ABSTRACT
Because of its remarkable ability to acquire antibiotic resistance and to survive in nosocomial environments, Acinetobacter baumannii has become a significant nosocomial infectious agent worldwide. Tigecycline is one of the few therapeutic options for treating infections caused by A. baumannii isolates. However, tigecycline resistance has increasingly been reported. Our aim was to assess the prevalence and characteristics of efflux-based tigecycline resistance in clinical isolates of A. baumannii collected from a hospital in China. A total of 74 A. baumannii isolates, including 64 tigecycline-nonsusceptible A. baumannii (TNAB) and 10 tigecycline-susceptible A. baumannii (TSAB) isolates, were analyzed. The majority of them were determined to be positive for adeABC, adeRS, adeIJK, and abeM, while the adeE gene was found in only one TSAB isolate. Compared with the levels in TSAB isolates, the mean expression levels of adeB, adeJ, adeG, and abeM in TNAB isolates were observed to increase 29-, 3-, 0.7-, and 1-fold, respectively. The efflux pump inhibitors (EPIs) phenyl-arginine-β-naphthylamide (PAβN) and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) could partially reverse the resistance pattern of tigecycline. Moreover, the tetX1 gene was detected in 12 (18.8%) TNAB isolates. To our knowledge, this is the first report of the tetX1 gene being detected in A. baumannii isolates. ST208 and ST191, which both clustered into clonal complex 92 (CC92), were the predominant sequence types (STs). This study showed that the active efflux pump AdeABC appeared to play important roles in the tigecycline resistance of A. baumannii. The dissemination of TNAB isolates in our hospital is attributable mainly to the spread of CC92.
INTRODUCTION
Because of its remarkable ability to acquire antibiotic resistance and to survive in nosocomial environments, Acinetobacter baumannii has successfully become a significant nosocomial infectious agent worldwide (1, 2). During recent decades, clinicians have witnessed dramatic increases in the rates of multidrug-resistant A. baumannii (MDRAB) (2). According to CHINET (antimicrobial resistance surveillance networks in China), the rates of A. baumannii resistance to the majority of antibiotics tested varied between 2.5% and 48.6% in 2000, whereas the resistance rates increased to approximately 50 to 60% in 2009 (2). The increasing prevalence of MDRAB has led to very limited therapeutic options, and tigecycline is considered one of the few therapeutic options (3, 4).
Tigecycline, a new class of glycylcyclines, is modified by addition of a 9-t-butyl-glycylamido side chain to minocycline (5). The drug binds to bacterial ribosomes with high affinity and therefore evades the major resistance mechanisms of tetracycline, retaining activity against a broad range of both Gram-positive and Gram-negative bacteria (4, 6), including multidrug-resistant isolates. However, tigecycline resistance has emerged recently and been detected during treatment with this agent (7–10).
Tigecycline nonsusceptibility in A. baumannii isolates has been associated with overexpression of a variety of efflux pumps. The major clinically relevant efflux pumps, such as AdeABC, AdeIJK, AdeFGH, AbeM, and AdeDE, have all been identified in A. baumannii. These efflux pumps display broad substrate specificity, and tigecycline is one such substrate (5, 9, 11–13).
The flavin-dependent monooxygenase TetX is a new resistance mechanism against tigecycline, which was detected only in Bacteroides fragilis strains and has not been isolated from any resistant clinical strains at present. The TetX protein can modify the narrow- and expanded-spectrum tetracyclines and requires NADPH, Mg2+, and O2 for its activity (14). TetX is able to accept tigecycline as a substrate as well, so bacterial strains harboring the tetX gene are highly resistant to tigecycline (15). The tetX1 gene is a tetX-orthologous gene and has 66% identity to the tetX gene (15, 16).
In this study, we focused on the roles of various efflux pumps and the tetX1 gene in tigecycline resistance in A. baumannii isolates. Comparisons of molecular and clinical characteristics were also performed between tigecycline-nonsusceptible A. baumannii (TNAB) isolates and tigecycline-susceptible A. baumannii (TSAB) isolates.
