Epidemiological Link Between Sewage And Human Methicillin Resistant

Published Date: 02 Nov 2017

Rahimi Fateh1, Bouzari Majid1*, Katouli Mohammad2, Pourshafie Mohammad Reza3*

Department of Biology, Faculty of Science, University of Isfahan, Iran

Faculty of Science, Health and Education, University of the Sunshine Coast, Queensland 4558, Australia

Department of Microbiology, Pasteur Institute of Iran

Short title: Sewage and human MRSA isolates

* Corresponding authors:

Majid Bouzari, Department of Biology, Faculty of Science, University of Isfahan, Hezarjereeb Street, Isfahan 81746-73441, Iran. Email: [email protected]

Mohammad R. Pourshafie, Department of Microbiology, Pasteur Institute of Iran, Pasteur Ave, Tehran, 13164, Iran. Email: [email protected]

Abstract

Methicillin resistant Staphylococcus aureus (MRSA) is known as a common pathogen in nosocomial and community-acquired infections. Sewage as an environmental reservoir may have a significant role in development and dissemination of antibiotic resistance in community. This study was undertaken to determine the epidemiological relatedness between the MRSA isolates of sewage and human infection isolates. We investigated the prevalence of methicillin resistant Staphylococcus aureus (MRSA) isolated from sewage (n = 653) and clinical (n = 489) samples, and the genetic linkage between the isolates was compared. Out of the total samples, 200 S. aureus (17.51%) from sewage (n = 100) and clinical (n = 100) isolates were found to be resistant to methicillin. The majority of the MRSA isolates from the two sources showed distinct phenotyping and genotyping patterns. At the same time, two common PhP patterns were found among the MRSA isolates obtained from sewage and human clinical isolates, suggestive of an epidemiological link.

Keywords: Methicillin resistant Staphylococcus aureus, sewage, Typing

1. Introduction

Staphylococcus aureus is known as a common pathogen in nosocomial and community-acquired infections (Lindsay and Holden, 2004). MRSA is resistant to all clinically used b-lactam antibiotics (Hanssen et al., 2006), and is epidemic in hospital environments in different parts of the world (Borjesson et al., 2010). mecA is the gene responsible for methicillin resistance in both MRSA and coagulase-negative staphylococci (CoNS). Staphylococcal cassette chromosome is known as the only mecA vector (Hanssen and Ericson, 2006). Due to the species-independent conservation of the gene complex, it is believed that transfer of the cassette chromosome occurs frequently (Borjesson et al., 2010).

Although, hospital acquired MRSA is one of the most frequent causes of hospital-associated infections, but community acquired MRSA has recently emerged as an additional threat, causing infections in healthy individuals with no health care-associated risk factors (Borjesson et al., 2010; Kluytmans-Vandenbergh, and Kluytmans, 2006). Moreover, it is believed that MRSA is an increasing problem in veterinary medicine (Rich, 2005), and animals can be a source of MRSA infections in humans (Lewis et al., 2008).

sewage as an environmental reservoir may have a significant role in development and dissemination of antibiotic resistance (Martinez, 2006), particularly as a large part of prescribed antibiotics end up in the environment in an active form (Halling-Sorensen et al., 1998; Kummerer et al., 2000). This includes the b-lactam antibiotics, which have been detected in both surface waters and wastewater systems (Watkinson et al., 2009).

The role of horizontal gene transfer in the spread of antibiotic resistance in the environment has been documented previously (Allen et al., 2010). Ohlesen and colleagues described the transfer of genes encoding antibiotic resistance by S. aureus in wastewater in vitro (Ohlsen et al., 2003). Furthermore, mecA has been detected in both hospital and municipal wastewater (Borjesson et al., 2009a; Borjesson et al., 2009b, Volkmann et al., 2004). Using both cultivation and molecular techniques, the presence of MRSA in municipal wastewater is shown (Borjesson et al., 2009b). Thus, sewage treatment plans (STPs) could be a potential source for dissemination and development of MRSA strains. This study was undertaken to determine the epidemiological relatedness between the MRSA isolates of sewage and human infection isolates.

2. Materials and Methods

2.1. Collection of sewage sample

Sampling was carried out during 2010. Sewage samples were collected from an urban STP located in the west of Tehran. Three samplings were done from this urban STP. Wastewater samples were collected into 250-mL sterile bottles. All samples were refrigerated and transported to the microbiology laboratory. Samples were diluted fivefold with phosphate-buffered saline before filtration on a 0.45-µm membrane (Millipore Corporation, Bedford, MA) and were cultured in Baird Parker agar (Merck KGaA, Darmstadt, Germany) plates as described before for enterococci (Rahimi et al., 2007; Talebi et al., 2007).

