ABSTRACT
Five OXA-232 carbapenemase-producing Klebsiella pneumoniae isolates, belonging to the pandemic clone sequence type 15 (ST15), were isolated from neonates and coproduced blaCTX-M-15 and blaSHV-1 genes. All isolates were resistant to ertapenem (MICs of >32 μg/ml) and meropenem (MICs of 4 to 8 μg/ml) and susceptible or intermediate to imipenem (MICs of 1 to 2 μg/ml). The blaOXA-232 gene was located on a ColE-type transformable plasmid of 6,141 bp. To the best of our knowledge, this is the first report of OXA-232 carbapenemase among clinical isolates in China.
KEYWORDS: Klebsiella pneumoniae, OXA-232, ST15, clonal dissemination, neonates
TEXT
Carbapenemase-producing Enterobacteriaceae isolates are usually extensively drug resistant, and infections caused by these pathogens present a serious clinical challenge, especially for pediatric patients (1). Based on the data of the CHINET antimicrobial resistance surveillance program for 2005 to 2014, the rates of Klebsiella pneumoniae resistance to imipenem and meropenem increased markedly, from 1.3% to 14.6% and from 0% to 15.2%, respectively (2). Production of Klebsiella pneumoniae carbapenemase (KPC), NDM-1-type metallo-β-lactamase, and oxacillinase is the main mechanism underlying resistance to carbapenems. The OXA-48 carbapenemase, which characteristically is highly resistant to ertapenem, was initially identified in 2001 in K. pneumoniae isolates from Istanbul, Turkey; since then, many variants of blaOXA-48 have been described, including blaOXA-162 from Turkey, blaOXA-163 from Argentina, blaOXA-181 from India, blaOXA-204 from Tunisia, and blaOXA-232 from France and Singapore (3–9). Here we report a small clonal dissemination of OXA-232 carbapenemase-producing K. pneumoniae clinical isolates, representing the first detection in China of Enterobacteriaceae clinical strains bearing blaOXA-232.
Five nonduplicate clinical strains of K. pneumoniae were isolated in April to June 2016 from five patients in the neonatal intensive care unit (NICU) of a university children's hospital in Shanghai, China. The presence of β-lactamase genes was confirmed by specific PCR and sequencing, as described previously (10–12). All K. pneumoniae isolates were positive for blaOXA-232, blaSHV-1, and blaCTX-M-15, belonging to a single pulsed-field gel electrophoresis (PFGE) type identified as sequence type 15 (ST15) by multilocus sequence typing (MLST) (Fig. 1A). The outer membrane porins (OMPs) were analyzed by SDS-PAGE (13), which revealed the presence of two main OMPs, OmpK35 and OmpK36 (Fig. 1B), in the five stable ertapenem-resistant isolates.
FIG 1.
(A) DNA fingerprinting of five OXA-232 carbapenemase-producing K. pneumoniae strains by PFGE. (B) OMP analysis of five OXA-232 carbapenemase-producing K. pneumoniae strains by SDS-PAGE.
Transformants harboring the plasmid with blaOXA-232 were selected on Mueller-Hinton agar containing 50 μg/ml ampicillin, with blaOXA-232 determination by PCR and DNA sequencing. All positive transformants were negative for blaSHV-1 and blaCTX-M-15. Plasmid DNA sequencing localized blaOXA-232 on a small plasmid of 6,141 bp, belonging to the ColE type. This targeted plasmid contained nine open reading frames, i.e., MobA, MobB, MobD, ΔMobC, ΔISEcp1, blaOXA-232, ΔLysR, ΔEreA, and RepA (Fig. 2). The results of antimicrobial susceptibility testing indicated that all clinical isolates were susceptible or intermediate to imipenem and susceptible to tigecycline and colistin but resistant to other antimicrobial agents. The ertapenem MICs of transformants increased at least 8-fold, compared with the recipients (Table 1).
FIG 2.
Overview of the target plasmid carrying blaOXA-232, from five OXA-232 carbapenemase-producing K. pneumoniae strains. MobC, transcription regulator ΔLysR, and erythromycin esterase ΔEreA, partial coding sequences; MobA, MobB, MobD, RepA, insertion sequence ΔISEcp1, and blaOXA-232, complete coding sequences.
TABLE 1.
