High-risk clones of extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolated from the University Hospital Establishment of Oran, Algeria (2011-2012)

Abstract

The purpose of the study was to characterize the resistome, virulome, mobilome and Clustered Regularly Interspaced Short Palindromic Repeats-associated (CRISPR-Cas) system of extended-spectrum β-lactamase producing Klebsiella pneumoniae (ESBL-KP) clinical isolates and to determine their phylogenetic relatedness. The isolates were from Algeria, isolated at the University Hospital Establishment of Oran, between 2011 and 2012. ESBL-KP isolates (n = 193) were screened for several antibiotic resistance genes (ARGs) using qPCR followed by Pulsed-Field Gel Electrophoresis (PFGE). Representative isolates were selected from PFGE clusters and subjected to whole-genome sequencing (WGS). Genomic characterization of the WGS data by studying prophages, CRISPR-Cas systems, Multi-Locus Sequence Typing (MLST), serotype, ARGs, virulence genes, plasmid replicons, and their pMLST. Phylogenetic and comparative genomic were done using core genome MLST and SNP-Based analysis. Generally, the ESBL-KP isolates were polyclonal. The whole genome sequences of nineteen isolates were taken of main PFGE clusters. Sixteen sequence types (ST) were found including high-risk clones ST14, ST23, ST37, and ST147. Serotypes K1 (n = 1), K2 (n = 2), K3 (n = 1), K31 (n = 1), K62 (n = 1), and K151 (n = 1) are associated with hyper-virulence. CRISPR-Cas system was found in 47.4%, typed I-E and I-E*. About ARGs, from 193 ESBL-KP, the majority of strains were multidrug-resistant, the CTX-M-1 enzyme was predominant (99%) and the prevalence of plasmid-mediated quinolone resistance (PMQR) genes was high with aac(6')-lb-cr (72.5%) and qnr's (65.8%). From 19 sequenced isolates we identified ESBL, AmpC, and carbapenemase genes: blaCTX-M-15 (n = 19), blaOXA-48 (n = 1), blaCMY-2 (n = 2), and blaCMY-16 (n = 2), as well as non-ESBL genes: qnrB1 (n = 12), qnrS1 (n = 1) and armA (n = 2). We found IncF, IncN, IncL/M, IncA/C2, and Col replicon types, at least once per isolate. This study is the first to report qnrS in ESBL-KP in Algeria. Our analysis shows the concerning co-existence of virulence and resistance genes and would support that genomic surveillance should be a high priority in the hospital environment.

Fig 1. Minimum Spanning Tree (MST) analysis of 19 isolates of K. pneumoniae based on 632 allelic profiles of cgMLST.

Each node within the tree represents a single isolate with a unique genotype. Selected nodes are labeled with corresponding STs. The length of branches linked two nodes indicates the number of allelic mismatches in a pairwise comparison. The oval discontinued dash in red, green, and blue demonstrate the isolates having ST37, ST1426, and ST147, respectively.

Fig 2. Minimum Spanning Trees (MSTs) analysis showing the distribution of MDR profiles, virulence factors, and infection types among 19 isolates of K. pneumoniae based on 632 allelic profiles of cgMLST.

Each node within the tree represents a single isolate with a unique genotype. Selected nodes are labeled with corresponding STs. The length of branches linked two nodes indicates the number of allelic mismatches in a pairwise comparison. Long branches are marked with dashed lines. Thick lines correspond to a short branch with less than 100 allelic mismatches. The grey zones surrounding nodes indicate that they belong to the same clonal group. Four MST graphs were generated separately based on the following associations. A: MST versus virulence genes, B: MST versus infection types, C: MST versus resistance genes profiles, D: MST versus MDR and virulence classes. MDR: Multi-Drug Resistance. UTI: Urinary Tract Infection.

Fig 3. Maximum likelihood tree created from concatenated SNPs sequences.

The numbers on the nodes represent the SNPs differences between isolates.

Fig 4. Heatmap with all results (resistance genes, virulence, MLST, K-types, replicons with the ML Tree from the SNP-based analysis) among the sequenced 19 isolates.

The scale is modified.

Fig 5. Architecture and organization of the CRISPR-Cas system in clinical ESBL-KP isolates.

A. The operon organization (cas genes), the leaders, classification, and core-promoter. The rows represent cas genes in different colors, the brown diamonds represent the spacer, and the blue rectangle represents the Direct Repeated (DR). B. Spacer map, their distribution, and the matching results. Spacers are represented in a box without repeats. Identical spacers are represented by the same ID and color. P: Plasmid, V: Virus or Phage, S: Self-target, RS: The reverse self-target, spacers in red had no homology. +: Longer spacer.