Background: Pseudomonas aeruginosa is one of the major agents of nosocomial infections in burn centres, particularly in Iran. Our aims were to find the drug susceptibleness and familial diverseness of P. aeruginosa isolated from burn patients.
Methods: 131 P. aeruginosa isolates were collected from the burn centre of Shahid Motahhari Hospital in Tehran. Phenotypic testing for drug susceptibleness was performed by disc diffusion method harmonizing to CLSI guidelines, and familial diverseness of all isolates was determined by pulsed-field gel cataphoresis ( PFGE ) technique.
Consequences: Antimicrobial susceptibleness proving showed that the bulk of strains of P. aeruginosa were immune to ceftizoxime ( 87 % ) and aztreonam ( 80.2 % ) but few were immune to imipenem ( 16 % ) and piperacillin/tazobactam ( 19.1 % ) . PFGE revealed 11 profiles which environmental strains were included in PFGE1 and PFGE7 forms. The major PFGE profile was PFGE1 ( n = 42, 32.1 % ) , that contains 18 ( 42.9 % ) MDR isolates and included an environmental MDR bacteria.
Decisions: Our findings highlighted that farther attending demands to be focused on disinfection of inanimate objects in the infirmary environment and controlled contact between staff and patients to cut down transmittal of P. aeruginosa in this burn unit.
Cardinal words: P. aeruginosa, Pulsed-field gel cataphoresis, DNA fingerprinting, Drug susceptibleness, Multidrug-resistance.
Introduction. Pseudomonas aeruginosa is one of the major agents of nosocomial infections in burn centres, particularly in Iran.1,2 This timeserving and extremely immune bacteria causes terrible jobs for hospitalized burn patients.3 Burn patients that infected by Pseudomonas aeruginosa, peculiarly multidrug-resistant ( MDR ) strains, normally discussed as a general complication. They are evidently at high hazard for hard to handle or untreatable infectious.4 Severely burn patients with immunological system defects develop dangerous infections often and so, this Gram-negative bacteria continues to be a general complication in burn related morbidity and mortality worldwide.5-7
MDR bacteriums have been report normally as the cause of nosocomial eruptions of infection in burn units ( BU ) or as colonisers of the lesions of burn patients. Resistance to many drugs has been reached to a distressing point in P. aeruginosa isolated from burn patients in Iran.8,9,10 Previous surveies confirmed opposition to many antibiotics used routinely for intervention of burn lesions infected by P. aeruginosa in Persian hospitals.2,11,12 For illustration, Hadadi et Al was shown P. aeruginosa isolates were opposition to ceftizoxime ( 99 % ) , Fortaz ( 59.6 % ) , ticarcilin ( 50 % ) , Rocephin ( 44.3 % ) and Cefobid ( 37.5 % ) 12 and in another survey, oppositions of 75 % for imipenem and 39 % for Cipro in P. aeruginosa isolated from nosocomial beginning was reported.11 It was showed P. aeruginosa as the chief infective agents in the Tohid Burn Center in Tehran with the frequence of 73.9 % and it was revealed that 95 % of isolates were immune to gentamicin, carbenicillin, co-trimoxazole, ceftizoxime and Achromycin, 90 % for amikacin and 82 % for ciprofloxacin.2 P. aeruginosa has been demonstrates as the taking cause of nosocomial infective in Persian burn units.1 Molecular epidemiologic surveies have really of import function in finding of transmittal paths of pathogen for managing of infection. This type of information can be used in clinical scenes to divide go oning epidemics of an infective agent from by the way increased infection rates. Deoxyribonucleic acid typing methods known as the most suited attacks for epidemiological study.13,14 Pulsed-field gel cataphoresis ( PFGE ) is one of the most powerful techniques that used as “ gilded criterion ” for typing of many micro-organisms like P. aeruginosa.15-17 In this survey, PFGE were applied for molecular typewriting and consequences were used for elaborate analyzing of the paths of P. aeruginosa colonisation in the BU. Since effectual direction of nosocomial infections, particularly in BU, needs to information about infection transmittal paths and drug susceptibleness of pathogens, this survey was conducted to look into antibiotic susceptibleness 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 attention burn unit of the Shahid Motahhari Hospital is a referral centre for patients with terrible burn hurt 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 hurts. 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 lesion swabs, blood, and biopsy specimens and environmental samples includedA H2O from spigots, antiseptics, hand-washing solutions and swabs from sinks, hydropathy equipment, floors and other moist surfaces with possible 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 processs. Each isolate originated from a individual settlement of each patient ‘s civilization and was identified as P. aeruginosa by API 20NE ( bioMerieux, Lyon, France ) . P. aeruginosa isolates were stored in Luria-Bertani stock medium ( Merck KGaA, Darmstadt, Germany ) incorporating 30 % glycerin at -80A°C.
