First Report of OXA-181-Producing Klebsiella pneumoniae in China

Abstract

We present here the first report of an OXA-181-producing Klebsiella pneumoniae isolated from the fecal specimen of a patient in China. The OXA-181-encoding gene bla_OXA-181 was located on a 51 kb IncX3-type plasmid. Conjugation assay and whole-genome sequencing analysis revealed that this transferrable plasmid in the K. pneumoniae isolate might have originated from Escherichia coli and have the potential to mediate the spread of bla_OXA-181.

Introduction

In 2001, a new carbapenem-hydrolyzing class D β-lactamase named OXA-48 was first identified in Klebsiella pneumoniae in Turkey.¹ Since this report, several variants of OXA-48 (including OXA-162, OXA-204, OXA-232, OXA-245, and OXA-181 et. al) have been identified in Enterobacteriaceae worldwide.^2,3 Of these variants, OXA-181, which contains four amino acid substitutions, was first reported in India in 2007⁴ and has since been identified, mainly in K. pneumoniae and E. coli, in several countries (UK, USA, and Denmark), showing a trend of increasing prevalence in in Enterobacteriaceae.^5,6 The gene encoding OXA-181, bla_OXA-181, is often found to be located on plasmids of incompatibility group (Inc) X that are defined as X3 type (IncX3).^7–9 These plasmids are known to disseminate various carbapenemase genes, including bla_KPC and bla_NDM.^10,11 To date, only two OXA-181-producing E. coli isolates have been reported in China, namely, in Sichuan and Henan.^12,13 Here, we present the first report of the identification of a K. pneumoniae isolate harboring bla_OXA-181 in China.

Materials and Methods

Clinical Isolate

A 60-year-old male patient was admitted to the general practice inpatient department of the Jinhua Municipal Central Hospital in Zhejiang Province, China, for 11 days in May 2019 due to headache and pain in the right finger with unknown causes. Clinical laboratory tests were conducted on the patient’s blood, urine, and fecal samples. These tests revealed no abnormal results and no infection symptoms were observed. Neither sputum culture nor blood culture was conducted and no antibiotic treatment was provided before or during hospitalization. The patient was discharged after the pain was alleviated. However, during the discharge screening, a K. pneumoniae isolate carrying bla_OXA-181 was recovered from the fecal sample on the MacConkey agar medium supplemented with 0.3 mg/L meropenem; the isolate was designated K. pneumoniae 709. The strain was identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) using a spectrometer from Bruker, Germany.

Antimicrobial Susceptibility Testing, Identification of Antibiotic Resistance Genes, and Conjugation Assay

Antimicrobial susceptibility of strain 709 and its conjugant, designated J709 was determined by the micro broth dilution method and interpreted according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). The resistance breakpoints from the CLSI were used for imipenem, meropenem, ertapenem, ceftazidime, cefotaxime, piperacillin/tazobactam, cefoperazone/sulbactam, ceftazidime/avibactam, cefepime, ciprofloxacin, amikacin, and aztreonam. The resistance breakpoints for polymyxin E and tigecycline were interpreted according to the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (available at http://www.eucast.org/clinical_breakpoints/). Polymerase chain reaction (PCR) was used to detect bla_OXA-181 using the following pair of primers: OXA48-F, 5ʹ-TCAGTAGCTGAACAGGAGGGA-3ʹ and OXA48-R, 5ʹ-TTCGAGCCGCAGACAATT-3ʹ. To determine the transferability of bla_OXA-181, a conjugation assay was performed using E. coli EC600 as the recipient strain. The conjugants were selected on a MacConkey agar medium containing 600 mg/L rifampicin and 1 mg/L meropenem. MALDI-TOF MS and PCR with the same primers as above were used to confirm the presumptive conjugant. Besides, the Multilocus sequence typing (MLST) - was performed using SRST2.¹⁴

Whole-Genome Sequencing and Plasmid Analysis

Genomic DNA was extracted by using the PureLink Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA). Whole genome sequencing was conducted using the Illumina HiSeq X10 (San Diego, CA, USA) and Nanopore MinION (Oxford, UK) sequencer platforms. The draft genomes were assembled using SPAdes v3.13.1.¹⁵ The complete plasmid sequence was annotated using RAST tool.¹⁶ Complete plasmid sequence alignment was conducted using BLAST Ring Image Generator (BRIG).¹⁷

Results and Discussion

In the antimicrobial susceptibility testing, 709 and J709 showed a low level of hydrolytic activity against carbapenems (Table 1), consistent with another report.¹⁸ Strains with low carbapenem MICs are often ignored and not referred for further investigations in clinical practice; therefore, bla_OXA-181 may have already spread in China despite there being only two reports from this country about strains harboring this gene.^12,13 In addition, the OXA-181-producing strains with high carbapenem MICs identified previously also carried additional resistance mechanisms, such as porin deficiency.^19,20 By contrast, isolate 709 was still susceptible to the other tested antimicrobial agents (cefotaxime, ceftazidime, aztreonam, amikacin, and ciprofloxacin).

