Diversity in the characteristics of Klebsiella pneumoniae ST101 of Human, environmental, and animal origin

BACKGROUND: Klebsiella pneumoniae ST101 is an emerging high-risk clone which exhibits extensive drug resistance. Bacterial strains residing in multiple hosts show unique signatures related to host adaptation. In this study, we assess the genetic relationship of K. pneumoniae ST101 isolated from hospital samples, the environment, community, and livestock using whole genome sequencing (WGS). MATERIALS AND METHODS: We selected ten K. pneumoniae ST101 strains from hospitalized patients in Italy (n = 3) (2014) and Spain (n = 5) (2015–2016) as well as Belgian livestock animals (n = 2) (2017–2018). WGS was performed with 2 × 250 bp paired-end sequencing (Nextera XT) sample preparation kit and MiSeq (Illumina Inc.). Long-read sequencing (Pacbio Sequel I) was used to sequence the two livestock strains and three Italian hospital-associated strains. Furthermore, a public ST101 sequence collection of 586 strains (566 hospital-associated strains, 12 environmental strains, six strains from healthy individuals, one food-associated strain and one pig strain) was obtained. BacPipe and Kleborate were used to conduct genome analysis. ISFinder was used to find IS elements, and PHASTER was utilized to identify prophages. A phylogenetic tree was constructed to illustrate genetic relatedness. RESULTS: Hospital-associated K. pneumoniae ST101 showed higher resistance scores than non-clinical isolates from healthy individuals, the environment, food and livestock (1.85 ± 0.72 in hospital-associated isolates vs. 1.14 ± 1.13 in non-clinical isolates, p < 0.01). Importantly, the lack of integrative conjugative elements ICEKp bearing iron-scavenging yersiniabactin siderophores (ybt) in livestock-associated strains suggests a lower pathogenicity potential than hospital-associated strains. Mobile genetic elements (MGE) appear to be an important source of diversity in K. pneumoniae ST101 strains from different origins, with a highly stable genome and few recombination events outside the prophage-containing regions. Core genome MLST based analysis revealed a distinct genetic clustering between human and livestock-associated isolates. CONCLUSION: The study of K. pneumoniae ST101 hospital-associated and strains from healthy individuals and animals revealed a genetic diversity between these two groups, allowing us to identify the presence of yersiniabactin siderophores in hospital-associated isolates. Resistance and virulence levels in livestock-associated strains were considerably lower than hospital-associated strains, implying that the public health risk remains low. The introduction of an ICEKp into animal strains, on the other hand, might pose a public threat over time

Genetic characterization of ESBL-producing and ciprofloxacin-resistant Escherichia coli from Belgian broilers and pigs

Background: The increasing number of infections caused by Escherichia coli resistant to clinically important antibiotics is a global concern for human and animal health. High overall levels of extended-spectrum beta-lactamase (ESBL)-producing and ciprofloxacin-resistant (ciproR) Escherichia coli in livestock are reported in Belgium. This cross-sectional study aimed to genotypically characterize and trace ESBL-and ciproR-E. coli of Belgian food-producing animals. Methods: A total of 798 fecal samples were collected in a stratified-random sampling design from Belgian broilers and sows. Consequently, 77 ESBL-E. coli and 84 ciproR-E. coli were sequenced using Illumina MiSeq. Minimum inhibitory concentration (MIC) for fluoroquinolones and cephalosporins were determined. Molecular in silico typing, resistance and virulence gene determination, and plasmid identification was performed. Scaffolds harboring ESBL or plasmid-mediated quinolone resistance (PMQR) genes were analyzed to detect mobile genetic elements (MGEs) and plasmid origins. Core genome allelic distances were used to determine genetic relationships among isolates. Results: A variety of E. coli sequence types (ST) (n = 63), resistance genes and virulence profiles was detected. ST10 was the most frequently encountered ST (8.1%, n = 13). The pandemic multidrug-resistant clone ST131 was not detected. Most farms harbored more than one ESBL type, with bla(CTX-M-1) (41.6% of ESBL-E. coli) being the most prevalent and bla(CTX M-15) (n = 3) being the least prevalent. PMQR genes (15.5%, n = 13) played a limited role in the occurrence of ciproR-E. coli. More importantly, sequential acquisition of mutations in quinolone resistance-determining regions (QRDR) of gyrA and parC led to increasing MICs for fluoroquinolones. GyrA S83L, D87N and ParC S80I mutations were strongly associated with high-level fluoroquinolone resistance. Genetically related isolates identified within the farms or among different farms highlight transmission of resistant E. coli or the presence of a common reservoir. IncI1-I(alpha) replicon type plasmids carried different ESBL genes (bla(CTX-M-1), bla(CTX-M-32) and bla(TEM-52C)). In addition, the detection of plasmid replicons with associated insertion sequence (IS) elements and ESBL/PMQR genes in different farms and among several STs (e.g., IncI1-I(alpha)/IncX3) underline that plasmid transmission could be another important contributor to transmission of resistance in these farms. Conclusion: Our findings reveal a multifaceted narrative of transmission pathways. These findings could be relevant in understanding and battling the problem of antibiotic resistance in farms

Harmonization of whole- genome sequencing for outbreak surveillance of Enterobacteriaceae and Enterococci

Whole-genome sequencing (WGS) is becoming the de facto standard for bacterial typing and outbreak surveillance of resistant bacterial pathogens. However, interoperability for WGS of bacterial outbreaks is poorly understood. We hypothesized that harmonization of WGS for outbreak surveillance is achievable through the use of identical protocols for both data generation and data analysis. A set of 30 bacterial isolates, comprising of various species belonging to the Enterobacteriaceae family and Enterococcus genera, were selected and sequenced using the same protocol on the Illumina MiSeq platform in each individual centre. All generated sequencing data were analysed by one centre using BioNumerics (6.7.3) for (i) genotyping origin of replications and antimicrobial resistance genes, (ii) core-genome multi-locus sequence typing (cgMLST) for Escherichia coli and Klebsiella pneumoniae and whole-genome multi-locus sequencing typing (wgMLST) for all species. Additionally, a split k-mer analysis was performed to determine the number of SNPs between samples. A precision of 99.0% and an accuracy of 99.2% was achieved for genotyping. Based on cgMLST, a discrepant allele was called only in 2/27 and 3/15 comparisons between two genomes, for E. coli and K. pneumoniae, respectively. Based on wgMLST, the number of discrepant alleles ranged from 0 to 7 (average 1.6). For SNPs, this ranged from 0 to 11 SNPs (average 3.4). Furthermore, we demonstrate that using different de novo assemblers to analyse the same dataset introduces up to 150 SNPs, which surpasses most thresholds for bacterial outbreaks. This shows the importance of harmonization of data-processing surveillance of bacterial outbreaks. In summary, multi-centre WGS for bacterial surveillance is achievable, but only if protocols are harmonized

Novel vancomycin resistance gene cluster in ST1486, Belgium, June 2021.

We identified a novel gene cluster in a clinical isolate with vancomycin minimum inhibitory concentration (MIC) of 4 µg/mL. The ligase gene, , was part of a operon cluster of 4,589 bp on a putative novel integrative conjugative element located in a ca 98 kb genomic region presumed to be acquired by horizontal gene transfer from and sp. 499. Screening for genes in strains with borderline susceptibility to vancomycin is important