JAC Advance Access originally published online on August 29, 2007
Journal of Antimicrobial Chemotherapy 2007 60(5):1163-1167; doi:10.1093/jac/dkm305
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High rates of resistance to colistin and polymyxin B in subgroups of Acinetobacter baumannii isolates from Korea
1 Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, Korea 2 Asian-Pacific Research Foundation for Infectious Diseases (ARFID), Seoul, Korea 3 Division of Infectious Diseases, Daegu Fatima Hospital, Daegu, Korea 4 Division of Infectious Diseases, Chonnam National University Medical School, Gwangju, Korea 5 Division of Infectious Diseases, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
* Correspondence address. Division of Infectious Diseases, Samsung Medical Center, Sungkyunkwan University, and Asian-Pacific Research Foundation for Infectious Diseases (ARFID), 50 Il-won dong, Gangnam-gu, Seoul 135-710, Korea. Tel: +82-2-3410-0320; Fax: +82-2-3410-0328; E-mail: jhsong{at}smc.samsung.co.kr
Received 25 April 2007; returned 21 June 2007; revised 9 July 2007; accepted 19 July 2007
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Abstract |
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Objectives: To investigate antimicrobial resistance in clinical isolates of Acinetobacter spp. from two Korean hospitals.
Methods: Two hundred and sixty-five isolates of Acinetobacter spp. from two Korean hospitals were collected and were identified to species level using partial rpoB gene sequences. Antimicrobial susceptibility testing was performed using a broth microdilution method.
Results: rpoB gene sequences indicated that 214 isolates (80.8%) were Acinetobacter baumannii, and allowed these to be classified into three subgroups (I, II and III); 142 isolates (53.6%) belonged to subgroup I, 54 (20.4%) to subgroup II and 18 (6.8%) to subgroup III. Forty-eight isolates (18.1%) and 74 isolates (27.9%) were resistant to polymyxin B and colistin, respectively. However, antimicrobial resistance rates varied markedly between subgroups. While A. baumannii subgroup I showed low resistance rates to polymyxin B and colistin (2.1% and 7.0%, respectively), subgroups II and III showed high resistance rates to these antibiotics (38.9% and 64.8% in subgroup II and 72.2% and 88.9%, in subgroup III, respectively). Multidrug resistance was also significantly more frequent in subgroup I (45.1%) than in subgroups II and III (13.0% and 16.7%, respectively).
Conclusions: Our data indicate that subgroup identification of A. baumannii may aid selection of appropriate antimicrobial agents for the treatment of Acinetobacter infections.
Keywords: rpoB , antimicrobial resistance , subgrouping
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Introduction |
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Acinetobacter species have increasingly been recognized as hospital-acquired pathogens mainly in immunocompromised patients and patients in intensive care units (ICUs).1 In ICUs, the prevalence of infections by Acinetobacter spp. currently account for 2% to 10% of all nosocomial Gram-negative bacterial infections in the United States and Europe.2 To date, 10 nomenspecies and 4 genomospecies have been isolated from humans; Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter lwoffii, Acinetobacter parvus, Acinetobacter radioresistens, Acinetobacter schindleri, Acinetobacter ursingii and genomospecies 3, 13TU, 10 and 11. Of these, A. baumannii, A. calcoaceticus and genomospecies 3 and 13TU are the most frequently isolated and clinically relevant.3 They are commonly referred to as the A. baumannii–A. calcoaceticus complex because they are genetically closely related and cannot be easily differentiated by phenotypic methods in the clinical microbiology laboratory.
Emergence of pandrug-resistant or multidrug-resistant (MDR) A. baumannii strains has become a serious clinical problem in many parts of the world, especially in some Asian countries.4 Antimicrobial options for the treatment of MDR A. baumannii are limited, including polymyxins or sulbactam.1 In this study, we identified Acinetobacter isolates from two Korean hospitals using partial rpoB gene sequencing, which has been used for the identification of several bacterial species,5 and tested their antimicrobial susceptibilities in vitro.