2.2. Collection of Clinical Sample

Clinical samples (n = 489) included in this study comprised specimens from patients admitted to one major hospital in Tehran during 2010. MRSA isolation from the clinical specimens was carried out by standard microbiological techniques. Briefly, clinical samples were inoculated on blood agar. The suspicious colonies from these cultures were Gram stained. Gram-positive cocci were tentatively identified as staphylococci on the basis of positive catalase test, negative oxidase reaction and growth in 10-15% sodium chloride. All isolates confirmed as S. aureus using mannitol fermentation, DNase and coagulase tests (Kateete et al., 2010). Moreover, sewage and human MRSA isolates reconfirmed using species-specific primers for nucA gene (see details below).

2.3. Antibiotic susceptibility tests

Susceptibility of S. aureus isolates to oxacillin (1 µg), as the surrogate for methicilin, was determined using disc diffusion method according to the guidelines of Clinical and Laboratory Standards Institute (CLSI) (2006). All methicillin resistant strains were collected and their MICs for oxacillin and vancomycin were determined using E-test (AB, Biomerieux, Marcy l'Etoile, France) according to the manufacturer's instructions. Susceptibility of the MRSA isolates to 17 commonly used antibiotics were determined using disc diffusion method according to the guidelines of CLSI (2006). These included kanamycin (30 μg), amikacin (30 μg), penicillin (5 μg), fusidic acid (10 μg), minocycline (30 μg), erythromycin (15 μg), clindamycin (2 μg), tobramycin (10 μg), rifampicin (2 μg), nitrofurantoin (50 μg), sulphamethoxazole-trimethoprime (1.25-23.75 μg), linezolid (10 μg), synercid (15 μg), chloramphenicol (30 μg), ciprofloxacin (30 μg), gentamicin (10 μg) and tetracycline (30 μg). The antibiotic discs were purchased from Mast Diagnostics (Merseyside, United Kingdom).

2.4. DNA extraction and PCR assay

DNA extraction was done using High Pure PCR Template Preparation kit (Roche, Mannheim, Germany) according to the instructions of the manufacturer with some modifications. The concentration of all extracted DNAs was determined using Nanodrop 1000 (NanoDrop, Wilmington, USA). One microliter of each DNA was used as template in PCR reaction.

PCR primers for detection of nucA and mecA genes were according to Du et al. (2002) and synthesized by Tib-Molbiol (Berlin, Germany). PCR mixture contained 10X PCR buffer, Tag DNA polymerase (0.5 U) (HT Biotechnology, Cambridge, United Kingdom), each primer (1.6 μM), MgCl2 (1.2 μM) and each of dNTPs (0.64 μM). The PCR cycles were as follows: an initial denaturation at 94°C for 3 min, with 30 cycles of denaturation at 94°C for 30s, annealing at 56°C for 30s and elongation at 72°C for 30s and final extension at 72°C for 5 min (Du et al. 2002). PCR products were electrophoresed on a 1.5% agarose gel in a 0.5X Tris-Borate-EDTA (TBE) buffer and stained in ethidium bromide and visualized under UV.

2.5. SCCmec and ccr typing

A multiplex PCR typing assay was used for typing of SCCmec gene which contained 8 pairs of primers including the unique and specific primers for SCCmec types and subtypes I, II, III, IVa, IVb, IVc, IVd, and V (Zhang et al., 2005). Another multiplex PCR assay was used for characterization of ccr gene complexes which employed four sets of primers specific for each of the ccr genes i.e. ccrAB-β2, ccrAB-α2, ccrAB-α3, and ccrAB-α4 (Zhang et al., 2005).

The multiplex PCR mixture contained 10X PCR buffer, Tag DNA polymerase (0.5 U) (HT Biotechnology, Cambridge, United Kingdom), each primer (1.6 μM), MgCl2 (1.2 μM) and each of dNTPs (0.64 μM). The PCR cycles were as follows: an initial denaturation at 94°C for 5 min, followed by 10 cycles of 94°C for 45 seconds, 65°C for 45 seconds, and 72°C for 1.5 min and another 25 cycles of 94°C for 45 seconds, 55°C for 45 seconds, and 72°C for 1.5 min, ending with a final extension step at 72°C for 10 min and followed by a hold at 4°C (Zhang et al., 2005).

