Abstract
Multidrug-resistant Acinetobacter baumannii strains have increasingly resulted in nosocomial outbreaks worldwide, leaving limited options for treatment. To date, little has been reported on the antimicrobial susceptibilities and genomic profiles of A. baumannii strains from hospital outbreaks in the Greater Los Angeles area. In this study, we examined the susceptibilities and genetic profiles of 20 nonduplicate isolates of A. baumannii from nosocomial outbreaks in Los Angeles County (LAC) and determined their mechanisms of fluoroquinolone resistance. Antibiotic susceptibility testing indicated that the majority of these LAC isolates were not susceptible to 14 of the 17 antibiotics tested, with the exception of doxycycline, minocycline, and tigecycline. In particular, all isolates were found to be resistant to ciprofloxacin. Genomic DNA analysis revealed eight epidemiologically distinct groups among these 20 A. baumannii isolates, consistent with antibiotic susceptibility profiles. Sequencing analysis confirmed that concurrent GyrA and ParC amino acid substitutions in the “hot spots” of their respective quinolone resistance-determining regions were primarily responsible for the high-level ciprofloxacin resistance of these isolates. Antibiotic susceptibility testing using two efflux pump inhibitors suggested that the presence of efflux pumps was only a secondary contributor to ciprofloxacin resistance for some of the isolates. In summary, the present study has revealed good correlation between the antibiotic susceptibility profiles and genetic fingerprints of 20 clinical isolates from nosocomial outbreaks in Los Angeles County and has determined their mechanisms of fluoroquinolone resistance, providing an important foundation for continued surveillance and epidemiological analyses of emerging A. baumannii isolates in Los Angeles County hospitals.
Acinetobacter baumannii, which was susceptible to many antibiotics 3 decades ago (3), is now a multidrug-resistant opportunistic human pathogen that is a frequent cause of nosocomial outbreaks worldwide (10, 18, 22, 42, 46, 50). The types of infections include pneumonia, urinary tract infection, endocarditis, surgical-site infection, meningitis, and septicemia (3, 10, 18, 22, 23, 25, 42, 46, 50). In Europe, A. baumannii accounts for as many as 10% of all infections caused by gram-negative bacteria seen in intensive care units (ICUs) (11), and in the United States, it accounts for 2.5% (16). Additionally, A. baumannii is increasingly recognized as an uncommon but increasingly important cause of community-acquired pneumonia, with a high mortality rate of 40% to 64% (2, 4, 8, 21, 53). In particular, increased incidences of A. baumannii infection have been reported among military personnel injured while deployed to Iraq and Afghanistan (12, 15, 41, 55). Currently there are A. baumannii strains that are resistant to all major antibiotic classes normally used to treat infections with this organism, including β-lactams, aminoglycosides, fluoroquinolones, chloramphenicol, tetracycline, and rifampin (9, 54). The prevalence of these multidrug-resistant A. baumannii strains leaves limited clinical options for treatment (44), underscoring the need to develop novel antibiotics for bacterial pathogens in general and gram-negative pathogens in particular.
There have been extensive surveillance and research efforts worldwide focusing on the antibiotic susceptibilities (11, 17, 18), genomic DNA profiles (19, 36, 41, 43), and mechanisms of resistance (14, 15, 20, 30) of A. baumannii clinical isolates. However, little has been reported on nosocomial-outbreak isolates of this bacterium from the Los Angeles metropolitan area. The availability of 20 nonduplicate nosocomial-outbreak isolates from Los Angeles County provided us an opportunity to determine whether the phenotypic profiles of these isolates correlate with their genetic fingerprints. In this communication, we examine the relationship between the antibiotic susceptibility profiles and genomic fingerprints, analyze the quinolone resistance-determining region (QRDR) sequences, and investigate the contribution of efflux pumps to fluoroquinolone resistance for these 20 clinical isolates.
MATERIALS AND METHODS
Bacterial strains and isolates.
All bacterial strains and clinical isolates are listed in Table 1. Quality control for susceptibility testing was carried out with four standard quality control reference species obtained from the American Type Culture Collection (ATCC, Manassas, VA): Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Enterococcus faecalis (ATCC 29212), and Staphylococcus aureus (ATCC 29213). A. baumannii strain ATCC 17978, type strain ATCC 19606T, and strain 15839 (ATCC item 202080, a patent deposit that is not available from the online catalog) were also acquired from ATCC and used for comparison to the A. baumannii clinical isolates. Twenty representative clinical isolates of A. baumannii (referred to below as LAC-1 to LAC-20) were isolated from hospital outbreaks over an 8-year period in Los Angeles County and were kindly provided by the Los Angeles County Public Health Laboratory. All 20 Los Angeles County A. baumannii clinical isolates were confirmed in our laboratory by using an API 20NE kit according to the manufacturer's protocols (bioMérieux, Durham, NC) and the temperature growth test (44°C). A. baumannii isolates AYE and SDF (9) were kindly provided by Didier Raoult of France.
TABLE 1.
