Genetic Basis of Multidrug Resistance in Acinetobacter Clinical Isolates in Taiwan

Bacterial strains and plasmids.A total of 75 nonduplicated Acinetobacter clinical isolates were collected from the National Taiwan University Hospital (Taipei, Taiwan) in 2006 and were identified by the API 20NE system. Genospecies were identified according to the 16S-23S rRNA gene intergenic spacer (ITS) region as described previously (20). Escherichia coli TOP10 and A. baumannii ATCC 15151 (CIP 70.10) were used as hosts for the cloning and expression experiments, respectively. Plasmid pCR2.1-TOPO (Invitrogen) was used as the cloning vector, and shuttle plasmid pAT801, with a PvuII fragment harboring the oriC of Acinetobacter from pW1277 (15) in pUc18, was obtained from P. Courvalin of Institut Pasteur and was used as the expression vector. E. coli and A. baumannii were cultured on Luria-Bertani broth (LB) and tryptic soy broth (TSB) plates, respectively, at 37°C for 20 h.

Antibiotic susceptibility testing.Antibiotic susceptibility testing was performed using the Vitek 2 automatic system (bioMérieux), with the AST-GN09 card for the identification of Gram-negative bacilli. The MIC of imipenem was further determined by Etest (AB Biodisk, Solna, Sweden). The criteria used were in accordance with the guidelines established by the Clinical and Laboratory Standards Institute (CLSI) (4).

Detection of antibiotic resistance genes.Genomic DNA was extracted as described previously (20). All primers used in this study are listed in Table 1. The QRDR of gyrA was amplified and sequenced by procedures similar to those used in our previous study (36). The gyrA QRDR was defined as gyrA nucleotide positions 241 to 243 in Acinetobacter spp., and it is equivalent to Ser83 of E. coli. Integrons were amplified and sequenced with primers derived from the 5′ and 3′ conserved segments (18), and additional internal primers were designed to ensure complete identification. The carbapenem resistance genes, including bla_IMP, bla_VIM, bla_OXA-23, bla_OXA-24-like, bla_OXA-51-like, and bla_OXA-58, were detected by using primers and PCR conditions described previously (29, 41). The presence of the ISAba1 element was detected by PCR in order to determine the proximity of this element to bla_OXA-23, bla_OXA-51-like, and bla_AmpC (5, 12, 39). The insertion sequence preceding the bla_OXA-58 gene was detected by PCR using a reverse primer of the bla gene and a forward primer of the IS (ISAba1, ISAba2, or ISAba3) (31).

Cloning of ISAba1-bla_OXA-23 and ISAba1-bla_OXA-66.For cloning and verification of the nucleotide sequences of ISAba1-bla_OXA-23 and ISAba1-bla_OXA-51-like, primers ISAba1-F and OXA23-R1 were used to amplify ISAba1-bla_OXA-23 from strain 6AB15, and primers ISAba1-F and OXA51-R1 were used to amplify ISAba1-bla_OXA-51-like from strain 6AB11. The PCR conditions for ISAba1-bla_OXA-23 and ISAba1-bla_OXA-51-like were as follows: preheating at 95°C for 2 min; 35 cycles consisting of 95°C for 10 s, 50°C for 10 s, and 72°C for 130 s; and a final extension at 72°C for 60 s. The two PCR products were separately ligated into the pCR2.1-TOPO vector, generating pCR2.1-OXA23 and pCR2.1-OXA51. The inserts of pCR2.1-OXA23 and pCR2.1-OXA51 were sequenced with an Applied Biosystems sequencer (ABI 3730). The nucleotide and deduced protein sequences were analyzed with software available on the website of the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov ).

Expression of bla_OXA-23 and bla_OXA-66.The sequence analysis identified bla_OXA-51-like from 6AB11 as bla_OXA-66. Fragments containing ISAba1-bla_OXA-23 and ISAba1-bla_OXA-66 were obtained from pCR2.1-OXA23 and pCR2.1-OXA51, respectively, by digestion with BamHI/EcoRI. BamHI/EcoRI-restricted ISAba1-bla_OXA-23 and BamHI/EcoRI-restricted ISAba1-bla_OXA-66 were ligated into BamHI/EcoRI-restricted pAT801, generating pOXA23 and pOXA66, respectively. pOXA23 and pOXA66 were separately transformed into A. baumannii ATCC 15151 by electroporation. The transformants were selected on LB plates containing 100 mg/liter ampicillin.

SDS-PAGE analysis.Bacterial cells were grown overnight at 37°C in LB, harvested by centrifugation at 8,000 × g for 5 min, and washed once with iced phosphate-buffered saline (PBS). The cells were resuspended in fresh PBS with 1 mM dithiothreitol (DTT) and 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and were disrupted by sonication. The crude cell extracts were clarified by centrifugation at 12,000 × g for 10 min at 4°C, and the supernatants were collected. The protein concentration of the crude cell extract was determined using a commercial bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL) with bovine serum albumin (BSA) (0.05 to 2 mg/ml) as the standard. The crude cell extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 4- to-20% gradient polyacrylamide gel, and protein patterns were detected by staining with Coomassie blue.