2.6. Prophage typing

Primers for detection of phage types 3A, 11, 77, 187 and Twort-like phages representative of phage serogroups A (SGA), B (SGB), F (SGF, with 2 subtypes SGFa/b), L (SGL) and D (SGD) were according to Pantucek et al. (2004) which code for tail fibers, hypothetical tail proteins, hypothetical tail proteins (packaging proteins) hypothetical capsid proteins, major capsid proteins, respectively.

A multiplex PCR reaction was mixed in a volume of 25μl consisting of 1µl of template DNA, 10X PCR buffer, Tag DNA polymerase (1 U), each primer (0.4 μM) (SGA1/2, SGB1/2, SGD1/2, SGL1/2, SGFb1/b2 and SGFa1/a2), MgCl2 (0.8 μM) and dNTP mix (100 μM). Each reaction mixture was then loaded into a thermal cycler for initial denaturation (5 min, 94º C) and 30 cycles of amplification consisting of denaturation (1 min, 94º C), annealing (1.5 min, 57º C), and chain extension (1.5 min, 70º C). Amplification was once again followed by a final chain extension cycle (3 min, 70º C).

2.7. Detection of pvl gene

For detection of pvl gene encoding PVL toxin among MRSA isolates, specific primers were used as described previously by McClure and colleagues (2006). PCR mixture contained 10X PCR buffer, Tag DNA polymerase (0.5 U) (HT Biotechnology, Cambridge, United Kingdom), each primer (1.6 μM), MgCl2 (1.2 μM) and each of dNTPs (0.64 μM). The PCR cycles were as follows: an initial denaturation at 94°C for 10 min, with 10 cycles of denaturation at 94°C for 45s, annealing at 55°C for 45s and elongation at 72°C for 75s; 25 cycles of denaturation at 94°C for 45s, annealing at 50°C for 45s and elongation at 72°C for 75s and final extension at 72°C for 10 min.

2.8. Biochemical Fingerprinting

The 200 MRSA isolates were typed using the PhP-RF plates (PhPlate AB, Stockholm, Sweden). These are microplates with four sets of dehydrated reagents that are used to measure the kinetics of bacterial metabolism of 23 substrates and a control, specifically chosen to differentiate between of S. aureus strains (Persson et al., 2006). For each bacterial isolate, it yields a biochemical fingerprint made of 23 quantitative data, which were used with the PhPlate software to calculate the level of similarity between the tested isolates and to identify different phenotypes. Preparation and inoculation of the plates were done according to the manufacturer’s instructions. The inoculated microplates were incubated at 37°C and the absorbance value (A620) of each well was measured at 16, 40, and 64 h (Talebi et al., 2007). The mean value of all three readings was calculated and the similarity between the strains was calculated as the correlation coefficient after pairwise comparison of the strains. The similarity matrix was then clustered according to the unweighted pair group method with arithmetic averages (UPGMA) to obtain a dendrogram. An identity level (ID level = 0.965) was set up for the system after testing five isolates in duplicate. The mean similarity between the compared isolated minus 2 standard deviations was taken as the ID level of the system. Isolates showing similarity to each other above this level were considered as identical (Common Biochemical Phenotypes: C-BPT). The diversity of the bacterial populations was calculated as Simpson’s index of diversity (Di) (Sneath and Sokal, 1973). The optical readings, calculation of correlation coefficients, diversity indexes, and clustering were performed using the PhPWin software (PhPlate Microplates Techniques AB, Sweden).

3. Results

3.1. Prevalence of MRSA isolates

A total of 653 of S. aureus isolates were collected from a STP in Tehran. One hundred (15.3%) MRSA strains were isolated from this STP. Of the total 489 clinical isolates, 100 (20.4%) strains were selected as MRSA. The clinical samples included 207 (42%) from wound, 96 (20%) urine, 69 (14%) sputum, 39 (8%) blood, 27 (6%) CSF, 26 (5%) nose and 25 (5%) abscess.

3.2. Antibiotic susceptibility testing

The frequency of MRSA isolated from different sources is shown in Fig. 1. Forty six percent of MRSA isolates were from wounds. The lowest number of MRSA isolates was associated with ear and eye infections. The frequency of MRSA isolates were 17, 14, 8 and 7 percent in urine, sputum, cerebrospinal fluid (CSF) and nose cultures, respectively.