Bacterial strains or isolates used
| Bacterial strain or isolate | Description | Sourcea |
|---|---|---|
| E. coli ATCC 25922 | Susceptibility reference strain | ATCC |
| P. aeruginosa ATCC 27853 | Susceptibility reference strain | ATCC |
| E. faecalis ATCC 29212 | Susceptibility reference strain | ATCC |
| S. aureus ATCC 29213 | Susceptibility reference strain | ATCC |
| A. baumannii | ||
| Strain 15839 | A. baumannii isolate (ATCC item 202080) | ATCC |
| ATCC 17978 | A. baumannii isolate | ATCC |
| ATCC 19606T | A. baumannii type strain | ATCC |
| LAC-1 | Outbreak strain isolated from hospital A in 1999 | LAC |
| LAC-2 | Outbreak strain isolated from hospital A in 1996 | LAC |
| LAC-3 | Outbreak strain isolated from hospital A in 1996 | LAC |
| LAC-4 | Outbreak strain isolated from hospital A in 1997 | LAC |
| LAC-5 | Outbreak strain isolated from hospital A in 1997 | LAC |
| LAC-6 | Outbreak strain isolated from hospital A in 2001 | LAC |
| LAC-7 | Outbreak strain isolated from hospital A in 2001 | LAC |
| LAC-8 | Outbreak strain isolated from hospital A in 2001 | LAC |
| LAC-9 | Outbreak strain isolated from hospital A in 2001 | LAC |
| LAC-10 | Outbreak strain isolated from hospital A in 2001 | LAC |
| LAC-11 | Outbreak strain isolated from hospital B in 2003 | LAC |
| LAC-12 | Outbreak strain isolated from hospital B in 2003 | LAC |
| LAC-13 | Outbreak strain isolated from hospital B in 2003 | LAC |
| LAC-14 | Outbreak strain isolated from hospital B in 2004 | LAC |
| LAC-15 | Outbreak strain isolated from hospital B in 2004 | LAC |
| LAC-16 | Outbreak strain isolated from hospital C in 1997 | LAC |
| LAC-17 | Outbreak strain isolated from hospital C in 1997 | LAC |
| LAC-18 | Outbreak strain isolated from hospital C in 1997 | LAC |
| LAC-19 | Outbreak strain isolated from hospital C in 1997 | LAC |
| LAC-20 | Outbreak strain isolated from hospital C in 1998 | LAC |
| AYE | Clinical strain obtained from the Raoult group | France |
| SDF | Clinical strain obtained from the Raoult group | France |
LAC, Los Angeles County Public Health Laboratory. None of the isolates came from the same patient.
Antibiotics and efflux pump inhibitors.
A panel of 17 antibiotics with known breakpoints (except for tigecycline, whose breakpoints are to be determined) was used to determine the antibiotic susceptibilities of A. baumannii strains and clinical isolates. All antibiotics (except for tigecycline) were purchased from commercial vendors. Cefotaxime, ceftazidime, ceftriaxone, doxycycline, gentamicin, levofloxacin, minocycline, piperacillin, and tetracycline were purchased from Sigma-Aldrich (St. Louis, MO). Ciprofloxacin was manufactured by Fluka BioChemika and purchased from Sigma-Aldrich (St. Louis, MO). Meropenem was from the U.S. Pharmacopeia (Rockville, MD) and was purchased through VWR International (Brisbane, CA). Amikacin, cefepime, gatifloxacin, imipenem, and tobramycin were purchased from Fisher Scientific (Tustin, CA). Tigecycline was kindly provided by Wyeth Research (Cambridge, MA). The efflux pump inhibitors Phe-Arg-β-naphthylamide dihydrochloride (PAβN) and 1-(1-naphthylmethyl)-piperazine (NMP) were purchased from VWR International.
Susceptibility testing.
The MICs of the 17 antibiotics (including 16 with established breakpoints against Acinetobacter spp.) against 20 nonduplicate A. baumannii clinical isolates obtained from nosocomial outbreaks in Los Angeles County (LAC-1 to LAC-20) were determined. For comparison, the susceptibilities of an additional five A. baumannii strains and isolates from locations other than Los Angeles County were also tested. Quality controls were carried out using ATCC reference strains (Table 1), and results were compared to Clinical and Laboratory Standards Institute (CLSI) MIC ranges (7) for quality control of susceptibility testing procedures. Antibiotic powders were dissolved in sterile deionized water or an appropriate solvent according to the manufacturer's recommendations. Test concentrations for antibiotics were 256 μg/ml, 128 μg/ml, 64 μg/ml, 32 μg/ml, 16 μg/ml, 8 μg/ml, 4 μg/ml, 2 μg/ml, 1 μg/ml, 0.5 μg/ml, and 0.25 μg/ml. The MIC method used was based on the microdilution procedures of the CLSI (6) with the following modifications. Each well of a 96-well microtiter plate (Costar 3795; Thermo Fisher Scientific, Tustin, CA) contained a total volume of 100 μl: 10 μl of the 10×-concentrated antibiotic dilution and 90 μl of 1.1× Mueller-Hinton medium with the bacterial inoculum. Antibiotics were serially diluted in stock plates with 5% dimethyl sulfoxide as the diluent. Samples were transferred to replicate plates using a Tomtec Quadra 3 robotic liquid handler (Tomtec, Hamden, CT). Microplates were stacked four high, covered in plastic wrap to reduce evaporation, and incubated at 35°C for 18 to 24 h. Plates were read visually using an inverted mirror to detect growth at the bottoms of wells. The lowest concentration of antibiotic that did not have visible bacterial growth was defined as the MIC (6).