Statistical analysis.Statistical analysis was performed using SPSS, version 10.0.7. Pearson's correlation coefficient was used to determine the relationships between the MIC distributions of antibiotic agents and antibiotic resistance genes. An r value of >0.7 (or <−0.7) and a P value of <0.05 were considered statistically significant.

Nucleotide sequence accession numbers.The nucleotide sequences of ISAba1-bla_OXA-23 and ISAba1-bla_OXA-66, reported in this paper, have been submitted to NCBI GenBank under accession numbers GQ849192 and GQ849191, respectively.

Fluoroquinolones.Eighty-five percent of A. baumannii isolates (45/53) were resistant to fluoroquinolones, and all of these contained a Ser83Leu mutation in GyrA, except for one ciprofloxacin-resistant isolate with Ser83 in GyrA that was susceptible to levofloxacin. Only 55% (24/44) of the A. baumannii isolates described above with a Ser83Leu mutation in GyrA were also resistant to levofloxacin, and 43% (19/44) were intermediate to levofloxacin. Most of the Acinetobacter genospecies 13TU isolates (90% [18/20]) were susceptible to ciprofloxacin, with Ser83 in GyrA. Two Acinetobacter genospecies 13TU isolates were resistant to ciprofloxacin; one had a Ser83Leu mutation and the other had a Ser83Phe mutation in GyrA, and the isolate with Ser83Leu in GyrA was also resistant to levofloxacin. Of the two Acinetobacter genospecies 3 isolates, the one with Ser83Leu in GyrA was resistant to ciprofloxacin and levofloxacin. The GyrA mutations of Acinetobacter spp. were significantly correlated with susceptibility to ciprofloxacin (r, 0.972; P, <0.001) and levofloxacin (r, 0.847; P, <0.001).

Aminoglycosides.Among the 75 Acinetobacter isolates, six distinct integrons were found, and their gene cassettes contained various aminoglycoside-modifying genes, including aacA4, aacC1, aac(6′)-II, aadA1, aadA2, and aadA4. Three integrons were detected in A. baumannii, including aacA4-catB8-aadA1 (2,381 bp), aacC1-orfX-orfX′-aadA1 (2,542 bp), and dhrfXII-orfF-aadA2 (1,873 bp), while three other integrons, including arr-3-aacA4 (1,395 bp), bla_VIM-11 (1,062 bp), and bla_IMP-1-aac(6′)-II-aadA4 (2,507 bp), were detected only in Acinetobacter genospecies 13TU. Twenty-eight percent (15/53) of A. baumannii isolates were resistant to amikacin and contained integrons. Furthermore, 81% (43/53) and 75% (40/53) of the A. baumannii isolates were resistant to gentamicin and tobramycin, respectively, and most of them contained integrons. In Acinetobacter genospecies 13TU, five isolates contained integrons, but some of them remained susceptible to aminoglycosides. No integrons were detected in the two Acinetobacter genospecies 3 isolates, and they were also susceptible to the three aminoglycosides analyzed. Consequently, the presence of integrons in Acinetobacter spp. was significantly correlated with susceptibility to gentamicin (r, 0.8; P, <0.001) and tobramycin (r, 0.841; P, <0.001) but not to amikacin (r, 0.403; P, <0.001).

Cephalosporins.Eighty-three percent (44/53) of A. baumannii isolates were resistant to ceftazidime, and all of them contained the ISAba1-bla_AmpC structure. The eight A. baumannii isolates lacking ISAba1 upstream of bla_AmpC were susceptible to ceftazidime, and the sole isolate intermediate to ceftazidime did not contain bla_AmpC. Seventy-nine percent (42/53) of A. baumannii isolates were resistant to cefepime, and they contained the ISAba1-bla_AmpC structure. In Acinetobacter genospecies 13TU, seven isolates were resistant or intermediate to ceftazidime, and two of these contained the ISAba1-bla_AmpC structure. Furthermore, these isolates were also not susceptible to cefepime and one ceftazidime-intermediate isolate harboring the ISAba1-bla_AmpC structure showed resistance to cefepime. In addition, one Acinetobacter genospecies 3 isolates without bla_AmpC was intermediate to ceftazidime and resistant to cefepime. The presence of ISAba1 upstream of bla_AmpC in Acinetobacter spp. was significantly correlated with susceptibility to ceftazidime (r, 0.861; P, <0.001) and cefepime (r, 0.725; P, <0.001).

