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Introduction

Acinetobacter baumannii (A. baumannii) has emerged as a major pathogen of nosocomial infections and is associated with high rates of morbidity and mortality in recent years (1,2). A nationwide surveillance program, including hospitals from 14 geographically different regions in China revealed that the ratio of A. baumannii is on the increase annually (3).

Carbapenem has good antibacterial activity against A. baumannii and was the first choice in treatment of infection caused by A. baumannii in the past years (4). However, the emergence of resistance to carbapenem was reported in 1991 (5), followed by similar reports from different parts of the world (6,7). In China, 57 and 61% of Acinetobacter spp. (A. baumannii accounted for 89.6%) showed resistance to imipenem and meropenem, respectively (3). International studies in China as well as in other parts of the world focused only on evaluating the resistance of A. baumannii to various antimicrobials (8–10). However, to the best of our knowledge, few studies have investigated the molecular mechanism underlying drug resistance. Additionally, no data are available on the epidemiological characteristics of imipenem-resistant A. baumannii in Shanghai.

Thus, A. baumannii clinical isolates were collected from three tertiary hospitals in Shanghai and their drug resistance pattern to a spectrum of antimicrobials, molecular mechanisms (including carbapenemase, efflux pumps and membrane proteins) behind their resistance and multilocus sequence analysis (MLST) were analyzed to assess their molecular epidemiology.

Materials and methods

Bacterial strains

During the period July, 2011 to June, 2012, 46 non-duplicate imipenem-resistant A. baumannii strains were collected from three tertiary hospitals located in Shanghai, China.

Reconfirmation of strains

The collected strains were subjected to gram staining, biochemical tests, and recA gene and 16S-23S rRNA gene intergenic spacer region to reconfirm them as A. baumannii (7).

Antimicrobial susceptibility and efflux phenotype tests

The collected A. baumannii isolates were subjected to an antimicrobial susceptibility test against imipenem, meropenem, amikacin, piperacillin, ceftazidime, cefotaxime, mino-cycline, ciprofloxacin, ampicillin/sulbactam, sulbactam, cefoperazone/sulbactam, piperacillin/tazobactam, colistin, tigecycline and trimethoprim/sulfamethoxazole using agar dilution method. Escherichia coli strain ATCC25922 and Pseudomonas aeruginosa (P. aeruginosa) strain ATCC27853 were used as reference strains.

Strains in which efflux pump operation was detected by agar dilution method where imipenem- and meropenem-resistant isolates were cultured in Mueller-Hinton agar contained the efflux pump inhibitor phenylalanine-arginine β-naphthylamide (PAβN) at a final concentration of 20 mg/l (11,12). A ≥4-fold reduction of imipenem or meropenem minimal inhibitory concentrations (MICs) in the presence of PAβN possessed an operating drug efflux pump.

Analysis of genes responsible for drug resistance, drug efflux and outer membrane protein

Polymerase chain reaction (PCR) was performed for the genes, blaKPC, blaIMP, blaNDM, blaOXA-51, blaOXA-23, blaOXA-24, blaOXA-58, blaSHV, blaGIM and blaVIM, CarO, adeA, adeB, adeC, adeS and adeR. Thus, obtained amplicons were subjected to sequencing analysis.

A fresh and pure bacterial colony was suspended in distilled water and boiled at 100°C for 15 min. After centrifu-gation at 8,000 × g for 15 min, 1 µl of the supernatant was used for PCR analysis with the primers (Table I). PCR was performed in a total volume of 50 µl containing 0.25 µl Taq DNA polymerase (Takara Bio, Inc., Tokyo, Japan), 5 µl 10X PCR buffer (Mg2+ Plus), 4 µl dNTP mixture (2.5 mM each), 2.5 µl DNA template, 1 µl of each primer (20 µM), and 36.25 µl ddH2O. The PCR thermal cycle consisted of initial denaturation at 94°C for 5 min, followed by 30 cycles of 94°C for 30 sec, annealing 55°C for 1 min and 72°C for 1 min and a final extension at 72°C for 7 min. The PCR products were electrophoresed in 1% agarose gel and visualized under ultraviolet light, and subsequently sequenced (Sangon Biotech Co., Ltd., Shanghai, China).

Table I Gene-specific primers used in this study.

Table I

Gene-specific primers used in this study.

