Asian Journal of Medical Sciences 4(5): 179-183, 2012 ISSN: 2040-8773 Maxwell Scientific Organization, 2012 Submitted: August 09, 2012 Accepted: September 17, 2012 Published: October 25, 2012 Identification of Multidrug-Resistant Genes in Acinetobacter baumannii in Sulaimani City-Kurdistan Regional Government of Iraq Aras A.K. Shali Department of Biology, School of Science, Faculty of Science and Educational Sciences, University of Sulaimani, Kurdistan Regional Government, Iraq Abstract: Acinetobacter baumannii is an opportunistic pathogen responsible for hospital-acquired infections. A. baumannii epidemics described world-wide were caused by few genotypic clusters of strains. The occurrence of epidemics caused by multi-drug resistant strains assigned to novel genotypes have been reported over the last few years. Multiple drug-resistant strains of Acinetobacter have created therapeutic problems worldwide. This study was conducted to determine the antimicrobial susceptibility patterns of bla OXA -type carbapenemases among isolates of Acinetobacter spp obtained from clinical specimens. Twenty one Acinetobacter isolates were identified at the species level and their susceptibilities to different antibiotics were determined using Vitek 2 system. Resistant percentages for all isolates were recorded; highest resistant rate was against ampicillin (100%) while lowest rate was against imipenem (57.1%). The MICs of imipenem for the resistant isolates were 16. All isolates show multi drug resistance to different antibiotics used. The Isolates were then subjected to multiplex-pcr targeting bla OXA genes. All strains of A. baumannii possess a bla OXA-51-like gene. The co-existence of bla OXA-51-like and bla OXA-23-like were detected in 17 strains while in 4 strains bla OXA-51-like was the only presented gene. Detection of bla OXA-51-like can be used as a simple and reliable method to differentiate A. baumannii strains from other species. Keywords: Bla OXA-51-like Acinetobacter baumannii, Kurdistan Acinetobacter baumannii, multidrug resistant A. baumannii INTRODUCTION Acinetobacter baumannii (AB) is a Gram-negative coccobacillus that is ubiquitous in fresh water and soil and is also found frequently as a skin and throat commensal in humans (Houang et al., 2001). Acinetobacter spp has become major pathogens in hospital-associated infections, especially in critical care settings such as Intensive Care Units (ICUs) and units for patients with severe burns (Murray and Hospenthal, 2008). They can survive in the hospital environment for long periods and have a remarkable propensity to develop resistance to multiple classes of antimicrobial agents (Boo et al., 2009). During the past decade, nosocomial outbreaks of Acinetobacter baumannii have been described with increasing frequency, occurring mostly in intensive care units, burn units and surgical wards (Mandell et al., 2000; Bergogne-Bérézin and Towner, 1996). Epidemic strains of Acinetobacter baumannii are often resistant to several antimicrobial drugs, which reduces treatment effectiveness. Nosocomial transmission is from patient to patient and associated with environmental reservoirs (Bergogne- Bérézin and Towner, 1996). In a Canadian study, Acinetobacter baumannii has been ranked as the 20 th most common organism identified from ICUs (Zhanel et al., 2008). Antimicrobial resistance is increasingly being reported in Acinetobacter baumannii, often leaving carbapenems as the only effective drug to treat severe infections (Pournaras et al., 2006). Resistance to carbapenems in the population of Acinetobacter strains is high with the majority of isolates showing multidrug resistance. On the other hand, carbapenem resistance has been observed on every continent; examples include the UK, Greece, North America, Asia-Pacific region and Iran (Pournaras et al., 2006; Turton et al., 2006a; Morgan et al., 2009; Mendes et al., 2009; Feizabadi et al., 2008). However, MDR (carbapenem resistant) Acinetobacter baumannii originating from injured Canadian military personnel returning from Afghanistan and Iraq have also been described (Tien et al., 2007). In 1993, the first of a novel group of narrowspectrum OXA-type β-lactamases was discovered in an imipenem-resistant A. baumannii strain from a patient in the Royal Infirmary of Edinburgh that was found to possess carbapenem hydrolyzing activity (Paton et al., 1993). Moreover, bla OXA-58 was found to be the likely cause of carbapenem resistance while another isolate had an insertion sequence element upstream of its intrinsic bla OXA-51 (McCracken et al., 2009). Polymerase Chain Reaction (PCR) testing of the synergy-positive isolates for carbapenemase genes was done by using consensus primers for bla IMP (Senda et al., 1996), bla VIM (Tsakris et al., 2000), bla SPM (Toleman et al., 2002), bla OXA-23-like, bla OXA-24-like, 179
