Dissemination and Genetic Structure of Carbapenemase Encoding Genes ( bla OXA-23 and bla OXA-24 ) in Acinetobacter baumannii from Southern Texas

Acinetobacter baumannii is one of the most important human pathogens causing a variety of nosocomial infections. Carbapenem antibiotics have been primarily used to treat the A. baumannii infections. However, carbapenem resistant A. baumannii producing carbapenemases causes serious treatment problems worldwide. Outbreaks of carbapenem resistant isolates have reported in some area of the United States, but their dissemination and genetic structure of the carbapenemase encoding genes are currently little known. To understand outbreaks, dissemination, and genetic structure of the carbapenemase encoding genes in Southern Texas, 32 clinical isolates collected from Austin and Houston, TX were characterized. Twenty-eight of 32 isolates were resistant to all tested β-lactam antibiotics including carbapenem (imipenem and meropenem). Three of them carried blaOXA-23 as a part of Tn2008 integrated into a known plasmid (pACICU2) and all others carried blaOXA-24 flanked by XerC/XerD-like recombinase binding sites that were adjoined by DNA sequences originated from multiple plasmids. Genotype analysis revealed that the 25 isolates carrying blaOXA-24 were all identical genotypes same as a representative isolate carrying blaOXA-24 from Chicago, IL but the 3 isolates carrying blaOXA-23 was a distinct genotype as compared with isolates carrying blaOXA-23 from Chicago, IL and Washington, D.C. Each of the blaOXA-23 and blaOXA-24 was transferred to carbapenem susceptible A. baumannii and E. coli with similar minimal inhibitory concentration (MIC) of carbapenem as that of their parental isolates but significantly lower levels of MIC in E. coli. Overall results suggest that a unique strain carrying blaOXA-23 and a similar strain carrying blaOXA-24 as seen in other geographic areas are currently disseminated in Southern Texas. Corresponding author.


Introduction
Acinetobacter baumannii is a gram-negative human pathogen characterizing strictly aerobic, non-pigmented, non-motile, non-fermenting, oxidase-negative, and catalase-positive coccobacillus [2].A major clinical impact of A. baumannii is hospital-acquired infections including ventilator-associated pneumonia, skin and soft-tissue infections, wound infections, urinary-tract infections, secondary meningitis, and bloodstream infections [2] [3].The treatment of this pathogen is very difficult due to its innate resistance to a number of commonly used antibiotics and an enormous capability to acquire resistance mechanisms to almost all commercially available antibiotics [4].
For the last decade A. baumannii has acquired resistance mechanisms to most of commercially available antibiotics.Carbapenem has been used to treat these multidrug resistant strains.However, carbapenem resistant clinical isolates of A. baumannii have been emerged and rapidly spread worldwide with serious treatment problem [5].Carbapenems (imipenem and meropenem) are broad-spectrum β-lactam antibiotics and the most important antimicrobial agents for treatment of multidrug resistant gram-negative pathogens including A. baumannii [6].Carbapenem resistant isolates of A. baumannii are usually resistant to all classes of β-lactams and also resistant to most of other classes of antibiotics [6].Carbapenem resistance in A. baumannii can be mediated by one or multiple mechanisms including enzymatic inactivation, active efflux, decreased membrane permeability, and modification of target sites.The most common carbapenem resistant mechanism is enzymatic inactivation by carbapenem-hydrolyzing β-lactamases (or carbapenemases) in clinical isolates of A. baumannii [2].
Outbreaks of carbapenem resistant A. baumannii have been increasingly reported and the carbapenem resistant strains rapidly spread among different cities of worldwide [9].In the United States, outbreaks of carbapenem resistant A. baumannii were first reported from New York City in 1994 [10].Study showed that carbapenem resistance of the isolates from New York City was caused by OXA-51-type carbapenemase encoding genes that were activated by insertion sequence (ISAba1) [11].Similar outbreaks of carbapenem resistant A. baumannii isolates associated with OXA-23-type and OXA-24-type were reported in Pittsburgh, PA and Chicago, IL [12] [13].Emergence of carbapenem resistant A. baumannii isolates associated with OXA-24-type and OXA-58type were also reported in Houston, TX as a part of SENTRY antimicrobial surveillance program [14].Carbapenem resistant A. baumannii isolates infected in U.S. military personnel combatting operations in Iraq, which were associated with OXA-23-type and OXA-58-type, were reported from the Walter Reed Army Medical Center, Washington, D.C. [15].
In this report, 32 clinical isolates of A. baumannii collected from Southern Texas (Austin and Houston) were characterized to understand outbreaks and dissemination of carbapenem resistant A. baumannii and genetic structure surrounding the carbapenemase encoding genes.Results revealed that carbapenem resistant A. baumannii isolates carried genes encoding OXA-23 (3 isolates) and OXA-24 (25 isolates).Genotype of the isolates carrying OXA-24 and genetic structure surrounding the bla OXA-24 were all identical and same as the isolate carrying bla OXA-24 from Chicago, IL.However, genotype of the isolates carrying OXA-23 and genetic structure surrounding the bla OXA-23 were distinct as compared with the isolates carrying bla OXA-23 from Chicago, IL and Washington, D.C.