A high level of resistance to ciprofloxacin (91%), erythromycin (90%), tobramycin (89%), kanamycin (88%), clindamycin (84%) and tetracycline (81%) was found among the clinical MRSA isolates. On the contrary, the level of resistance to nitrofurantoin (3%) and fusidic acid (2%) was low. Also, none of the clinical isolates showed resistance to vancomycin, synercid and linezolid. About 80% of the human MRSA isolates were concomitantly resistant to Cip/E/Tn/K/CD/T. Similarly, the number of MRSA strains isolated from sewage that was concomitantly resistant to these six antibiotics was a little bit more (Fig. 1). The most significant difference between the levels of antibiotic resistance was observed with chloramphenicol, fusidic acid and nitrofurantoin. The level of resistance in the sewage isolates was 2, 0 and 0%, respectively, as compared to 0, 2 and 3% in the human clinical isolates. All clinical and sewage MRSA isolates were resistant to penicillin.

Seven clinical isolates (7%) and 6 sewage isolates (6%), were susceptible to all antibiotics except for penicillin. Moreover, 18 and 13% of clinical and sewage isolates, respectively, showed resistance to all different antibiotics except for nitrofurantoin, fusidic acid, synercid, chloramphenicol, linezolid and vancomycin.

The oxacillin MIC for all MRSA isolated from humans and sewage showed that all isolates were resistant to oxacillin (MIC≥4 µg/ml) (Table 1). Moreover, 49% of clinical isolates and 58% of sewage isolates showed high level resistance (MIC≥256 µg/ml) to oxacillin. Also, 7% of clinical isolates and 6% of sewage isolates showed low resistance to oxacillin (MIC=4 µg/ml).

3.3. Prophage typing

In this study we could detect all different prophage types except for SGL and SGD in a single PCR reaction (Table 2). SGF prophage type, with two subtypes, was the dominant type and 100% of clinical and sewage MRSA isolates shared it’s specific gene. Moreover, the prevalence of SGA prophage type among the clinical isolates in comparison of sewage isolates was significant different. Also, 39 and 56% of clinical and sewage isolates contained SGB prophate type, respectively.

Multiplex-PCR reaction including SGA, SGB and SGF prophage sero-groups and sub-group SGFa was amplified successfully (data not shown). PCR reaction showed that all isolates contained at least 1 prophage sero-groups and 2 sub-groups. SGF serotype was present in 100% of the MRSA isolates, also SGFa and SGFb was the dominant (100%) sub-types among the isolates. According to the types of the prophage, 4 different patterns were identified among 200 MRSA isolates. Pattern 4 was the predominant pattern (52%) among all MRSA isolates. Pattern 3 with SGB, SGF prophage and its two sub-groups constituted 41.5% of the isolates. The prevalence of patterns 3 and 4 between clinical and sewage isolates was completely different. Prophage patterns 3 and 4 were the dominant prophage types among clinical and sewage MRSA isolates, respectively. All MRSA contained SGA prophage type (6.5%) showed resistance only to penicillin and also low resistance to oxacillin (MIC=4µg/ml).

3.4. Biochemical Fingerprinting

The results of PhP typing for the clinical and sewage MRSA isolates showed the presence of diverse (diversity index, Di = 0.975) PhP types among the isolates from both origins. The diversity index (Di) for clinical and STPs isolates were 0.95 and 0.96, respectively. PhP typing discriminated the 200 isolates into 29 types with 13 single types (6.5%) and the remaining 16 common types (CTs) constituting 93.5% of the MRSA isolates (Fig. 2). The results showed that each common BPT (C-BPT) comprised two to ninety eight strains. The CT11 contained the highest number of MRSA strains (98 isolates, 49%) and was the most common type amongst the MRSA isolates. The least number of strains belonged to CT3, CT4, CT6, CT8, CT9, CT10, CT12, CT13 and CT15 containing 2 isolates (1%). Amongst the Single Types, 11 and 2 belonged to clinical and sewage MRSA isolates, respectively. Some of the isolates collected from the clinical and STPs were found to have the same C-BPT (i.e., CT2, CT11). In general, isolates in the same PhP type showed different prophage types and also different antibiotic resistance patterns, there is no relatedness between their clonal dissemination and also their prophage and antibiotic resistance patterns (i.e., ST2, ST3).

3.5. SCCmec and ccr typing

One hundred and eighty seven MRSA isolates were shown to carry SCCmec type III and were PCR positive with the ccrAB-α4 specific primers indicating the presence of type 3 ccr. Moreover, 13 isolates which showed low resistance to oxacillin (MIC=4 µg/ml) carried SCCmec type IV and also showed type 2 ccr. Amongst these, 54 and 46% isolated from clinical and sewage samples, respectively. Moreover, all 7 clinical isolates shared SCCmec type IVa. However, one sewage MRSA isolate (6.7%) shared SCCmec type IVc. All 7 clinical isolates of MRSA which shared SCCmec type IV and contained SGA prophage type, isolated from wound (71%), urine (29%), respectively.

