|Year : 2015 | Volume
| Issue : 1 | Page : 87-91
Clonal evolution multi-drug resistant Acinetobacter baumannii by pulsed-field gel electrophoresis
P Mohajeri1, A Farahani2, MM Feizabadi3, B Norozi4
1 Department of Microbiology, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
2 Department of Microbiology, Faculty of Medicine; Student Research Committee, Kermanshah University of Medical Sciences, Kermanshah, Iran
3 Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
4 Student Research Committee, Kermanshah University of Medical Sciences, Kermanshah, Iran
|Date of Submission||25-May-2013|
|Date of Acceptance||24-Feb-2014|
|Date of Web Publication||5-Jan-2015|
Department of Microbiology, Faculty of Medicine; Student Research Committee, Kermanshah University of Medical Sciences, Kermanshah
Source of Support: None, Conflict of Interest: None
Background: Acinetobacter baumannii is usually multi-drug resistant (MDR), including third generation cephalosporins, amino glycosides and fluoroquinolone. Resistance to these antibiotics is mediated by multiple factors such as: lactamases, efflux pumps and other mechanisms of resistance. Pulsed-field gel electrophoresis (PFGE) was then used to investigate the genetic relationships among the MDR isolates. Aim: The aim of this study was to determine MDR isolates and the existence of OXAs genes among MDR isolates of A. baumannii collected from Kermanshah hospitals in west of Iran. Materials and Methods: Forty-two MDR A. baumannii were collected from patients at Kermanshah hospitals. The isolates were identified by biochemical tests and API 20NE kit. The susceptibility to different antibiotics by disk diffusion method was determined. Polymerase chain reaction (PCR) was performed for detection of blaOXA-23-like , blaOXA-24-like , blaOXA-51-like and blaOXA-58-like betalactamase genes in isolates and clonal relatedness was done by PFGE (with the restriction enzyme ApaI) and patterns analyzed by Bionumeric software. Results: This study showed high resistant to ciprofloxacin, piperacillin, ceftazidime and also resistant to other anti-microbial agents and more spread blaOXA-23-like gene (93%) in MDR isolate. The PFGE method obtained six clones: A (10), B (9), C (5), D (4), E (11) and F (3) that clone E was outbreak and dominant in different wards of hospitals studied. Conclusion: An isolate from the emergency ward of these hospitals had indistinguishable isolates PFGE profile and similar resistance profile to isolates from intensive care unit (ICU), suggesting likely transmission from ICU to emergency via patient or hospital staff contact.
Keywords: Acinetobacter baumannii, multi-drug resistant, OXA-type, Pulsed-field gel electrophoresis
|How to cite this article:|
Mohajeri P, Farahani A, Feizabadi M M, Norozi B. Clonal evolution multi-drug resistant Acinetobacter baumannii by pulsed-field gel electrophoresis. Indian J Med Microbiol 2015;33:87-91
|How to cite this URL:|
Mohajeri P, Farahani A, Feizabadi M M, Norozi B. Clonal evolution multi-drug resistant Acinetobacter baumannii by pulsed-field gel electrophoresis. Indian J Med Microbiol [serial online] 2015 [cited 2019 Jul 18];33:87-91. Available from: http://www.ijmm.org/text.asp?2015/33/1/87/148390
| ~ Introduction|| |
Acinetobacter baumannii has emerged as leading opportunistic pathogens and nosocomial pathogen worldwide.  This isolates is among the most common isolates from intensive care units (ICUs) in most hospitals in Iran. Infection with A. baumannii is becoming a serious problem, particularly in Iran. ,,, A. baumannii has emerged as a common worldwide problem as a nosocomial pathogen in patients and high infection-associated morbidity and mortality. ,, Most of the isolates are resistant to multiple anti-microbial agents such as penicillin's, β-lactamase, aminoglycosides, fluoroquinolone and carbapenems, which are the drugs of choice for treatment of the infection. In the past decades A. baumannii has emerged as opportunistic pathogen, especially as multi-drug resistant (MDR) to the major agents used to treat nosocomial infections. , A. baumannii are usually MDR, including third generation cephalosporins, aminoglycosides and fluoroquinolons,  which also spread infection with MDR A. baumannii clones reported worldwide. , There are several mechanisms for MDR. Transfer of antibiotic resistance genes by chromosomes, plasmids and transposone.  It seems that new anti-microbial agents that are effective against the bacteria activity may not be available in the near future and this subject is much more important than anti-microbial agents.  Often susceptible only to carbapenems (imipenem, meropenem), though resistant strains are increasingly reported. , A. baumannii possesses a large number of β-lactamases that inactivate cephalosporins and carbapenems (imipenem, meropenem). However, the most widespread β-lactamases with carbapenemase activity in A. baumannii are class D β-lactamases (OXA-type enzymes), which are frequently identified in this species. ,
The aim of this study was to understand the molecular epidemiology of MDR A. baumannii by pulsed-field gel electrophoresis (PFGE) method. We report evaluation clones in west of Iran and to determine the distribution of pulsotypes. Also our isolates for the presence of genes blaOXA-23-like , blaOXA-24-like , blaOXA-51-like and blaOXA-58-like by PCR is determined.