Clinical Isolates and Culture Conditions
Thirty-two clinical isolates of A. baumannii were collected from Southern Texas (24 isolates from Clinical Bacteriology Laboratory Services Section, Texas Department of State Health Services, Austin, TX; 8 isolates from Pathology Department of Baylor College of Medicine, Houston, TX).All isolates were collected from individual patients and from August 2010 to July 2013.Two carbapenem resistant A. baumannii collected from Chicago, IL (C1: H26, OXA-23 positive; C2: AC1204, OXA-24 positive) and one carbapenem resistant A. baumannii AB0057 collected from Washington, D.C. (OXA-23 positive; a complete genome sequence strain) were obtained from Lolans et al. [12] and Adams et al. [16], respectively.A. baumannii ATCC 19606 was also included as a reference strain in this study.To ensure identity of A. baumannii, gyrB multiplex PCR was performed for each of the isolates as described [17] and their identities were confirmed by DNA sequence determination (data not shown).The clinical isolates were routinely grown in Luria-Bertani (LB) broth or LB agar plates.

Antibiotic Susceptibility Testing
All antibiotics used in this study were purchased from Sigma (Sigma, St. Louis, MO) except for meropenem (U S Pharmacopeia, Rockville, MD).Antibiotic susceptibility was determined by minimal inhibitory concentrations (MICs) using the broth dilution method as described [18].Each antibiotic was added to divalent cation-adjusted Mueller-Hinton (MH) (Oxoid, Ogdensburg, New York) broth (pH 7.0) to achieve serial two-fold dilutions between 0.25 and up to 128 µg/mL using sterile 17 × 100 mm snapped-cap Falcon culture tubes (1 mL/tube; Fisher Scientific).Fresh overnight cultures of each isolate were diluted in saline to an optical density at 600 nm of 0.1 to 0.12 (approximately 1 × 10 8 viable cells per mL).A portion of the adjusted cell suspension (3 μL for ~10 5 cells) was inoculated to each MH broth containing antibiotics as indicated.The cell cultures were then incubated overnight (16 to 18 hrs) at 37˚C without shaking.MIC was defined as the lowest concentration that completely inhibited growth of the inoculums.MIC values were confirmed by three independent experiments.Antibiotic resistant breakpoints were used as suggested by Clinical and Laboratory Standards Institute (CLSI) cited by Peleg et al. [2].

Detection of Genes Encoding Carbapenemases and an Insertion Sequence Element (ISAba1)
Presence of genes encoding carbapenemases known as Ambler Class A β-lactamase (bla KPC2 ), Ambler Class B metallo β-lactamase (bla IMP , bla GIM , bla SIM , bla SPM , and bla VIM ), and Ambler Class D β-lactamase or oxacillinase (bla OXA-23-type , bla OXA-24-type , bla OXA-51-type , and bla OXA-58-type ) was examined by multiplex PCR methods as described [1] [19].The presence of an insertion sequence element (ISAba1) and its location in the upstream of bla OXA-51-type was also examined by PCR methods as described [8].All PCR amplified fragments from clinical isolates were used to determine DNA sequences of both strands from commercial DNA sequencing services (GENEWIZ, Inc., South Plainfield, NJ).

