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Introduction. Pseudomonas aeruginosa is one of the major agents of nosocomial infections in burn centers, especially in Iran.1,2 This opportunistic and highly resistant bacterium causes severe problems for hospitalized burn patients.3 Burn patients that infected by Pseudomonas aeruginosa, particularly multidrug-resistant (MDR) strains, commonly discussed as a general complication. They are obviously at high risk for difficult to treat or untreatable infectious.4 Severely burn patients with immunological system defects develop life-threatening infections frequently and so, this gram-negative bacterium continues to be a general complication in burn related morbidity and mortality worldwide.5-7
MDR bacteria have been report commonly as the cause of nosocomial outbreaks of infection in burn units (BU) or as colonizers of the wounds of burn patients. Resistance to many drugs has been reached to a worrying point in P. aeruginosa isolated from burn patients in Iran.8,9,10 Previous studies confirmed resistance to many antibiotics used routinely for treatment of burn wounds infected by P. aeruginosa in Iranian hospitals.2,11,12 For example, Hadadi et al was shown P. aeruginosa isolates were resistance to ceftizoxime (99%), ceftazidime (59.6%), ticarcilin (50%), ceftriaxone (44.3%) and cefoperazone (37.5%) 12 and in another study, resistances of 75% for imipenem and 39% for ciprofloxacin in P. aeruginosa isolated from nosocomial source was reported.11 It was showed P. aeruginosa as the main infectious agents in the Tohid Burn Center in Tehran with the frequency of 73.9% and it was revealed that 95% of isolates were resistant to gentamicin, carbenicillin, co-trimoxazole, ceftizoxime and tetracycline, 90% for amikacin and 82% for ciprofloxacin.2 P. aeruginosa has been demonstrates as the leading cause of nosocomial infectious in Iranian burn units.1 Molecular epidemiologic studies have very important role in determination of transmission routes of pathogen for managing of infection. This type of information can be used in clinical settings to separate continuing epidemics of an infectious agent from incidentally increased infection rates. DNA typing methods known as the most suitable approaches for epidemiological study.13,14 Pulsed-field gel electrophoresis (PFGE) is one of the most powerful techniques that used as "gold standard" for typing of many microorganisms like P. aeruginosa.15-17 In this study, PFGE were applied for molecular typing and results were used for detailed analyzing of the routes of P. aeruginosa colonization in the BU. Since effective management of nosocomial infections, especially in BU, needs to information about infection transmission routes and drug susceptibility of pathogens, this study was conducted to investigate antibiotic susceptibility and molecular epidemiology of P. aeruginosa isolated in BU of Shahid Motahhari Hospital, one of the referral burn unit in Tehran (Iran), between February 2008 and June 2008.
MATERIALS AND METHODS
Sampling and patients demographics: The intensive care burn unit of the Shahid Motahhari Hospital is a referral center for patients with severe burn injury in Tehran, Iran. Between February 2008 and June 2008, 129 P. aeruginosa isolates from burn patients and 2 isolates from Hospital environment were collected. Patients were hospitalized in BU had different types of burn injuries. They include; 14 (10%) under 15 year-old and 126 (90%) over 15 year-old patients and 108 (77.1%) were male and 32 (22.9%) were female. The clinical samples included burn wound swabs, blood, and biopsy specimens and environmental samples included water from faucets, antiseptics, hand-washing solutions and swabs from sinks, hydrotherapy equipment, floors and other damp surfaces with potential for cross-contamination throughout the burn unit.
Bacterial analysis: All samples were cultured on the Mueller-Hinton agar and the P. aeruginosa were isolated from samples by standard microbiology procedures. Each isolate originated from a single colony of each patient's culture and was identified as P. aeruginosa by API 20NE (bioMérieux, Lyon, France). P. aeruginosa isolates were stored in Luria-Bertani broth medium (Merck KGaA, Darmstadt, Germany) containing 30% glycerol at -80°C.
Drug susceptibility testing: Drug susceptibility testing and interpretation was performed according to CLSI guidelines.18 Tests were done for all isolates by disk diffusion method for thirteen antimicrobial agents including, amikacin, aztreonam, cefotaxime, ceftazidime, ceftizoxime, ciprofloxacin, gentamicin, imipenem, kanamycin, meropenem, piperacillin, piperacillin-tazobactam and tetracycline (Mast Diagnostics, Mast Group Ltd, Merseyside, UK). MDR P. aeruginosa isolates were resistant to ceftazidime and at least three of following antibiotics; amikacin, aztreonam, ciprofloxacin, gentamicin, imipenem, piperacillin and aminoglycosides. P. aeruginosa ATCC 27853 was used as control.
