Genomic Characterisation of Community and Hospital Acquired Carbapenem Resistant Enterobacteriaceae

Abstract

Carbapenems are considered last-line beta-lactams for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, their activity is compromised by the rising prevalence of carbapenem-resistant Enterobacteriaceae (CRE), which are especially marked in the Indian subcontinent. In Pakistan, previous reports have warned about the possible spread of CRE in the community, but data are still partial. This study was carried out to analyse the prevalence of CRE, the genetic characterisation, and phylogenetic links among the spreading CRE in the community and hospital settings. In this cohort study, we collected 306 rectal swabs from patients visiting Benazir Bhutto hospital, Rawalpindi. We obtained a second sample from 94 patients during their hospital stay in whom carriage was negative at admission. CREs were screened by using ertapenem-supplemented MacConkey agar. Identification was performed by using conventional biochemical tests, confirmation was done by using Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS) and genomes were sequenced using Illumina chemistry. Antibiotic resistance genes, plasmid incompatibility groups, and Escherichia coli phylogroups were determined in silico. Sequence types were determined by using multilocus sequence typing (MLST) tool. Study findings showed that the prevalence of CRE in the community was 15.68% (48/306), while carbapenemase-producing Enterobacteriaceae (CPE) carriage observed was 14.4% (44/306 samples). CRE carriage among hospital-acquired patients was 7.5% (7/94 samples). All CRE isolates were tested against commonly used antimicrobial groups. The highest resistance was observed against penicillins, penicillins/β-lactamase inhibitors, cephalosporins, carbapenems, quinolones and sulfonamides. The most effective antibiotics found against CRE isolates were colistin, amikacin and gentamicin. Based on antimicrobial susceptibility profiling, CRE isolates were grouped into two categories, extensively-drug resistant (XDR), and multi-drug resistant (MDR), based on their resistance phenotypes. There were no isolates that exhibited pan-drug resistance (PDR), while 64 (82%) isolates were grouped into XDR and 14 (18%) isolates into the MDR category. The most common carbapenemase-encoding gene was blaNDM-5 (n=58) followed by blaNDM Abstract x

1 (n=7), blaNDM (non-assigned variant, n=4), blaOXA-181 (n=3), blaOXA-232 (n=3) and blaNDM 7 (n=1). Among the hospital-associated strains (n=9), eight carried blaNDM-5 and one carried blaNDM-1. Forty-three strains were found to harbour an extended-spectrum β-lactamase (ESBL)-encoding gene, mainly CTX-M-15 ESBLs. The major plasmid-mediated quinolone resistance.(PMQR) genes identified were qnrS1 (n=12) and qepA (n=3). Consistent with their resistance phenotype, 66/68 E. coli had mutations in topoisomerases known to confer resistance to quinolones. Multiple aminoglycoside resistance genes (ARG) coding for enzymes were also identified in 77/78 CRE strains. The sul (dihydropteroate synthase) and dfr (dihydrofolate reductase) resistance genes that confer cotrimoxazole resistance were present in 67 and 73 strains, respectively. In the current study we also reported first detection of mcr-1 from the gut of outpatient (individual who visit healthcare facility for medical treatment or consultation, but do not require an overnight stay), in Rawalpindi. Most of the community (55/69, 80%), and hospital-acquired CPE were E. coli (8/9, 89%), and the genomic analysis revealed a pauciclonal diffusion of E. coli with ST167 (n=14), 405 (n=10), 940 (n=8), 648 (n=6) and 617 (n=5). Phylogenetic analysis revealed that the CRE isolates within clonal groups were genetically close. We identified 27 plasmid replicons in the 78 genomes. The IncX3 and IncFII incompatibility groups commonly associated with blaNDM were present in 23% (18/78) and 41% (32/78) strains, respectively. The carriage of CRE in a Pakistani urban community was high and driven by the dissemination of some E. coli clones, with ST167 being the most frequent. The high CRE carriage in the community poses a serious public health threat and calls for the implementation of adequate preventive measures