MATERIALS AND METHODS
Bacterial strains and patients.From January 2012 to December 2012, a total of 74 nonduplicate A. baumannii isolates were collected in the First Affiliated Hospital, School of Medicine, Zhejiang University, a tertiary care academic medical center with 2,500 beds in use. Bacterial identification was performed by the Vitek 32 system (bioMérieux, France). A case patient was defined as a patient with A. baumannii isolated from clinical specimens during the study period.
Retrospective observational cohort study.We performed a retrospective observational cohort study of TNAB and TSAB patients. Detailed clinical information on case patients was collected from their medical records, and follow-up was performed until discharge from our hospital or death. Isolates identified within the first 72 h after admission were characterized as imported from another hospital, while patients with A. baumannii isolated >72 h after admission were regarded as having received horizontal transmission during the current hospitalization. The clinical and microbiological diagnosis of infections was performed in accordance with the criteria from the Centers for Disease Control and Prevention (CDC)/National Healthcare Safety Network (17). The study was approved by the Institutional Review Board of our hospital.
Antimicrobial susceptibility testing.Susceptibility testing of 19 antimicrobials was performed by the broth microdilution method. Results were interpreted according to the breakpoints suggested by the Clinical and Laboratory Standards Institute (CLSI) (18). With regard to tigecycline, susceptibility/resistance breakpoints were interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria (susceptible, ≤1 mg/liter; and resistant, ≥4 mg/liter) (19). Escherichia coli ATCC 25922 and A. baumannii ATCC 19606 were used as reference strains.
PCR and nucleotide sequencing.The presence of a variety of resistance determinants was conducted by PCR with specific primers as reported previously (13), including primers for efflux system genes (adeA, adeB, adeC, adeR, adeS, adeK, adeJ, adeE, adeF, adeH, and abeM), β-lactamase genes (blaOXA, blaAmpC, blaSHV, blaPER, blaIMP, blaVIM, blaSIM, and blaNDM), and two specific resistance genes (tetX and tetX1). PCR products were sequenced and analyzed with the NCBI BLAST program (www.ncbi.nlm.nih.gov/blast/).
Real-time RT-PCR.The expression levels of the adeB, adeJ, adeG, and abeM genes were assessed using reverse transcription-PCR (RT-PCR). DNase-treated RNA templates were extracted by means of a Qiagen RNeasy kit (Qiagen), and afterward, cDNA was synthesized using a PrimeScript RT-PCR kit (TaKaRa Bio) following the manufacturer's instructions. Real-time PCR assays were performed by using a DNA Engine Opticon 2 real-time PCR detection system (Bio-Rad) with a SYBR Premix Ex Taq kit (TaKaRa Bio). The 16S rRNA gene was used as a housekeeping gene to normalize the expression of target genes. Expression analysis was carried out by determining the relative expression of the mRNA compared with that of A. baumannii ATCC 19606.
Determination of efflux pump activity.Efflux pump activity was determined for each strain. MICs of tigecycline in the presence of the following efflux pump inhibitors (EPIs) were determined by the broth microdilution method: carbonyl cyanide 3-chlorophenylhydrazone (CCCP), phenyl-arginine-β-naphthylamide (PAβN), 1-(1-naphthylmethyl)-piperazine (NMP), reserpine, and verapamil (Sigma). CCCP, PAβN, NMP, reserpine, and verapamil were added to the broth at final concentrations of 5 μg/ml, 70 μg/ml, 100 μg/ml, 50 μg/ml, and 100 μg/ml, respectively. A significant inhibition effect was defined as a 4-fold or greater decrease in the MIC values in the presence of EPIs (13, 20).