3.6. Detection of pvl gene

The results of detection of pvl gene among MRSA isolates showed that only 13 (6.5%) MRSA isolates were PCR positive with the pvl gene. These MRSA isolates showed susceptibility to all antibiotics tested except for penicillin. The presence of pvl gene among the MRSA isolates, was limited to isolates shared type 2 ccr, also contained SGA prophage type and also showed low level oxacillin resistance (MIC=4 µg/ml).

4. Discussion

In this study we showed that the prevalence of MRSA isolates in a hospital in Tehran was 20.4%. This rate of resistance is less than other reports from Iran (Fatholahzadeh et al., 2008; Japoni et al., 2011; Rahimi et al., 2009). Moreover, in previous studies we showed that prevalence of MRSA isolates in different hospitals in Tehran ranged between 19.8 to 25.5% (Rahimi et al., 2012; Rahimi et al., 2013). These differences between our studies and other reports from Iran, could be due to geographical locations, population, methods and techniques used, hygiene measures in hospitals, and antibiotic usage in different hospitals.

Our study shows that MRSA isolates could be found in municipal STPs over the time. In this study, for the first time in Iran we detected a high number of MRSA of diverse PhP types in municipal STP (Fig. 2), showing that it was not a specific lineage that survived in this environment. Our findings is contrary to other studies, which have shown that S. aureus either has a low prevalence or does not survive or present in sewage (Savichtcheva et al., 2007, Schwartz et al., 2003; Shannon, et al., 2007; Volkmann et al., 2004). A possible explanation for these findings, is the modified protocol for isolation of S. aureus strains as we used previously for isolation of enterococci from sewage (Rahimi et al., 2007; Talebi et al., 2007).We expected a high prevalence of MRSA in sewage, given that Iran has a high clinical prevalence of MRSA (Fatholahzadeh et al., 2008; Japoni et al, 2011; Rahimi et al., 2009; Rahimi et al., 2012; Rahimi et al., 2013). The results may, therefore, indicate that STPs are a potential reservoir for MRSA. On the other hand, it is likely that a large part of the MRSA isolated from this sewage originates from the population connected to the STP, as most of the PhP types have been isolated in clinical settings in Tehran (Fig. 2). Furthermore, the sewage MRSA flora changed slightly with each sampling, indicating that the findings are partly dependent upon the recent flora transiting the STP. In addition, the sewage MRSA isolates had a high genetic diversity, harbored mostly SCCmec type III, type 3 ccr and also showed resistance to different classes of antibiotics other than beta-lactam antibiotics, which is comparable to clinical isolates found in clinical isolates, indicating their possible hospital origin. However, there are indications that some MRSA strains may be resident in the STP and ⁄ or better adapted to the sewage environment, e.g. isolates of CT2 and CT11.

The prevalence of MRSA isolates from patients and the environment is different in Europe and the United States. In the United States, MRSA isolates are restricted to hospitalized patients, whereas in a European country (Sweden) MRSA have been isolated from STPs (Borjesson et al., 2010). As of now, there is no information available about the interrelation of the MRSA in sewage and human clinical isolates in Iran.

The results of PhP typing showed that most of the isolates were found at different sampling occasions from STP, indicating a high prevalence of MRSA isolates (i.e., PhP types C2, C11) in the municipal STP.

PhP analysis revealed an extensive diversity among the isolates. The isolates from the STPs were more homogeneous than the isolates from clinical samples. The presence of dominant MRSA PhP types among the human isolates was consistent with the fact that there had been high MRSA outbreaks in Tehran and consequently predominant bacterial clone dissemination.

In general, the majority of the MRSA isolates showed distinct PhP types as an indication of lack of a close genetic relationship between the isolates from the human and STP. Our results showed that some of the isolates were found in different sources, indicating high prevalence of certain MRSA isolates (C2, C11) in hospital and STP where samples were taken. Moreover, the majority of isolates from the two sources may have acquired oxacillin resistance elements independently, possibly by horizontal transfer. Nevertheless, PhP types 2 and 11 were the only pattern shared by the clinical and STP isolates, which may imply that this PhP types were genetically stable, even in the harsh conditions of STP. The presence of two common PhP types, CT 2 and CT11 with 50 and 98 isolates respectively, may be noteworthy, considering the fact that only a total of 200 MRSA isolates from the clinical sources and STPs were studied. Furthermore, this PhP types were recovered in different samplings from STPs, which further supports the spread of these clonal types in Tehran.