The results were analyzed by Chi-square test using SSPS 16 software. Continuous variables were compared with one-way analysis of variance. Variables were analyzed by Chi-square test. A P < 0.05 was considered to indicate significance.
| ~ Materials and Methods|| |
Isolates of Acinetobacter obtained from blood, urine and sputum were collected from two hospitals in Kermanshah between March 2010 and December 2011. The isolates were identified by conventional biochemical methods and confirmed by API 20NE kit (version 6.0, bio-Mérieux, Marcy L'Etoile, France).  We did the Kirby-Bauer method of disk diffusion for all isolates with CLSI guidelines  for: amikacin (AN: 30 μg), ceftriaxone (CRO: 30 μg), ciprofloxacin (CIP: 5 μg), trimethoprim/sulfamethoxazole (TS: 30 μg), gatifloxacin (GAT: 5 μg), colistin (CL: 10 μg), gentamicin (GM: 10 μg), imipenem (IMP: 10 μg), meropenem (MEM: 10 μg), piperacillin (PRL: 100 μg), polymyxin B (PB: 300 unit), levofloxacin (LVF: 5 μg), minocyclin (MIN: 30 μg), mezlocillin (MEZ: 75 μg), tetracycline (TET: 30 μg), tobramycin (TOBI: 10 μg), cefepime (CEF: 30 μg), cephpodoxime (CPD: 10 μg), cefotaxime (CTX: 30 μg), ceftazidime (CAZ: 30 μg), rifampicin (RF: 5 μg) (MAST, Merseyside, UK).
A. baumannii ATCC 19606 were used as control.
Determination of OXA type by PCR
PCR was carried out for amplification of blaOXA-23-like , blaOXA-24-like , blaOXA-51-like and blaOXA-58-like gene in MDR isolates. Primers for resistance genes were designed as previously described for PCR amplification. 
Pulsed-field gel electrophoresis
These strains were collected from two hospitals from where the most collected isolates came from. A total of 42 MDR A. baumannii were analysed by PFGE using the methods as described by Durmaz et al.  We used A. baumannii ATCC 19606 for external reference. Genotyping of all organisms were performed with ApaI (New England Biolabs) digestion. The Lambda Ladder PFG Marker (NEB, US) was used as a molecular size marker. Electrophoresis in a pulsed-field electrophoresis system (Chef Mapper; Bio-Rad Laboratories, Hercules, CA, USA) was by programming two states with the following conditions: temperature 14°C; voltage 6 V/cm; switch angle, 120°; switch ramp 2.2-35 s for 19 h. The gels were stained with ethidium bromide and patterns were photographed with UV gel Doc (BIO-RAD, USA) [Figure 1]. The DNA banding patterns were analyzed using Bionumeric 7.0 software (Apllied maths NV, St-Martens-Latem Beligum). The PFGE DNA patterns obtained were interpreted and compared as described by Tenover et al. 
|Figure 1: CHEF Profiles of A. baumannii strains isolated from different patients. Lateral lanes contain Lambda Ladder PFG Marker|
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| ~ Results|| |
Anti-microbial susceptibility test and PCR
A total of 42 A. baumannii isolates with MDR phenotype were highly resistant to ciprofloxacin, piperacillin, ceftazidime [Table 1]. These isolates were susceptible to colistin, polymyxin and tigecylcine. MDR isolates were mainly from sputum (85.7%) and blood (14.3%), from male patients (69%) aged 32 ± 23 years and female (31%) aged 28 ± 27 years, and from patients hospitalized in ICUs (95.2%) and emergency services (4.8%). Following PCR, MDR isolates were identified 92.9% the blaOXA-23-like and 26.2% blaOXA-24-like gene and also totally isolates were identified as carrying the blaOXA-51-like gene and none carried the blaOXA-58-like gene. Some isolates carrying both blaOXA-24-like -blaOXA-23-like 26.2% [Figure 2].
|Figure 2: Of right to left: Lane 1&8: Ladder 100 bp, lane2: blaOXA-23- like, lane 4: blaOXA-24-like, lane 6: blaOXA-51-like|
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pulsed-field gel electrophoresis
Based on Tenover's criteria,  the PFGE patterns of 42 isolates were clustering into six clones (pulsotype): A (10), B (9), C (5), D (4), E (11) and F (3) [Figure 3]. All isolates belonging to the same genotype had similar phenotypes, such as resistance pattern, distribution gene (bla OXA genes) and source of isolation. They showed co-resistance to anti-microbial agents including amikacin, piperacillin, ceftazidime, cefepime, cefotaxime, meropenem, polymyxin B and colistin [Table 1]. Clone E was dominant and widespread in different wards and hospitals. Clone E was dominant in different parts of our hospital and similarity in this clone was about 98%. Other clones especially clones A and B were in next dominant clones after clone E. Clones D and F exist only in male [Table 2]. Most pan drug resistant (PDR) isolates to clones A and E [Table 3].