Molecular typing by MLST and PFGE. (i) MLST.Multilocus sequence typing (MLST) was performed as described previously (21). Isolates were assigned to sequence types (STs) by using tools on the A. baumannii MLST database (http://pubmlst.org/abaumannii/). The eBURST algorithm (version 3; http://eburst.mlst.net/) was used to assign STs to clonal complexes (CCs).
(ii) PFGE.Chromosomal DNA was prepared from A. baumannii isolates and digested with ApaI (TaKaRa Bio) as reported previously (22). Pulsed-field gel electrophoresis (PFGE) results were interpreted by the criteria of Tenover et al. (23).
Statistical analysis.SPSS 17.0 (SPSS Inc., Chicago, IL) was used for all statistical analyses. Comparative analyses were executed by χ2 or Fisher exact tests for categorical variables and by the Student t test for continuous variables. All tests were 2-tailed, and P values of <0.05 are considered statistically significant.
RESULTS
Clinical characteristics.A total of 74 A. baumannii isolates, including 64 TNAB and 10 TSAB isolates, were identified during the study period. Available medical records were collected from 66 patients. Among them, 56 were confirmed to be TNAB patients, and the rest were TSAB patients. Comparisons of various clinical characteristics between TNAB and TSAB patients are summarized in Table 1. A majority of patients were severely ill, and nearly half of the patients were from the intensive care unit (ICU). Sputum was the most common source of A. baumannii isolates from both TNAB patients and TSAB patients. Most clinical isolates were acquired at our hospital, but 7 (12.5%) TNAB isolates and 1 (10%) TSAB isolate were imported. It is noteworthy that only 7 patients had been given therapies with tigecycline during the hospital stay (six TNAB patients and one TSAB patient).
Univariate analysis of clinical characteristics of patients infected with TNAB and TSAB isolatesa
Antimicrobial susceptibility and detection of β-lactamase genes.Susceptibility profiles of TNAB and TSAB isolates are presented in Table 2. All these A. baumannii isolates were multidrug resistant, and they showed sufficient susceptibility only to colistin (98.4% for TNAB and 100% for TSAB). In all 74 clinical A. baumannii isolates, tigecycline MICs ranged from 0.5 to 8 μg/ml, with a MIC50 of 4 μg/ml and a MIC90 of 4 μg/ml as well. With regard to minocycline, a relatively high percentage of A. baumannii isolates were still susceptible (40.6% for TNAB and 40% for TSAB), whereas all isolates showed high-level resistance to chloromycetin and penicillin antibiotics. However, TNAB exhibited much severer multidrug resistance phenotypes than TSAB. In addition to quinolone antibiotics (1.6% for TNAB versus 20% for TSAB; P < 0.05), TNAB isolates also displayed significantly lower susceptibility rates to imipenem (0% for TNAB versus 30% for TSAB; P < 0.01) and the third-generation cephalosporins (0 to 1.6% for TNAB versus 20 to 30% for TSAB; P < 0.05).
Susceptibility profiles of 64 TNAB and 10 TSAB isolates
All isolates carried the blaOXA-51-like gene, followed by blaOXA-23-like (90.6% in TNAB and 80% in TSAB). None of the 74 isolates had ISAba1 upstream of blaOXA-51-like genes, while the ISAba1 element was found upstream of blaOXA-23-like genes in 21.9% of TNAB isolates and 20% of TSAB isolates. The ampC gene was detected in 37.5% of TNAB isolates and 70% of TSAB isolates, and upstream ISAba1 was found in 12.5% of TNAB isolates and 30% of TSAB isolates. Other genes (blaOXA-24-like, blaOXA-58-like, blaOXA-143-like, blaIMP, blaPER, blaSHV, blaNDM, blaSIM, blaTEM, and blaVIM) were negative in all of these isolates.