Gene Cloning Experiment
Genomic DNA from A. baumannii isolates A21 (bla OXA-23 positive) and H1 (bla OXA-24 positive) was extracted by Qiagen DNeasy kit (Valencia, CA).The genomic DNA was completely digested by BamHI or EcoRI and ligated to pGH240 (a bla gene of pBluescriptSK+ [Stratagene, La Jolla] was destroyed by inserting a chloramphenicol resistant gene cassette [20] into ScaI site) digested by BamHI or EcoRI, respectively.E. coli DH5α was then transformed with the ligation mixture by a standard chemical transformation method.Transformed E. coli DH5α carrying bla OXA-23 or bla OXA-24 was selected on LB agar plates containing chloramphenicol (15 μg/mL) and Ampicillin (100 μg/mL).Insert DNA fragments from the recombinant plasmids were confirmed by BamHI or EcoRI digestion and used to determine DNA sequences of both strands from commercial DNA sequencing services (GENEWIZ, Inc., South Plainfield, NJ).

Construction of Rifampicin Resistant A. baumannii ATCC 19606 and Transfer of Carbapenem Resistance
Carbapenem susceptible A. baumannii ATCC 19606 grown in LB broth was concentrated at 10 10 cells and spread on LB agar plates containing 50 μg/mL of rifampicin for 24 hours and one of colonies grown on the agar plates was used to grow in LB broth containing 50 μg/mL of rifampicin for 24 hours.The cells grown in LB broth containing 50 μg/mL of rifampicin were spread on agar plates containing 100 μg/mL of rifampicin.One of the colonies grown on the agar plates containing 100 μg/mL of rifampicin was subcultured on LB agar plates without any antibiotic and passaged three times on the same LB agar plates.The cells on the third passage was used to confirm stability of rifampicin resistance (MIC > 100 μg/mL) and used for a recipient strain.Two carbapenem resistant clinical isolates (A21 for OXA-23 positiveand H1 for OXA-24 positive) were confirmed as rifampicin susceptible (MIC 0.5 μg/mL) and used as donor strains for conjugative transfer of carbapenem resistance.Mating conditions for the donor and recipient strains were essentially identical as described previously [21] except for an extended-mating period (16 to 18 hours).The mating mixtures were spread on LB agar plates containing meropenem (5 μg/mL) or carbenicillin (100 μg/mL) and rifampicin (100 μg/mL).Individual transconjugants (3 colonies) were used for phenotype analysis and genomic DNA extraction.

Transformation of A. baumannii
Electroporation for transformation of A. baumannii ATCC 19606 was performed as described [22].Briefly, electrocompetent cells were prepared by washing the cells (OD 600 of 0.8) 3 times with 10% (v/v) glycerol and concentrating the cells at 2 × 10 10 cells per mL of 10% (v/v) glycerol.Plasmid DNA (up to 3 μg in 40 μL) extracted by Qiagen kit (Valencia, CA) mixed with the electrocompetent cells and placed in a pre-chilled sterile electroporation cuvette (2 mm electrode gap, Bio-Rad) following immediately pulse by using a Bio-Rad Gene Pulser (2.5 kV, 200 Ω, and 25 μF).The mixture was incubated at 37˚C for 1 hr with 1 mL of LB broth and spread the cells on agar plate containing carbenicillin (100 μg/mL) or meropenem (5 μg/mL).Natural transformation of carbapenem susceptible A. baumannii ATCC 19606 was also alternatively performed as described [23].Briefly, 100 ng of plasmid DNA was added in a mixture containing 50 μL of fresh LB broth and 50 μL of culture in a stationary phase, followed by incubation at 37˚C for 1 hour and plating on LB agar plates containing carbenicillin (100 μg/mL) or meropenem (5 μg/mL).As a control for the natural competency of A. baumannii ATCC 19606, kanamycin resistant plasmid (pJHCMW1) was used for the natural transformation by selection on LB agar plates containing kanamycin (25 μg/mL) [23] [24].

