A Case of IMP-4-, OXA-421-, OXA-96-, and CARB-2-Producing Acinetobacter pittii Sequence Type 119 in Australia

Abstract

An IMP-4-producing Acinetobacter pittii strain coproducing oxacillinases was isolated from a leg wound of a 67-year-old female patient. Identification to the species level by rpoB and gyrB sequencing and multiplex-PCR-based analysis revealed that the isolate was A. pittii. Whole-genome sequencing of this A. pittii isolate determined the presence of bla_OXA-96, bla_CARB-2, and a novel bla_OXA-421 gene. The position of this novel bla_OXA-421 gene was similar to that of bla_OXA-51 in A. baumannii, downstream of the phosphinothricin N-acetyltransferase gene and upstream of fxsA in the chromosome. This A. pittii isolate was found to belong to sequence type 119 (ST119). Here, we report the first isolation of IMP-4-producing A. pittii ST119 with a novel bla_OXA-421 gene from a patient in Australia and characterize its draft genome.

CASE REPORT

A 67-year-old diabetic woman suffered a fall leading to a displaced distal spiral tibial plateau fracture. In the weeks prior to the fall, she had received multiple antimicrobials (clindamycin, lincomycin, cephalexin, ciprofloxacin, and ceftazidime) for an infected hematoma of the breast and a series of lower respiratory tract infections. The patient underwent definitive repair of the fracture but postoperatively developed osteomyelitis. Debridement of the leg wound was performed. Acinetobacter species and vancomycin-resistant Enterococcus strains were isolated from the tissue removed. This Acinetobacter species (CR12-42) was carbapenem resistant. Despite ongoing antibiotic treatment, the patient's leg required amputation in March 2013, after continuous inflammation, infections for more than 5 months, and an episode of severe Clostridium difficile infection resulting in colectomy. The leg infection was resolved by the amputation.

The initial identification of this Acinetobacter species was done by Vitek 2. Antimicrobial susceptibility testing by Vitek 2 (bioMérieux) showed resistance to carbapenems, ceftazidime, ceftriaxone, cefepime, gentamicin, tobramycin, trimethoprim-sulfamethoxazole, ticarcillin-clavulanic acid, and ciprofloxacin according to the EUCAST standard (1). The isolate was referred to our laboratory at the University of Queensland Centre for Clinical Research. The Acinetobacter isolate was identified to the species level by a gyrB multiplex PCR, which revealed that CR12-42 was Acinetobacter pittii (2). Partial rpoB sequencing (3) confirmed that CR12-42 was A. pittii.

Phenotypic characterization to determine the class of carbapenemase was performed as previously described (4–6). The A. pittii isolate showed a metallo-β-lactamase phenotype by producing a larger inhibition zone around carbapenem disks with EDTA than around carbapenem disks alone (>5-mm breakpoint increase in the size of the inhibition zone). The isolate also produced a positive result in the modified Hodge and Carba NP tests for carbapenemase production. MICs were determined with Etest (bioMérieux). The isolate was resistant to all of the carbapenems tested, i.e., ceftazidime, cefotaxime, cefepime, cefoxitin, ticarcillin-clavulanic acid, trimethoprim-sulfamethoxazole, and ciprofloxacin (Table 1). Interestingly, this A. pittii isolate was susceptible to tetracycline, minocycline, colistin, and tigecycline (Table 1).

TABLE 1.

MICs of antimicrobials for A. pittii CR12-42 as determined by Etest

Antimicrobial(s)	MIC (mg/liter)	Interpretationa
Ertapenem	>32	Resistant
Imipenem	24	Resistant
Meropenem	12	Resistant
Doripenem	>32	Resistant
Cefepime	64	Resistant
Ceftazidime	>256	Resistant
Cefotaxime	>32	Resistant
Ceftriaxone	>32	Resistant
Cefuroxime	>256	Resistant
Cefoxitin	>256	Resistant
Piperacillin-tazobactam	12	Resistant
Ampicillin-sulbactam	2	Susceptibleb
Ticarcillin-clavulanic acid	256	Resistant
Piperacillin	>256	Resistant
Amikacin	12	Intermediate
Gentamicin	>256	Resistant
Netilmicin	24	Resistant
Ciprofloxacin	3	Resistant
Tetracycline	0.75	Susceptibleb
Minocycline	0.023	Susceptibleb
Trimethoprim-sulfamethoxazole	>32	Resistant
Colistin	0.094	Susceptible
Tigecycline	0.094	Susceptible

^aUnless noted otherwise, MIC interpretations are based on EUCAST criteria (1).