PFGE method. PFGE was performed according to previously described protocol by Gautom with some modifications 16. Briefly, P. aeruginosa bacteria were grown overnight on Mueller-Hinton agar plates and then suspended directly with sterile cotton swabs in about two to three ml of TE buffer (100 mM Tris and 100 mM EDTA). The cell suspensions were adjusted with TE buffer to 20% transmittance by using a Bio-Rad spectrophotometer (Bio-Rad Laboratories, Hercules, California, USA). Aliquots of 100 µl of the cell suspensions were transferred to 1.5 ml microcentrifuge tubes. Lysozyme and proteinase K were added to a final concentration of 1 mg/ml each and mixed by pipetting. The bacterial suspensions were incubated at 37°C for 10 to 15 min. Multi-purpose (MP) agarose (Roche Diagnostics GmbH, Mannheim, Germany) was prepared in water to a final concentration of 1.2% and maintained at 55°C in a water bath. Following the lysozyme-proteinase K incubation, 7 µl of 20% sodium dodecyl sulfate and 140 µl of 1.2% MP agarose were mixed with each bacterial suspension with the help of a pipette. This bacterium-agarose mixture was immediately added to plug molds (Pharmacia Biotech, Sweden). The plugs were allowed to solidify for 5-10 min at 4°C and then transferred to 2-ml round-bottom tubes containing 1.5 ml of ESP buffer (0.5 M EDTA, pH 9.0; 1% sodium lauryl sarcosine; 1 mg of proteinase K per ml). These were incubated in water bath at 55°C for 2 h. After the completion of proteolysis, the plugs were transferred to 50-ml tubes containing 8 to 10 ml of sterile, preheated (50°C) distilled water and incubated for 10 min at 50°C with gentle mixing in a shaker water bath. Subsequently, four 50°C washes were done in a shaker water bath for 15 min each with 8 to 10 ml of preheated (50°C) TE buffer (10 mM Tris, pH 8.0; 1 mM EDTA, pH= 8). The plugs were then cooled to room temperature in TE buffer. At this point, they could be used immediately or stored for 3 to 4 weeks at 4°C in 1 ml of TE. For restriction endonuclease digestion, two 1-mm thick slices of each plug were incubated at 37°C for 3 h with 50 U of XbaI enzyme, in 100 ml of the appropriate (1X) restriction enzyme buffer.
MP agarose at a concentration of 1.2% provided the desired resolution of DNA fingerprints. The plug slices of the samples were loaded and electrophoresed in 1.2% MP agarose with 2.5 liters of standard 0.5X TBE running buffer. The electrophoresis was performed with the Gene Navigator System (Pharmacia Biotech, Sweden). Electrophoresis run conditions were designed for a run time of 24 h; in these runs, the initial and final switch times were 5 s and 90 s, respectively; all other parameters remained identical with those of the standard procedure.
Following electrophoresis, the gels were stained for 20 min in 500 ml of sterile distilled water containing 50 µl of ethidium bromide (10 mg/ml) and destained in three washes of 30 min each in one liter of distilled water. The gels were analyzed under UV transilluminator (UVItec, Cambridge, UK) and TIFF files were saved for analyzing with GelCompar software (Applied Maths, Kortrijk, Belgium). Cluster analysis of the Dice similarity indices based on the unweighted pair group method using arithmetic averages (UPGMA) was done to generate a dendrogram describing the relationship among pulsotypes. A difference of at least one restriction fragment in the profiles was considered the criterion for discriminating between clones. Visual analysis was done based on Tenover criteria, too.19
Drug susceptibility testing: Drug susceptibility tests by disc diffusion method have showed many isolates were resistant to ceftizoxime (87%), aztreonam (80.2%), kanamycin (79.4%), tetracycline (78.6%) and ceftazidime (74.8%) but few isolates were resistant to imipenem (16%), piperacillin/tazobactam (19.1%) and amikacin (35.1%). 42 MDR P. aeruginosa isolates were recovered from clinical samples and one isolate was recovered from environment. The results of drug susceptibility tests are showed in Table 1.
PFGE fingerprinting: Genotyping by PFGE reveals different profiles (Figure 1) that by GelCompar software classified in eleven profiles, PFGE1 to PFGE11 (Table 2). Five PFGE profiles included MDR strains that were resistant to multiple classes of antibiotics; these MDRs were resistant to similar classes of drugs. PFGE1 with 42 isolates was the major PFGE group that including 18 MDR clinical samples isolates and one environmental MDR strain. PFGE2 profile has 23 isolates and 13 MDR isolates and PFGE3 profile has 17 isolates with 6 MDR strains. Other profiles comprising PFGE4, 5, 6, 7, 8, 9, 10, and 11 have 13, 8, 4, 9, 5, 5, 3, and 2 isolates respectively. PFGE1 to PFGE5 profiles were included MDR isolates (table 2). Two environmental isolates (E1 and E2) were classified in PFGE1 and PFGE7 profiles, respectively. It was showed that E1 was a MDR isolate.
