Browsing by Author "Phelan, Jody"
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- Genomic epidemiology of carbapenemase producing Klebsiella pneumoniae strains at a northern portuguese hospital enables the detection of a misidentified Klebsiella variicola KPC-3 producing strainPublication . Perdigão, João; Caneiras, Cátia; Elias, Rita; Modesto, Ana; Spadar, Anton; Phelan, Jody; Campino, Susana; Clark, Taane G.; Costa, Eliana; Saavedra, Maria José; Duarte, AidaThe evolutionary epidemiology, resistome, virulome and mobilome of thirty-one multidrug resistant Klebsiella pneumoniae clinical isolates from the northern Vila Real region of Portugal were characterized using whole-genome sequencing and bioinformatic analysis. The genomic population structure was dominated by two main sequence types (STs): ST147 (n = 17; 54.8%) and ST15 (n = 6; 19.4%) comprising four distinct genomic clusters. Two main carbapenemase coding genes were detected (blaKPC-3 and blaOXA-48) along with additional extended-spectrum β-lactamase coding loci (blaCTX-M-15, blaSHV-12, blaSHV-27, and blaSHV-187). Moreover, whole genome sequencing enabled the identification of one Klebsiella variicola KPC-3 producer isolate previously misidentified as K. pneumoniae, which in addition to the blaKPC-3 carbapenemase gene, bore the chromosomal broad spectrum β-lactamase blaLEN-2 coding gene, oqxAB and fosA resistance loci. The blaKPC-3 genes were located in a Tn4401b transposon (K. variicola n = 1; K. pneumoniae n = 2) and Tn4401d isoform (K. pneumoniae n = 28). Overall, our work describes the first report of a blaKPC-3 producing K. variicola, as well as the detection of this species during infection control measures in surveillance cultures from infected patients. It also highlights the importance of additional control measures to overcome the clonal dissemination of carbapenemase producing clones.
- Methylation analysis of Klebsiella pneumoniae from Portuguese hospitalsPublication . Spadar, Anton; Perdigão, João; Phelan, Jody; Charleston, James; Modesto, Ana; Elias, Rita; Sessions, Paola Florez de; Hibberd, Martin L.; Campino, Susana; Duarte, Aida; Clark, Taane G.Klebsiella pneumoniae is an important nosocomial infectious agent with a high antimicrobial resistance (AMR) burden. The application of long read sequencing technologies is providing insights into bacterial chromosomal and putative extra-chromosomal genetic elements (PEGEs) associated with AMR, but also epigenetic DNA methylation, which is thought to play a role in cleavage of foreign DNA and expression regulation. Here, we apply the PacBio sequencing platform to eight Portuguese hospital isolates, including one carbapenemase producing isolate, to identify methylation motifs. The resulting assembled chromosomes were between 5.2 and 5.5Mbp in length, and twenty-six PEGEs were found. Four of our eight samples carry blaCTX-M-15, a dominant Extended Spectrum Beta Lactamase in Europe. We identified methylation motifs that control Restriction–Modification systems, including GATC of the DNA adenine methylase (Dam), which methylates N6-methyladenine (m6A) across all our K. pneumoniae assemblies. There was a consistent lack of methylation by Dam of the GATC motif downstream of two genes: fosA, a locus associated with low level fosfomycin resistance, and tnpB transposase on IncFIB(K) plasmids. Overall, we have constructed eight high quality reference genomes of K. pneumoniae, with insights into horizontal gene transfer and methylation m6A motifs.
- A phylogenomic approach for the analysis of colistin resistance-associated genes in Klebsiella pneumoniae, its mutational diversity and implications for phenotypic resistancePublication . Elias, Rita; Spadar, Anton; Phelan, Jody; Melo-Cristino, José; Lito, Luís; Pinto, Margarida; Gonçalves, Luísa; Campino, Susana; Clark, Taane G.; Duarte, Aida; Perdigão, JoãoThe emergence of carbapenemase-producing Klebsiella pneumoniae strains has triggered the use of old antibiotics such as colistin. This is driving the emergence of colistin resistance in multidrug-resistant strains that underlie life-threatening infections. This study analyses the mutational diversity of 22 genes associated with colistin resistance in 140 K. pneumoniae clinical isolates integrated in a high-resolution phylogenetic scenario. Colistin susceptibility was accessed by broth microdilution. A total of 98 isolates were susceptible and 16 were resistant, 10 of which were carbapenemase producers. Across the 22 genes examined, 171 non-synonymous mutations and 9 mutations associated with promoter regions were found. Eighty-five isolates had a truncation and/or deletion in at least one of the 22 genes. However, only seven mutations, the complete deletion of mgrB or insertion sequence (IS)-mediated disruption, were exclusively observed in resistant isolates. Four of these (mgrB Ile13fs, pmrB Gly207Asp, phoQ His339Asp and ramA Ile28Met) comprised novel mutations that are potentially involved in colistin resistance. One strain bore a ISEcp1-blaCTX-M-15::mgrB disruption, underlying co-resistance to third-generation cephalosporins and colistin. Moreover, the high-resolution phylogenetic context shows that most of the mutational diversity spans multiple phylogenetic clades, and most of the mutations previously associated with colistin resistance are clade-associated and present in susceptible isolates, showing no correlation with colistin resistance. In conclusion, the present study provides relevant data on the genetic background of genes involved with colistin resistance deeply rooted across monophyletic groups and provides a better understanding of the genes and mutations involved in colistin resistance.
- Whole-genome sequencing resolves a polyclonal outbreak by extended-spectrum beta-lactam and carbapenem-resistant Klebsiella pneumoniae in a Portuguese tertiary-care hospitalPublication . Perdigão, João; Modesto, Ana; Pereira, A. L.; Neto, O.; Matos, V.; Godinho, A.; Phelan, Jody; Charleston, James; Spadar, Anton; Sessions, Paola Florez de; Hibberd, Martin; Campino, Susana; Costa, A.; Fernandes, F.; Ferreira, F.; Correia, A. B.; Gonçalves, Luisa; Clark, Taane G.; Duarte, AidaKlebsiella pneumoniae has emerged as an important nosocomial pathogen, with whole-genome sequencing (WGS) significantly improving our ability to characterize associated outbreaks. Our study sought to perform a genome-wide analysis of multiclonal K. pneumoniae isolates (n=39; 23 patients) producing extended spectrum beta-lactamases and/or carbapenemases sourced between 2011 and 2016 in a Portuguese tertiary-care hospital. All isolates showed resistance to third-generation cephalosporins and six isolates (five patients) were also carbapenem resistant. Genome-wide-based phylogenetic analysis revealed a topology representing ongoing dissemination of three main sequence-type (ST) clades (ST15, ST147 and ST307) and transmission across different wards, compatible with missing links that can take the form of undetected colonized patients. Two carbapenemase-coding genes were detected: blaKPC-3, located on a Tn4401d transposon, and blaGES-5 on a novel class 3 integron. Additionally, four genes coding for ESBLs (blaBEL-1, blaCTX-M-8, blaCTX-M-15 and blaCTX-M-32) were also detected. ESBL horizontal dissemination across five clades is highlighted by the similar genetic environments of blaCTX-M-15 gene upstream of ISEcp1 on a Tn3-like transposon. Overall, this study provides a high-resolution genome-wide perspective on the epidemiology of ESBL and carbapenemase-producing K. pneumoniae in a healthcare setting while contributing for the adoption of appropriate intervention and prevention strategies.