In vitro activities of antimicrobial agents against K. pneumoniae isolates and their transformants
| Strain | β-Lactamase(s) | MIC (mg/liter)a |
|||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ETP | IPM | MEM | CSL | CZO | CXM | CTX | CAZ | CMZ | TGC | FEP | PIP | TZP | CIP | LEV | GEN | AMK | CST | ATM | CHL | MNO | SXT | ||
| KP1 | OXA-232, SHV-1/CTX-M-15 | 32 | 1 | 4 | 128/64 | >32 | >64 | >32 | >32 | 32 | 1 | >32 | >256 | >256/4 | >8 | >16 | >32 | >128 | ≤1 | >64 | >32 | 16 | >8/152 |
| KP1-T | OXA-232 | 0.25 | 0.5 | 0.06 | 8/4 | 8 | 16 | ≤0.25 | ≤0.25 | 32 | ≤0.25 | ≤0.5 | 256 | 64/4 | ≤0.25 | ≤0.5 | ≤2 | ≤8 | ≤1 | ≤2 | ≤8 | ≤2 | ≤1 |
| KP2 | OXA-232, SHV-1/CTX-M-15 | 32 | 2 | 8 | 128/64 | >32 | >64 | >32 | >32 | 16 | 1 | >32 | >256 | >256/4 | >8 | >16 | >32 | >128 | ≤1 | >64 | >32 | 16 | >8/152 |
| KP2-T | OXA-232 | 0.5 | 0.5 | 0.06 | 16/8 | 8 | 8 | ≤0.25 | ≤0.25 | 8 | ≤0.25 | ≤0.5 | 128 | 32/4 | ≤0.25 | ≤0.5 | ≤2 | ≤8 | ≤1 | ≤2 | ≤8 | ≤2 | ≤1 |
| KP3 | OXA-232, SHV-1/CTX-M-15 | 64 | 1 | 4 | >128/64 | >32 | >64 | >32 | >32 | 32 | 2 | >32 | >256 | >256/4 | >8 | >16 | >32 | >128 | ≤1 | >64 | >32 | 16 | >8/152 |
| KP3-T | OXA-232 | 0.25 | 0.5 | 0.06 | 16/8 | 8 | 8 | ≤0.25 | ≤0.25 | ≤0.5 | ≤0.25 | ≤0.5 | 256 | 64/4 | ≤0.25 | ≤0.5 | ≤2 | ≤8 | ≤1 | ≤2 | ≤8 | ≤2 | ≤1 |
| KP4 | OXA-232, SHV-1/CTX-M-15 | 32 | 1 | 4 | >128/64 | >32 | >64 | >32 | >32 | ≤0.5 | 1 | >32 | >256 | >256/4 | >8 | >16 | >32 | >128 | ≤1 | >64 | >32 | 16 | >8/152 |
| KP4-T | OXA-232 | 0.25 | 0.5 | ≤0.03 | 16/8 | 8 | 8 | ≤0.25 | ≤0.25 | ≤0.5 | ≤0.25 | ≤0.5 | >256 | 64/4 | ≤0.25 | ≤0.5 | ≤2 | ≤8 | ≤1 | ≤2 | ≤8 | ≤2 | ≤1 |
| KP5 | OXA-232, SHV-1/CTX-M-15 | 64 | 2 | 4 | 128/64 | >32 | >64 | >32 | >32 | ≤0.5 | 1 | >32 | >256 | >256/4 | >8 | >16 | >32 | >128 | ≤1 | >64 | >32 | 16 | >8/152 |
| KP5-T | OXA-232 | 0.25 | 0.5 | 0.06 | 16/8 | 8 | 8 | ≤0.25 | ≤0.25 | 1 | ≤0.25 | ≤0.5 | 128 | 64/4 | ≤0.25 | ≤0.5 | ≤2 | ≤8 | ≤1 | ≤2 | ≤8 | ≤2 | ≤1 |
| E. coli DH5α | ≤0.03 | 0.125 | ≤0.03 | ||||||||||||||||||||
ETP, ertapenem; IPM, imipenem; MEM, meropenem; CSL, cefoperazone-sulbactam; CZO, cefazolin; CXM, cefuroxime; CTX, cefotaxime; CAZ, ceftazidime; CMZ, cefmetazole; TGC, tigecycline; FEP, cefepime; PIP, piperacillin; TZP, piperacillin-tazobactam; CIP, ciprofloxacin; LEV, levofloxacin; GEN, gentamicin; AMK, amikacin; CST, colistin; ATM, aztreonam; CHL, chloramphenicol; MNO, minocycline; SXT, trimethoprim-sulfamethoxazole.
Differing from blaOXA-181 by one amino acid substitution, blaOXA-232 was initially isolated from Enterobacteriaceae collected from patients in France who had traveled previously to India and then was sporadically detected in Klebsiella pneumoniae in Singapore, the United States, and South Korea (9, 14, 15). To some extent, the first description of blaOXA-232 in K. pneumoniae clinical isolates highlights the potential risk of spreading in pediatric hospitals and the importance of carbapenem-resistant Enterobacteriaceae (CRE) surveillance indicated by ertapenem resistance.
