrospective analysis of microbiological quality of water from hot water distribution systems in Polish hospitals, determined in 2009-2013 at certified laboratories of the Sanitary-Epidemiological Surveillance. The results were kindly provided by the hospitals’ administration upon request, for which the authors would like to express their deepest gratitude. The study material included samples of hot water from internal distribution systems in 379 Polish hospitals, collected by the State Sanitary Inspectorate. Results: Based on the results of microbiological analyses, we were able to estimate Legionella spp. colonization rates in hospital water distribution systems and to assess the activities undertaken by the State Sanitary Inspectorate to reduce excessive bacterial counts in this reservoir. Microbiological analyses conducted in 2009-2013, i.e. after implementing a statutory obligation to monitor Legionella spp. in hospital hot water distribution systems, showed elevated bacterial counts in 3.92% to 12.7% of the samples. These findings justify further microbiological monitoring of hospital water distribution systems. Discussion: The prevalence of the most pathogenic serotype SG1 in hot water distribution systems of Polish hospitals is relatively low compared to other European countries. To maintain this favorable status, hospital water systems should be monitored not only for the presence of Legionella pneumophila, but also for the prevalence of serogroup 1 L. pneumophila.

Molecular characterization of nontuberculous mycobacteria in hospital waters: a two-year surveillance study in Tehran, Iran.

Microbiological control of hospital waters as one of the main sources of nontuberculous mycobacteria (NTM) is important for the prevention of NTM-associated illness. This study aimed to investigate the prevalence of NTM in the hospital water systems of Tehran, Iran. A total of 218 samples from different hospital waters (i.e., tap water and medical devices such as humidifying cup of oxygen manometer, dialysis devices, nebulizers, and dental units) were included in this study. Phenotypic and molecular tests were used to identify the isolated organisms to species level. Of 218, 85 (39.0%) samples at 37 °C and 87 (40.0%) samples at 25 °C were identified as NTM. Using hsp65-sequencing method, Mycobacterium lentiflavum was the most frequently encountered, followed by M. gordonae and M. paragordonae. No significant difference was seen in frequency and species in mycobacteria isolated at 37 °C and 25 °C temperatures. Humidifying cup of oxygen manometer had the most contaminated water among the investigated water distribution systems in hospitals. Isolation of NTM from hospital water sources is a serious public health problem in Iran and merits further attention by health authorities. Establishment of microbiological monitoring systems for hospital waters and expanding the number of facilitated laboratories are strongly recommended.

1726. A Random Forest Prediction Model Accurately Identifies Periods at Increased Risk for Positive Legionella Cultures in a Hospital Water Distribution System

BackgroundHospitals devote considerable resources to water distribution system (WS) surveillance and remediation for Legionella in an effort to reduce risk of transmitting Legionnaires disease (LD). There are no models that accurately predict periods of greatest risk for Legionella culture positivity (cx +) within a WS. Our goal was to build and validate a model based on weather and water parameters that predicted Legionella cx+ in our hospital WS.MethodsOne liter water samples from fixtures at 2 campuses were cultured for Legionella on BCYE plates with cysteine as part of infection prevention protocols. Logistic regression (LR) and random forest (RF) models included daily hospital WS measurements and Pittsburgh FAA weather observation station data. Training and validation used 2014–2015 and 2016–2017 data, respectively. Models predicted a first +cx within 14 day windows.ResultsCxs were defined as + by loop-day, if any cx from within a unique WS loop was + for Legionella on a given day. Of the 7,272 water samples, 5,304 were collected from 16 buildings on 2 campuses in which ≥1 cx + was obtained. A total of 1,262 WS loop-days were collected over 339 unique days from these buildings. Details on training and validation data sets appear in figure. Overall, water was Legionella cx + on 3% of loop-days. Models predicted positivity if risk was >6%. The LR model comprised of independent predictors of cx + had sensitivity/specificity of 44%/80% (AUC: 0.715; misclassification error: 0.21), and PPV/NPV of 9%/97% in the validation data set. The RF model comprised of the same predictors had sensitivity/specificity of 100%/98% (AUC: 1.0; misclassification error: 0.02), and PPV/NPV of 67%/100%. The most important RF variables in the validation data set were WS temperature and minimum pH over the 7 days prior to cx.ConclusionAn RF model using water and weather data was validated as an accurate predictor of new Legionella cx+ within a hospital WS. Most importantly, NPV for the model was 100%, meaning that no positive Legionella cxs were recovered during periods identified as low-risk. The RF model is a powerful tool for most efficiently directing resources to Legionella surveillance and LD prevention. Disclosures All authors: No reported disclosures.

