Polyclonal spread of bla OXA-23 and bla OXA-58 in Acinetobacter baumannii isolates from Argentina

Background: In order to study the enzymatic carbapenem resistance mechanisms in Acinetobacter baumannii isolates from Argentina, we performed molecular characterization on 41 epidemiologically unrelated strains isolated from 1995 to 2006 with diminished susceptibilities to imipenem and meropenem. Methodology: Acinetobacter baumannii isolates were identified with the ARDRA technique. The total genomic DNA was used to detect each carbapenem β-lactamase gene described so far in this species and those insertion sequences usually associated to carbapenem β-lactamase genes (ISAba1, 2, 3, 4 and IS18) by the PCR technique with specific primers. Results: 26 out of 41 Acinetobacter baumannii isolates with diminished susceptibilities to carbapenems harboured the blaOXA-23 gene. The blaOXA-58 was detected in 13 out of 41 isolates. ISAba1 was always located upstream blaOXA-23. All isolates containing the blaOXA-58 gene showed ISAba3 downstream of the carbapenemase, while 4 isolates had a second copy of the ISAba3 upstream of the gene. Conclusion: Enzymatic carbapenem resistance in Acinetobacter baumannii was found in 88% of 41 non-epidemiologicallyrelated strains mediated by the polyclonal spread of the blaOXA-23 and blaOXA-58 genes. The genetic structures surrounding the oxacillinase genes found in our bacterial isolates revealed a particular epidemiology in our geographical region. This data suggests the need of local molecular surveillance to help control multirresistance Acinetobacter baumannii infections.


Introduction
Acinetobacter baumannii (AB) is an opportunistic pathogen that causes a wide variety of serious infections that frequently occur in severely ill intensive care unit (ICU) patients with chronic illnesses or prolonged hospitalizations [1].As a distinctive feature, it is a multidrug resistant species with the ability to acquire determinants of resistance to several antimicrobial agents, including β-lactam mechanisms [1].Infections are often difficult to treat since carbapenems are now almost always the drug of choice for the treatment of Acinetobacter infections.

Carbapenem-resistant
Acinetobacter baumannii (CRAB) strains were first isolated from clinical samples in South America during the 1990s [2].Nosocomial infection outbreaks caused by CRAB isolates are now documented worldwide [3].Recent reports also showed that the spread of the -lactamase-mediated carbapenem resistance is the most common mechanism found in CRAB isolates carried out by the class B (MBL) (IMP-like, VIM-like, and SIM-1 enzymes) or carbapenemhydrolyzing class D -lactamases (CHDLs) (OXA-23, -24, -51 and -58 -related families) [3].
The widespread dissemination of bla OXA-23 among different clones of A. baumannii in Colombia and Brazil has been documented [4,5]; previous studies from our laboratory also showed that the bla OXA-51 -type genes are ubiquitous in the AB genome [6].Moreover, we found that the bla OXA-51 -type alleles varied within a strain and were found in different PFGE clones, either susceptible or resistant to carbapenems [6].Recently, it has been observed that the insertion of ISAba1 upstream of both genes may provide a promoter to enhance gene expression, potentially contributing to increase the levels of resistance to carbapenemes [1,7].The goal of the present study was to survey the presence of enzymatic mechanisms in 41 epidemiologically unrelated AB isolates with diminished susceptibilities to carbapenems.

Bacterial strains and growth conditions
A total of 41 epidemiologically unrelated AB isolates were collected from 1995 to 2006 in 3 hospitals in Buenos Aires City, Argentina (Table 1).Most of them were obtained from respiratoty secretions (n = 16) and blood (n = 15) samples from ICUs, but also from catheters (n = 5), urine (n = 3) and others (n = 2).The isolates were identified at species level by phenotypic scheme [8] and amplified ribosomal DNA restriction analysis (ARDRA) that consists of amplification of the 16S rRNA-gene followed by restriction fragment length polymorfism [9].Isolates were stored at -70º C in Brain Heart Infusion (BHI) (Difco Laboratories, Detroit, USA) and supplemented with 10% glycerol until used.

Antimicrobial susceptibility testing
The MIC to imipenem and meropenem was performed by agar dilution method according to CLSI recommendations (Table 1) [10].

DNA techniques
Total DNA extraction and PCR reactions were performed as described by Sambrook et al. [11].Specific primers used for detecting the carbapenem -lactamase genes and the insertion sequence (IS) elements are listed in Table 2.The Taq polimerase enzyme (Invitrogen, Carlsbad, CA) was used to amplify the different bla genes.PCR DNA products were analysed by conventional agarose gel electrophoresis.PCR products were purified using the QIAquick kit according to the manufacturer´s instructions (Qiagen Inc., Studio City, Calif.).Genotype was determined by PFGE.The clones belonged to clones previously described in hospitals from Argentina, clones I, III, and IV [12]. 2 -, +, negative or positive, respectively, for the presence of bla OXA-23 by PCR reaction with specific primers. 3-, +, negative or positive, respectively, for the presence of bla OXA-58 by PCR reaction with specific primers. 4The MIC was evaluated for imipenem in µg/ml. 5The MIC was evaluated for meropenem in µg/ml. 6-, +, negative or positive, respectively, for the microbiological disk assay performed with the method described by Bou et al. [13] that is a modification of Masuda method.Sequencing of all positive reactions was performed on both DNA strands using the ABI Prism 3100 BioAnalyzer equipment.The nucleotide sequences were analyzed using the Genetics Computer Group (GCG) software and the NCBI/NLM Blast V2.0 software (URL: http: //www.ncbi.nlm.nih.gov/BLAST/).
Genotypes were defined by macrorestriction.Genomic DNAs embedded in agarose plugs were obtained as previously described [12].DNA was digested with 20 U ApaI (Promega Corporation, Madison, WI, USA), and digests were separated by PFGE (CHEF-DR III system, Bio-Rad, Richmond, CA, USA).Running conditions were 24 hours at 6 V/cm and 13°C, with pulse time from 1 second to 30 seconds.