bla OXA-58-like (Poirel et al., 2005) and bla OXA-51-like (Héritier et al., 2005). In two Greek hospitals, 5 Metallo-Β-Lactamase (MBL) -positive Acinetobacter baumannii isolates were found. The isolates were unrelated and carried bla VIM-1 in a class 1 integron; bla OXA-51- and bla OXA-58-like carbapenemase genes were also detected (Athanassios et al., 2006). Two classes of molecular carbapenemase, classes B and D have been found among strains of Acinetobacter. The enzymes in class D (OXA enzymes) have emerged as the major carbapenemases in the world, although metallo enzymes are mainly prevalent in East Asia (Livermore, 2002). OXA enzymes (encoded by bla OXA genes) can be sub classified into eight distinct subgroups: OXA- 23-like, OXA-24-like, OXA-51-like and OXA-58-have been identified in Acinetobacter spp. Reports from different countries have shown that bla OXA-51- type genes are intrinsically harbored by A. baumannii isolates and they support the presence of a direct reservoir of β-lactam-resistance genes within the nosocomial environment (Livermore, 2002; Brown and Amyes, 2006). Detection of bla OXA-58-like can be used as a simple and reliable method to differentiate A. baumannii strains from other species (Feizabadi et al., 2008; Turton et al., 2006b). Furthermore, Acinetobacter spp isolates that gave a band for bla OXA- 51-like identified as A. baumannii (Turton et al., 2006a). Hence this study was designed to identify Acinetobacter spp using molecular methods to species level. MATERIALS AND METHODS Microbiological investigations: This study was carried out at Biotechnology Research Laboratory in the Department of Biology, School of Science, Faculty of Science and Educational Sciences-University of Sulaimani. A total of 21 bacterial isolates were recovered from clinical specimens (burn and urine) in Central Health Laboratory and Burn and Plastic Surgery Hospital/Emergency in Sulaimani city during the period from February, 2010 to March, 2012. Presumptive identification based on culture characteristics and gram stain. Standard identification, confirmation and complete methods were conducted including using the commercial identification systems; API 20E (biomérieux, France) and Vitek 2 system (biomérieux, France). Non Acinetobacter baumannii isolates were excluded from the study. Confirmatory identification and speciation was carried out by PCR for the detection of the bla OXA-51- and bla OXA-58-like carbapenemase genes, as described by Turton et al. (2006b) and Feizabadi et al. (2008). Antimicrobial susceptibility testing and MIC: Antimicrobial susceptibility test was performed and MICs were determined using Vitek 2 susceptibility test system (biome rieux) for the following antimicrobial agents: Ampicillin, Amoxicillin/ Clavulanic Acid, Pipracillin/Tazobactam, Cefazolin, Cefepime, Imipenem, Amikacin, Gentamicin, Ciprofloxacin, Nitrofurantoin, Trimethoprime, Cefoxitin, Ceftazidim and Cefotaxim. PCR amplification of bla OXA genes: A single bacterial colony cultured previously on nutrient agar was suspended in 50 µl ddh 2 O. The cells then disrupted by heating for 10 min at 99 C in PCR machine. The samples were centrifuged at 13000 rpm for 10 min (Person et al., 2007) and 5 µl of the supernatant was used as template in the PCR reactions. To amplify the genes encoding carbapenemases, a multiplex-pcr assay was run using the primers bla OXA-51-like (353 bp: 5 -TAA TGC TTT GAT CGG CCT TG-3 and 5 -TGG ATT GCA CTT CAT CTT GG-3 ), bla OXA -23- like (501 bp: 5 -GAT CGG ATT GGA GAA CCA GA-3 and 5 - ATT TCT GAC CGC ATT TCC AT-3 ), bla OXA -24- like (246 bp: 5 -GGT TAG TTG GCC CCC TTA AA-3 and 5 -AGT TGA GCG AAA AGG GGA TT-3 ) and bla OXA-58-like (599 bp: 5 -AAG TAT TGG GGC TTG TGC TG-3 and 5 -CCC CTC TGC GCT CTA CAT AC-3 ) as described by Turton et al. (2006a) and Niel et al. (2006). Amplification was performed in a final volume of 20 µl according to instruction manual (Cinnagen, Iran), containing Master max (4 µl). Forward and Reverse primer (each 0.6 