Detection of pump genes and tetX and tetX1 genes.adeB was detected in 54 (84.4%) TNAB isolates and 8 (80%) TSAB isolates. However, only 40 (62.5%) TNAB isolates and 6 (60%) TSAB isolates carried the adeABC genes. Among the 40 adeABC-positive TNAB isolates, 34 isolates cocarried the regulatory system adeRS genes, while all 6 adeABC-positive TSAB isolates were positive for the adeRS genes. The adeIJK, adeFGH, and abeM genes were detected in 54 (84.4%), 57 (89.1%), and 61 (95.3%) TNAB isolates, respectively. In contrast, all of them were detected in 7 (70%) TSAB isolates. Specifically, adeE was detected in only one (10%) TSAB isolate, which possessed adeABC simultaneously. No adeE gene was found in TNAB strains. Apart from these pump genes, tetX1 was revealed in 12 (18.8%) TNAB isolates, whereas it was negative in all TSAB isolates. None of these isolates was identified to carry the tetX gene. The PCR products of tetX1 were purified and sequenced.
Assessment of adeABC, adeIJK, adeFGH, and abeM expression.Real-time RT-PCR showed that the mean relative expression of adeB, adeJ, adeG, and abeM was observed to increase to approximately 29-fold, 3-fold, 0.7-fold, and 1-fold higher, respectively, in TNAB isolates (means ± standard deviation [SD], 66.26 ± 82.53, 2.97 ± 4.57, 2.85 ± 10.11, and 4.89 ± 19.58) than in TSAB isolates (2.32 ± 1.18, 3.64 ± 8.25, 0.89 ± 0.82, and 4.38 ± 8.21).
Efflux mechanism.All isolates grew well in the presence of 5 μg/ml CCCP, 70 μg/ml PAβN, 100 μg/ml NMP, 50 μg/ml reserpine, and 100 μg/ml verapamil. The effects of the EPIs on tigecycline MICs are shown in Table 3. When 5 μg/ml CCCP was present, 4- to 8-fold decreases of MICs were observed in 8 (12.5%) TNAB isolates and 3 (30%) TSAB isolates. Addition of PAβN (70 μg/ml) led to 4- to 8-fold reductions of the tigecycline MICs in 7 (10.9%) TNAB isolates and 3 (30%) TSAB isolates. A 4-fold decrease was observed in only one TNAB isolate (1.6%) and one TSAB isolate (10%) after the addition of 50 μg/ml reserpine. However, no obvious inhibitory effects on tigecycline MICs were observed in the presence of NMP and verapamil. In contrast, after the 100-μg/ml verapamil exposure, a 4-fold increase of tigecycline MICs occurred in 10 (15.6%) TNAB isolates and 2 (20%) TSAB isolates.
Reduction in MIC after addition of EPIsa
Molecular typing by PFGE and MLST.Molecular typing and pump genes are shown in Table 4. PFGE analysis showed 19 different pulsotypes (PTs), designated A to S (Fig. 1). Among them, 13 (17.6%) isolates belonged to PTA, 10 (13.5%) belonged to PTB, and 8 (10.8%) belonged to PTC. By MLST, 10 different STs and 8 novel STs (designated STn1 to STn 8) were identified. ST208 and ST191 were the predominant STs, comprising 20 (27.0%) isolates and 19 (25.7%) isolates, respectively. By means of the eBURST algorithm, 65 isolates belonging to ST208, ST191, ST451, ST368, ST381, or STn1-8 were clustered into a clonal complex, with ST208 being the predicted founder. The remaining 9 isolates belonged to 3 singletons (Fig. 2).
STs, PFGE pulsotypes, and distribution of resistance determinants in 64 TNAB and 10 TSAB isolates
PFGE patterns of clinical isolates of A. baumannii in this study, representing all pulsotypes of A. baumannii isolates. Lanes A to S, pulsotypes A to S; m, Salmonella enterica serotype Braenderup strain H9812 marker, digested with XbaI (TaKaRa Bio).
Population snapshot of A. baumannii isolates in this study and existing isolates in the MLST database by use of the eBURST algorithm.