PFGE analysis of genomic DNA.
A. baumannii genomic DNA was isolated according to the procedures of McDougal et al. (24), and bacterial genomic DNA plugs were prepared according to the instructions for the Bio-Rad (Hercules, CA) bacterial DNA plug kit. Pulsed-field gel electrophoresis (PFGE) was performed as described by Peleg and colleagues (31).
Sequencing of the QRDRs of the gyrA and parC genes.
The A. baumannii gyrA and parC genes from 20 Los Angeles County isolates, French strain AYE, strain 15839, and ATCC 19606T were amplified via colony PCR. Briefly, one or two healthy colonies of an isolate were resuspended in 100 μl DNase/RNase-free H2O (Invitrogen, Carlsbad, CA). The cell suspension (1 μl) was combined with 20 μl Pfu Turbo DNA polymerase Master Mix (Stratagene, San Diego, CA), 0.4 μl forward primer (120 ng/μl), 0.4 μl reverse primer (120 ng/μl), and 28.2 μl DNase/RNase-free H2O. PCRs were performed in the GeneAmp PCR system, model 9700 (Applied Biosystems, Foster City, CA). For gyrA, the following parameters were used: an initial template denaturation at 95°C for 1 min; 36 cycles consisting of 30 s of denaturation at 95°C, 30 s of annealing at 52°C, and 2 min of extension at 72°C; and a final extension at 72°C for 10 min. For parC, PCR conditions consisted of an initial template denaturation at 95°C for 2 min; 36 cycles of 1 min of denaturation at 95°C, 1 min of annealing at 60°C, and 2 min of extension at 72°C; and a final extension at 72°C for 10 min. The PCR primers for the amplification and sequencing of the gyrA QRDR are as follows: forward, 5′-AAATCTGCTCGTGTCGTTGG-3′; reverse, 5′-GCCATACCTACAGCAATACC-3′. The PCR primers for the amplification and sequencing of the parC QRDR are derived from the QRDR sequence of the parC gene of A. baumannii ATCC 17978 (38) and are as follows: forward, 5′-AAGCCCGTACAGCGCCGTATT-3′; reverse, 5′-AAAGTTATCTTGCCATTCGCT-3′. Amplified products were confirmed by agarose gel electrophoresis using a GeneRuler 1-kb DNA Ladder Plus from Fermentas Life Sciences (Hanover, MD) to estimate PCR fragment sizes, followed by cleanup and purification using a QiaQuick PCR purification kit (Qiagen, Valencia, CA). Sequencing reactions were carried out using BigDye Terminator, version 3.1 (Applied Biosystems, Foster City, CA). PCR conditions for sequencing with BigDye are as follows: an initial denaturation of 96°C for 1 min; 25 cycles consisting of 10 s of denaturation at 96°C, 5 s of annealing at 50°C, and 4 min of extension at 60°C; and a final extension at 4°C for 4 min. Both strands of each amplified DNA were sequenced using forward and reverse primers. DNA sequences obtained were initially aligned with known sequences by using the BLASTX option (at the NCBI website) to generate amino acid alignment within the QRDRs. Sequence comparisons were made to the wild-type A. baumannii GyrA (GenBank accession no. X82165) and ParC (GenBank accession no. X95819) QRDRs (48, 49).
Effects of efflux pump inhibitors on ciprofloxacin resistance.
Susceptibility to ciprofloxacin in the presence of efflux pump inhibitors was tested as described under “Susceptibility testing” above, except for the presence of 100 μg/ml of the efflux pump inhibitor PAβN or NMP. Specifically, susceptibility to ciprofloxacin was tested in parallel in the presence or absence of the efflux pump inhibitors. Following the addition of ciprofloxacin and the bacterial cell inoculum, 2 μl of the 5-mg/ml stock of either PAβN or NMP was added to the microplate wells (total volume, 100 μl). The rest of the procedures were carried out as described above.
RESULTS
Susceptibility testing.