Previously reported K. pneumoniae with blaOXA-232 belonged to ST14 and ST231 and Escherichia coli belonged to ST2968 (3, 9), while the dominant epidemic ST in our study was ST15. As a successful international clone, ST15 (as well as ST14 and ST231) K. pneumoniae has been identified in various countries in association with a variety of β-lactamases, mainly including NDM-1, CTX-M-15, KPC-2, and OXA-48-like (16, 17). In our study, blaCTX-M-15 results were negative among transformants but positive in donor strains, demonstrating that blaCTX-M-15 and blaOXA-232 were located in different plasmids. Compared with two K. pneumoniae isolates (RAN and DEL) detected in France (3), the K. pneumoniae isolates in our study showed similar drug resistance profiles, with ertapenem MIC values of >32 μg/ml, and both remained susceptible to colistin and tigecycline. However, K. pneumoniae RAN was also resistant to imipenem and meropenem, with MIC values of >32 μg/ml, which differed from the findings in our study (MIC values of 1 to 2 μg/ml for impenem and 4 to 8 μg/ml for meropenem), despite identical structures of the ColE-type plasmid. To date, there have been limited data regarding the clinical features, treatment, and prognosis of patients infected with isolates with blaOXA-232. In this study, all patients were treated with meropenem plus other antimicrobial agents, and four recovered, although one died (Table 2). According to the report by Tzouvelekis et al. (18), the therapeutic efficacy of carbapenems for patients infected with carbapenemase-positive K. pneumoniae would increase from 66.7% with a MIC of 8 μg/ml to 71.4% with a MIC of 4 μg/ml, indicating that carbapenems could provide some therapeutic benefit in infections caused by strains like ours.
TABLE 2.
Clinical features of patients infected or colonized with OXA-232 carbapenemase-producing K. pneumoniae isolates
| Patient no. | Age | Sex | Underlying condition | Days of hospital stay before infection/total days | Specimen | Invasive procedurea | Prior antibiotic exposure | Antimicrobial therapy | Infection or colonization | Status at hospital discharge |
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 4 mo | Male | Neonatal pulmonary hypoplasia, neonatal pneumonia, septicemia | 1/180 | Sputum | Endotracheal intubation | SAM, MEM, VRO, CRO, IPM | SAM, MEM, FOS, SCF, VRO, CAZ, AMK, CIP | Infection | Died |
| 2 | 35 d | Male | Congenital laryngeal cartilage dysplasia, malrotation | 26/46 | Sputum | Endotracheal intubation, PICC | CDZ, CPC, SAM, MEM | FOS, MEM, SCF | Infection | Recovered |
| 3 | 4 d | Male | Neonatal respiratory distress syndrome, persistent pulmonary hypertension of newborn, congenital heart disease | 4/14 | Sputum | Endotracheal intubation | SAM, CTX | SAM, MEM | Infection | Recovered |
| 4 | 16 d | Male | Hyperbilirubinemia, hemolytic disease of newborn | 14/23 | Urine | None | SAM | SAM | Colonization | Recovered |
| 5 | 4 mo | Male | Neonatal pulmonary hypoplasia, anemia, neonatal abdominal distension | 98/114 | Urine | UVC, endotracheal intubation, intestinal surgery | SAM, CPC | MEM, FOS | Infection | Recovered |
PICC, peripherally inserted central catheter; UVC, umbilical venous catheter; AMK, amikacin; CDZ, cefodizime; CPC, cefepime; CIP, ciprofloxacin; CTX, cefotaxime; CAZ, ceftazidime; SAM, ampicillin-sulbactam; MEM, meropenem; IPM, imipenem; VRO, vancomycin; CRO, ceftriaxone; FOS, fosfomycin; SCF, cefoperazone-sulbactam.
Additionally, it was demonstrated that a novel OmpK36 variant was associated with ertapenem resistance, reduced susceptibility to meropenem, and susceptibility to imipenem (13). In this study, sequencing of the ompK35 and ompK36 genes from five strains showed less than 80% and 90% amino acid identities with other K. pneumoniae OmpK35 and OmpK36 proteins (GenBank accession numbers KX528035.1, GU945359.1, GQ485678.1, HM000077.1, and KY019192.1), respectively. The differences in amino acid sequences may reveal OmpK35 and OmpK36 protein variants in these strains and may explain the carbapenem susceptibility test results, similar to the previous description.
Here, we highlight the emergence and outbreak potential of K. pneumoniae carrying a novel OXA-48-like variant in a health care setting. Although there is limited evidence for the clonal dissemination of blaOXA-232, we speculate that low levels of hand hygiene compliance and failure of contact precautions might have contributed to the infection or colonization of five patients during hospitalization. Active surveillance for CRE should be performed to restrict the spread of these multidrug-resistant clinical isolates, especially for pediatric patients.
Accession number(s).
The complete nucleotide sequence of the plasmid carrying blaOXA-232 is available in GenBank under accession number KY454616.
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (grant 81273559).
The funders had no role in study design, data collection and analysis, the decision to publish, or preparation of the manuscript.
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