Correspondence: Mahnaz Nikaeen, Department of Environmental Health Engineering, School of Health, Isfahan University of Medical Sciences, Hezar Jerib Avenue, 8174673461 Isfahan, Iran. Tel.: +983117922660; fax: +983116682509; e-mail: Nikaeen@hlth.mui.ac.ir
Pseudomonas aeruginosa has emerged as a major pathogen in nosocomial infections. Biofilm formation allows the microorganism to persist in hospital water systems for extended periods, which have been associated with nosocomial infections. The aim of this study was to evaluate the frequency of P. aeruginosa colonization of hospital tap waters by nested PCR assay. A total of 44 water samples were collected from 11 hospitals and analyzed for the presence of Pseudomonas spp. and P. aeruginosa by specific primer sets of 16S rRNA gene. Some physicochemical parameters and heterotrophic plate count (HPC) of samples for possible association with P. aeruginosa contamination were also determined. The nested PCR revealed 32% of the water samples being positive for P. aeruginosa. From the 11 hospitals surveyed, 82% (9 of 11) of the hospitals water systems were positive for P. aeruginosa. No correlation was seen between the presence of P. aeruginosa and HPC as well as physicochemical parameters. Identification of contaminated sources could be a key priority in waterborne nosocomial infections. PCR assay was used in the study provides simple, rapid, and reliable identification of P. aeruginosa in hospital water systems, which could eliminate the infections of P. aeruginosa through implementation of immediate control measures.
Pseudomonas spp., ubiquitous Gram negative bacilli, are found in natural waters such as lakes and rivers. On account of their tolerance to a wide variety of physical conditions and minimal nutrition requirements, Pseudomonas also can colonize biofilms in manmade systems such as drinking water. Pseudomonas aeruginosa is a major human opportunistic pathogen species of this group, which can cause a wide range of infections (Lavenir et al., 2007; Mena & Gerba, 2009). In particular, P. aeruginosa has emerged as one of the most important pathogens of nosocomial infections (Trautmanna et al., 2006; Mena & Gerba, 2009), which typically infects the pulmonary tract, urinary tract, burn, and wounds. Immunocompromised patients are particular at increased risk to acquire such infections (Anaissie et al., 2002; Trautmanna et al., 2006). Pseudomonas aeruginosa continues to be one of the most important causes of wound or pulmonary nosocomial infections, with significant mortality rate of up to 1400 deaths per year of nosocomial pneumonias in the United States (Anaissie et al., 2002). The common route of acquiring infection is contaminated water (Mena & Gerba, 2009). Patient exposure occurs while showering, bathing, and drinking and through contact with medical equipment rinsed with contaminated tap water (Anaissie et al., 2002). However, lung exposure from inhaling aerosols from contaminated water systems appears to carry the greatest health risk (Mena & Gerba, 2009).
In several studies, the roll of tap water as the source of P. aeruginosa colonization and infections was demonstrated (Trautmann et al., 2005; Kerr & Snelling, 2009; Mena & Gerba, 2009). In recent studies have shown that there is a close genotypic proximity of clinical and tap water isolates (Trautmann et al., 2000; Reuter et al., 2002; Blanc et al., 2004; Rogues et al., 2007). During a 7-month period, Trautmann et al. (2000) observed that 29% (5/17) of patients in a surgical intensive care unit (ICU) were infected with P. aeruginosa genotypes that were the same as those detected in the unit's tap water. Because of the ongoing increase in immunocompromised patients and inherently resistance of P. aeruginosa to many antibiotics, hospitals are often encountered with the problem of Pseudomonas infections management as an important public health concern (Trautmann et al., 2005; Kerr & Snelling, 2009). Therefore, minimizing exposure of high-risk patients by eliminating the contamination sources underscores the importance of a fast and reliable detection method of this bacterium in water systems. Culture technique currently is in use for P. aeruginosa identification. However, it is a labor intensive method and takes a long time (Al-Qadiri et al., 2006). Furthermore, culture method fails to detect bacteria that become nonculturable in response to environmental stresses. Currently, PCR-based methods would represent a valuable tool for identification of this bacterium, which could improve the speed, specificity, and sensitivity of P. aeruginosa detection in ecological and diagnostic studies (Spilker et al., 2004).