Drug susceptibleness testing: Drug susceptibleness testing and reading was performed harmonizing to CLSI guidelines.18 Trials were done for all isolates by disc diffusion method for 13 antimicrobic agents including, amikacin, Azactam, Claforan, Fortaz, ceftizoxime, Cipro, Garamycin, imipenem, Kantrex, meropenem, Pipracil, piperacillin-tazobactam and Achromycin ( Mast Diagnostics, Mast Group Ltd, Merseyside, UK ) . MDR P. aeruginosa isolates were immune to ceftazidime and at least three of following antibiotics ; amikacin, Azactam, Cipro, Garamycin, imipenem, Pipracil and aminoglycosides. P. aeruginosa ATCC 27853 was used as control.
PFGE method. PFGE was performed harmonizing to antecedently described protocol by Gautom with some alterations 16. Briefly, P. aeruginosa bacteriums were adult nightlong on Mueller-Hinton agar home bases and so suspended straight with unfertile cotton swabs in approximately two to three milliliter of TE buffer ( 100 millimeter Tris and 100 millimeters EDTA ) . The cell suspensions were adjusted with TE buffer to 20 % transmission by utilizing a Bio-Rad spectrophotometer ( Bio-Rad Laboratories, Hercules, California, USA ) . Aliquots of 100 Aµl of the cell suspensions were transferred to 1.5 milliliters microcentrifuge tubings. Lysozyme and protease K were added to a concluding concentration of 1 mg/ml each and assorted by pipetting. The bacterial suspensions were incubated at 37A°C for 10 to 15 min. Multi-purpose ( MP ) agarose ( Roche Diagnostics GmbH, Mannheim, Germany ) was prepared in H2O to a concluding concentration of 1.2 % and maintained at 55A°C in a H2O bath. Following the lysozyme-proteinase K incubation, 7 Aµl of 20 % Na dodecyl sulphate and 140 Aµl of 1.2 % MP agarose were assorted with each bacterial suspension with the aid of a pipette. This bacterium-agarose mixture was instantly added to stop up casts ( Pharmacia Biotech, Sweden ) . The stoppers were allowed to solidify for 5-10 min at 4A°C and so transferred to 2-ml round-bottom tubings incorporating 1.5 milliliter of ESP buffer ( 0.5 M EDTA, pH 9.0 ; 1 % Na lauryl sarcosine ; 1 milligram of protease K per milliliter ) . These were incubated in H2O bath at 55A°C for 2 h. After the completion of proteolysis, the stoppers were transferred to 50-ml tubings incorporating 8 to 10 milliliter of sterile, preheated ( 50A°C ) distilled H2O and incubated for 10 min at 50A°C with soft commixture in a shaker H2O bath. Subsequently, four 50A°C washes were done in a shaker H2O bath for 15 min each with 8 to 10 milliliter of preheated ( 50A°C ) TE buffer ( 10 millimeter Tris, pH 8.0 ; 1 millimeter EDTA, pH= 8 ) . The stoppers were so cooled to room temperature in TE buffer. At this point, they could be used instantly or stored for 3 to 4 hebdomads at 4A°C in 1 milliliter of TE. For limitation endonuclease digestion, two 1-mm thick pieces of each stopper were incubated at 37A°C for 3 H with 50 U of XbaI enzyme, in 100 milliliter of the appropriate ( 1X ) limitation enzyme buffer.
MP agarose at a concentration of 1.2 % provided the coveted declaration of Deoxyribonucleic acid fingerprints. The stopper pieces of the samples were loaded and electrophoresed in 1.2 % MP agarose with 2.5 litres of standard 0.5X TBE running buffer. The cataphoresis was performed with the Gene Navigator System ( Pharmacia Biotech, Sweden ) . Electrophoresis run conditions were designed for a run clip of 24 H ; in these tallies, the initial and concluding switch times were 5 s and 90 s, severally ; all other parametric quantities remained indistinguishable with those of the standard process.
Following cataphoresis, the gels were stained for 20 min in 500 milliliter of sterile distilled H2O incorporating 50 Aµl of ethidium bromide ( 10 mg/ml ) and destained in three washes of 30 min each in one litre of distilled H2O. The gels were analyzed under UV transilluminator ( UVItec, Cambridge, UK ) and TIFF files were saved for analysing with GelCompar package ( Applied Maths, Kortrijk, Belgium ) . Cluster analysis of the Dice similarity indices based on the unweighted brace group method utilizing arithmetic norms ( UPGMA ) was done to bring forth a dendrogram depicting the relationship among pulsotypes. A difference of at least one limitation fragment in the profiles was considered the standard for know aparting between ringers. Ocular analysis was done based on Tenover standards, too.19
Drug susceptibleness testing: Drug susceptibleness trials by disc diffusion method have showed many isolates were immune to ceftizoxime ( 87 % ) , aztreonam ( 80.2 % ) , kanamycin ( 79.4 % ) , Achromycin ( 78.6 % ) and Fortaz ( 74.8 % ) but few isolates were immune 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 consequences of drug susceptibleness trials are showed in Table 1.
