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- Methylation analysis of Klebsiella pneumoniae from Portuguese hospitalsPublication . Spadar, Anton; Perdigão, João; Phelan, Jody; Charleston, James; Modesto, Ana; Elias, Rita; De Sessions, Paola; 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; 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.
- Genomic epidemiological analysis of Klebsiella pneumoniae from Portuguese hospitals reveals insights into circulating antimicrobial resistancePublication . Spadar, Anton; Phelan, Jody; Elias, Rita; Modesto, Ana; Caneiras, Cátia; Marques, Cátia; Lito, Luís; Pinto, Margarida; Cavaco‑Silva, Patrícia; Ferreira, Helena; Pomba, Constança; Silva, Gabriela J. Da; Saavedra, Maria José; Melo‑Cristino, José; Duarte, Aida; Campino, Susana; Perdigão, JoãoKlebsiella pneumoniae (Kp) bacteria are an increasing threat to public health and represent one of the most concerning pathogens involved in life-threatening infections and antimicrobial resistance (AMR). To understand the epidemiology of AMR of Kp in Portugal, we analysed whole genome sequencing, susceptibility testing and other meta data on 509 isolates collected nationwide from 16 hospitals and environmental settings between years 1980 and 2019. Predominant sequence types (STs) included ST15 (n = 161, 32%), ST147 (n = 36, 7%), ST14 (n = 26, 5%) or ST13 (n = 26, 5%), while 31% of isolates belonged to STs with fewer than 10 isolates. AMR testing revealed widespread resistance to aminoglycosides, fluoroquinolones, cephalosporins and carbapenems. The most common carbapenemase gene was blaKPC-3. Whilst the distribution of AMR linked plasmids appears uncorrelated with ST, their frequency has changed over time. Before year 2010, the dominant plasmid group was associated with the extended spectrum beta-lactamase gene blaCTX-M-15, but this group appears to have been displaced by another carrying the blaKPC-3 gene. Co-carriage of blaCTX-M and blaKPC-3 was uncommon. Our results from the largest genomics study of Kp in Portugal highlight the active transmission of strains with AMR genes and provide a baseline set of variants for future resistance monitoring and epidemiological studies.
- Large-scale genomic analysis of global Klebsiella pneumoniae plasmids reveals multiple simultaneous clusters of carbapenem-resistant hypervirulent strainsPublication . Spadar, Anton; Perdigão, João; Campino, Susana; Clark, Taane G.Background Klebsiella pneumoniae (Kp) Gram-negative bacteria cause nosocomial infections and rapidly acquire antimicrobial resistance (AMR), which makes it a global threat to human health. It also has a comparatively rare hypervirulent phenotype that can lead to severe disease in otherwise healthy individuals. Unlike classic Kp, canonical hypervirulent strains usually have limited AMR. However, after initial case reports in 2015, carbapenem-resistant hypervirulent Kp has increased in prevalence, including in China, but there is limited understanding of its burden in other geographical regions. Methods Here, we examined the largest collection of publicly available sequenced Kp isolates (n=13,178), containing 1603 different sequence types (e.g. ST11 15.0%, ST258 9.5%), and 2174 (16.5%) hypervirulent strains. We analysed the plasmid replicons and carbapenemase and siderophore encoding genes to understand the movement of hypervirulence and AMR genes located on plasmids, and their convergence in carbapenem-resistant hypervirulent Kp. Results We identified and analysed 3034 unique plasmid replicons to inform the epidemiology and transmission dynamics of carbapenem-resistant hypervirulent Kp (n=1028, 7.8%). We found several outbreaks globally, including one involving ST11 strains in China and another of ST231 in Asia centred on India, Thailand, and Pakistan. There was evidence of global flow of Kp, including across multiple continents. In most cases, clusters of Kp isolates are the result of hypervirulence genes entering classic strains, instead of carbapenem resistance genes entering canonical hypervirulent ones. Conclusions Our analysis demonstrates the importance of plasmid analysis in the monitoring of carbapenem-resistant and hypervirulent strains of Kp. With the growing adoption of omics-based technologies for clinical and surveillance applications, including in geographical regions with gaps in data and knowledge (e.g. sub-Saharan Africa), the identification of the spread of AMR will inform infection control globally.