MICs were determined for the panel of 17 antibiotics against the 20 A. baumannii clinical isolates obtained from nosocomial outbreaks in Los Angeles County (Table 1). Based on the MICs obtained, the clinical isolates and strains of A. baumannii were designated susceptible, intermediate, or resistant to any 1 of the 17 antibiotics tested (Fig. 1), according to established breakpoint values (7), except for tigecycline. Since no tigecycline breakpoints are available for Acinetobacter spp., U.S. FDA tigecycline susceptibility breakpoints for Enterobacteriaceae (≤2 μg/ml), applied to Acinetobacter spp. (Tygacil package insert [June 2005]; Wyeth Pharmaceuticals Inc., Philadelphia, PA), were used here. These U.S. FDA breakpoints for tigecycline have also been used by several recent studies (17, 39). The distributions of antibiotic MICs (Table 2) and susceptibility designations (Fig. 1) indicated that the majority of A. baumannii nosocomial-outbreak isolates from Los Angeles County were not susceptible to the 17 antibiotics tested, except for doxycycline, minocycline, and tigecycline. Specifically, no isolate was susceptible to gentamicin, yet 25% (5/20) were susceptible to two other aminoglycosides tested (amikacin and tobramycin) (Table 2; Fig. 1). With regard to carbapenems (imipenem and meropenem), 50% and 45% of the isolates, respectively, were susceptible. Additionally, none of the 20 isolates were susceptible to either piperacillin or the four cephems (cefotaxime, ceftriaxone, cefepime, and ceftazidime), although there were a few “intermediate” isolates (Table 2; Fig. 1). The fact that all 20 isolates were found to be resistant to ciprofloxacin was especially noteworthy. Even with the newer fluoroquinolones (gatifloxacin and levofloxacin), only 1 of the 20 isolates (LAC-4) was still susceptible (Table 2; Fig. 1). The only antibiotics tested that still exhibited consistent potency were doxycycline, minocycline, and tigecycline: 80% of the isolates were still susceptible to these three drugs (Table 2; Fig. 1).
FIG. 1.
Susceptibility profiles of A. baumannii isolates. Isolates were designated susceptible (S), intermediate (I), or resistant (R) according to the antibiotic breakpoint guidelines of the CLSI (7) for Acinetobacter spp. (except for tigecycline). Since breakpoints for tigecycline are not yet available from the CLSI, the U.S. FDA tigecycline susceptibility breakpoints listed for Enterobacteriaceae (≤2 μg/ml), applied to Acinetobacter spp. (Tygacil package insert [June 2005]; Wyeth Pharmaceuticals Inc., Philadelphia, PA), were used in the interim. Abbreviations: AMK, amikacin; FEP, cefepime; CTX, cefotaxime; CAZ, ceftazidime; CRO, ceftriaxone; CIP, ciprofloxacin; DOX, doxycycline; GAT, gatifloxacin; GEN, gentamicin; IPM, imipenem; LVX, levofloxacin; MEM, meropenem; MIN, minocycline; PIP, piperacillin; TET, tetracycline; TIG, tigecycline; TOB, tobramycin.
TABLE 2.
Distribution of MICs of 17 antibiotics against 20 clinical isolates of A. baumannii
| Antibiotica | No. of A. baumannii isolates with the following MIC (μg/ml):
|
|||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | >256 | |
| Amikacin | 3 | 1 | 1 | 1 | 6 | 3 | 3 | 2 | ||||
| Gentamicin | 3 | 5 | 5 | 2 | 2 | 1 | 2 | |||||
| Tobramycin | 6 | 3 | 4 | 4 | 2 | 1 | ||||||
| Imipenem | 6 | 4 | 7 | 3 | ||||||||
| Meropenem | 4 | 6 | 2 | 8 | ||||||||
| Piperacillin | 1 | 2 | 1 | 1 | 15 | |||||||
| Cefepime | 1 | 3 | 4 | 4 | 7 | 1 | ||||||
| Cefotaxime | 2 | 5 | 4 | 9 | ||||||||
| Ceftazidime | 2 | 1 | 2 | 3 | 1 | 11 | ||||||
| Ceftriaxone | 2 | 1 | 5 | 12 | ||||||||
| Ciprofloxacin | 1 | 2 | 4 | 6 | 7 | |||||||
| Gatifloxacin | 2 | 1 | 4 | 5 | 7 | 1 | ||||||
| Levofloxacin | 2 | 3 | 5 | 6 | 1 | 3 | ||||||
| Doxycycline | 2 | 5 | 1 | 2 | 6 | 4 | ||||||
| Minocycline | 7 | 1 | 4 | 4 | 2 | 2 | ||||||
| Tetracycline | 3 | 1 | 3 | 5 | 4 | 4 | ||||||
| Tigecycline | 3 | 6 | 7 | 4 | ||||||||
The antibiotics are grouped by chemical class.
Certain LAC isolates had identical or highly similar antibiotic susceptibility profiles (Fig. 1). Isolates with identical or similar profiles include the following: LAC-5 and LAC-8; LAC-7, LAC-9, and LAC-10; LAC-11 to LAC-14; and LAC-16, LAC-17, LAC-18, and LAC-20.