The aim of this study was to evaluate the frequency of P. aeruginosa colonization of hospital tap water by PCR method. Some physicochemical parameters and heterotrophic plate count (HPC) of water samples for possible association with P. aeruginosa contamination were also determined.
A total of 44 water samples were collected in sterilized 500-mL glass bottles from 11 hospitals of Isfahan University of medical sciences, Iran. Water samples were taken from tap water outlets at four different points of each hospital including ICU, operating room, surgical ward, and patient's bathroom. In the study, HPC bacteria were also determined with R2A agar and incubation at 35 °C for 48–72 h.
Water samples were concentrated by membrane filtration (0.22 μm, 47 mm diameter; Millipore). Membrane filters were washed in the sterile phosphate buffer solution, shaken for 30 min and then centrifuged.
To extract DNA, the resuspended pellets were frozen in liquid nitrogen and heated in boiling water for three times. The DNA was further extracted and purified using Promega DNA Extraction Kit (Promega Wizard® Genomic DNA Purification Kit, Madison, WI) according to manufacturer's instruction. The purified DNA was finally recovered in 25 μL of distilled water and was used in PCR assay.
In the first PCR step, a c. 1420-bp fragment of 16S rRNA gene region of bacteria was amplified using the bacterial primer set Eubac 27F and 1492R (Table 1) to check the nucleic acid extraction as well as the presence of inhibitors. For the detection of Pseudomonas spp. and P. aeruginosa, nested PCR technique was applied to increase the sensitivity. In the second PCR step, Pseudomonas spp.- and P. aeruginosa-specific primer sets were used (Table 1; Spilker et al., 2004).
Table 1. Primers used for detection of bacteria
Sequence (5′ to 3′)
Product size (bp)
16S rRNA gene
Pseudomonas spp. F
Pseudomonas spp. R
16S rRNA gene
P. aeruginosa F
P. aeruginosa R
16S rRNA gene
The PCR amplification was conducted in a total volume of 25 μL consisting of 2.5 μL of 10X PCR buffer, 0.2 μM of each primer, 0.2 mM of each dNTPs, 1.5 units of Taq DNA polymerase, and 1 μL of DNA. All PCR assays contained a positive and a negative control.
PCR was performed with an initial denaturation step for 5 min at 95 °C, 30 cycles of 94 °C for 45 s, 55 °C for 1 min, and 72 °C for 1.30 min and a final extension step at 72 °C for 5 min. PCR products were visualized by agarose gel electrophoresis using 1.5% gels stained with ethidium bromide. Gels were viewed on a UV transilluminator (UV Tech, France), and DNA fragment sizes were compared with the 100-bp ladder DNA.
Physical and chemical analyses
A water sample was also taken from each water tap for physicochemical analyses. pH (Corning pH Meter) and residual free chlorine (METERRC) were determined at the time of sample collection. Concentrations of iron, manganese, and zinc were measured by flame atomic absorption spectrophotometer (Perkins-Elmer 2380). Turbidity was also measured (Eutech Instruments Turbidimeter TN-100).