Antibiotic Susceptibility of Clinical Isolates of A. baumannii
MIC levels to multiple antibiotics were determined for the 32 clinical isolates collected from Austin and Houston, TX including 3 representative carbapenem resistant clinical isolates from Chicago, IL, Washington, D.C., and the reference A. baumannii ATCC 19606.Results revealed that 28 isolates (87%) were resistant to all tested β-lactams including carbapenem (imipenem and meropenem).Four isolates were resistant to polymyxin B (MIC ≥ 8 μg/mL) and 3 of them were resistance to all tested antibiotics including polymyxin B (Table 1).The 3 isolates from Chicago, IL and Washington, D.C. were also resistant to all tested antibiotics except for polymyxin B. The reference strain ATCC 19606 was susceptible to all tested antibiotics (Table 1).
Presence of the insertion sequence element (ISAba1) at upstream of the gene encoding OXA-51-type requires for production of significant carbapenem resistance in A. baumannii [8].To understand involvement of the OXA-51-type in carbapenem resistance, presence andlocation, if any, of the ISAba1 was examined from the isolates as described [8].Results revealed that the ISAba1 was detected in all isolates except for 8 isolates from Houston, TX.However, none of them was preceded to the gene encoding OXA-51-type.

Genotype Analysis of Clinical Isolates Carrying Carbapenemase Encoding genes
To understand dissemination of carbapenemase encoding genes among the clinical isolates, two PCR-based genotype methods (REP-PCR and RAPD-PCR) proved as similar discrimination power as a pulse field gel electrophoresis (PFGE) method [25] were used.REP-PCR profiles for the 3 isolates carrying OXA-23-type from Austin, TX (A6, A15, A21) showed an identical pattern (Figure 1, lane 3) which was different from that of isolate carrying OXA-23-type from Washington, D.C. or from Chicago, IL (Figure 1, lane 1 and 2).REP-PCR profiles for all isolates carrying OXA-24-type from Austin and Houston, TX and Chicago, IL showed an identical pattern (Figure 1, lanes 4 and 5); interestingly, this genotype pattern was very similar as that of the isolate carrying OXA-23-type from Chicago, IL (Figure 1, lane 2).
RAPD-PCR profiles for the 3 isolates carrying OXA-23-type from Austin, TX showed an identical pattern (Figure 1, lane 8) which was different from that of isolate carrying OXA-23-type from Washington, D.C. or from Chicago, IL (Figure 1, lanes 6 and 7).As shown in REP-PCR profiles, RAPD-PCR profiles for all isolates carrying OXA-24-type from Austin and Houston, TX and Chicago, IL showed an identical pattern (Figure 1, lanes 9 and 10).The two genotype methods produced repeatedly consistent results, suggesting that a unique isolate carrying OXA-23-type is currently propagated in Austin, TX but the isolate carrying OXA-24-type in Austin and Houston, TX may be clonally disseminated from isolates carrying OXA-24-type in Chicago, IL or vice versa.
EcoRI fragment (6285-bp) from total DNA of the clinical isolate H1 conferred resistance to ampicillin (100 μg/mL) in E. coli DH5α and the insert DNA was used for DNA sequencing analysis.As shown in Figure 2(b), the gene encoding OXA-24 was flanked by XerC/XerD-like recombinases binding sites and surrounded by DNA sequences originated from multiple plasmids.Deduced amino acid sequence of the gene was 100% identical as previously reported OXA-24 (GenBank accession number GU199040) [13].A newly designed forward PCR primer for upstream 500-bp from the start codon of OXA-24 (5'-AGGTGTGCATCATACTTCTA-3') and a reverse primer for downstream 500-bp from the stop codon of OXA-24 (5'-TAAAGAATCAGATCGATTTT-3') were used to amplify a full length of bla OXA-24 from all the other OXA-24 positive isolates (24 isolates).DNA sequence analysis showed that all PCR fragments were 100% identical as that from the isolates H1.
Results revealed that countless transconjugants from each conjugation mating mixture grew on LB agar plates containing carbenicillin (100 μg/mL) or meropenem (5 μg/mL) and rifampicin (100 μg/mL) suggesting carbapenem resistance can be transferrable and may be mediated by a plasmid-specific gene.
To test whether the plasmid-specific gene is associated with carbapenem resistance plasmid DNA extracted from the clinical isolates (A21 and H1) was used for transformation of the carbapenem susceptible ATCC 19606 with selection on LB agar plates containing carbenicillin (100 μg/mL) or meropenem (5 μg/mL).Results revealed that each plasmid DNA from the carbapenem resistant clinical isolates produced countless transformants on the agar plates.Presence of the genes encoding OXA-23 and OXA-24 in the transconjugants and transformants (3 colonies for each) was confirmed by PCR methods as done above.

