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Abstract
Acinetobacter species are non-fermentative Gram-negative coccobacilli and they have emerged as important nosocomial pathogens which are associated with the significant multidrug resistance in recent years. Carbapenem-resistant A. baumannii (CRAB) and pandrug-resistant A. baumannii (PDRAB) were reported in 1991 and 1998, respectively. Fifty-eight isolates of Acinetobacter species recovered from a university hospital between August 2004 and March 2005 were investigated for the existence of CRAB, PDRAB, extended-spectrum β-lactamase (ESBL)-producing Acinetobacter and examined for their phenotypic and genotypic characteristics. Genomospecies of Acinetobacter species were determined by amplified rDNA restriction analysis (ARDRA) and antimicrobial susceptibility test was performed with 13 kinds of antimicrobial agents. Metallo-β-lactamase (MBL) producers were screened by modified hodge test and confirmed by imipenem-EDTA disk synergy test. Detection of blaIMP-1, blaVIM-2, blaTEM, and blaPER-1 was performed by PCR. Genomic DNAs were analyzed by pulsed-field gel electrophoresis (PFGE). Among 58 isolates of Acinteobacter species, 40 isolates were identified as genospecies 2 (A. baumannii), 9 were 13TU, 5 were A. phenon 6/ct, and 4 were Acinetobacter genospecies 3 by ARDRA. Thirteen isolates were confirmed as MBL-producers and blaIMP-1 and blaVIM-2 were carried by 5 and 8 isolates of them, respectively. MBL-producers were mostly 13TU, A. phenon 6/ct 13TU, and Acinetobacter genospecies 3 and they were susceptible to ciprofloxacin and ampicillin-sulbactam. BlaPER-1 was carried by thirteen isolates and 12 isolates of them were PDRAB showing resistance to all antimicrobial agents tested, including ceftazidime, cefepime, aztreonam, ciprofloxacin, amikacin, gentamicin, ampicillin-sulbactam, and imipenem. In conclusion, most MBL-producers belonged to 13TU, A. phenon 6/ct 13TU, and Acinetobacter genospecies 3 which were susceptible to ciprofloxacin and ampicillin-sulbactam, whereas 12 of 13 PER-1-producers were PDRAB originated from the same clone.
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Figure 1.
Restriction patterns obtained after restriction digestion with CfoI, AluI, MboI, RsaI, and MspI for amplified 16S rDNA of different Acinetobacter species. Numbers below each lane correspond to arbitrarily assigned ARDRA pattern numbers for each enzyme (http://users.ugent.be/∼mvaneech/ARDRA/Acinetobacter.html).
Figure 2.
Dendrogram generated by Gel Compar II software showing the relatedness of Acinetobacter baumannii determined by PFGE.
Table 1.
Oligonucletide primers used for detection of β-lactamase genes
| Primers | Tma (°C) | Nucleotide sequence | References | Expected amplicon size (bp) |
|---|---|---|---|---|
| TEM-sence | 5′-ATA AAA TTC TTG AAG ACG AAA-3′ | |||
| TEM-antisence | 50 | 5′-GAC AGT TAC CAA TGC TTA ATC-3′ | AB103506 | 1080 |
| SHV-sence | 5′-TGG TTA TGC GTT ATA TTC GCC-3′ | |||
| SHV-antisence | 55 | 5′-GGT TAG CGT TGC CAG TGC T-3′ | AY223863 | 869 |
| CTX-M-sence | 5′-CGC TTT ATG CGC AGA CGA-3′ | |||
| CTX-M-antisence | 55 | 5′-GAT TCT CGC CGC TGA AGC-3′ | AY847147 | 785 |
| OXA-1-sence' | 5′-AGC CGT TAA AAT TAA GCC C-3′ | |||
| OXA-1-antisence | 55 | 5′-CTT GAT TGA AGG GTT GGG CG-3′ | AV162283 | 908 |
| PER-1-sence | 5′-ATG AAT GTC ATT ATA AAA GC-3′ | |||
| PER-1-antisence | 50 | 5′-AAT TTG GGC TTA GGG CAA GAA A-3′ | Z21957 | 925 |
| VIM-2-sence | 5′-ATT GGT CTA TTT GAC CGC GTC-3′ | |||
| VIM-2-antisence | 54 | 5′-TGC TAC TCA ACG ACT GAC CG-3′ | DQ153217 | 780 |
| IMP-1-sence | 5′-CTA CCG CAG CAG ACT CTT TGC-3′ | |||
| IMP-1-antisence | 55 | 5′-GAA CAA CCA GTT TTG CCT TAC C-3′ | AB162949 | 591 |
Table 2.