P. aeruginosa infection is a major cause of mortality and morbidity in hospitalized patients in developing countries.1 One of the most important aspects for choosing efficient method to prevention this infection is determination of relationship between genotype and drug susceptibility. In this study, relationship between isolates, genotypes and drug susceptibility patterns of P. aeruginosa isolates were investigated. The results may useful for achieving an appropriate approach to elimination infections. PFGE analysis is one of the best genotyping methods for P. aeruginosa fingerprinting and sometimes mentioned as "gold-standard" method for this bacterium.13 We used PFGE for typing all P. aeruginosa isolates obtained from hospitalized patients in Shahid Motahari BU and environmental isolates, too. All of P.aeruginosa isolates were typeable and 11 PFGE profiles were identified. They were analyzed for any possible relationship to environmental and/or MDR isolates.
MDR bacteria have commonly been reported as the cause of nosocomial outbreaks of infection in BUs or as colonizers of the wounds of burn patients.1,11 P. aeruginosa has been demonstrated to be a leading cause of nosocomial infections in Iranian burn patients and antimicrobial resistance has reached a critical point.2,8-10 In previous studies, resistance to many antibiotics that usually used to treatment of burn injuries infected by P. aeruginosa in Iranian hospitals were showed1,2,8-12. For example in one study, resistance amount of P. aeruginosa isolates to ceftizoxime, ceftazidime, ticarcilin, ceftriaxone, and cefoperazone were 99%, 59.6%, 50%, 44.3%, and 37.5%, respectively.2 In another study, 75% resistance for imipenem and 39% for ciprofloxacin in P. aeruginosa isolated from nosocomial source were showed.12 It was showed P. aeruginosa as the main infectious agents, in the Tohid Burn Center in Tehran, with the frequency of 73.9 and it was reveal that these isolates were resistance over 95% for gentamicin, carbenicillin, co-trimoxazole, ceftizoxime and tetracycline, 90% for amikacin and 82% for ciprofloxacin.2
Drug susceptibility tests of P. aeruginosa isolates were done and some isolates that resistant with many antibiotics were determined. Forty-three MDR isolates with five PFGE profiles (PFGE1-PFGE5) were found in this study. These results reveal different potential sources for MDR isolates, which may have endogen or exogen. Our results have been shown two environment sources for FGE1 and PFGE7 profiles that found in the tap water and sink drains, respectively. . However, we may be missed some important outsources agents for other PFGE patterns in this study.
In this study some isolates including the MDR PFGE1 strains, showed resistance to amikacin, aztreonam, ceftazidime, imipenem, meropenem, and piperacillin, which are the first-line antibiotics that were used in BU. This may illustrates the importance of the selective pressure of antibiotics in the emergence and selection of MDR epidemic strains. Nowadays, outbreaks with MDR P. aeruginosa strains have become rather frequent and the persistence of MDR P. aeruginosa clone in BUs have been reported.10,11
P. aeruginosa colonization may originate from endogenous sources such as intestinal tract or from exogenous sources such as contaminated equipment or other colonized patients with P. aeruginosa. Understanding the routes of colonization is critical to the development of efficient preventive measures against infection. Even if the overall rate of P. aeruginosa colonization is not significantly reduced, it is important to recognize cross-infecting strains, especially if they exhibit resistance to a variety of antibiotics and give rise to severe infections. Colonized patients represent a continuous reservoir of (epidemic) strains from which other patients can be colonized via cross-acquisition. In contrast, with some studies,3 we isolated two P. aeruginosa strains from the inanimate hospital environment that were important source of patients' infections. The large number of unique genotypes observed in the patients, however, suggests that most of patients were colonized from an exogenous source. On the other hand, 42 patients were colonized with the PFGE1 strain, 23 patients were colonized with the PFGE2 strain and 17, 13, 8, 4, 9, 5, 5, 3 and 2 patients were colonized with PFGE3 to PFGE11 isolates, respectively. There was very little overlap between the patients, at the time of hospital admission and hospitalized period. In addition a thorough survey of the inanimate hospital environment successes to identify two ongoing reservoirs of PFGE1 and PFGE7 strains, that found in the tap water and sink drains, respectively.
Several studies have demonstrated that cross-acquisition can play an important part in the epidemiology of nosocomial colonization and infection with P. aeruginosa.14,20-22 Nikbin et al. have shown that a few isolates were distributed widely at two hospitals and environment in Tehran.21
In this study, transmission of some patients with PFGE1 and PFGE7 profiles, may be originated from the environmental sources and other isolates may be originated from staff hands, some equipment or other unknown sources in the BU. These results emphases the importance of doing routine drug susceptibility tests and molecular fingerprinting to monitoring routes of infections and changes in drug resistance in infectious agents for successful management of infections.
In conclusion, our findings show that environmental sources may have significant role in transmission of P. aeruginosa in this BU. This study highlighted the need for further attention to disinfection of inanimate objects in the hospital environment to limit transfer of P. aeruginosa in this BU; moreover, use of some antimicrobial agents must be restricted due to existence of high resistance to them.