Expression of Carbapenemase Encoding Genes in A. baumannii and E. coli
To understand roles of the carbapenemase encoding genes in β-lactam susceptibility the transconjugants of rifampicin resistant A. baumannii ATCC 19606 and the transformants of E. coli DH5α carrying bla OXA-23 and bla OXA-24 were used to measure β-lactam susceptibility.As shown in Table 3, MIC levels to carbapenem (imipenem and meropenem) and penicillin (carbenicillin) were significantly increased (up to 64-fold) in both A. baumannii ATCC 19606 and E. coli DH5α for both bla OXA-23 and bla OXA-24 .However, MIC levels to monobactam (aztreonam) and cephalosporin (ceftazidime) were slightly increased or no difference in both A. baumannii ATCC 19606 and E. coli DH5αfor both bla OXA-23 and bla OXA-24 .These results suggest that the transconjugants of A. baumannii ATCC 19606 are similar MIC levels for both bla OXA-23 and bla OXA-24 as that of their clinical isolates while the transformants of E. coli DH5α are significantly lower MIC levels for both genes than that of their clinical isolates.

Discussion
Outbreaks of carbapenem associated multidrug resistant A. baumannii may be the most problematic issue to treat the infected patients since the only viable therapeutic option for this infection is polymyxins [9], but polymyxins cause neuro-and nephro-toxicity [27] and polymyxin selection pressure can readily develop polymyxin resistant A. baumannii [28] [29].In this study, most of the isolates showed carbapenem associated multidrug resistance, which was similar as other areas of the United States and worldwide [9] [11] [30], and more sadly some of them conferred intermediate levels of polymyxin resistance.Outbreaks of polymyxin associated carbapenem resistant A. baumannii should alert in this area.
Carbapenem resistance of the isolates in Southern Texas was associated with bla OXA-23 and bla OXA-24 .The carbapenem resistant isolates reported previously in Houston, TX carried bla OXA-24 and bla OXA-58 [14].However, the bla OXA-58 was not detected in this study; instead, bla OXA-23 was newly detected in the isolates from Austin, TX.In the United States, all known OXA-type carbapenemase encoding genes are currently associated with carbapenem resistance in clinical isolates.Carbapenem resistant isolates associated with OXA-23-type, OXA-  [31].The carbapenem resistant isolates from New York City harbored genes encoding OXA-51-type which were fused by ISAba1 providing promoter function for the genes [11].OXA-51-type carbapenemase is intrinsic to all A. baumannii and usually not associated with carbapenem resistance unless authentic expression of the carbapenemase [2].All isolates carrying bla OXA-24 including the representative isolate carrying bla OXA-24 from Chicago, IL were identical genotype.The isolate carrying bla OXA-24 (or bla OXA-40 ) from Chicago, IL was one of predominantly (97%) circulating isolates [12].Therefore, this result suggests that a similar genotype strain carrying bla OXA-24 is currently disseminating in Chicago, IL and Southern Texas.Interestingly, this genotype isolate carrying bla OXA-24 was very similar genotype as that of the isolate carrying bla OXA-23 from Chicago, IL, suggesting the similar genotype isolate may carry two different types of carbapenemase encoding genes in Chicago, IL.It is consistent to the note that a limited number of genotypic clusters of carbapenem associated multidrug resistant isolates of A. baumannii are successfully spread among hospitals of different cities and countries [9].However, genotype of the isolates carrying bla OXA-23 in Southern Texas was unmatched to any of the isolates from Chicago, IL and