It was apparent that the additional A. baumannii isolates (AYE and SDF) and strains (strain 15839, ATCC 17978, and type strain ATCC 19606T), obtained from a variety of sources, exhibited varied antibiotic susceptibilities (Fig. 1). AYE, the multidrug-resistant French isolate, was susceptible to only five antibiotics: imipenem, meropenem, doxycycline, minocycline, and tigecycline. In contrast, the other French isolate, SDF, was susceptible to all antibiotics except piperacillin and tetracycline (intermediate). Strain 15839 was highly resistant, with susceptibility observed toward only four antibiotics: imipenem, meropenem, minocycline, and tigecycline. ATCC 17978 was susceptible to all 17 breakpoint antibiotics. The type strain (ATCC 19606T) was susceptible to 10 of the 17 antibiotics, intermediate to 6 antibiotics, and resistant to only 1 antibiotic, gentamicin (Fig. 1). MICs obtained with appropriate quality control reference strains (P. aeruginosa ATCC 27853 and E. coli ATCC 25922) were within acceptable quality control ranges (7). For example, the MICs in our tests with P. aeruginosa ATCC 27853 were 2, 16, 0.25, and 0.5 μg/ml for amikacin, cefotaxime, ciprofloxacin, and meropenem, respectively, values well within the acceptable ranges listed (7). Additionally, the MICs of cefepime, cefotaxime, ceftazidime, piperacillin, and imipenem for strain AYE were >256, >256, >256, 256, and 1 μg/ml, respectively, values similar to the MICs (512, >512, 512, 256, and 1 μg/ml for the same antibiotics) reported previously by Poirel and coworkers for the same strain (32).
Genomic DNA profiles.
To determine genomic DNA fingerprint profiles for A. baumannii isolates and strains, genomic DNA restriction digestion by ApaI endonuclease, followed by PFGE, was performed. To interpret chromosomal restriction digest profiles, the criteria of Tenover et al. (40) were used. Based on PFGE analysis of genomic DNA, the 20 clinical isolates from nosocomial outbreaks in Los Angeles County can be divided into eight distinct groups, each of which consists of one or more isolates with similar or identical profiles: LAC-1 to LAC-3; LAC-5 and LAC-8; LAC-7, LAC-9, and LAC-10; LAC-11, LAC-12, and LAC-14 (with LAC-13 possibly related); LAC-16 to LAC-20; LAC-4; LAC-6; and LAC-15 (Fig. 2A and B). Specifically, LAC-1 to LAC-3 are closely related, with three or fewer bands differing between any two isolates (Fig. 2A). LAC-5 and LAC-8 appear to be identical (no DNA bands differ) (Fig. 2A). In addition, LAC-7, LAC-9, and LAC-10 appear to be identical (Fig. 2A). Moreover, LAC-11, LAC-12, and LAC-14 appear to be identical, and LAC-13 is possibly related, having four fragments that differ (Fig. 2B). Finally, LAC-16 to LAC-20 are closely related based on PFGE fingerprints, and LAC-17 to LAC-19 are indistinguishable (Fig. 2B). The remaining three isolates (LAC-4, LAC-6, and LAC-15) are genetically unrelated based on the PFGE fingerprints (Fig. 2A and B). Genomic restriction digest profiling of other isolates and strains indicated that A. baumannii strain 15839, ATCC 17978, and ATCC 19606T all have unique PFGE fingerprint profiles and are distinguishable from all the LAC isolates (Fig. 2C). The genomic profiles for the French isolates AYE and SDF were also unique, both different from each other and different from the other isolates (Fig. 2A, B, and C).
FIG. 2.
Genomic DNA PFGE fingerprint profiles for A. baumannii isolates from Los Angeles County and other isolates/strains. (A) Lanes L, λ DNA ladder; lanes 1 to 10, LAC-1 to LAC-10, respectively. (B) Lanes L, λ DNA ladder; lanes 11 to 20, LAC-11 to LAC-20, respectively. (C) Lanes, from left to right, show the λ DNA ladder, AYE, SDF, strain 15839, ATCC 17978, and ATCC 19606T.
Sequence analysis of the gyrA and parC QRDRs.
Results from susceptibility testing indicated that all 20 LAC isolates were uniformly resistant to ciprofloxacin (Fig. 1). To determine whether the ciprofloxacin resistance was due to changes in the structure of the fluoroquinolone protein targets (DNA gyrase and DNA topoisomerase IV), the QRDRs of the gyrA and parC genes were PCR amplified and subsequently sequenced. The PCR-amplified DNA products for the QRDRs of the gyrA (Fig. 3A) and parC (Fig. 3B) genes of select clinical isolates were shown to be consistent with the respective lengths of the amplicons. Sequencing results (Table 3) showed that all the A. baumannii clinical isolates from Los Angeles County had a point mutation on the gyrA gene that converted the serine at position 83 (Ser-83) to leucine (Leu) in GyrA, a change that is consistent with a fluoroquinolone-resistant phenotype. No additional amino acid sequence changes were observed for the GyrA polypeptide in these clinical isolates, not even at other “hot spot” amino acid positions (Gly-81, Ala-84, and Glu-87) known to contribute to fluoroquinolone resistance (Table 3). Sequencing of the parC genes indicated that 19 of the 20 clinical isolates from Los Angeles County had mutations in the parC gene that caused an amino acid change in either Ser-80 (TCG) or Glu-84 (GAA), but not in both, in ParC. Among these 19 isolates, 8 had leucine replacing Ser-80, 4 had phenylalanine replacing Ser-80, and 7 had lysine replacing Glu-84. Amino acid substitutions in both the GyrA and ParC polypeptides are consistent with a high-level fluoroquinolone-resistant phenotype (48).
FIG. 3.