Results and discussion
Pseudomonas aeruginosa has been increasingly recognized for its ability to cause nosocomial infection outbreaks with significant morbidity and mortality (Kerr & Snelling, 2009; Mena & Gerba, 2009). Biofilm formation in water systems allows the microorganisms to persist in hospital water systems for extended periods, which has been associated with nosocomial infections (Anaissie et al., 2002; Lavenir et al., 2007; Kerr & Snelling, 2009). Therefore, hospital populations of P. aeruginosa represent an important public health concern (Lavenir et al., 2007). In this study, water samples were taken from tap water outlets of 11 hospitals at four different points including ICU, operating room, surgical ward, and patient's bathroom. Pseudomonas spp. were detected in 66% (29 of 44 samples) of the hospital water taps. The nested PCR with specific primers of P. aeruginosa revealed 32% (14 of 44 samples) of the tap water samples being positive for P. aeruginosa. This corresponds with 82% (9 from 11) of the hospitals water system being positive for P. aeruginosa (Table 2). Figure 1 shows The PCR products analysis for Pseudomonas detection with specific primers by agarose gel electrophoresis. The study by Trautmann et al. (2000) showed that 49 of 72 water samples (68%) taken from water taps in surgical and medical ICUs were positive for P. aeruginosa (Trautmann et al., 2000). In the study of Rogues et al. (2007) also, P. aeruginosa was found in 11.4% of 484 tap water samples taken from patient rooms and in 5.3% of 189 other tap water samples in a medical ICU (Rogues et al., 2007). Reuter et al. (2002) also found P. aeruginosa in 150 (58%) of 259 tap water samples taken from patient rooms (Reuter et al., 2002). Although the rate of Pseudomonas contamination of water samples in hospitals is different, all studies show that water taps in hospitals often colonized with P. aeruginosa and could provide evidence of the need to improve monitoring and control strategies.
Table 2. Results of nested PCR assay for detection of Pseudomonas spp. and Pseudomonas aeruginosa in hospital water taps
Number of positive samples (%)
Despite concerns regarding the increase in Pseudomonas nosocomial infections (Trautmanna et al., 2006), the significance of rapid and reliable detection method of this pathogen in hospital water systems as an important transmission route has received relatively little attention. Culture technique, based on our knowledge, was used in all published frequency data of P. aeruginosa in hospital waters. The potential for misidentification of P. aeruginosa in the presence of other Pseudomonas spp. by culture-based methods (Spilker et al., 2004) could hamper identification of contamination sources and management of infection prevention. Choosing of 16S rRNA gene-based PCR assay with high specificity provided rapid and reliable identification of P. aeruginosa (Spilker et al., 2004). In the study, furthermore, high sensitivity of rRNA gene could improve the sensitivity of detection (Yamamoto et al., 1993). Although PCR assay could detect dead cells, there may be a risk of overestimation of positive samples, but the ability of P. aeruginosa to form biofilm on water systems lead to disinfectant resistance of this bacterium (Kerr & Snelling, 2009), and it is likely that water samples contain no dead cells.
Several studies showed that decontamination of water taps by control measures result in a significant decrease in P. aeruginosa clinical infections (Trautmann et al., 2005). Indeed, identification of contaminated sources could be a key priority which eliminates the potential exposure of at risk patients through implementation of immediate control measures. The nested PCR assay is a qualitative method that only recognized the presence or absence of P. aeruginosa in water systems. However, According to the guidelines of the German Ministry of Environment, the target level of P. aeruginosa in medical high-risk areas is 0 cfu per 100 mL water (Whapam, 2008) which stresses the importance of rapid and sensitive detection method of P. aeruginosa in water samples, not quantitative method.
Some studies showed a significant association between Legionella as another nosocomial infection pathogen contamination and HPC in water systems (Edagawa et al., 2008). Legionella spp. are one of the most well-known Gram negative bacteria which could colonize biofilm in hospital water systems such as Pseudomonas. Mean concentration of heterotrophic bacteria was 41 cfu mL−1. However, our study showed no relationship between the HPC and the presence of P. aeruginosa. The results of the physicochemical characteristics of water samples are presented in Table 3. No correlation was also seen between the presence of P. aeruginosa and physicochemical parameters.
Table 3. Physicochemical characteristics of the water samples
Residual chlorine (mg L−1)
Fe (mg L−1)
Mn (mg L−1)
Zn (mg L−1)
A key factor in an effective prevention strategy of waterborne nosocomial infections is to identify the contamination sources and implement effective control measures. The detection procedure used in this study provides several advantages over traditional method. 16S rRNA gene-based PCR assays provide simple, rapid, and reliable identification of P. aeruginosa in hospital water systems. This technique offers also a cost-effective monitoring strategy which consequently with implementation of immediate control measures eradicates the clinical infections of waterborne P. aeruginosa particularly for high-risk patients.
This research was conducted with funding from the vice chancellery for research at the Isfahan University of Medical Sciences (Grant no. 387346) as a part of MSc. dissertation.