Washington, D.C., indicating that a distinct genotype strain was currently propagated in Southern Texas.The bla OXA-23 in Southern Texas was carried on Tn2008 while the same genes from Washington, D.C. and Chicago, IL were carried on Tn2006 and others.This observation suggests that the different vehicles for the carbapenemase encoding genes may be delivered by different genotype strains.The bla OXA-23 carried on Tn2008 was reported in clinical isolates from Pittsburgh, PA [26]; thus, it is interesting to test whether the isolate carrying bla OXA-23 from Pittsburgh, PA has a similar genotype as the isolate carrying bla OXA-23 from Southern Texas.
The genetic structures surrounding the genes of bla OXA-23 and bla OXA-24 were reported in clinical isolates from Pittsburgh, PA [13] [26].In this study, genetic structure surrounding the bla OXA-23 was essentially identical as that from Pittsburgh, PA, characterizing that the gene was carried on Tn2008 integrated into plasmid pACICU2 [26] [32].However, genetic structure surrounding the bla OXA-24 from Southern Texas was different from that of Pittsburgh, PA.The bla OXA-24 from Southern Texas, which was flanked by XerC/XerD-like recombinase binding site, was surrounded by A. venetianus plasmid pAV2 [33] and A. baumannii plasmids p1ABAYE and p2ABSDF [34] while the bla OXA-24 from Pittsburgh, PA was surrounded by A. venetianus plasmid pAV2 [13] [33].XerC/ XerD-like DNA sequences are recognized by recombinase and adjoining by different DNA modules in different Acinetobacter plasmids [35], suggesting that XerC/XerD-mediated recombination events may direct multiple genetic motifs from multiple plasmids to be surrounded by a specific gene (or locus) as shown in this case of bla OXA-24 from Southern Texas.
The genes of bla OXA-23 and bla OXA-24 have been identified in chromosome and/or plasmid [2].Two copies of bla OXA-23 were also identified in both chromosome and plasmid in a single isolate of A. baumannii [36].Theoretically, even more than two copies of genes for bla OXA-23 or bla OXA-24 would be also possible since the vehicles for the genes are usually large sizes of conjugative plasmids that can be integrated (or excised) into (or out) chromosome and, if the genes are carried on transposons, they would be also transposed to other incompatibility group of plasmids in a single strain.In this report, we confirmed that both bla OXA-23 and bla OXA-24 from the clinical isolates were transferrable to the carbapenem susceptible A. baumannii ATCC 19606, indicating that at least one copy of each gene was carried on a plasmid DNA, which was also supported by the genetic structure for the genes that were surrounded by DNA sequences from plasmids as mentioned above.Both genes of bla OXA-23 and bla OXA-24 on the plasmid DNA were expressed in the recipient A. baumannii at the similar MIC levels as their parental isolates.However, expression of the same genes cloned into a commercial plasmid vector in E. coli DH5α was significantly lower MIC levels than that of their parental clinical isolates of A. baumannii.Similar results were also observed in other studies [13] [37], suggesting that expression of the bla OXA-23 and bla OXA-24 may be hindered at the level of transcription and/or translation in E. coli.

Table 1 .
Antibiotic susceptibility of clinical isolates of A. baumannii.
A gene encoding OXA-24-type was detected in 26 of the 36 isolates (17 from Austin, TX, 8 from Houston, TX, and a positive control from Chicago, IL) (Table2).None of the other carbapenemase encoding gene was detected from any isolate in this study.

Table 2 .
Detection of carbapenemase-encoding genes in clinical isolates of A. baumannii.