Colony PCR amplification of the QRDRs of the gyrA (A) and parC (B) genes in A. baumannii isolates. Lanes 1, DNA ladder. (A) Lanes 2 to 7, LAC-1 to LAC-6, respectively. (B) Lanes 2 to 5, LAC-1 to LAC-4, respectively.
TABLE 3.
GyrA and ParC amino acid substitutionsa due to point mutations in the QRDRs of the gyrA and parC genes in clinical isolates of A. baumannii
| Strain or isolate | Ciprofloxacin MIC (μg/ml) | Change in the following GyrA amino acid:
|
Change in the following ParC amino acid:
|
||||
|---|---|---|---|---|---|---|---|
| Gly-81 (GGT) | Ser-83 (TCA) | Ala-84 (GCT) | Glu-87 (GAA) | Ser-80 (TCG) | Glu-84 (GAA) | ||
| LAC-1 | 128 | − | Leu (TTA) | − | − | − | Lys (AAA) |
| LAC-2 | 32 | − | Leu (TTA) | − | − | − | − |
| LAC-3 | 64 | − | Leu (TTA) | − | − | − | Lys (AAA) |
| LAC-4 | 16 | − | Leu (TTA) | − | − | − | Lys (AAA) |
| LAC-5 | 64 | − | Leu (TTA) | − | − | Leu (TTG) | − |
| LAC-6 | 128 | − | Leu (TTA) | − | − | Phe (TTT) | − |
| LAC-7 | 128 | − | Leu (TTA) | − | − | Leu (TTG) | − |
| LAC-8 | 256 | − | Leu (TTA) | − | − | Leu (TTG) | − |
| LAC-9 | 256 | − | Leu (TTA) | − | − | Phe (TTT) | − |
| LAC-10 | 128 | − | Leu (TTA) | − | − | Phe (TTT) | − |
| LAC-11 | 256 | − | Leu (TTA) | − | − | Leu (TTG) | − |
| LAC-12 | 256 | − | Leu (TTA) | − | − | Leu (TTG) | − |
| LAC-13 | 128 | − | Leu (TTA) | − | − | Leu (TTG) | − |
| LAC-14 | 128 | − | Leu (TTA) | − | − | Leu (TTG) | − |
| LAC-15 | 128 | − | Leu (TTA) | − | − | Phe (TTT) | − |
| LAC-16 | 128 | − | Leu (TTA) | − | − | − | Lys (AAA) |
| LAC-17 | 256 | − | Leu (TTA) | − | − | − | Lys (AAA) |
| LAC-18 | 256 | − | Leu (TTA) | − | − | − | Lys (AAA) |
| LAC-19 | 32 | − | Leu (TTA) | − | − | Leu (TTG) | − |
| LAC-20 | 128 | − | Leu (TTA) | − | − | − | Lys (AAA) |
| AYE | 128 | − | Leu (TTA) | − | − | Leu (TTG) | − |
| ATCC 19606 | 2 | − | − | − | − | − | − |
| Strain 15839 | 256 | − | Leu (TTA) | − | − | Leu (TTG) | − |
Effects of efflux pump inhibitors on ciprofloxacin resistance.
In addition to drug target protein modification, another possible mechanism of fluoroquinolone resistance is the presence of efflux pumps that can remove fluoroquinolones from the cell. To determine if and to what degree efflux pumps are involved in the ciprofloxacin resistance phenotype among the LAC isolates, susceptibility to ciprofloxacin in the presence of one of the two efflux pump inhibitors at 100 μg/ml (30) was tested. Susceptibility testing results with and without an efflux pump inhibitor indicated that most of the isolates become less resistant (two- to eightfold) to ciprofloxacin in the presence of either PAβN or NMP (Table 4). Based on a fourfold or greater reduction in the MIC as the criterion for significance (30), the ciprofloxacin MICs for 6 of the 20 isolates (LAC-7, LAC-15 through LAC-18, and LAC-20) decreased significantly (four- to eightfold) in the presence of NMP (Table 4). Interestingly, six isolates (LAC-2, LAC-12 through LAC-15, and LAC-20; four are different from those above) also exhibited significantly reduced resistance to ciprofloxacin in the presence of PAβN (Table 4). The susceptibilities of two isolates (LAC-6 and LAC-14) to ciprofloxacin remained unchanged in the presence of NMP. Additionally, the ciprofloxacin susceptibilities of two other isolates (LAC-1 and LAC-16) remained unchanged in the presence of PAβN (Table 4). The majority of clinical isolates (14 of 20) exhibited no change or a twofold change in susceptibility to ciprofloxacin in the presence of either efflux pump inhibitor. Even those isolates for which the MIC was four- or eightfold lower in the presence of an efflux pump inhibitor were still classified as resistant based on ciprofloxacin breakpoints. These results indicated that efflux pumps contributed to ciprofloxacin resistance for some of the LAC isolates but were not the primary contributors to ciprofloxacin resistance.
TABLE 4.