Legionella pneumophila recurrently isolated in a Spanish hospital: Two years of antimicrobial resistance surveillance

ObjectivesThe aim of this study was to monitor the spread, persistence and antibiotic resistance patterns of Legionella spp. strains found in a hospital water distribution system. These environmental studies are intended to help detect the presence of antibiotic resistant strains before they infect patients. Methods: Antimicrobial surveillance tests were performed at 27 different sampling points of the water network of a large Spanish hospital over two years. Water samples were screened for Legionella according to ISO 11731:2007. Legionella spp. isolates were identified by serotyping and by mass spectrometry (MALDI-ToF). Epidemiological molecular typing was done by Pulse-Field Gel Electrophoresis (PFGE) and by Sequence-Based Typing (SBT). Antibiotic susceptibility tests were performed using disk diffusion and ETEST®. ResultsLegionella spp. were recurrently isolated for 2 years. All isolates belonged the same group, L. pneumophila serogroups 2–14. Isolates were all attributed by SBT to sequence type (ST) ST328, although PFGE revealed 5 different patterns. No significant change in antibiotic susceptibility could be observed for this study period, irrespectively of the method used. ConclusionColonization of water systems by Legionella spp. is still occurring, although all the prevention rules were strictly followed. Antibiotic resistance monitoring may help us to find resistance in bacteria with environmental reservoirs but difficult to isolate from patients. The knowledge of the antibiotic susceptibility in environmental strains may help us to predict changes in clinical strains. This study might also help reconsidering Legionnaires’ disease (LD) diagnostic methods. L. pneumophila serogroups 2–14 present all along the time of the investigation in the water distribution system can cause LD. However, they may not be detected by routine urine tests run on patients, thereby missing an ongoing LD infection.

KESEPANCARAN (HOMOLOGI) LEGIONELLA PNEUMOPHILA JARINGAN DISTRIBUSI AIR DAN PNEUMONIA NOSOKOMIAL

L. pneumophila is one of the nosocomial pneumonia causes that contaminated hospital water distribution system. The aim of this study was to determine the homology between L. pneumophila 16S rRNA base sequence found in the water distribution systemand the sequence derived from the sputum of nosocomial pneumonia patients identified at RSHS Bandung as well as the homology of L. pneumophila 16S rRNA found in the same system network. The study also include the nosocomial pneumonia patients at RSHSBandung with L. pneumophila from GenBank. The research using descriptive bioinformatics BLAST method by comparative analyticapproach, which performed from April 2006 to February 2008. The material consists of 60 biofilm samples from water distributionsystem and pneumonia nosocomial patient's sputum is positive L.pneumophila from water distribution system in her/his room. Inthe result was found: out of the 60 biofilm samples from the water distribution system, there are seven (7) L. pneumophila positivePCR and culture. During the 12 months of observation, there is only one (1) out of 31 pneumonia nosocomial patients with positively L. pneumophila PCR and culture. The conclusion so far can be mentioned that: The water distribution system in RSHS for patient roomsmay become the source for nosocomial pneumonia transmission of L. pneumophila and also was detected a new species of L. pneumophilathat is genetically different from that has been found in GenBank.

Identification of nontuberculous mycobacteria isolated from hospital water by sequence analysis of the hsp65 and 16S rRNA genes.