µl) mixed with 5 µl DNA sample and completed to 20 µl by ddh 2 O. The thermocycler run was programmed at 94 C for 5 min followed by 30 cycles of 25s at 94 C, 40s at 53 C, 50s at 72 C and a final cycle of 6 min at 72 C (Feizabadi et al., 2008). RESULTS Results obtained from standard identification methods show that all isolates were Acinetobacter baumannii. The result of antimicrobial susceptibility test show that all strains (n = 21) were resistant Table1: Percentage of antimicrobial resistance and MICs of A. baumannii isolates to different antibiotics Antimicrobial agent Resistance (%) MIC range (µg/ml) Ampicillin 100 16-32 Amoxicillin/lavulanic acid 85.7 4-32 Pipracillin/tazbactam 90.4 128 Cefazolin 100 64 Cefeime 90.4 64 Imipenem 57.1 16 Amikacin 66.6 64 Gentamicin 85.7 16 Ciprofloxacin 95.2 64-320 Nitrofurantion 95.2 512 Trimethopreime 95.2 4 Cefoxitin 100 64 Ceftazidim 85.7 64 Cefotaxim 85.7 64 180
Fig. 1: Detection of genes encoding OXA carbapenemase in Acinetobacter baumannii by multiple PCR A-Lane 1: Ladder (1500bp); Lane -ve: (ddh 2 O); Lane 3-13: Acinetobacter baumannii; B-Lane L: Ladder (1500bp); Lane 11-21: Acinetobacter baumannii; Lane -ve: Negative control- Pseudomonas aeruginosa. Table 2: Distribution of two bla OXA alleles and determination of MICs for imipenem, piperacillin/tazobactam and cefotaxime among carbapenem resistance A. baumannii showing resistance to these antimicrobial agents by Vitek 2 system Acinetobacter spp Bla OXA allele No. of strain No. of resistant strains Antibiotic MIC (μg/ml) A. baumannii bla OXA-51/OXA-23 17 10, 15, 14 Imipeneme, Piperacillin/ 16 128 tazobactam/cefotaxime A. baumannii bla OXA-51 4 2, 3, 3 Imipeneme, Piperacillin/ tazobactam/cefotaxime to ampicillin (MIC 32), while 57.1% (n = 12) of the strains showed resistant to imipenem (MIC 16), these strains considered as carbapenem resistant A. baumannii, about 90% (n = 19) strains were resistant to Pipracillin/Tazobactam (MIC 128) whereas 85.71% (n = 18) of the strains showed resistance to cefotaxim (MIC 16-64) (Table 1). Multiplex PCR results revealed that all strains possess a bla OXA-51-like gene (amp icon size: 353 bp). The co-existence of bla OXA-51-like and bla OXA-23-like (amp icon size: 501 bp) were detected in 17 strains, in which 58.8% of them (n = 10) were resistant to imipenem, while in 4 strains bla OXA-51-like gene was only present (Fig. 1 and Table 2). Pseudomonas aeruginosa used as negative control (Feizabadi et al., 2008). DISCUSSION Accurate identification and typing of bacterial isolates are essential, particularly when determining strains involved in hospital outbreaks. Inappropriate infection control measures and inaccurate antibiotic usage are highly potential factors that might increase 181 16-64 8 128 16-64 the prevalence and spread of antibiotic resistant A. Baumannii isolates (Dhabaan et al., 2011). Class D β-lactamase-mediated resistance to β-lactams has been increasingly reported during the last decade. Those enzymes also known as oxacillinases or OXAs are widely distributed among Gram negatives. Genes encoding class D β-lactamases are known to be intrinsic in many Gram-negative rods, including Acinetobacter baumannii and Pseudomonas aeruginosa, but play a minor role in natural resistance phenotypes (Niel et al., 2006). The OXAs are characterized by an important genetic diversity and a great heterogeneity in terms of β-lactam hydrolysis spectrum. The acquired OXAs possess either a narrow spectrum or an expanded spectrum of hydrolysis, including carbapenems in several instances. Acquired class D β-lactamase genes are mostly associated to class 1 integron or to insertion sequences (Poirel et al., 2010). However, bla OXA-51-like was sought in clinical strains of Acinetobacter baumannii in a multiplex PCR, which also detects bla OXA-23-like. All isolates that gave a band for bla OXA-51- like identified as A. baumannii. This gene was detected in all of 21 strains of A. baumannii but not in the