Table 1.

Susceptibilities of the K. pneumoniae 709, Conjugant J709 and Recipient EC600

Strains	MIC (mg/L)
	IPM	MEM	ETP	CAZ	CTX	TZP	SCF	CAV	FEP	PE	TGC	CIP	AK	ATM
709	8	4	8	≤2	≤4	32/4	≤8/4	≤0.5/4	≤4	≤0.5	≤0.25	≤1	≤4	≤4
J709	4	2	4	≤2	≤4	≤8/4	≤8/4	≤0.5/4	≤4	≤0.5	≤0.25	2	≤4	≤4
EC600	≤1	≤1	≤2	≤2	≤4	≤8/4	≤8/4	≤0.5/4	≤4	≤0.5	≤0.25	≤1	≤4	≤4

Abbreviations: IMP, imipenem; MEM, meropenem; ETP, ertapenem; CAZ, Ceftazidime; CTX, Cefotaxime; TZP, Piperacillin/Tazobactam; SCF, Cefoperazone/Sulbactam; CAV, ceftazidime/avibactam; FEP, Cefepime; PE, polymyxin E; TGC, tigecycline; CIP, ciprofloxacin; AK, amikacin; ATM, aztreonam.

To characterize the genetic environment of bla_OXA-181 and the molecular type of K. pneumoniae 709, whole-genome sequencing was performed. bla_OXA-181 has been detected in various K. pneumoniae strains (ST43, ST147, ST836, ST11, ST61, ST25, ST307, ST709, etc.)^5,20,21 worldwide. Multilocus sequence typing (MLST) analysis indicated that K. pneumoniae 709 belonged to a novel sequence typing with one allele differing from ST432. Further analysis indicated that bla_OXA-181, together with the quinolone resistance gene qnrS1, was located on a 51 kb IncX3-type plasmid, designated by pKP709-OXA-181. The complete sequence of this plasmid has been deposited in GenBank under accession number MN227183. The conjugation assay revealed that pKP709-OXA-181 was a conjugative plasmid and BLAST analysis showed that the plasmid was identical to the E. coli plasmids pEC21-OXA-181 (GenBank accession number MG893567)¹³ and pOXA181_EC14828 (GenBank accession number KP400525)¹² (100% coverage and 99% sequence similarity) reported from China (Figure S1). This finding suggested that our K. pneumoniae plasmid pKP709 might have been derived from E. coli. Furthermore, pKP709-OXA-181 also harbored ISEcp1, an efficient genetic vehicle for disseminating clinically significant extended-spectrum β-lactamases, upstream of bla_OXA-181, as previously reported.^13,22 In this isolate, the qnrS1 gene, which was located between a truncated IS2 insertion sequence and a Tn3-like transposon, did not confer resistance to fluoroquinolones (Table 1). The genetic context of the bla_OXA-181 gene was the same as that of pEC21-OXA-181 as described by Qin et al.¹³ These findings suggest that close surveillance of resistance strains in the human gut flora should be included as a routine clinical practice to prevent occurrence of infections, especially among immunocompromised patients.

Conclusion

In summary, we present here the first report of an OXA-181-producing K. pneumoniae in China. The genetic environment of bla_OXA-181 is identical to a previously described E. coli plasmid, indicating that the K. pneumoniae strain might have acquired the gene from E. coli via the transferable IncX3-type plasmid. IncX3-type plasmids harboring bla_OXA-181 could become the main vehicle for the spread of bla_OXA-181 in future in China. Moreover, because the gastrointestinal tract is a major reservoir of antibiotic resistance genes, screening of fecal samples for bla_OXA-181 is recommended to prevent its possible rapid dissemination via the IncX3-type plasmid.

Ethics and Consent Statement

The conduction of this research and publication of case details were approved by the Ethics Committee of Jinhua Municipal Central Hospital (2019-135-001) and the written informed consent was acquired from the patient to have the case details and any accompanying images published.

Disclosure

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.