OXA-181-Like Carbapenemases in Klebsiella pneumoniae ST14, ST15, ST23, ST48, and ST231 from Septicemic Neonates: Coexistence with NDM-5, Resistome, Transmissibility, and Genome Diversity

Abstract

Studies on the epidemiology and genomes of isolates harboring OXA-48-like genes in septicemic neonates are rare. Here, isolates producing these carbapenemases which emerged and persisted in an Indian neonatal unit were characterized in terms of their resistome, transmissibility, and genome diversity. Antibiotic susceptibility and whole-genome sequencing were carried out. The sequence types, resistome, virulome, mobile genetic elements, and transmissibility of carbapenem-resistant plasmids were evaluated. Core genome analysis of isolates was shown in a global context with other OXA-48-like carbapenemase-harboring genomes, including those from neonatal studies. Eleven OXA-48-like carbapenemase-producing Klebsiella pneumoniae (bla_OXA-181, n = 7 and bla_OXA-232, n = 4) isolates belonging to diverse sequence types (ST14, ST15, ST23, ST48, and ST231) were identified. bla_{OXA-181/OXA-232} and bla_NDM-5 were found in a high-risk clone, ST14 (n = 4). bla_{OXA-181/OXA-232} were in small, nonconjugative ColKP3 plasmids located on truncated Tn2013, whereas bla_NDM-5 was in self-transmissible, conjugative IncFII plasmids, within truncated Tn125 Conjugal transfer of bla_{OXA-181/OXA-232} was observed in the presence of bla_NDM-5 The study strains were diverse among themselves and showed various levels of relatedness with non-neonatal strains from different parts of the world and similarity with neonatal strains from Tanzania and Ghana when compared with a representative collection of carbapenemase-positive K. pneumoniae strains. We found that bla_{OXA-181/OXA-232}-harboring isolates from a single neonatal unit had remarkably diverse genomes, ruling out clonal spread and emphasizing the extent of plasmid spreading across different STs. This study is probably the first to report the coexistence of bla_OXA-181/232 and bla_NDM-5 in neonatal isolates.IMPORTANCE Neonatal sepsis is a leading cause of neonatal mortality in low- and middle-income countries (LMICs). Treatment of sepsis in this vulnerable population is dependent on antimicrobials, and resistance to these life-saving antimicrobials is worrisome. Carbapenemases, enzymes produced by bacteria, can make these antimicrobials useless. Our study describes how OXA-48-like carbapenemases in neonatal septicemic Klebsiella pneumoniae shows remarkable diversity in the genomes of the strains and relatedness with strains from other parts of world and also to some neonatal outbreak strains. It is also the first to describe such resistance due to coproduction of dual carbapenemases, (OXA)-48 and New Delhi metallo-β-lactamase-5, in Klebsiella pneumoniae from neonatal settings. Carbapenemase genes situated on plasmids within high-risk international clones, as seen here, increase the ease and transfer of resistant genetic material. With the WHO treatment protocols not adequately poised to handle such infections, prompt attention to neonatal health care is required.

Keywords: ColKP3; India; NDM-5; OXA-181/232; WGS; core genome; dual carbapenemases; neonates; sepsis.

FIG 1

Pulsed-field gel electrophoresis with XbaI macrodigestion of bla_OXA-181-like-harboring K. pneumoniae isolated from blood of septicemic neonates. Lanes 1, 7, 10, and 15: M (marker) Salmonella serotype Braenderup H9812 as reference standard; lane 2: Kp1; lane 3: Kp2; lane 4: Kp3; lane 5: Kp4; lane 6: Kp5; lane 8: Kp6; lane 9: Kp7; lane 11: Kp8; lane 12: Kp9; lane 13: Kp10; lane 14: Kp11. Sequence types found in the strains are listed above each strain. ST, sequence type; CC, clonal complexes.

FIG 2

Schematic presentation of MGEs associated with bla_OXA-181/232 and bla_NDM-5 in the K. pneumoniae strains isolated from neonates. Heterogeneity of the genetic environment found in the studied carbapenemases: (a) bla_OXA-181/232 (Kp2-Kp4, Kp6-Kp11), (b) bla_OXA-181 in Kp1, and (c) genetic environment of transposon 125 (Tn125) harboring bla_NDM-5 (Kp3, Kp9-Kp11). Genes and their corresponding transcription orientations are indicated by horizontal arrows. Target site duplications (ATATA) generated by the insertion of Tn2013 are indicated by white triangles. mobA, mobB, mobC, and mobD, mobilization relaxosome proteins; ΔlysR, truncated LysR-type transcriptional regulator; ΔereA, truncated erythromycin esterase; repA, replicase; tnpA, transposase; IS, insertion sequence; ble_MBL; bleomycin resistance gene; trpF, N-(5′-phosphoribosyl) anthranilate isomerase; tat, twin-arginine translocation pathway signal sequence protein; Hypo. protein, hypothetical protein; Δ, denotes deletion or truncation.

FIG 3

Diagrammatic representation of class 1 integron found in the strains under study. arr-2, ADP-ribosyl transferase; qacEΔ1, quaternary ammonium compound resistance protein; sul1, sulfonamide resistant dihydropteroate synthase; orfΔ5, an open reading frame of unknown function; aadA1e and aadA2, aminoglycoside adenyltransferase; gcuF, DUF1010 domain-containing protein; dfrA12 and dfrA14b, dihydrofolate reductases type-A; S.ma.I2, group IIc intron; aacA4′-17, aminoglycoside 6′-N-acetyltransferase; ereA3, erythromycin esterase; cmlA1g, chloramphenicol resistance gene; attI, site of recombination; intI1, integrase gene; attC, site of attenuation; P_i, promoter of integrase; CS, conserved sequence.

FIG 4

Core genome phylogeny of 197 Klebsiella pneumoniae isolates using Roary (v3.12.0) and FastTree (v2.1.11). Isolates are colored at the endpoint according to country, and the outer ring abbreviation is labeled according to the sample source. The additional two outer rings denote the presence of bla_NDM and bla_OXA-48-like antibiotic resistance genes. Clades containing isolates from this study are highlighted in teal, and light blue clade highlights indicate K. pneumoniae neonatal sepsis isolates from other studies. The year of sample collection for isolates in this study has been added external to the tree phylogeny.

FIG 5

Core genome SNP phylogeny of EN5153 (Kp1) with other ST48 neonatal isolates. An outgroup rooted tree was built using the most distant isolate from the Mash genome estimation analysis (an isolate from London, submitted to the NCBI database in 2018). Isolates beginning with ERR are from other ST48 neonatal isolates and another isolate submitted to NCBI on 2014.