- Genomic analysis of hypervirulent Klebsiella pneumoniae reveals potential genetic markers for differentiation from classical strainsPublication . Spadar, Anton; Perdigão, João; Campino, Susana; Clark, TaaneThe majority of Klebsiella pneumoniae (Kp) infections are nosocomial, but a growing number of community-acquired infections are caused by hypervirulent strains (hvKp) characterised by liver invasion and rapid metastasis. Unlike nosocomial Kp infections, hvKp are generally susceptible to antibiotics. Due to the rapid progression of hvKp infections, timely and accurate diagnosis is required for effective treatment. To identify potential drivers of the hypervirulent phenotype, we performed a genome-wide association study (GWAS) analysis on single nucleotide variants and accessory genome loci across 79 publicly available Kp isolates collected from patients’ liver and a diverse global Kp dataset (n = 646). The GWAS analysis revealed 29 putative genes (P < 10–10) associated with higher risk of liver phenotype, including hypervirulence linked salmochelin iro (odds ratio (OR): 29.8) and aerobactin iuc (OR: 14.1) loci. A minority of liver isolates (n = 15, 19%) had neither of these siderophores nor any other shared biomarker, suggesting possible unknown drivers of hypervirulence and an intrinsic ability of Kp to invade the liver. Despite identifying potential novel loci linked to a liver invasive Kp phenotype, our work highlights the need for large-scale studies involving more sequence types to identify further hypervirulence biomarkers to assist clinical decision making.
- Emergence of KPC-3- and OXA-181-producing ST13 and ST17 Klebsiella pneumoniae in Portugal: genomic insights on national and international disseminationPublication . Elias, Rita; Spadar, Anton; Hendrickx, Antoni P A; Bonnin, Remy A; Dortet, Laurent; Pinto, Margarida; Phelan, Jody E; Portugal, Isabel; Campino, Susana; Silva, Gabriela Jorge da; Clark, Taane; Duarte, Aida; Perdigão, JoãoBackground Carbapenem-resistant Klebsiella pneumoniae (CRKP) strains are of particular concern, especially strains with mobilizable carbapenemase genes such as blaKPC, blaNDM or blaOXA-48, given that carbapenems are usually the last line drugs in the β-lactam class and, resistance to this sub-class is associated with increased mortality and frequently co-occurs with resistance to other antimicrobial classes. Objectives To characterize the genomic diversity and international dissemination of CRKP strains from tertiary care hospitals in Lisbon, Portugal. Methods Twenty CRKP isolates obtained from different patients were subjected to WGS for species confirmation, typing, drug resistance gene detection and phylogenetic reconstruction. Two additional genomic datasets were included for comparative purposes: 26 isolates (ST13, ST17 and ST231) from our collection and 64 internationally available genomic assemblies (ST13). Results By imposing a 21 SNP cut-off on pairwise comparisons we identified two genomic clusters (GCs): ST13/GC1 (n = 11), all bearing blaKPC-3, and ST17/GC2 (n = 4) harbouring blaOXA-181 and blaCTX-M-15 genes. The inclusion of the additional datasets allowed the expansion of GC1/ST13/KPC-3 to 23 isolates, all exclusively from Portugal, France and the Netherlands. The phylogenetic tree reinforced the importance of the GC1/KPC-3-producing clones along with their rapid emergence and expansion across these countries. The data obtained suggest that the ST13 branch emerged over a decade ago and only more recently did it underpin a stronger pulse of transmission in the studied population. Conclusions This study identifies an emerging OXA-181/ST17-producing strain in Portugal and highlights the ongoing international dissemination of a KPC-3/ST13-producing clone from Portugal.