Microbiological test for hydrolytic activity
The microbiological disk assay was performed with the method described by Bou et al., [13] which is a modification of the Masuda method.

Antimicrobial susceptibility pattern of A. baumannii isolates.
Isolates with imipenem or meropenem MIC of 4-8 µg/ml were considered to be low-level resistant in agreement with Afzal-Shah et al. [14].The obtained range of the MIC for imipenem was 8-64 µg/ml and for meropenem was 2-32 µg/ml.The MIC90 for imipenem was 32 µg/ml, and for meropenem was 16 µg/ml.

Study of clonal relationships
Three different clones were delineated in the 41 AB isolates by the PFGE method and they were identified as I, III, and IV [12] (Table 1).
During 1996, the clone IV was recovered with a susceptible imipenem MIC90 of 0.5 µg/ml from H1 [12].In the present study, the same clone IV was found in some isolates of hospital H3 with an MIC for imipenem of 32 µg/ml (Table 1), and from hospital H1 with an MIC for imipenem of 16 µg/ml (Table 1).
The study of the enzymatic inactivation of imipenem and/or meropenem exhibited 4 nonhydrolizing strains over 41 AB isolates, performed by a modification of the Masuda method [13] (Table 1).

Detection of carbapenemases by PCR and sequencing
We searched for the presence of carbapenemase genes within the 41 AB isolates by PCR reactions with specific primers.CHDLs genes were detected in all isolates that were positive for the Masuda test (Table 1).PCR results only indicate the presence of particular genetic determinants but not their expression.Therefore, it must be considered that some of the bla OXA genes found in this study could not be expressed, yielding unexpected values of MIC.We found the presence of bla OXA-23 gene in 26 out of 41 AB isolates (Table 1).The bla OXA-58 , recently characterized [3,15], was also detected in 13 out of 41 isolates.No other carbapenemase gene was found except for the bla OXA-51 -like genes harboured by all AB isolates.Although both genes, bla OXA-23 and bla OXA-58 , were found in the AB394 and AB311 isolates that belong to clone I, the MIC values were not increased (Table 1).In this regard, multicopy bla OXA-58 gene was found as a source of high-level resistance to carbapenems in AB isolates from Italy [16]; we found isolates with a MIC value of ≤ 16 µg/ml in our AB collection (n=11), which is in agreement with the presence of only one copy of bla OXA-58 in these strains [16].
The highest MICs observed in this study (imipenem 64 µg/ml) corresponded to three clone I isolates (AB305, AB320 and AB321) that carry the bla OXA-23 gene alone.In addition, a MIC of 32 µg/ml for imipenem was obtained for some clones, also carrying the bla OXA-23 gene.Whereas this may suggest that the bla OXA-23 gene yields elevated MIC values, we found several AB isolates, also harbouring the bla OXA-23 gene, showing a MIC value of 8 µg/ml.On the other hand, we found four AB isolates with MIC values of 8-16 µg/ml, which did not exhibit any hydrolyzing activity (Table 1).Our data strongly suggest that the presence of other factors, such as porin enzymatic deficiency and/or efflux mechanism may contribute in a multifactorial way to the carbapenem resistance.

Genetic location of the bla OXA-23 and bla OXA-58 genes
Previous studies showed that the CHDLs genes were found in several genetic contexts surrounded by different IS that are responsible for their spreading and regulation [3,15,17].
We searched for the presence of ISAba1, 2, 3, 4 and IS18 upstream and downstream of every CHDL gene identified in our isolates.PCR amplification with specific primers and sequence analysis revealed that ISAba1 was located upstream of all bla OXA-23 genes and upstream of a bla OXA-51 -type gene (AB316), the bla OXA-66 allele (GenBank nº EF051061).The contribution of ISAba1 upstream of the bla OXA-66 gene to the imipenem/meropenem resistance (CIM values of 16/8 µg/ml respectively) could not be evaluated since the AB316 isolate also harbours the bla OXA-23 gene.ISAba1 was previously described upstream of bla OXA-58 in one isolate from Turkey [3].Conversely, we did not find in any case ISAba1 upstream bla OXA-58 in our isolates.All isolates containing the bla OXA-58 gene showed ISAba3 downstream of the carbapenemase (n=14), and only 4 isolates (AB1400, AB1420, AB1525, and AB311) had a second copy of the ISAba3 upstream of the gene (GenBank nº DQ987830).Previous reports showed that the bla OXA-58 gene could be found embedded in various genetic platforms [3,15].Because none of the bla OXA genes were found adjacent to ISAba2, IS18 or ISAba4 in the AB isolates from our study, it can be presumed that other unknown structures might be in the boundaries of these CHDLs.

Discussion
AB is one of the most frequently gram-negative bacilli isolated from nosocomial infections in our country [18].Nowadays, over 80% of AB isolates are resistant to extended-spectrum cephalosporins and the imipenem resistance increased from 5% to 54% in the period 2000-2004 [18].In this regard, the differences observed in the molecular epidemiology of CHDLs in AB isolates detected in this study when compared to other reports [3,15,17] reveal the need of continuous molecular surveillance in order to prevent a higher dissemination of CRAB epidemic multiresistant clones emphasizing control of barrier precaution measures and antibiotic overuse.Moreover, the data provided at epidemiological and phenotypical level of AB isolates from our country [18], previous molecular studies [2,4,12,15,619,13,16,20] as well as the findings of the present work, suggest that treatment options should be redefined according to local epidemiology.

Table 2 .
Primers used in this study.