Carbapenems.Forty-five percent (24/53) of A. baumannii isolates were resistant or intermediate to imipenem and meropenem; most (88% [21/24]) of them contained an ISAba1-bla_OXA-51-like structure. Among the remaining isolates without ISAba1 upstream of bla_OXA-51-like, one isolate containing ISAba1-bla_OXA-23 and one containing bla_OXA-24-like were resistant to carbapenems, while one isolate, intermediate to carbapenems, had no detectable carbapenemase genes. The presence of ISAba1 upstream of bla_OXA-51-like in A. baumannii was highly correlated with susceptibility to imipenem (r, 0.753; P, <0.001) and meropenem (r, 0.764; P, <0.001). In addition, among the five Acinetobacter genospecies 13TU isolates harboring ISAba3-bla_OXA-58, one isolate containing only ISAba3-bla_OXA-58 was susceptible to carbapenems, while of the other four isolates, containing additional carbapenemase genes, two isolates with bla_IMP-1 were resistant to carbapenems, one isolate with ISAba1-bla_OXA-23 was intermediate to carbapenems, and one isolate with bla_VIM-11 was intermediate to imipenem but susceptible to meropenem. Furthermore, all of the five Acinetobacter genospecies 13TU isolates harboring ISAba3-bla_OXA-58 were also resistant to ceftazidime and cefepime, and only one of them, harboring bla_IMP-1, contained ISAba1-bla_AmpC. A total of 15 Acinetobacter genospecies 13TU and 2 Acinetobacter genospecies 3 isolates harbored no carbapenem resistance genes that we detected, and all of them were susceptible to carbapenems.

Cloning of ISAba1-bla_OXA-23 and ISAba1-bla_OXA-66.DNA sequence analysis indicated that the insert of pCR2.1-OXA23, with a 2,036-bp fragment size, was 99% identical to ISAba1-bla_OXA-23 in A. baumannii (GenBank accession number EF127491) and the insert of pCR2.1-OXA51 revealed a 2,012-bp fragment 100% identical to ISAba1-bla_OXA-66 in A. baumannii (GenBank accession number DQ923479).

Expression of bla_OXA-23 and bla_OXA-66 in A. baumannii ATCC 15151.The crude cell extracts of two transformants, A. baumannii ATCC 15151(pOXA23) and A. baumannii ATCC 15151(pOXA66), were analyzed by SDS-PAGE (Fig. 1). The protein patterns of clinical isolates 6AB11 and 6AB15 (Fig. 1, lanes 1 and 2) were significantly different from that of A. baumannii ATCC 15151 (lane 3), while the protein pattern of the A. baumannii transformant ATCC 15151(pAT801) (lane 4) was identical to that of A. baumannii ATCC 15151. Furthermore, the protein patterns of the two transformants A. baumannii ATCC 15151(pOXA23) and A. baumannii ATCC 15151(pOXA66) were very similar to that of A. baumannii ATCC 15151(pAT801) except for the conspicuous expression of two proteins of 30 and 32 kDa (Fig. 1, lanes 5 and 6).

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FIG. 1.

Expression of bla_OXA-23 and bla_OXA-66 from A. baumannii ATCC 15151. Lane 1, A. baumannii 6AB11 (ISAba1-bla_OXA-66); lane 2, A. baumannii 6AB15 (ISAba1-bla_OXA-23 and bla_OXA-66); lane 3, A. baumannii ATCC 15151; lane 4, A. baumannii ATCC 15151(pAT801); lane 5, A. baumannii ATCC 15151(pOXA23); lane 6, A. baumannii ATCC 15151(pOXA66). The arrows in lanes 5 and 6 indicate the increased expression of 30-kDa and 32-kDa proteins in two transformants. The molecular masses of the size markers (lane M) are given on the left.

Antibiotic susceptibilities of two transformants.We chose the wild-type A. baumannii strain ATCC 15151 as the host for investigation of the possible roles of ISAba1-bla_OXA-23 and ISAba1-bla_OXA-66. The susceptibilities of the isolates to β-lactams are shown in Table 4. Clinical strains 6AB11 and 6AB15 were resistant to all antibiotics tested. A. baumannii ATCC 15151 was susceptible to most of the antibiotic agents, and only the MICs of ampicillin, ampicillin-sulbactam, and piperacillin were increased for the transformant A. baumannii ATCC 15151(pAT801), harboring the AmpR-selectable marker from pAT801. The imipenem and meropenem MICs for transformants A. baumannii ATCC 15151(pOXA23) and ATCC 15151(pOXA66) were significantly increased (64-fold) over that for A. baumannii ATCC 15151(pAT801). Furthermore, the piperacillin-tazobactam MICs for those two transformants were increased 4- to 32-fold over that for A. baumannii ATCC 15151(pAT801). Only for ATCC 15151(pOXA23) was the cefepime MIC increased (16-fold) over that for the reference strain. ATCC 15151(pOXA23) and ATCC 15151(pOXA66) were susceptible to ciprofloxacin, levofloxacin, amikacin, gentamicin, and tobramycin (data not shown).