^bAmpicillin-sulbactam, tetracycline, and minocycline MIC interpretations are based on CLSI criteria (33).

Carbapenem resistance in Acinetobacter species is commonly associated with the presence of carbapenem-hydrolyzing class D β-lactamase- or oxacillinase-encoding genes such as bla_OXA-23 and bla_OXA-51 in Acinetobacter baumannii (7, 8). A PCR assay and sequencing for all of the bla_OXA genes frequently present in Acinetobacter species, i.e., bla_OXA-23-like, bla_OXA-51-like, bla_OXA-40-like, and bla_OXA-58-like, were performed (7–9). The isolate was positive for the bla_OXA-58-like subclass and negative for other subclasses of bla_OXA. A PCR assay for ISAba1, the common insertion element in A. baumannii, was also negative. A PCR assay and sequencing for other carbapenemase-encoding genes (10, 11), i.e., bla_IMP, bla_NDM, bla_KPC, and bla_VIM, were positive for bla_IMP-4. A prepared pair-ended library of the whole genomic DNA was sequenced via Illumina MiSeq to further characterize the resistance mechanisms of A. pittii CR12-42 and to analyze its genome.

Whole-genome DNA sequencing produced a total of 138,932,382 paired-end reads with 30× average coverage. We used the CLC genomic workbench version 7.5 (CLC Bio, Aarhus, Denmark) for de novo assembly with a 500-bp minimum threshold resulting in 127 contigs. The draft genome consisted of 4,372,178 nucleotides and was annotated by rapid annotations using subsystems technology (RAST) (12). RAST annotation showed that Acinetobacter calcoaceticus PHEA-2 (score, 503) and Acinetobacter sp. strain SH024 (score, 436) are the two closest neighbors of A. pittii CR12-42. Our isolate was related to only one other A. pittii strain, TG6411, but with a lower score of 221. A total of 13 A. pittii draft genomes have been described in the BioProject (http://www.ncbi.nlm.nih.gov/bioproject/); however, draft genomes of only three isolates were published, including one draft genome of an NDM-1-producing A. pittii strain from China (13).

In silico identification of CR12-42 to the species level by using rpoB and gyrB showed it to be 100% identical to A. pittii. A. pittii belongs, together with Acinetobacter nosocomialis, within the A. calcoaceticus-baumannii complex and was formerly named Acinetobacter genomic species 3 (14). In silico analysis of A. baumannii multilocus sequence typing (MLST) by the Pasteur scheme (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Abaumannii.html) identified A. pittii CR12-42 as being of sequence type 119 (ST119). The alleles found were cpn-60 (n = 36), fusA (n = 20), gltA (n = 38), pyrG (n = 16), recA (n = 38), rplB (n = 18), and rpoB (n = 20). It has been reported that MLST by the Pasteur scheme is capable of providing the ST of A. pittii (15). The clinical significance of A. pittii ST119 is indicated by the fact that it has been reported to be the predominant clone among the A. pittii strains (18 out of 25) isolated in four hospitals in Japan (16). Interestingly, these Japanese A. pittii isolates possessed a different bla_IMP variant, bla_IMP-19 (16). Of note, A. pittii ST119 has not been reported previously in Australia.

The resistance genes were screened with ResFinder (17). The β-lactamase-encoding genes bla_IMP-4, bla_OXA-96, and bla_CARB-2 were identified. bla_OXA-96 has a single nucleotide difference (a guanine-for-adenine substitution at position 483) from bla_OXA-58. bla_OXA-96 had been reported within an A. baumannii isolate from Singapore that also harbored bla_OXA-23 and bla_OXA-64 (18). In our isolate, bla_OXA-96 had a genetic context similar to that of bla_OXA-58, which was bracketed by ISAba3 (GenBank accession number JX968506) (Fig. 1).

FIG 1.

Genetic contexts of the four β-lactamase-encoding genes in A. pittii CR12-42.

In addition, a novel bla_OXA gene, bla_OXA-421, was identified (Fig. 1). This gene had a genetic environment identical to that of the chromosomal bla_OXA-51 gene in A. baumannii (19), which includes two genes that are usually present upstream and downstream of bla_OXA-51 in A. baumannii, the phosphinothricin N-acetyltransferase-encoding gene and fxsA, respectively. bla_OXA-421 has 95% identity with the previously reported bla_OXA gene (GenBank accession number CP002177, locus tag BDGL_000903) from the genome of A. calcoaceticus PHEA-2 (20), which is the closest neighbor of our CR12-42 isolate, as previously mentioned. The second closest relative of bla_OXA-421 was bla_OXA of Acinetobacter oleivorans, with 89% similarity (GenBank accession number CP002080, locus tag AOLE_1170) (21). The other bla_OXA genes similar to bla_OXA-421 were bla_OXA-324, bla_OXA-325, bla_OXA-326, bla_OXA-332, and bla_OXA-354 (88 to 89% similarity), which were recently identified in A. calcoaceticus (22). The carbapenemase activity of OXA-421 warrants further investigation.