Although the isolates in the same CT (i.e, CT2 and CT11) had the same SCCmec type but the presence of different prophage and antibiotic resistance pattern in the sewage MRSA isolates may suggest the acquisition of new traits by these isolates for surviving in the sewage. Obtaining these characteristics may, in turn, allow better fitness and increase the chance of passing of this clone of bacteria to other milieu such as surface water, as indicated by others (Borjesson et al., 2010).

Here we could detect 4 different prophage patterns consisted of 3 serotypes and 2 sub-types among the clinical and sewage isolates. Reports from other countries have indicated different prophage patterns amongst their MRSA isolates. Pantuceck and colleagues in Czech Republic (Pantucek et al., 2004), Workman and colleagues in USA (Workman et al., 2006), and our previous reports in Iran (Rahimi et al., 2012; Rahimi et al., 2013) have reported 9, 10, 8 and 4 prophage patterns among their S. aureus isolates, respectively.

In addition, these investigators have also reported different dominant phage patterns. SGA (human isolates), SGF and SGFb (human isolates), and SGA (coastal water isolates) prophages were shown to be the dominant prophages in Czeck Republic, Iran, and USA, respectively. The difference in phage patterns in these studies could be, in part, due to different ecological settings and geographical locations where these studies were performed. In this study, on the other hand, 32 and 51% of clinical and sewage isolates, respectively contained SGB, SGF, SGFa and SGFb prophage pattern. Similar prophage patterns were seen in our previous study which further suggests the circulation of some MRSA clonal types in community, different hospitals and also sewage treatment plants in Tehran.

Furthermore, it was noted that the isolates with SGA prophage type (prophage patterns 1 and 2) were STs whereas the MRSA strains lacking SGA (i.e. prophage patterns 3 and 4) were CTs. This result is suggestive that the SGA may affect the dissemination of MRSA as the CT by maintaining them as STs.

Here we also showed that the frequency of SCCmec type III was 93.5% followed with 6.5% of SCCmec type IV, while Japooni and colleagues could isolate strains with SCCmec types II, III, IVa, IVc, IVd and V in the south of Iran (Japoni et al., 2011). It might be due in part to higher number of outpatients in comparison to inpatients in that study, and also the differences in geographical regions the studies have been done.

In this study, all MRSA isolates (clinical and sewage) shared SCCmec type IV (a or c) showed susceptibility to all classes of antibiotics except for penicillin. It is in contrast to other studies, suggesting that SCCmec type IV strains can acquire resistance to nonb-lactam antibiotics in order to survive in the hospital environment or through exposure to the antibiotics. It might be due in part to their new distribution from community to hospital.

The prevalence of community-acquired (CA)-MRSA in clinical isolates (7%) was higher than the prevalence found earlier in Tehran (Fatholahzadeh et al., 2008). Prior antibiotic usage was a major risk factor for the development of MRSA infections, probably due to promoting the selection of resistant strains.

The high prevalence of MRSA infections is an important healthcare burden, associated with increased mortality and morbidity. Effective infection control programs, including early detection of MRSA carriers, pre-emptive isolation, new decontamination regimens, restriction of certain antibiotic classes, environmental control and adequate hand hygiene are needed to control the spread of MRSA.

PVL is an exoprotein of S. aureus that has been linked to furuncles, cutaneous abscesses, severe necrotic skin infections and severe necrotising pneumonia. Five percent of the MRSA isolates of our study were PVL positive. While PVL is associated with an increased incidence of the above-mentioned S. aureus infections, the result is not surprising, since other investigators suggested that, PVL is not an important virulence factor in the pathogenesis of staphylococcal bacteremia (Alp et al., 2009).

5. Conclusion

In conclusion, the presence of MRSA with PhP type 3 and SCCmec type III in 49% of MRSA isolates may provide evidence for the epidemiological link between the isolates from the STP reservoirs and human infections. Moreover, a possible transmission route for this clone from hospital patients via urban sewage to surface water may occur. The presence of this clone in surface water is under investigation.

Acknowledgment

This research funded, in part, by an operating grant of the Dean of Research and Graduate Studies at the University of Isfahan, and a grant from Ministry of Health of Iran, Deputy of research and innovation.