Emergence of OXA-232-producing hypervirulent Klebsiella pneumoniae ST23 causing neonatal sepsis

Sir, Hypervirulent Klebsiella pneumoniae (hvKP) is an emerging pathogen causing several community-acquired invasive life-threatening infections in healthy and adult individuals.1–3 A carbapenem-resistant hvKP (CR-hvKP) pathotype has also recently been reported.4–6 However, hvKP/CR-hvKP strains causing neonatal sepsis have not been reported yet. This communication reports genome-level characterization of an OXA-232-producing hvKP ST23 K1 strain causing neonatal septicaemia. During a retrospective study, a hypermucoviscous neonatal septicaemic K. pneumoniae strain EN5275 was detected. The neonate (single, term, male and with low birth weight) was delivered at home and was on cow's milk since birth. Subsequently, 3 days after birth, in view of poor feeding, the neonate was referred to the level III unit of a tertiary care hospital in India in January 2016. The mother had a history of foul-smelling amniotic fluid. On admission, the neonate was pale, lethargic, tachypnoeic and was diagnosed with symptomatic hypoglycaemia and culture-positive sepsis. As empirical treatment, piperacillin/tazobactam and netilmicin were given. The baby stayed at the unit for 22 days before taking a high-risk discharge. As the neonate was referred to the unit from a rural area, a sample of the cow's milk could not be obtained. The consumption of cow's milk after birth could be a possible route of strain acquisition. However, this could not be substantiated due to the unavailability of a milk specimen. EN5275 was XDR (Table 1) and exhibited high MICs of carbapenems (>32 mg/L for both ertapenem and meropenem). However, MBLs and KPCs were not detected phenotypically, indicating the presence of other carbapenemases. Microbiological and genome-level characteristics of CR-hvKP strain EN5275 PIP, piperacillin; CTX, cefotaxime; FOX, cefoxitin; ATM, aztreonam; ETP, ertapenem; MEM, meropenem; AMK, amikacin; GEN, gentamicin; CIP, ciprofloxacin; ncRNA, non-coding RNA; RND, resistance-nodulation-division; HTH, helix-turn-helix. Microbiological and genome-level characteristics of CR-hvKP strain EN5275 PIP, piperacillin; CTX, cefotaxime; FOX, cefoxitin; ATM, aztreonam; ETP, ertapenem; MEM, meropenem; AMK, amikacin; GEN, gentamicin; CIP, ciprofloxacin; ncRNA, non-coding RNA; RND, resistance-nodulation-division; HTH, helix-turn-helix. PCR, Sanger sequencing and MLST showed that EN5275 was an ST23 strain and harboured blaOXA-232, a class D carbapenemase gene. WGS was performed on an Illumina NextSeq 500 platform (Illumina Inc., San Diego, CA, USA) (see the Supplementary methods available at JAC Online). The draft genome of EN5275 (accession number VINI00000000) consisted of ∼6 Mb with 56.13% GC content and 139 contigs. Genomic features are depicted in Table 1. A total of 22 antimicrobial resistance genes, 18 heavy metal resistance genes, 14 efflux pump and regulator genes and 8 plasmid replicons [Col440I, Col440II, ColKP3, ColRNAI, IncFIB(pQil), IncFII(K), IncA/C2 and IncX3] were identified in the EN5275 genome (Table 1). blaOXA-232 was found to be located on a ColKP3-type plasmid (∼6 kb) and was bracketed by truncated ISEcp1 and ΔlysR, consistent with other reports.7,8 Recently, an occurrence of OXA-232-producing K. pneumoniae ST15 harbouring a pLVPK-like virulence plasmid was reported from elderly patients.8 Here, our study, probably for the first time, reports a case of neonatal septicaemia caused by an OXA-232- and RmtF-producing hvKP ST23 strain EN5275. Since K1-ST23 strains are often found to be carrying pLVPK-like plasmids,3 the occurrence of blaOXA-232 in EN5275 possibly suggests the acquisition of resistance genes thereafter.9 No other cases of neonatal sepsis due to hvKP ST23 were detected in the current setting until the end of December 2016. EN5275 harboured K1 and O1V1 loci (Figure S1), one incomplete Edwardsiella phage PEi20 (Figure S2) and two complete CRISPR arrays (Figure S3). The EN5275 genome carried 86 putative virulence factors (Table 1) including several hypervirulent molecular markers (magA, rmpA, rmpA2, iroBCDEN and iucABCDiutA), clearly depicting EN5275 as hypervirulent. Multiple type VI secretion system (T6SS) loci have also been identified in the EN5275 genome. PCR and Sanger sequencing confirmed the presence of resistance and virulence determinants in the strain. CR-hvKP ST23 strain EN5275 was found to be hypermucoviscous, possessed significant biofilm-forming (OD595 = 0.902) ability and was resistant to the bactericidal activity of normal human serum (100% viable counts after 3 h) (see the methods section of the Supplementary data available at JAC Online). The negatively stained TEM image of EN5275 revealed the presence of a prominent layer of hypercapsule with clear indication of a fine meshwork, indicating its strong biofilm-forming capability (Figure S4). Comparative BLAST+ and BLASTN analysis of the EN5275 genome with other ST23 reference hvKP genomes (from China and Taiwan) revealed high genomic similarity (in silico DNA–DNA hybridization values >99.5%), suggesting that the ST23 hvKP strains possibly share a common ancestry (Table S1 and Figure S5). Due to the short-read output, we were unable to retrieve the complete virulence plasmid sequence from the EN5275 genome. The occurrence of pLVPK-associated markers (rmpA, rmpA2, iroBCDEN and iucABCDiutA) and progressiveMauve10 alignment between EN5275 and pLVPK (GenBank accession AY378100) confirmed the presence of a pLVPK-like sequence in the EN5275 genome (Figure S6). Analysis of transformants revealed that blaOXA-232 (as the sole resistance determinant) was located on a non-conjugative ColKP3-type plasmid and also displayed susceptible MICs towards all tested antimicrobials except for ertapenem (3 mg/L). To the best of our knowledge, this is the first report of a CR-hvKP ST23 strain causing neonatal sepsis and also speculates a plausible route of strain infiltration from the community. Further investigation and active surveillance of CR-hvKP strains are required to prevent these perilous strains from disseminating. We thank the staff of the Department of Neonatology, Institute of Postgraduate Medical Education & Research and SSKM Hospital for their support. This study was supported by the Indian Council of Medical Research (ICMR) intramural fund. S.M. and S.N. were supported by fellowships from ICMR. None to declare.