DISCUSSION
Nowadays, due to the wide dissemination of multidrug-resistant A. baumannii (MDRAB) in Chinese hospitals, treatment of A. baumannii infections has become a serious clinical concern. Tigecycline is regarded as the last resort in the control of clinical infections caused by MDRAB. In our study, among the 56 TNAB patients, only 6 patients had a history of tigecycline therapy, while the remaining TNAB patients had never been treated with tigecycline. This phenomenon is in accordance with a previous study that demonstrated that selective pressure caused by other antibiotics may lead to nonsusceptibility to tigecycline (24). Another notable feature was that a higher percentage of TNAB patients than TSAB patients were treated with carbapenems during the last month before A. baumannii was isolated, which is consistent with the observation that use of carbapenems might induce resistance not only to carbapenems but also to many other antibiotics, including tigecycline. This means that carbapenems might be a potent inducer of multidrug resistance in A. baumannii and therefore should be used in a more reasonable and effective way to reduce the spread of MDRAB (25).
It is reported that reduced susceptibility to tigecycline appears to be mediated in part by the active efflux systems (5, 9, 12, 20, 26). In our study, a majority of both TNAB and TSAB isolates were demonstrated to carry adeABC, adeIJK, adeFGH, and abeM genes. These results were contrary to a previous observation that adeABC genes were not expressed in tigecycline-susceptible A. baumannii strains but were detected only in MDR isolates (27). A possible explanation is that these efflux systems are intrinsic in A. baumannii and that A. baumannii isolates might become multidrug resistant by overexpression of these efflux pumps when exposed to their substrates. The comparisons of expression levels of adeB between TNAB and TSAB isolates indicated that overexpression of the AdeABC efflux pump is a prevalent mechanism in A. baumannii that led to decreased susceptibility to tigecycline (5, 9). However, no statistically significant differences were found in the mean expression levels of adeJ, adeG, and abeM between TNAB and TSAB isolates, which suggested that expression levels of the adeJ, adeG, and abeM genes did not correlate with tigecycline resistance. Nevertheless, we could not exclude the possibility that the adeIJK, adeFGH, and abeM efflux pumps might play a part role in tigecycline resistance.
Although a mean 29-fold higher level of adeABC expression was observed in TNAB isolates than in TSAB isolates, which is in contrast to the report by Bratu et al. (20), we found no obvious correlation between the MICs of tigecycline and the expression levels of adeB in our study (data not shown). This might suggest that other mechanisms of tigecycline resistance can be involved. Hou et al. described that the adeE gene coexists with the adeB gene in a small number of A. baumannii isolates (13). In our study, one TSAB isolate was identified as cocarrying adeB, adeF, and adeE genes simultaneously.
Apart from the active efflux systems, the tetX gene and its orthologous tetX1 gene can contribute partly to decreased tigecycline susceptibility as well (14–16). Although the presence of tetX and tetX1 genes is responsible for elevated MICs of tigecycline, the direct predictive value for tigecycline resistance could be observed only for the presence of the tetX1 gene (16). In this study, the tetX1 gene was detected in 12 TNAB isolates, indicating that the tetX1 gene might also play a role in reduced tigecycline susceptibility in clinical A. baumannii isolates.
In the present study, we used five EPIs to assess the activity of efflux pumps. EPIs are drugs that can reverse resistance patterns by blocking bacterial pumps and preventing discharge of certain antibiotics. In our study, PAβN and CCCP partially restored susceptibility in both TNAB and TSAB isolates, which could indirectly prove the overexpression of efflux pumps and also indicated that other mechanisms might account for tigecycline resistance in A. baumannii isolates (5). Moreover, the strains affected by CCCP were not affected by PAβN (data not shown). A possible explanation is that CCCP and PAβN have different specificities toward various efflux pumps. Meanwhile, with the addition of CCCP or PAβN, no significant reduction in MICs of tigecycline was observed between the TNAB and TSAB isolates, further indicating that these efflux pumps on which CCCP and PAβN act may be intrinsic in A. baumannii species. Verapamil has been reported to inhibit several bacterial ABC efflux pumps in Mycobacterium tuberculosis isolates (28). In this study, the MICs of tigecycline were elevated in some A. baumannii isolates in the presence of verapamil, which indicated that verapamil acted as an antagonist against tigecycline. In accordance with a previous report (24), in our study, NMP did not achieve a significant reduction in tigecycline MICs either, so a combination of tigecycline and NMP should not be suggested. Reserpine was reported to decrease the MICs of nalidixic acid, ciprofloxacin, and norfloxacin in A. baumannii isolates (29), whereas it had few effects on the MICs of tigecycline in the present study. This is concordant with the suggestion that as different antibiotics may have different binding sites on the efflux pump, the EPIs might interfere with them in diverse manners (30).
