Pulsed Field Gel Electrophoresis & Plasmid Profile of Pseudomonas Aeruginosa at Two Hospitals in Tehran, Iran
By Nikbin, V S Abdi-Ali, A; Feizabadi, M M; Gharavi, S
Background & objectives: Pseudomonas aeruginosa is a common cause of nosocomial infections and exhibits innate resistance to a wide range of antibiotics. This study was undertaken to determine the resistance patterns of P. aeruginosa isolates recovered from patients at two hospitals in Tehran, to investigate the presence of plasmids and to genetically characterize them by pulsed field gel electrophoresis (PFGE). Methods: The susceptibility of 104 isolates of P. aeruginosa to 13 different antibiotics was determined by agar disk diffusion method. The alkaline lysis method was used for plasmid extraction. PFGE technique was optimized for DNA fingerprinting of isolates.
Results: The isolates showed resistance to 13 different antibiotics ceftizoxime (99%), lomefloxacin (94.3%), ceftazidime (59.6%), ticarcillin (50%), ceftriaxone (44.3%), cefoperazone (37.5%), tobramycin (34.6%), piperacillin and gentamicin (33.7%), carbenicillin (25%), amikacin (22%), ciprofloxacin (15.4%) and imipenem (2.9%). Plasmids were detected in 31 isolates (29.8%) that produced 15 different patterns. In total, 84 DNA banding patterns were detected by PFGE. The dominant PFGE type, Pattern A with 14 isolates was found at both hospitals. The remaining isolates were grouped in B, C, D and PF1-PF80. The majority of isolates with the identical plasmid profiles and resistance patterns produced closely related DNA fingerprints by PFGE.
Interpretation & conclusions: Isolates in pattern A were distributed widely at both hospitals and the environment. Absence of plasmids in majority of isolates indicated low typeability and discriminatory power of this technique.
Key words Antibiotics – plasmids – pulsed-field gel electrophoresis – Pseudomonas aeruginosa
Pseudomonas aeruginosa is a major opportunistic pathogen causing high mortality, particularly in patients with suppressed immunity, traumatic wounds, burns, cystic fibrosis, metabolic disorders and malignancies1,2. These bacteria remain for long periods in soaps, sponges, sinks, oral thermometers, inhalatory equipments, dialysis fluids, water taps, and clothes etc3. They are extremely resistant to disinfectants and can contaminate certain compounds and solutions4.
Infections with P. aeruginosa in particular multidrug resistant (MDR) strains, are widespread among Iranian hospitals3. To trace the source of outbreaks at hospitals, typing of bacterial isolates with sensitive molecular techniques such as pulsed-field gel electrophoresis (PFGE) is necessary6,7. Previous studies have shown that Iranian isolates of P. aeruginosa from burn patients belonged to various serovars5. However, little information is available on their phenotypes and genetic background.
We therefore undertook this study to genetically characterize clinical and environmental isolates of P. aeruginosa by plasmid profiling and PFGE analysis. The results obtained with molecular typing were compared with those of antimicrobial susceptibility testing.
Material and Methods
A total of 104 isolates of P. aeruginosa (96 clinical, 8 environmental isolates) were included in this study. These isolates were cultured from urine, sputum, blood, stool, wound and burned patients during September 2005 to May 2006 at Mehr (n=73) and Mostafa-Khomeini teaching hospitals (n=23) in Tehran. These hospitals were located in the same area of Tehran, however, the first one was a private hospital. The environment isolates recovered from sinks, hands, disinfectant solutions, oxygen tubes and beds by swabbing at Mostafa-Khomeini (n=6) and Mehr (n=2) hospitals, were also included P. aeruginosa was identified on the basis of pigment production, oxidase test, glucose fermentation, hydrolysis of arginine, nitrate production and growth on acetamide agar8,9.
The isolates were tested for antimicrobial susceptibility using agar disk diffusion method10. Various antimicrobial agents ([mu]g) used were amikacin (30), gentamicin (10), tobramycin (10), ceftazidime (30), ceftriaxone (30), ceftizoxime (30), cefoperazone (75), carnbenicillin (100), piperacillin (100), Ticarcillin (75), ciprofloxacin (5), lomefloxacin (10) and imipenem (10) (Hi-media, Mumbai, India). P. aeruginosa ATCC 27853 strain (Pasteur Institute, Tehran, Iran) was used as control.