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Materials and methods |
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Bacterial isolates
A total of 265 non-duplicate clinical isolates of Acinetobacter spp., which were collected from two tertiary-care hospitals in Korea, were tested in the study. One hundred and six isolates were from blood of patients in the Chonnam National University Hospital (CNUH; Gwangju) during the period from March 2002 to May 2006. The other 159 isolates from various specimens were collected at the Samsung Medical Center (SMC; Seoul) between January and May 2006; 76 isolates from sputum, 31 from tracheal aspirate, 7 from bile, 6 isolates from blood, pus and urine, respectively, and other various specimens. We also included three strains representing European clones I (RUH875), II (RUH134) and III (RUH5875).
To identify Acinetobacter species and to analyse the intraspecific variation of A. baumannii, we determined the partial rpoB gene sequence of 265 isolates of Acinetobacter spp. using primers Ac1055F (GTGATAARATGGCBGGTCGT) and Ac1598R (CGBGCRTGCATYTTGTCRT).5 We obtained unambiguous 468 bp sequences from all isolates, which included one of the variable regions of the rpoB gene, zone 2.5
Antimicrobial susceptibility testing
In vitro susceptibility testing was performed with all isolates of Acinetobacter spp. using the broth microdilution method according to CLSI guidelines.6 Fourteen antimicrobial agents were tested: imipenem, meropenem, polymyxin B, colistin, tetracycline, ciprofloxacin, rifampicin, amikacin, cefepime, ceftriaxone, cefoperazone/sulbactam, ceftazidime, piperacillin/tazobactam and ampicillin/sulbactam. The interpretive criteria used were those established in CLSI standard M100-S16.6 Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922 and ATCC 35218 and Pseudomonas aeruginosa ATCC 27853 were used as control strains. MDR was defined in accordance with Paterson.7
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Results |
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Based on partial (468 bp) rpoB gene sequences, we identified 12 Acinetobacter species (8 nomenspecies and 4 genomospecies) among the 265 isolates. Of these, 214 isolates (80.8%) were classified as A. baumannii. Genomospecies 3 was the second most common species (24 isolates, 9.1%). Other species were represented by one to five isolates, and six isolates could not be assigned to species level although they clustered within the genus Acinetobacter in the rpoB gene tree. A. calcoaceticus and A. parvus were found only in the CNUH, and Acinetobacter baylyi, A. haemolyticus, A. junii, Acinetobacter tjernbergiae, A. ursingii and genomospecies 11 were isolated only in the SMC. Isolates within the same species or subgroup in A. baumannii showed rpoB sequence divergence of <2%, equivalent to no more than nine nucleotide changes.
The 214 A. baumannii isolates were further divided into three subgroups, subgroups I, II or III, based on phylogenetic clustering (Figure 1): 142 (66.4%) isolates belonged to subgroup I; 54 isolates (20.4%) to subgroup II; and 18 isolates (6.8%) to subgroup III. A. baumannii CIP 70.34 (=ATCC 19606), which is the type strain of A. baumannii, and three representative strains of European clones I, II and III also belonged to subgroup I. Subgroup I included 47 isolates (44.3%) from the CNUH and 95 isolates (59.7%) from the SMC. While 17 isolates were grouped into subgroup III in the CNUH, only one isolate from SMC belonged to this subgroup.
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For all isolates of Acinetobacter spp., resistance rates to polymyxin B and colistin were 18.1% and 30.6%, respectively (Table 1). MIC90s of polymyxin B and colistin were 8 and 32 mg/L, respectively. Resistance rates to carbapenems were 8.3% (imipenem) and 11.7% (meropenem). Resistance rates and MIC90s of the other antimicrobials are shown in Table 1.
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Antimicrobial resistance rates varied by subgroups of A. baumannii (Table 1). Resistance rates to polymyxin B and colistin in A. baumannii subgroup I were only 2.1% and 7.0%, respectively. They were also low in genomospecies 3 (4.2% and 8.3%, respectively). However, they were very high in A. baumannii subgroups II and III. Polymyxin B resistance rates of A. baumannii subgroups II and III were 38.9% and 72.2%, respectively. In addition, most isolates of A. baumannii subgroups II and III were resistant to colistin (resistance rates, 64.8% and 88.9%, respectively). MIC90s of polymyxin B and colistin were also markedly different among A. baumannii subgroups and genomospecies 3 (Table 1).