Effects of efflux pump inhibitors on susceptibility to ciprofloxacin
| Isolate | MIC (μg/ml)a
|
||
|---|---|---|---|
| Ciprofloxacin | Ciprofloxacin + NMP | Ciprofloxacin + PAβN | |
| LAC-1 | 128 | 64 (2) | 128 |
| LAC-2 | 32 | 16 (2) | 8 (4) |
| LAC-3 | 64 | 32 (2) | 32 (2) |
| LAC-4 | 16 | 8 (2) | 8 (2) |
| LAC-5 | 64 | 32 (2) | 32 (2) |
| LAC-6 | 128 | 128 | 64 (2) |
| LAC-7 | 128 | 32 (4) | 64 (2) |
| LAC-8 | 256 | 128 (2) | 128 (2) |
| LAC-9 | 256 | 128 (2) | 128 (2) |
| LAC-10 | 128 | 64 (2) | 64 (2) |
| LAC-11 | 256 | 128 (2) | 128 (2) |
| LAC-12 | 256 | 128 (2) | 32 (8) |
| LAC-13 | 128 | 64 (2) | 32 (4) |
| LAC-14 | 128 | 128 | 32 (4) |
| LAC-15 | 128 | 32 (4) | 32 (4) |
| LAC-16 | 128 | 16 (8) | 128 |
| LAC-17 | 256 | 64 (4) | 128 (2) |
| LAC-18 | 256 | 64 (4) | 128 (2) |
| LAC-19 | 32 | 16 (2) | 16 (2) |
| LAC-20 | 128 | 16 (8) | 32 (4) |
Values in parentheses are reductions (n-fold) in the MIC after the addition of the indicated efflux pump inhibitor.
DISCUSSION
A. baumannii is now a multidrug-resistant opportunistic human pathogen that is frequently involved in outbreaks of nosocomial infections (10, 18, 22, 42, 46, 50) and is an important cause of community-acquired pneumonia (2, 4, 8, 21, 53). In addition to its significance in infections in civilian settings, this bacterium has also been involved in wound infections among military personnel (12, 15, 41, 55). Although nosocomial infections caused by A. baumannii have been reported worldwide (10, 23, 25, 46, 50) and throughout the United States (18, 34), very little has been reported on the antibiotic susceptibility, genetic profiles, and molecular characteristics of hospital-acquired A. baumannii isolates in the Greater Los Angeles area. The availability of 20 Los Angeles County nosocomial-outbreak isolates has provided us with an opportunity to examine their antibiotic susceptibilities and molecular characteristics, such as genomic profiles, antibiotic resistance determinants, and mechanism of resistance.
It is alarming that all 20 clinical isolates are multidrug resistant. Only minocycline, doxycycline, and the recently approved drug tigecycline maintained antimicrobial activity against the majority of the 20 isolates (80%). Imipenem and meropenem were active against 50% and 45%, respectively, of the LAC isolates. It was encouraging that the newest antibiotic approved by the FDA, tigecycline, is still fairly active against all 20 A. baumannii LAC isolates, with MICs of ≤4 μg/ml. These results are similar to published reports for clinical isolates from other geographic regions (5, 13, 28, 37, 39, 51). However, although tigecycline is active in vitro, recent reports indicate that it is far less effective in vivo, with cases of resistance increasingly being reported for A. baumannii (26, 31, 33).
The antibiotic susceptibility profiles were found to correlate well with the genomic profiles of these isolates (Fig. 4). For example, isolates LAC-5 and LAC-8, which had highly similar antibiotic susceptibility profiles (Fig. 1), exhibited identical PFGE patterns (Fig. 2A) and contained identical amino acid substitutions due to mutations in the QRDRs of the gyrA and parC genes (Table 3). In addition, isolates LAC-16, LAC-17, and LAC-18 all had similar antibiotic susceptibility profiles (Fig. 1), highly similar genomic DNA fingerprints by PFGE (Fig. 2B), and identical amino acid substitutions at position 84 of ParC (Glu-84 to Lys-84) (Table 3). These results support the epidemiological relationships of the isolates with identical or similar genomic fingerprints and antibiotic susceptibility profiles. Based primarily on the genomic DNA profiles, it was apparent that there are eight epidemiologically distinct lineages of A. baumannii among these 20 clinical isolates: LAC-1 to LAC-3; LAC-4; LAC-5 and LAC-8; LAC-6; LAC-7, LAC-9, and LAC-10; LAC-11 to LAC-14; LAC-15; and LAC-16 to LAC-20. The division of these epidemiological lineages appears to be supported by the antibiotic susceptibility profiles (Fig. 4).
FIG. 4.
Correlation of antibiotic susceptibility with genomic profiles. Boxes with various patterns represent different clusters of strains (or individual strains) among the 20 outbreak isolates from Los Angeles County hospitals. For genomic profiles, strains for which three or fewer DNA fragments are different after ApaI digestion of their genomic DNA are grouped as at least closely related (including indistinguishable strains) (boxes with identical patterns), as proposed by Tenover and colleagues (40). Strains for which four to six fragments are different are considered possibly related (40) (boxes with similar patterns [e.g., LAC-13 versus LAC-14]), while those with seven or more fragments different are deemed unrelated (40) and thus are represented by different patterns (e.g., the matrix pattern of LAC-7 versus the vertical-stripe pattern of LAC-16). Similarly, for susceptibility patterns, strains for which three or fewer susceptibility designations (R, S, or I in Fig. 1) are different have identical patterns, while strains for which four to six susceptibility designations are different have similar patterns (e.g., LAC-19 versus LAC-20). Strains for which seven or more susceptibility designations are different have different patterns.