|Table 2: Comparison of PFGE pattern with blaOXA genes, wards, Hospitals and Sex of isolates MDR A. baumannii |
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|Table 3: Dispersal of PFGE pattern with anti - microbial susceptibility of isolates MDR A. Baumannii |
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|Figure 3: Pulsed-field gel electrophoresis dendrogram of MDR Acinetobacter baumannii|
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| ~ Discussion|| |
Anti-microbial resistance is common among A. baumannii isolates. It is also noteworthy to highlight the fact that Acinetobacter strains are naturally resistant to cephalosporins owing to production of a cephalosporinase (AmpC) that is known to hydrolyze amino-penicillins and first-, second- and third-generation Cephalosporins.  MDR A. baumannii strains have been widely reported all over the world, such as Spain, Palestine, Europe, North America, Russia, Brazil, Tahiti, China and also recently reported in Iran. ,,, MDR strains often spread, causing outbreaks throughout entire cities, countries and continents.  This study is the first such report of MDR A. baumannii isolated in Kermanshah Hospitals in west of the I.R. The study showed low susceptibility rates to most of the clinically available anti-microbial agents for the treatment of infections caused by A. baumannii except for polymyxin B, colistin and tigecylcine that was maintained well, these antibiotics can be helpful in treating infections by A. baumannii. A similar increase in the resistance of A. baumannii isolates to most of the clinically available anti-microbial agents from 2006 to 2010 was reported in Iran. , Different mechanisms are involved in the resistance to imipenem (or meropenem) in A. baumannii. β-lactamase is one important factor. blaOXA-23-like was the most prevalent carbapenemase among imipenem-resistant isolates in Iran.  Carbapenem resistance is becoming more and more common among A. baumannii isolates in I.R. hospitals in recent years. ,,, In this study, 93% of MDR A. baumannii carrying blaOXA-23-like and 26.2% of isolates carrying blaOXA-24-like . All isolates were carriers for genes encoding the enzyme blaOXA-51-like , an enzyme that naturally exists in A. baumannii and confirming their identity as A. baumannii. The blaOXA-58-like is not found among the isolates. The blaOXA-23-like was associated with Carbapenemase (P = 0.003) because the blaOXA-23-like gene associated with Tn2006 is capable of transposing from bacteria to bacteria and could be located on the plasmid  and also class 1 integrons plays an important role in spread of antibiotic resistance in MDR A. baumannii For better understanding the epidemiology and spread of bacteria, particular in outbreaks of nosocomial infection, a number of molecular typing systems have been developed.  PFGE analysis has been used with excellent data in epidemiologic studies of numerous bacterial outbreaks and is gold standard for molecular epidemiologic.  The results of this study support the contention that clonal spread was the main reason for the increasing trend of MDR resistance in our hospital. It is increasingly recognized that A. baumannii is clonal relatedness in different wards of hospitals and that a large part of MDR A. baumannii in west of Iran. This study was conducted to understand the molecular epidemiology of MDR A. baumannii in hospitals, with the aim of identifying predominant clonal lineages currently circulating in this area. Isolates in this study with PFGE profiles E, A and B were believed to be endemic in the ICU and emergency area throughout the years.
| ~ Conclusion|| |
An isolate from the emergency of these hospitals had an indistinguishable isolates PFGE profile and similar resistance profile to isolates from ICU, suggesting likely transmission from ICU to emergency via patient or hospital staff contact, however, the direction was unknown. Early recognition of the presence of MDR A. baumannii clones is useful for preventing their spread within the hospital environment,  for this point sampling via patient or hospital staff in my study should be controlled by several time in during year of study. In conclusion, A. baumannii isolates from the ICU and emergency at Kermanshah from March 2010 to December 2011 had high resistance rates to the majority of anti-microbials commonly used in hospitals. Only tigecylcine and colistin remains effective for the treatment of infections caused by MDR A. baumannii. PFGE analysis confirmed a persistent clone of MDR A. baumannii in the ICU and emergency environment. Overall, our study indicated that MDR A. baumannii strains are spreading in west of Iran.
| ~ Acknowledgements|| |
This work was performed in partial fulfilment of the requirements for MSc thesis in medical microbiology (Abbas Farahani). The authors would like to acknowledge Kermanshah University of Medical Sciences. The study was financially supported by the Kermanshah University of Medical Sciences for grant 93045. We are very grateful to Yohei Doi for critically reviewing the original version of the manuscript.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]