ARDRA profiles of Acinetobacter species
| Genomic species | Pattern with enzymea | No. of isolates | Reference strain | ||||
|---|---|---|---|---|---|---|---|
| CfoI | AluI | MboI | RsaI | MspI | |||
| 2 (A. baumannii) | 1 | 1 | 1 | 2 | 3 | 39 | ATCC 17904 |
| 1 | 1 | 1 | 2 | 1 | 1 | ATCC 19606 | |
| 3 | 2 | 1 | 3 | 1 | 3 | 5 | ATCC 19004 |
| 13TU | 2 | 1 | 1 | 1 | 1 | 3 | ATCC 17903 |
| 2 | 1 | 1 | 1 | 3 | 4 | 100 | |
| 2 | 1 | 1 | 1 | 1+3 | 2 | RUH 2624 | |
| A. phenon 6/ct 3TU | 3 | 1 | 3 | 1 | 3 | 4 | MGH 99896, 5804 |
Table 3.
Antimicrobial resistance for clinical isolates of Acinetobacter species
| Antibiotics | No. of resistant strains | Total (n= 58) | |||
|---|---|---|---|---|---|
| A. baumannii (n=40) | 13TU (n=9) | A. phenon 6/ct 13TU (n=5) | Acinetobacter genospecies 3 (n=4) | ||
| piperacillin | 38 | 4 | 4 | 3 | 49 (84.5%) |
| ticarcillin | 38 | 4 | 2 | 2 | 46 (79.3%) |
| amikacin | 39 | 7 | 2 | 3 | 51 (87.9%) |
| gentamicin | 40 | 8 | 5 | 3 | 56 (96.6%) |
| tobramycin | 40 | 7 | 5 | 3 | 55 (94.8%) |
| ciprofloxacin | 39 | 1 | 0 | 0 | 40 (69.0%) |
| tazobactam | 35 | 2 | 1 | 2 | 40 (69.0%) |
| ceftazidime | 39 | 7 | 0 | 3 | 49 (84.5%) |
| cefepime | 35 | 3 | 0 | 3 | 41 (70.7%) |
| aztreonam | 36 | 5 | 0 | 2 | 43 (74.1%) |
| imipenem | 13 | 5 | 5 | 2 | 25 (43.0%) |
| meropenem | 14 | 4 | 1 | 2 | 21 (36.2%) |
| amp/sula | 38 | 2 | 0 | 0 | 40 (69.0%) |
Table 4.
Antimicrobial susceptibility to imipenem, modified Hodge test, and detection of metallo-β-lactamases and β-lactamases among clinical isolates of Acinetobacter spp.
Table 5.
Antimicrobial susceptibilities of β-lactamase or metallo-β-lactamase producing Acinetobacter species
| Antibiotics | MICa (μg/ml) for isolates | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| blaPER-1 positive (n=13) | blaTEM-1 positive (n=27) | blaVIM-2 positive (n=8) | blaIMP-1 positive (n=5) | |||||||||
| Range | MIC50 | MIC90 | Range | MIC50 | MIC90 | Range | MIC50 | MIC90 | Range | MIC50 | MIC90 | |
| piperacillin | 32∼512 | 256 | 512 | 512∼>512 | >512 | >512 | 32∼256 | 64 | 256 | 16∼256 | 64 | 256 |
| amikacin | 64∼128 | 64 | 64 | 4∼>256 | >256 | >256 | 32∼128 | 64 | 128 | 64∼256 | 256 | 256 |
| gentamicin | >64 | >64 | >64 | >64 | >64 | >64 | 32∼>64 | >64 | >64 | 8∼64 | 32 | 64 |
| tobramycin | >64 | >64 | >64 | 64∼>64 | >64 | >64 | 32∼>64 | 64 | >64 | 64∼>64 | >64 | >64 |
| ciprofloxacin | 0.5∼>16 | >16 | >16 | 16∼>16 | >16 | >16 | <0.25∼0.5 | 0.25 | 0.5 | <0.25∼2 | 0.25 | 2 |
| amp-sulb | 16∼128 | 32 | 128 | 16∼>128 | 64 | 128 | 4∼32 | 8 | 32 | 1∼16 | 2 | 16 |
| ceftazidime | >128 | >128 | >128 | 16∼>128 | >128 | >128 | 16∼64 | 32 | 64 | 128∼>128 | >128 | >128 |
| cefepime | >128 | >128 | >128 | 16∼128 | 64 | 128 | 4∼16 | 16 | 16 | 16∼128 | 64 | 128 |
| aztreonam | >128 | >128 | >128 | 16∼128 | 64 | 128 | 32∼64 | 32 | 64 | 16∼32 | 16 | 32 |
| imipenem | 1∼16 | 16 | 16 | 2∼8 | 2 | 8 | 16∼64 | 16 | 64 | 16∼64 | 32 | 64 |
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