Genome Sequencing of an Extended Series of NDM-Producing Klebsiella pneumoniae Isolates from Neonatal Infections in a Nepali Hospital Characterizes the Extent of Community- versus Hospital-Associated Transmission in an Endemic Setting

Maximum-likelihood phylogeny of all sequenced Klebsiella pneumoniae study isolates (deduplicated by individual). Isolates in red represent those originally considered part of case clusters on the basis of clinical suspicion and susceptibility testing. Additional closely genetically related clusters of nonoutbreak strains are indicated with curly brackets. “CA” denotes community-associated strains, and “HA” hospital-associated strains; the time interval listed is that spanning the isolation dates of clustered isolates. Symbols indicating bootstrap support at nodes are defined in the key.

Resistance genotypes and susceptibility phenotypes as determined by the Phoenix automated system are shown for all sequenced Klebsiella pneumoniae isolates (A) and for all sequenced non-Klebsiella pneumoniae isolates (B). Copy numbers calculated for bla_NDM, where present, are also shown. Susceptibility categories were determined by Phoenix in accordance with EUCAST breakpoints. S, susceptible; I, intermediate; R, resistant; NA, no result available; AMP, ampicillin-amoxicillin; AMC, amoxicillin-clavulanate; TZP, piperacillin-tazobactam; CXM, cefuroxime; CRO, ceftriaxone; CAZ, ceftazidime; CIP, ciprofloxacin; ERT, ertapenem; MEM, meropenem; ATM, aztreonam; GEN, gentamicin; AMK, amikacin; SXT, trimethoprim-sulfamethoxazole; CST, colistin.

(A) Nucleotide and amino acid level variations in outbreak isolates with respect to the sequences of the PMK1 reference chromosome and pPMK1-B and pPMK1-NDM. del, deletion; NA, unannotated hypothetical protein; -, stop codon. The order of the isolates approximates the outbreak time frame but also accommodates the grouping of isolates that share identical genetic sequences based on mapping to the PMK1 reference chromosome (i.e., 0 SNV differences; shown in boxes with black borders). Brown-shaded chromosomal positions represent noncoding positions, gray-shaded positions represent positions in plasmids. Nonsynonymous, persistent mutations occurred in ppx (an exopolyphosphatase), azlC (an azaleucine resistance protein), adaA (a methyltransferase), gcd (a quinoprotein glucose dehydrogenase), bmR (a transcriptional repressor), oprD (an outer membrane porin), ssuC (an alkane sulfonate transporter subunit), and virB4 (a type IV secretion system). (B) Phylogenetic tree summarizing chromosomal genetic relationships between all outbreak isolates. Colored nodes represent sampled isolates and black nodes unsampled intermediates; node colors are defined in the key. Each solid branch represents a single SNV, with branch colors indicating mutation types as follows: orange, nonsynonymous; green, synonymous; gray, intergenic; gray dashed line, 121,366-bp deletion; black dashed line, 21 single-nucleotide variants (SNVs).

Transmission network inferred using Outbreaker. The sizes of the nodes reflect the numbers of secondary cases per infected individual. The colors of nodes reflect the sampling date as indicated in the key at the upper left. Shading of arrows represents the degree of posterior support for the transmission link, as indicated in the key at the bottom left.