negative control (Fig. 1). This result agrees with that of Turton et al. (2006b). Although it is clear that bla OXA-51- like genes are present in the vast majority of isolates of A. baumannii, there has been some debate as to whether they are present in all isolates of this species (Brown and Amyes, 2006). Furthermore, strains that considered as carbapenem resistant (MIC 16) possess both bla OXA- 51-like and bla OXA-23-like genes were highly resistant to imipenem than the strains show intermediate resistant to imipenem (n = 2, MIC = 8) that have only bla OXA-51- like which describe the lower MIC range (Table 2). However, intermediate ranges of susceptibility are considered as resistant (Raffaele et al., 2004). These results provide evidences that detection of bla OXA-51-like can be used as a simple and reliable way for identifying A. baumannii. It has been found that bla OXA-51-like exists in all isolates of A. baumannii and those strains that show carbapenem resistance are almost possess bla OXA-51-like and bla OXA-23-like genes. ACKNOWLEDGMENT Special thanks to Mrs. Paywast Jamal Jalal for her assistance as well as Central Health Laboratory and Burn and Plastic Surgery Hospital/Emergency in Sulaimani city for collaboration. REFERENCES Athanassios, T., I. Alexandros, P. Spyros, S.T. Leonidas, S. Danai, J.L. Nicholas and N.M. Antonios, 2006. VIM-1 Metallo- β-lactamase in Acinetobacter baumannii. Emerg. Infec. Diseas., 12(6): 981-983. Bergogne-Bérézin, E. and K.J. Towner, 1996. Acinetobacter spp. as nosocomial pathogens: Microbiological, clinical and epidemiological features. Clin Microbiol. Rev., 9: 148-165. Boo, T.W., F. Walsh and B. Crowley, 2009. Molecular characterization of carbapenem resistant Acinetobacter species in an Irish university hospital: predominance of Acinetobacter genomic species 3.J. Med. Microbiol., 58: 209-216. Brown, S. and S.G.B. Amyes, 2006. OXA (beta)- lactamases in Acinetobacter: the story so far. J. Antimicrob. Chemother., 57: 1-3. Dhabaan, G.N., H. Hamimah and M.A. Shorman, 2011. Emergence of extensive drug-resistant Acinetobacter baumannii in North of Jordan. Afric. J. Microbiol. Res., 5(9): 1070-1075. Feizabadi, M.M., B. Fathollahzadeh, M. Tahirekalani, M. Rasoolinejad, N. Sadighifard, M. Aligholi, S. Soroush and S. Mohammadi-Yegane, 2008. Antimicrobial susceptibility patterns and distribution of blaoxa genes among Acinetobacter spp. Isolated from patients at Tehran hospitals. Jpn. J. infect. Dis., 61: 274-278. Asian J. Med. Sci., 4(5): 179-183, 2012 182 Héritier, C., L. Poirel, P.E. Fournier, J.M. Claverie, D. Raoult and P. Nordmann, 2005. Characterization of the naturally occurring oxacillinase of Acinetobacter baumannii. Antimicrob. Agents Chemother., 49: 4174-4179. Houang, E.T., Y.W. Chu, C.M. Leung, C.M. Leung, K.Y. Chu, J. Berlau, K.C. Ng and A.F.B. Cheng, 2001. Epidemiology and infection control implications of Acinetobacter spp in Hong Kong. J. Clin. Microbiol., 39: 228-234. Livermore, D.M., 2002. The impact of carbapenemases on antimicrobial development and therapy. Curr. Opin. Investig. Drugs., 3: 218-224. Mandell, G.L., J.E. Bennett and R. Dolin, 2000. Principles and Practice of Infectious Diseases. 5th Edn., Churchill Livingstone, Philadelphia, 23: 39-44. McCracken, M., M. DeCorby, J. Fuller, V. Loo, D.J. Hoban, G.G. Zhanel and M.R. Mulvey, 2009. Identification of multidrug- and carbapenemresistant Acinetobacter baumannii in Canada: results from can ward 2007. J. Antimicrob. Chemother., 64: 552-555. Mendes, R.E., J.M. Bell, J.D. Turnidge, M. Castanheira and R.N. Jones, 2009. Emergence and widespread dissemination of OXA-23, -24/40 and -58 carbapenemases among Acinetobacter spp. in Asia- Pacific nations: Report from the Sentry Surveillance Program. J. Antimicrob. Chem., 63: 55-59. Morgan, D.J., S.A. Weisenberg, M.H. Augenbraun, D.P. Calfee, B.P. Currie, E.Y. Furuya, R. Holzman, M.C. Montecalvo, M. Phillips, B. Polsky and K.A. Sepkowitz, 2009. Multidrug-resistant Acinetobacter baumannii in New York City-10 years into the epidemic. Infect. Cont. Hosp Epidemiol., 30: 196-197. Murray, C.K. and D.R. Hospenthal, 2008. Acinetobacter infection in the ICU. Crit Care Clin., 24: 237-248. Niel, W., J.E. Matthew, M.C. Juliana, J.F. Turton, M.E. Ward, S. Brown, S.G.B. Amyes and D.M. Livermore, 2006. Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int. J. Antimicrob. Agents., 27: 351-356. Person, S., K.E.P. Olsen, F. Scheutz, K.A. Krogfelt and P. Gerner-Smidt, 2007. A method for fast and simple detection of major diarrhoegenic Escherichia coli in the routine diagnostic laboratory. J. Clin. Microbio. Infec., 13(5): 516-524. Poirel, L., S. Marqué, C. Héritier, C. Segonds, G. Chabanon and P. Nordmann, 2005. OXA-58: A novel class D β-lactamase involved in resistance to carbapenems in Acinetobacter baumannii. Antimicrob. Agents Chemother., 49: 202-208.
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