Spread of Plasmid-Encoded NDM-1 and GES-5 Carbapenemases among Extensively Drug-Resistant and Pandrug-Resistant Clinical Enterobacteriaceae in Durban, South Africa

Abstract

Whole-genome sequence analyses revealed the presence of bla_NDM-1 (n = 31), bla_GES-5 (n = 8), bla_OXA-232 (n = 1), or bla_NDM-5 (n = 1) in extensively drug-resistant and pandrug-resistant Enterobacteriaceae organisms isolated from in-patients in 10 private hospitals (2012 to 2013) in Durban, South Africa. Two novel NDM-1-encoding plasmids from Klebsiella pneumoniae were circularized by PacBio sequencing. In p19-10_01 [IncFIB(K); 223.434 bp], bla_NDM-1 was part of a Tn1548-like structure (16.276 bp) delineated by IS26 The multireplicon plasmid p18-43_01 [IncR_1/IncFIB(pB171)/IncFII(Yp); 212.326 bp] shared an 80-kb region with p19-10_01, not including the bla_NDM-1-containing region. The two plasmids were used as references for tracing NDM-1-encoding plasmids in the other genome assemblies. The p19-10_01 sequence was detected in K. pneumoniae (n = 7) only, whereas p18-43_01 was tracked to K. pneumoniae (n = 4), Klebsiella michiganensis (n = 1), Serratia marcescens (n = 11), Enterobacter spp. (n = 7), and Citrobacter freundii (n = 1), revealing horizontal spread of this bla_NDM-1-bearing plasmid structure. Global phylogeny showed clustering of the K. pneumoniae (18/20) isolates together with closely related carbapenemase-negative ST101 isolates from other geographical origins. The South African isolates were divided into three phylogenetic subbranches, where each group had distinct resistance and replicon profiles, carrying either p19-10_01, p18-10_01, or pCHE-A1 (8,201 bp). The latter plasmid carried bla_GES-5 and aacA4 within an integron mobilization unit. Our findings imply independent plasmid acquisition followed by local dissemination. Additionally, we detected bla_OXA-232 carried by pPKPN4 in K. pneumoniae (ST14) and bla_NDM-5 contained by a pNDM-MGR194-like genetic structure in Escherichia coli (ST167), adding even more complexity to the multilayer molecular mechanisms behind nosocomial spread of carbapenem-resistant Enterobacteriaceae in Durban, South Africa.

Keywords: Enterobacteriaceae; GES-5; NDM-1; carbapenemases; plasmid-mediated resistance.

FIG 1

Phylogenetic branch of K. pneumoniae containing the South African isolates (n = 18). Global phylogeny from genome assembly data sets (n = 1,148) downloaded from the PATRIC database (https://www.patricbrc.org/) was revealed by rapid core genome multialignment (https://github.com/marbl/parsnp) and metadata (isolation country and MLST) coupled with the use of Phandango (https://github.com/jameshadfield/phandango/wiki). The selected branch shows the isolate assembly identifier (ID) for the downloaded samples (lilac) and isolate ID for the samples from this study (yellow), for which subgroup (I, II, or III) and carbapenemase-encoding determinants are indicated. Color codes show isolation country and MLST for each isolate.

FIG 2

Tracking of plasmid p18-43_01 in NDM-1-encoding CPE isolates (n = 24). The map was constructed using BRIG software. The concentric circles represent comparisons between p18-43_01 and, starting with the inner circle, genome assemblies from Enterobacter species (strain ID 950178628, 953099839, 941713674, 950180354, 953102574, 939705067, and 19870317), C. freundii (944526466), S. marcescens (945174350, 945154301, 950145377, 950172946, 950174583, 950166381, 950165859, 950164094, 950163360, 950196656, and 950005607), K. michiganensis (939742031), and K. pneumoniae (941530379, 950118422, 950117510, and 950142398). Color codes are given for each species and for DNA identity, ranging from 70 to 100%, as indicated. Plasmids with extensive homology to p18-43_01, including pRJF866 (NC_025184.1), plasmid1 (CP009116.1), pKPC_CAV1217 (CP018675.1), and p19-10_01 (this study), were included in the BLAST comparisons and are represented as circles according to the given color codes. The outer black and red circles represent the p18-43_01 reference sequence and its annotated coding DNA sequence (CDS), respectively. Black transverse lines mark the ends of homology between p18-43_01 and p19-10_01 or pRJF866.

FIG 3

Tracking of plasmid p19-10_01 in subgroup II K. pneumoniae. The map was constructed using BRIG software. The concentric blue circles represent BLAST comparisons between p19-10_01 and genome assemblies from K. pneumoniae 960186733, 951362657, 951363981, 951373950, 950173000, 950171785, and 951384356, starting with the innermost circle. Color codes are for DNA identity, ranging from 70 to 100%, as indicated. Plasmids with regions homologous to the reference, including pKp848CTX (NC_024992.1), pPMK-1A (NZ_CP008930.1), pNDM-HK (NC_019063.1), and p18-43_01 (this study), were included in the BLAST comparisons and are represented as circles according to the given color codes. The outer black and red circles represent the p18-43_01 reference sequence and its annotated CDS, respectively. The Tn1548 region is marked, with the CDS shown in purple. The black transverse lines mark the ends of homology between p19-10_01 and p18-43_01.