In the present study, MLST and PFGE were performed for molecular typing. ST208 and ST191 were the most prominent sequence types, which is not completely concordant with previous observations that ST92 was the dominant clone of A. baumannii isolates in mainland China (31–34). However, both ST208 and ST191 are single-locus variants (SLVs) of ST92. It has been reported that ST92 is found in more than 30 countries (35), and isolates belonging to ST92 which are resistant to all available antimicrobial agents, including colistin, polymyxin B, and tigecycline, have been found in South Korea (36). In our study, ST208 and ST191 isolates were all multiresistant isolates as well. It is noteworthy that 3 TNAB isolates (2 belonging to ST208 and 1 belonging to ST191) were imported from other local hospitals, indicating the emergence of interhospital dissemination of MDR A. baumannii. In addition, both ST208 and ST191 belong to European clone II and clonal complex 92 (CC92) (34, 37). CC92 is the most widely distributed clonal complex worldwide, encompassing over 50 STs identified from many countries, including several from China (31–35). The results of our study suggest that CC92 also plays an important role in A. baumannii infections in our hospital.
PFGE and MLST showed similar discriminatory powers (21, 38). However, the PFGE profiles did not fully correlate with the MLST results in the present study. This indicates that there has been a more rapid increase in the genetic diversity indexed by PFGE than the changes of housekeeping genes applied for MLST under selective pressure of antimicrobial agents. Another explanation is the persistent high-level endemicity of A. baumannii in our hospital, which would undoubtedly lead to various PFGE profiles of A. baumannii isolates during their dissemination, while the relatively conserved housekeeping genes for MLST might suffer rather fewer effects.
Our results are concordant with the observations that the blaOXA-23 carbapenemase gene with upstream ISAba1 is disseminated worldwide and is also prevalent in China (31–33).
Conclusions.In conclusion, we have documented the emergence and wide dissemination of tigecycline-nonsusceptible A. baumannii isolates in a Chinese university hospital. The diffusion of tigecycline-nonsusceptible A. baumannii isolates is attributable mainly to the spread of CC92. Efflux-mediated mechanisms, including high-level expression of AdeABC, seem to play an important role in reduced tigecycline susceptibility, while active AdeIJK, AdeFGH, and AbeM did not appear to be a major reason for tigecycline resistance. Both of the putative EPIs, PAβN and CCCP, could partially reverse the resistance to tigecycline, while the remaining EPIs had no obvious effects. This is concordant with the view that different antimicrobial agents have various binding sites on efflux pumps, so these EPIs might be active in diverse manners (30). Last but not least, to our knowledge, this is the first report that the tetX1 gene was detected in tigecycline-nonsusceptible A. baumannii isolates.
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (grant 30972592) and the Chinese High Tech Research & Development (863) Program (grant 2011AA020104).
We thank the Acinetobacter baumannii MLST website (http://pubmlst.org/abaumannii/) at the University of Oxford for coding our MLST alleles and profiles.
FOOTNOTES
- Received 12 August 2013.
- Returned for modification 21 September 2013.
- Accepted 10 October 2013.
- Accepted manuscript posted online 28 October 2013.
- Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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