Alkaline lysis method was used for plasmid extraction11. Plasmid DNA was electrophoresed in 0.7 per cent agarose gel. Escherichia coli DH5a (Fermentas, Lithuania) lacking plasmid and E. coli TG1 harbouring 100 kb plasmid (Amersham International, Herpenden, UK ) were used as controls. Cosmid pVFsp4 (40 kb)12 and plasmids pCMV62 (10 kb)13, pSDK (5 kb)14 and pUC19 (2.6 kb)15 were mixed and used as size marker. The plasmid sizes were calculated by Labworks software (UVP.INK, UK).
The method described by Corona-Nakamura et al3 was used to extract genomic DNA for PFGE analysis with minor modifications. Briefly, bacterial suspension was prepared by harvesting of bacterial colonies directly from overnight incubated culture on Muller-Hinton agar. The colonies were suspended in saline-EDTA buffer (75 mM NaCl and 25 mM EDTA, pH 7.5) and adjusted to a concentration of 10^sup 9^ cfu/ ml. Cell pellet was obtained by centrifuging at 9300 x g for 2 min, and re-suspended in saline-EDTA buffer, mixed with equal volume of 1.6 per cent low melting point agarose. The mixture was allowed to solidify in a 100 ml plug mold. The agarose plug was incubated for 24 h at 37 [degrees]C in 500 [mu]l of lysis buffer (6 mM TrisHCl, pH 7.6, 0.1 M EDTA, 1 M NaCl, 0.5 per cent Brij 58, 0.4 per cent sodium deoxycholate, 0.5 per cent sodium lauryl sarcosine (sarcosyl), lysozyme, 1 mg/ml). Following this step, the lysis buffer was replaced by 500 [mu]l of proteinase K buffer (1% sodium lauryl sarcosine, 0.5 M EDTA, pH 9, proteinase K (50 [mu]g/ml). This solution was incubated with gentle shaking at 50[degrees]C for 20 h. The plugs were washed 6 times for 30 min at 37 [degrees]C with 10 ml of Tris-EDTA buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA) after which the plugs can be stored for several months. A slice of each plug (4mm x 4mm in size) was cut and incubated for 18-20 h with 30U of Dra I (Fermentase, Lithuania) in the 100 [mu]l of appropriate restriction buffer. Large restriction fragments were separated by Field Inversion Gel Electrophoresis (FIGE) (Faculty of Engineering at Tehran University, Tehran). A pulsed field marker (Sigma, St. Louis, USA) was used as the molecular size marker. The gel was stained in ethidium bromide solution (0.5 [mu]g/ml) and photographed with Bio-Doc system (UVP, UK). All reagents for DNA extraction were purchased from Sigma unless stated.
The criteria of Tenover et al16,17 were used to interpret the PFGE banding patterns. Isolates are genetically indistinguishable or identical, if their restriction patterns demonstrated the same number of bands with the same apparent size were designated as genomic pattern A. Isolates were considered to be closely related if their PFGE patterns showed two to three band differences, consistent with a single genetic event, and were classified as type A1. Isolates were possibly related if their PFGE patterns showed four to six band differences, consistent with two independent genetic events and were classified as type A2. They were considered to be unrelated (UR) if their PFGE patterns showed seven or more band differences, consistent with three or more independent genetic events, and were designated as type B, C, D, E if several isolates had the same pattern.
Statistical analysis: Data for plasmid, and genomic DNA patterns of the clinical and environmental isolates were evaluated by Spearman and t-test (SPSS version 13 Inc., Chicago, IL).
Results
The resistance pattern to different antibiotics for the 104 isolates was as follows: ceftizoxime (99%), lomefloxacin (94.3%), ceftazidime (59.6%), ticarcillin (50%), ceftriaxone (44.3%), cefoperazone (37.5%), tobramycin (34.6%), piperacillin and gentamicin (33.7%), carbenicillin (25%), amikacin (22%), ciprofloxacin (15.4%), imipenem (2.9%). Based on the results of drug susceptibility testing, 24 different antibiotypes numbered from 1 to 24 were found. The resistant patterns of selected 9 antibiotypes including 2, 12, 15, 18, 19, 21, 22, 23 and 24 are shown in the Table. Antibiotype 2 consisted of 40 isolates that showed resistance to ceftizoxime and lomefloxacin. It was the most prevalent antibiotype among the isolates. The remaining antibiotypes consisted of 1-10 isolates were resistant to different antibiotics (Table).
Plasmids were detected in 31 isolates (29.8%). They produced 15 different patterns giving 1, 2, 3 or 7 DNA bands in agarose gel after electrophoresis. The sizes of plasmids varied from 1.7 to 100 kb (Fig. 1).
The resistance patterns in isolates showing plasmid profiles 14 and 15 were not found among other isolates (Table). The association between presence of certain plasmids and resistance to antibiotics was significant. (P < 0.05). Up to 61.8 per cent of plasmid containing isolates were resistant to at least 8 different antibiotics.