Nontuberculous mycobacteria (NTM) have emerged as an important cause of opportunistic nosocomial infections. NTM has frequently been isolated from hospital water distribution systems. The aim of this study was to survey the risk of NTM infections and determine the prevalence of NTM species in the hospital water distribution systems in Tabriz, Iran. One hundred and twenty samples of water from different sources of Tabriz hospitals were collected. The samples were filtered through 0.45-µm pore size membranes and decontaminated with 0.01% cetylpyridinium chloride. The sediment was inoculated onto Lowenstein-Jensen medium and incubated for 8 weeks. For identification to the species level, partial sequence analysis of the hsp65 and 16S rRNA genes were used. NTM were detected in 76 (63.3%) of 120 samples. Potentially pathogenic mycobacteria and saprophytic mycobacteria were isolated. Mycobacterium gordonae was the only single species that was present in all types of water. The prevalence of NTM in Tabriz hospitals' water compared with many investigations on hospital waters was high. This indicates that the immunocompromised patients and transplant recipients are at risk of contamination which necessitates considering decontamination of water sources to prevent such potential hazards.

Evaluation of timing of re-appearance of VBNC Legionella for risk assessment in hospital water distribution systems.

In this study we estimated the presence of Legionella species, viable but non-culturable (VBNC), in hospital water networks. We also evaluated the time and load of Legionella appearance in samples found negative using the standard culture method. A total of 42 samples was obtained from the tap water of five hospital buildings. The samples were tested for Legionella by the standard culture method and were monitored for up to 12 months for the appearance of VBNC Legionella. All the 42 samples were negative at the time of collection. Seven of the 42 samples (17.0%) became positive for Legionella at different times of monitoring. The time to the appearance of VBNC Legionella was extremely variable, from 15 days to 9 months from sampling. The most frequent Legionella species observed were Legionella spp and L. anisa and only in one sample L. pneumophila srg.1. Our study confirms the presence of VBNC Legionella in samples resulting negative using the standard culture method and highlights the different time to its appearance that can occur several months after sampling. The results are important for risk assessment and risk management of engineered water systems.

Coexistence of free-living amoebae and bacteria in selected South African hospital water distribution systems.

Pathogenic free-living amoebae (FLA), such as Naegleria fowleri, Balamuthia mandrillaris and Acanthamoeba species isolated from aquatic environments have been implicated in central nervous system, eye and skin human infections. They also allow the survival, growth and transmission of bacteria such as Legionella, Mycobacteria and Vibrio species in water systems. The purpose of this study was to investigate the co-occurrence of potentially pathogenic FLA and their associated bacteria in hospital water networks in Johannesburg, South Africa. A total of 178 water (n = 95) and swab (n = 83) samples were collected from two hospital water distribution systems. FLA were isolated using the amoebal enrichment technique and identified using PCR and 18S rDNA sequencing. Amoebae potentially containing intra-amoebal bacteria were lysed and cultured on blood agar plates. Bacterial isolates were characterized using the VITEK®2 compact System. Free-living amoebae were isolated from 77 (43.3 %) of the samples. Using microscopy, PCR and 18S rRNA sequencing, Acanthamoeba spp. (T3 and T20 genotypes), Vermamoeba vermiformis and Naegleria gruberi specie were identified. The Acanthamoeba T3 and T20 genotypes have been implicated in eye and central nervous system infections. The most commonly detected bacterial species were Serratia marcescens, Stenotrophomonas maltophilia, Delftia acidovorans, Sphingomonas paucimobilis and Comamonas testosteroni. These nosocomial pathogenic bacteria are associated with systematic blood, respiratory tract, the urinary tract, surgical wounds and soft tissues infections. The detection of FLA and their associated opportunistic bacteria in the hospital water systems point out to a potential health risk to immune-compromised individuals.Electronic supplementary materialThe online version of this article (doi:10.1007/s00436-016-5271-3) contains supplementary material, which is available to authorized users.