A previous surveillance study of hundreds of A. baumannii isolates from clinical infections in the United States (34) reported a steady increase in the frequency of ciprofloxacin resistance among A. baumannii isolates (from 25% in 1999 to 45% in 2004). Another surveillance report (18) indicated that at least 45% of A. baumannii isolates, obtained from both non-ICU and ICU patients between 1998 and 2001, were resistant to ciprofloxacin. Our results with all 20 nonduplicate A. baumannii clinical isolates were more alarming: 100% were resistant to ciprofloxacin (Fig. 1). This may be due, in part, to the fact that our isolates were obtained from A. baumannii hospital outbreaks, while the surveillance reports included both outbreak and sporadic isolates.
A major mechanism of fluoroquinolone resistance in gram-negative bacteria involves changes in the structure (and hence in the affinity to the drugs) of the drug targets DNA gyrase (encoded by the gyrA and gyrB genes) and DNA topoisomerase IV (encoded by the parC and parE genes) (1, 35, 45). Specifically, amino acid substitutions at certain positions in subunits A (GyrA and ParC) of both DNA gyrase and DNA topoisomerase IV, due to point mutations in the QRDRs of the genes encoding these two polypeptides, have been found to contribute to fluoroquinolone resistance. In A. baumannii, the most frequent amino acid substitutions occur at position 83 (Ser-83) of GyrA (20, 49, 52) and at position 80 (Ser-80) of ParC (20, 48, 52). While changes in GyrA are necessary for moderate levels of fluoroquinolone resistance among clinical isolates of A. baumannii, concurrent modifications in the ParC polypeptide are required in order to achieve high levels of fluoroquinolone resistance (48). Among LAC isolates, the majority (except LAC-4) had ciprofloxacin MICs equal to or greater than 32 μg/ml (Table 3); 15 isolates (75%) had MICs of 128 μg/ml or higher, indicating that these isolates are highly resistant to fluoroquinolones. Our sequencing results revealed that almost all isolates (except LAC-2) had concurrent mutations in the gyrA and parC genes that resulted in Ser-83-to-Leu-83 substitutions in GyrA and one of three types of ParC substitutions at either Ser-80 or Glu-84 (Table 3). These results further confirmed the observation by Vila and coworkers (48) that both GyrA and ParC mutations are necessary to render A. baumannii highly resistant to quinolones. Interestingly, among the 12 LAC isolates with Ser-80 changes, the incidence of Ser-80-to-Phe-80 substitutions was significantly higher (4/12 [33%]) than that reported elsewhere (52).
Multidrug efflux pumps have been recognized as a mechanism of resistance in gram-negative bacteria (27, 29, 47). Efflux pump inhibitors have been shown to reverse multidrug resistance in A. baumannii isolates (30, 31). In particular, Peleg and colleagues (31) exposed a tigecycline-susceptible clinical isolate to increasing concentrations of tigecycline and isolated various mutant strains with elevated resistance to tigecycline and several other antibiotics. In the presence of PAβN, the tigecycline susceptibility of the mutant strain returned to the level for the parental strain. However, the presence of PAβN reduced ciprofloxacin MICs no more than fourfold (31). Consistent with the findings of Peleg and coworkers, the ciprofloxacin MICs for most of our 20 A. baumannii outbreak isolates (19/20) did not change more than fourfold in the presence of PAβN. In a study comparing the effects of PAβN and NMP on efflux pumps (30), NMP was found to be more active than PAβN in reducing ciprofloxacin MICs for a number of clinical isolates and their mutant strains. Our results with slightly more isolates (n = 20) found no significant difference between the abilities of these two efflux pump inhibitors to reduce ciprofloxacin MICs but indicated that these two inhibitors might affect different types of efflux pumps, since there was little overlap among the isolates for which MICs were reduced significantly (i.e., fourfold or more) (Table 4).
In conclusion, the 20 A. baumannii clinical isolates obtained from nosocomial outbreaks in Los Angeles County appeared to have originated from eight epidemiologically distinct lineages, as evidenced primarily by PFGE fingerprinting analysis, supported by phenotypic and other molecular characteristics. The uniform ciprofloxacin resistance of the 20 clinical isolates was due primarily to point mutations within QRDRs in the gyrA and parC genes. Efflux pumps did not appear to contribute significantly to the fluoroquinolone resistance observed for these isolates.
Acknowledgments
Funding for this project has been provided by an NIH MBRS-SCORE grant (S06GM008101) and by the NIH RIMI Program (P20MD001824) to H. H. Xu. D. Contreras thanks the National Institutes of Health for a MARC Traineeship through grant T34 GM 08228.
We are grateful to Wyeth for the generous gift of tigecycline and to Didier Raoult of France for sharing A. baumannii isolates AYE and SDF. We thank two anonymous reviewers for their critiques, which helped to improve the manuscript.
Footnotes
Published ahead of print on 4 June 2008.
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