We identified 88 MDR Acinetobacter isolates (33.2%) (Table 2); most (64 isolates, 72.7%) belonged to subgroup I. The MDR rate was significantly higher in A. baumannii subgroup I (45.1%) followed by A. baumannii subgroups II (13%) and III (16.7%) (P < 0.001) (Table 2). Polymyxin B and colistin showed good activity against MDR A. baumannii subgroup I isolates but they showed poorer activity against MDR A. baumannii isolates belonging to subgroups II and III.
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Discussion |
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Acinetobacter spp. have become important nosocomial pathogen due to the emergence and rapid spread of MDR strains.1 Very few antimicrobial agents can be reliably used for effective therapy of MDR Acinetobacter infections.
In this study, we identified Acinetobacter species based on rpoB gene analysis. Although we used only partial rpoB gene sequences, we could identify most isolates of Acinetobacter species.5 Based on the identification using the rpoB gene, we have identified the first isolates of A. baylyi and A. tjernbergia from patient specimens: these species had previously been identified from activated sludge plants.8 Our results may indicate that more diverse Acinetobacter species have the potential to infect humans.
In this study, three subgroups were identified within A. baumannii based on rpoB gene sequences. These subgroups were phylogenetically distinct from one another, although they clustered into a single clade robustly (Figure 1). More interestingly, subgrouping of A. baumannii isolates based on partial rpoB gene sequences correlated with antimicrobial resistance profiles. While most isolates belonging to subgroup I were susceptible to polymyxin B and colistin, many isolates of subgroups II and III were resistant to these agents (Table 1). Emergence and spread of polymyxin-resistant Acinetobacter spp. poses a serious therapeutic concern, because no antimicrobial agents except tigecycline are available for treatment of MDR Acinetobacter infections.9 Colistin or polymyxin B resistance in Acinetobacter spp. is rare worldwide.10 Due to many colistin- or polymyxin B-resistant isolates belonging to the A. baumannii subgroups II and III, colistin or polymyxin B resistance rates among Acinetobacter spp. were 30.6% and 18.1% in this study. This means that accurate identification of Acinetobacter spp. is needed to select appropriate antimicrobial agents, although most colistin-resistant isolates of subgroups II and III were susceptible to other antimicrobials. In addition, higher rates of resistance to colistin may also be relevant in clinical settings, because it is less toxic than polymyxin B.
Carbapenems also showed different resistance profiles among A. baumannii subgroups. Unlike polymyxins, imipenem and meropenem showed good in vitro activities against isolates of A. baumannii subgroup II. Out of 83 polymyxin-resistant isolates, only 5 and 7 isolates were resistant to imipenem or meropenem (6.0% and 8.4%, respectively).
Reports of a number of outbreaks of nosocomial infections caused by Acinetobacter isolates might indicate a propensity to dissemination within hospitals.1 Thus, polymyxin resistance in Acinetobacter spp. could increase within a short time, although in our collection it was restricted to particular A. baumannii subgroups. The increasing trend of resistance to colistin or polymyxin B, which is now considered often to be the last choice for treatment of Acinetobacter infections, warrants continuous surveillance.
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Funding |
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This study was done with the support of the Asian-Pacific Research Foundation of Infectious Diseases (ARFID; Seoul, Korea).
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Transparency declarations |
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None to declare.
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Acknowledgements |
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Drs Je Chul Lee (Kyungpook National University, Korea) and Lenie Dijkshoorn (Leiden University Medical Centre, The Netherlands) provided isolates of European clones.
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References |
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La Scola B, Gundi VAKB, Khamis A, et al. Sequencing of the rpoB gene and flanking spacers for molecular identification of Acinetobacter species. J Clin Microbiol (2006) 44:827–32.
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Carr EL, Kampfer P, Pater BK, et al. Seven novel species of Acinetobacter isolated from activated sludge. Int J Syst Evol Microbiol (2003) 53:953–63.
9 Reis AO, Luz DA, Tognim MC, et al. Polymixin-resistant Acinetobacter spp. Isolates: what is next? Emerg Infect Dis (2003) 9:1025–7.[Web of Science][Medline]
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