The bla_IMP-4 gene in A. pittii CR12-42 was located inside a class 1 integron. Downstream from bla_IMP-4 were qacG2 and the aminoglycoside and chloramphenicol resistance genes aacA4 and catB2 (Fig. 1). This genetic context of bla_IMP-4 in CR12-42 was found to be identical to that in an IMP-4-producing A. baumannii strain from Singapore (GenBank accession number DQ532122) (18). bla_IMP-4 has also been reported in Acinetobacter junii from Australia; however, the genetic context was not characterized (23). Our genetic context was also similar to that of bla_IMP-4 in the IncHI2-type plasmid from Enterobacter cloacae and Escherichia coli and the IncL/M plasmid carrying bla_IMP-4 in an E. cloacae strain from Australia (24, 25). However, the plasmid backbone of these sequences could not be identified within our draft genome. Further investigation is needed to determine if bla_IMP-4 is located on a plasmid or the chromosome of CR12-42.

A carbenicillinase gene, bla_CARB-2 was identified with ResFinder. bla_CARB-2, which was also designated bla_PSE-1, was first reported in Pseudomonas aeruginosa (26). The genetic context of bla_CARB-2 in CR12-42 was also potentially a class 1 integron with a truncated integrase (intI1) located upstream of bla_CARB-2 (Fig. 1). Other resistance genes found in this strain included sul1 (sulfonamide resistance), msr(E) and mph(E) (macrolide resistance), and aac-3-IId (aminoglycoside resistance). Consistent with this, the A. pittii strain was resistant to gentamicin and tobramycin but susceptible to amikacin. Of note, no 16S rRNA methylase was found in this isolate.

Regardless of its resistance to multiple antimicrobials, A. pittii CR12-42 remained susceptible to tetracycline and minocycline, which was consistent with the absence of a tetracycline resistance gene within the draft genome. In addition, the MIC of ampicillin-sulbactam remained low (2 mg/liter), despite the presence of multiple carbapenemase-encoding genes. Further, sulbactam is known to have activity against A. baumannii (27). In a study by Higgins et al., the ampicillin-sulbactam MIC₅₀ of 115 A. baumannii strains was 2 mg/liter (27). Ampicillin-sulbactam susceptibility was also shown in the majority of the previously reported A. pittii ST119 strains harboring bla_IMP-19 (94%) in Japan (16). In addition, 94% of these were susceptible to minocycline, similar to the antimicrobial phenotype of CR12-42 (16). Apart from the difference in bla_IMP variants, CR12-42 has an antimicrobial phenotype and genotype identical to those of A. pittii ST119 from Japan.

IMP-producing Enterobacteriaceae strains have been frequently reported in Australia. Although OXA-23-like is the main subclass of carbapenemases identified in A. baumannii, IMP-4 is occasionally identified in A. pittii in locations such as Hong Kong and Singapore (18, 28). Other variants of bla_IMP, such as bla_IMP-1, bla_IMP-8, bla_IMP-11, and bla_IMP-19, have been described in A. pittii in Southeast Asia (16, 29, 30). A. pittii has also recently been reported to produce NDM (31, 32).

Generally, A. baumannii is considered the most important and the most prevalent Acinetobacter species causing infections. However, A. pittii has caused hospital outbreaks in The Netherland and China (32, 33) and was reported as the most common Acinetobacter species causing nosocomial infections in Germany (34). Our study illustrates the emergence of a multidrug-resistant A. pittii strain in Australia. Therefore, accurate identification to the species level and characterization of the prevalence of A. pittii among the Acinetobacter species isolated in our region and its antibiotic resistance warrant further investigation.

This work was approved by the Royal Brisbane and Women's Hospital Human Research Ethics Committee (HREC/13/QRBW/391: epidemiology, clinical significance, treatment, and outcome of infections by carbapenem-resistant Enterobacteriaceae and Acinetobacter species in Queensland). This project is registered as BioProject PRJNA255268 and BioSample SAMN03003652.

Nucleotide sequence accession numbers.

The GenBank accession number of bla_OXA-421 is KM401566. The GenBank accession number of the draft genome of A. pittii CR12-42 is JQNT00000000.