All isolates were typed by PFGE. Tatally, 84 patterns were found among isolates. Pattern A consisted of 14 isolates and was found as the dominant type at both hospitals. Of the 14 isolates in this pattern, 7 were identical and the remaining were closely related (genomic pattern A1) (Fig. 2). Other strains were grouped in patterns B, C, D and PF1-PF80 (Fig. 2). Patterns PF1-PF80 each contained single isolate. Isolates within clone A were highly resistant to antibiotics (Table). Four isolates in clone A belonged to the Mostafa-Khomeini Hospital and ten belonged to Mehr Hospital. Two isolates had originated from the first hospital environment.
The majority of PFGE patterns, plasmid profiles and antibiotypes found in this study were hospital specific with the exception of, PFGE patterns A which was found at both hospitals.
The association between isolates with pattern A and antibiotype 19 was significant (P<0.05).
Discussion
The use of broad spectrum antibiotics such as ceftazidime in hospitals exerts selective pressure on bacteria, thereby promoting infections by multi-drug resistant strains18,19. Our findings showed that the most useful antibiotics for infections caused by P. aeruginosa were imipenem and ciprofloxacin. Resistance to some antibiotics such as imipenem, amikacin, gentamicin, tobramycin, ceftazidime showed increases in comparison with previous studies in different countries20-23.
Isolates grouped in the main antibiotype (antibiotype 2) consisting of 40 isolates were resistant only to lomefloxacin and ceftizoxime. Since these isolates were distinct by PFGE, it appears that similarity in management and treatment protocols caused the antibiotype 2 to emerge as the main resistant pattern at both the hospitals.
Bactericidal effect of lomefloxacin against Pseudomonas spp. and P. aeruginosa is moderate to poor and it is less active than ciprofloxacin24. We found similar results with the Iranian isolates.
P. aeruginosa isolates from burn patients had been shown to express higher level of resistance to antibiotics comparing to isolates from other patients7, 25, 26. In our study, isolates from burn wounds (n=2) were resistant to all antibiotics, too.
Analysis of clinical isolates by molecular techniques can be helpful in studying the epidemiology of outbreak strains, and in confirming their clonality27. No single technique has proven completely satisfactory and reliable28. The use of plasmid profiling as an epidemiological tool has gained impetus during the past 3-4 yr29. Plasmid DNA was detected in only 29.8 per cent isolates in our study. Therefore it is postulated that most of the resistance genes in P. aeruginosa are mostly chromosomal. The absence of plasmids from the majority of isolates showed low typeability and discriminatory power of this technique. However, 61.8 per cent of isolates containing plasmids were resistance to at least 8 antibiotics, indicating the importance of presence of plasmids in MDR strains in particular those showing resistance to all antibiotics. The relationship between certain plasmids and resistance to some antibiotics had been reported previously5, 30- 32.
PFGE showed that isolates in pattern A were distributed widely at both hospitals and the environment. In other studies33, although no source of transmission could be found, PFGE demonstrated that inter- hospital transmission with pattern A might have occurred prior to our study. Isolates grouped in one pattern by PFGE may belong to different antibiotype or plasmid profile, since different patients may not comply with drug regimen perfectly and resistant mutant may be selected. However, the majority of isolates that were closely related by PFGE produced similar plasmid profiles and antibiotic resistance patterns. It is suggested that the accuracy of tracing of nosocomial isolates of P. aeruginosa increased when two methods of antimicrobial susceptibility testing and plasmid profiling were used together.
In conclusion, the results obtained in this study, show that it is necessary to focus on tracing the source of infections, control of nosocomial infections and therefore, design of strategies to diminish the nonspecific use of broad spectrum antibiotics in the hospitals.
Acknowledgment
Authors thank Dr Mofidi, Mehr hospital and A. Shahin, Mostafa- Khomeini hospital, Tehran for collecting the isolates.
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V.S. Nikbin#, A. Abdi-Ali*, M.M. Feizabadi+ & S. Gharavi
Department of Biology, Faculty of Science, Alzahra University & * Department of Microbiology, School of Medicine, Medical Sciences, University of Tehran, Tehran, I.R., Iran
Received September 6, 2006
# Present address: Department of Microbiology, Pasteur Institute of Iran, Pasteur Street, Tehran, Iran
Reprint requests: Dr A. Abdi Ali, Department of Biology, Faculty of Sciences, Alzahra University, Tehran, Iran
e-mail: abdialya@alzahra.ac.ir
Copyright Indian Council of Medical Research Aug 2007
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