Genes	Primer sequences
recA	F: CCTGAATCTTCTGGTAAAAC R: GTTTCTGGGCTGCCAAACATTAC
ITS	F: CATTATCACGGTAATTAGTG R: AGAGCACTGTGCACTTAAG
blaKPC	F: TTACTGCCCGTTGACGCCCAATCC R: TCGCTAAACTCGAACAGG
blaIMP	F: AACCAGTTTTGCCTTACCAT R: CTACCGCAGCAGAGTCTTTG
blaNDM	F: CCGCCCAGATCCTCAACT R: ATCAGGCAGCCACCAAAA
blaOXA-51	F: TAATGCTTTGATCGGCCTTG R: TGGATTGCACTTCATCTTGG
blaOXA-23	F: GATCGGATTGGAGAACCAGA R: ATTTCTGACCCATTTCCAT
blaOXA-24	F: GGTTAGTTGGCCCCCTTAAA R: AGTTGAGCGAAAAGGGGATT
blaOXA-58	F: AAGTATTGGGGCTTGTGCTG R: CCCCTCTGCGCTCTACATAC
blaSHV	F: GGTTATGCGTTATATTCGCC R: TTAGCGTTGCCAGTGCTC
blaGIM	F: AGAACCTTGACCGAACGCAG R: ACTCATGACTCCTCACGAGG
blaVIM	F: TCCACGCACTTTCATGACGA R: AGACGTGCGTGACAACTCAT
adeA	F: GAAATCCGTCCGCAAGTC R: ACACGCACATACATACCC
adeB	F: AAAGACTTCAAAGAGCGG R: TCACGCATTGCTTCACCC
adeC	F: ATTTCAGGTCGTAGCATT R: CTTGATAAGTAGAGTAGGGATT
adeS	F: ACTGTTATCTTCTGTGGCTGTA R: GTGGACGTTAGGTCAAGTTCTG
adeR	F: AAACGGTTGGGAAGTATTA R: ATGGCTATCTACGGTTCG
CarO	F: AAGGAGAAAACGATGA R: TTATTACGTGGTTATGG
gltA	F: AATTTACAGTGGCACATTAGGTCCC R: GCAGAGATACCAGCAGAGATACACG
gyrB	F: TGAAGGCGGCTTATCTGAGT R: GCTGGGTCTTTTTCCTGACA
gdhB	F: ACCACATGCTTTGTTATG R: GTTGGCGTATGTTGTGC
recA	F: CCTGAATCTTCYGGTAAAAC R: GTTTCTGGGCTGCCAAACATTAC
cpn60	F: GGTGCTCAACTTGTTCGTGA R: CACCGAAACCAGGAGCTTTA
Gpi	F: GAAATTTCCGGAGCTCACAA R: TCAGGAGCAATACCCCACTC
rpoD	F: ACCCGTGAAGGTGAAATCAG R: TTCAGCTGGAGCTTTAGCAAT

MLST

Seven housekeeping genes including homologous recombination factor (recA), citrate synthase (gltA), DNA gyrase subunit (gyrB), glucose-6-phosphate isomerase isomerase (gpi), glucose dehydrogenase B (gdhB), 60-kDa chaperonin (cpn60), and RNA polymerase 70 factor (rpoD) were amplified in PCR using relevant primers (Table I) and appropriate thermal conditions. The amplicons were sequenced and the sequences were submitted to the MLST database (http://pubmlst.org.net) to compare them with sequences submitted from other parts of the world. Each strain was then characterized by a pattern of numbers defining its allelic profile.

Results

Antimicrobial susceptibility

A. baumannii resistant to imipenem simultaneously showed resistance to several other common antimicrobials. The resistance rate was >80% for all the antimicrobials except minocycline and colistin. Antibiotic susceptibility of the 46 clinical isolates is shown in Table II. Thirteen imipenem-resistant A. baumannii isolates were positive for efflux pump.

Table II The drug-resistant rates of imipenem-resistant Acinetobacter baumannii.

Table II

The drug-resistant rates of imipenem-resistant Acinetobacter baumannii.

Drug	Resistance rate No. of resistant strains (%)
Meropenem	41 (89)
Amikacin	38 (83)
Piperacillin	46 (100)
Ceftazidime	46 (100)
Minocycline	34 (74)
Ciprofloxacin	45 (98)
Ampicillin/sulbactam	43 (93)
Piperacillin/tazobactam	46 (100)
Colistin	1 (2)
Trimethoprim/sulfamethoxazole	43 (93)
Cefotaxime	45 (98)

Detection of genes involved in drug resistance, drug efflux and outer membrane protein

Of the various drug resistance genes tested, blaOXA-51 was present in 46 isolates, blaOXA-23 gene was present in 44 isolates and blaNDM gene was found in only one strain. Other drug-resistant genes including blaKPC, blaIMP, blaOXA-24, blaOXA-58, blaSHV, blaGIM and blaVIM were not detected in the isolates.

Of the five genes associated with the drug efflux pump tested, all five were found to be present in the isolates. Several mutations were found in the sequences of adeS gene in isolates with efflux phenotype. Differences were observed at three places when nucleotide sequences were translated into an amino acid sequence. This amino acid sequence was then compared to the amino acid sequence of the reference strain ATCC17978 (Fig. 1).