PFGE fingerprinting: Genotyping by PFGE reveals different profiles ( Figure 1 ) that by GelCompar package classified in 11 profiles, PFGE1 to PFGE11 ( Table 2 ) . Five PFGE profiles included MDR strains that were immune to multiple categories of antibiotics ; these MDRs were immune to similar categories 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 consisting PFGE4, 5, 6, 7, 8, 9, 10, and 11 have 13, 8, 4, 9, 5, 5, 3, and 2 isolates severally. PFGE1 to PFGE5 profiles were included MDR isolates ( table 2 ) . Two environmental isolates ( E1 and E2 ) were classified in PFGE1 and PFGE7 profiles, severally. 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 of import facets for taking efficient method to prevention this infection is finding of relationship between genotype and drug susceptibleness. In this survey, relationship between isolates, genotypes and drug susceptibleness forms of P. aeruginosa isolates were investigated. The consequences may useful for accomplishing an appropriate attack 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, excessively. 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 bacteriums have normally been reported as the cause of nosocomial eruptions of infection in BUs or as colonisers of the lesions of burn patients.1,11 P. aeruginosa has been demonstrated to be a prima cause of nosocomial infections in Persian burn patients and antimicrobic opposition has reached a critical point.2,8-10 In old surveies, opposition to many antibiotics that normally used to intervention of burn hurts infected by P. aeruginosa in Persian infirmaries were showed1,2,8-12. For illustration in one survey, opposition sum of P. aeruginosa isolates to ceftizoxime, Fortaz, ticarcilin, Rocephin, and Cefobid were 99 % , 59.6 % , 50 % , 44.3 % , and 37.5 % , respectively.2 In another survey, 75 % opposition for imipenem and 39 % for Cipro in P. aeruginosa isolated from nosocomial beginning were showed.12 It was showed P. aeruginosa as the chief infective agents, in the Tohid Burn Center in Tehran, with the frequence of 73.9 and it was reveal that these isolates were opposition over 95 % for Garamycin, carbenicillin, co-trimoxazole, ceftizoxime and Achromycin, 90 % for amikacin and 82 % for ciprofloxacin.2
Drug susceptibleness trials 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 survey. These consequences reveal different possible beginnings for MDR isolates, which may hold monocot or dicot. Our consequences have been shown two environment beginnings for FGE1 and PFGE7 profiles that found in the tap H2O and sink drains, severally. . However, we may be missed some of import outsources agents for other PFGE forms in this survey.
In this survey some isolates including the MDR PFGE1 strains, showed opposition to amikacin, aztreonam, Fortaz, imipenem, meropenem, and Pipracil, which are the first-line antibiotics that were used in BU. This may exemplify the importance of the selective force per unit area of antibiotics in the outgrowth and choice of MDR epidemic strains. Presents, eruptions with MDR P. aeruginosa strains have become instead frequent and the continuity of MDR P. aeruginosa ringer in BUs have been reported.10,11
P. aeruginosa colonisation may arise from endogenous beginnings such as enteric piece of land or from exogenic beginnings such as contaminated equipment or other colonised patients with P. aeruginosa. Understanding the paths of colonisation is critical to the development of efficient preventative steps against infection. Even if the overall rate of P. aeruginosa colonisation is non significantly reduced, it is of import to acknowledge cross-infecting strains, particularly if they exhibit opposition to a assortment of antibiotics and give rise to terrible infections. Colonized patients represent a uninterrupted 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 infirmary environment that were of import beginning of patients ‘ infections. The big figure of alone genotypes observed in the patients, nevertheless, suggests that most of patients were colonized from an exogenic beginning. On the other manus, 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, severally. There was really small convergence between the patients, at the clip of hospital admittance and hospitalized period. In add-on a thorough study of the inanimate infirmary environment successes to place two ongoing reservoirs of PFGE1 and PFGE7 strains, that found in the tap H2O and sink drains, severally.
Several surveies have demonstrated that cross-acquisition can play an of import portion in the epidemiology of nosocomial colonisation and infection with P. aeruginosa.14,20-22 Nikbin et Al. have shown that a few isolates were distributed widely at two infirmaries and environment in Tehran.21
In this survey, transmittal of some patients with PFGE1 and PFGE7 profiles, may be originated from the environmental beginnings and other isolates may be originated from staff custodies, some equipment or other unknown beginnings in the BU. These consequences emphases the importance of making everyday drug susceptibleness trials and molecular fingerprinting to supervising paths of infections and alterations in drug opposition in infective agents for successful direction of infections.
In decision, our findings show that environmental beginnings may hold important function in transmittal of P. aeruginosa in this BU. This survey highlighted the demand for farther attending to disinfection of inanimate objects in the infirmary environment to restrict transportation of P. aeruginosa in this BU ; furthermore, usage of some antimicrobic agents must be restricted due to existence of high opposition to them.
We thank Sara Amiri for adept proficient aid and computing machine analysis and Dr. Mohammad Ali Bahar for his support to obtaining samples. This survey was supported by National Institute of Genetic Engineering and Biotechnology and Shahed University.