Figure 1 (A) Comparison of nucleotide sequence of adeS gene between resistant strain and reference strain ATCC17978. (B) Comparison of amino acid sequence of adeS gene between resistant strain and reference strain ATCC17978.

Similarly, the nucleotide sequence of the outer membrane protein encoding gene CarO, when compared with the nucleotide sequence of reference strain ATCC17978, harbored mutations that were reflected in the amino acid sequence (Fig. 2).

Figure 2 (A) Comparison of nucleotide sequence of CarO gene between resistant and reference strain ATCC17978. (B) Comparison of amino acid sequence of CarO gene between resistant and reference strain ATCC17978.

Genotyping of isolates by MLST

The MLST analysis revealed that the isolates were clustered in 11 different genotypes or STs. The ST208 genotype was shared by the majority of isolates (58.7%, 27/46), followed by ST191 (10.9%, 5/46) and ST451 (6.5%, 3/46). We also detected some other STs shared by certain isolates such as ST75 (2.1%, 1/46), ST90 (4.2%, 2/46), ST92 (2.1%, 1/46), ST108 (2.1%, 31/46), ST109 (2.1%, 1/46), ST172 (2.1%, 1/46), ST368 (4.2%, 2/46) and ST69 (4.2%, 2/46). These STs were grouped into the three clonal complexes, CC92, CC109 and CC28.

Discussion

A. baumannii develops resistance to imipenem through a variety of mechanisms. Carbapenemase is an important factor responsible for imipenem resistance. In the present study, common carbapenemases were detected in the isolates, including blaOXA-51, blaOXA-23, blaOXA-24, blaOXA-58, blaKPC, blaIMP, blaSHV, blaGIM, blaNDM and blaVIM. OXA-type enzymes are naturally present in Acinetobacter spp. and are usually expressed in small amounts (13). The expression of such genes is markedly higher under the effect of a strong promoter (insertion sequence ISAba1 is the most shared) and induce drug resistance only when combined with a reduction in outer membrane permeability and/or activation of the efflux pump (14). In the present study, blaOXA-51 and blaOXA-23 genes were prevalent among the isolates, results that are consistent with other reports (15–17). Carbapenemases that are different from OXA, such as KPC, IMP, SHV, GIM, NDM and VIM have strong carbapenem-hydrolysing activity (14). However, such types of carbapenemases were rarely detected in A. baumannii. The blaNDM gene was identified in only one strain while the remaining resistant genes were not detected. NDM was first identified in Escherichia coli and Klebsiella pneumoniae in 2008 in India (18). This finding was followed by reports on NDM-producing P. aeruginosa, Enterobacter cloacae, Citrobacter freundii and Enterococcus faecium (19–23). In China, NDM-producing A. baumannii was first reported in 2011. Of the anti microbials tested one NDM-positive isolate in the present study was identified that was multidrug-resistant, and only susceptible to amikacin, colistin and minocycline.

Drug efflux systems including AdeABC, AdeIJK, AdeDE and AdeXYZ (RND family) have been found in A. baumannii. Of these, the AdeABC efflux system is common in A. baumannii (24). This efflux pump, together with other resistant mechanisms, can lead to high-level imipenem resistance. Although mediated by the substrate, its expression may increase when a single point mutation occurs in the adeR or adeS gene (25). PAβN was proven to be an effective inhibitor of drug efflux. In the present study, adeA, adeB and adeC were present in all of the isolates because when PAβN was added the MICs inherent to imipenem in 13 isolates were decreased. The adeS gene differed from the adeS of standard strain and this is the possible reason for increased drug efflux associated with drug resistance.

Few studies concerning the impact of changes on membrane proteins in A. baumannii are available. In 2002, a laboratory in Argentina advocated for the first time that inducible resistance by imipenem can trigger loss of a 29-kDa membrane protein. In 2005, the same laboratory furthering their study, demonstrated that the outer membrane protein is encoded by the CarO gene and when there is an insertion mutation or any other mutation in the CarO gene makes it off and thus the strain become resistant to certain drugs (26). In the present study, the sequence of CarO gene had nucleotide insertions, deletions and point mutations in comparison with the standard strains and there were also differences in their nucleotide and amino acid sequences.

In summary, for a global epidemiologic analysis, a comparison of the results between different laboratories is required. MLST is a powerful tool used to transfer typing data and compare results via relevant databases. The MLST analysis revealed that the major epidemic clone of A. baumannii in Shanghai was ST208 (CC92 clone complex), which differed from the results obtained in other regions in China (27).

Acknowledgments

The project was supported by a grant from the Natural Science Foundation of Shanghai Science and Technology Committee (no. 12ZR1426200), the Medical Guide Program of Shanghai Science and Technology Committee (no. 14411962900), Key project of Shanghai Municipal Health and Family Planning Commission (no. 201540367) and Central Universities Basic Research Program (no. 1511219024).

References