Biochemistry and Pharmacology - Research Publications
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ItemMaking microbial genomics work for clinical and public health microbiology(MICROBIOLOGY SOC, 2022-09)
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ItemGenomic epidemiology and temperature dependency of hypermucoviscous Klebsiella pneumoniae in Japan.(Microbiology Society, 2022-05)Klebsiella pneumoniae (Kp) has emerged as a global life-threatening pathogen owing to its multidrug resistance and hypervirulence phenotype. Several fatal outbreaks of carbapenem-resistant hypervirulent Kp have been reported recently. Hypermucoviscosity (HMV) is a phenotype commonly associated with hypervirulence of Kp, which is usually regulated by rmpA or rmpA2 (regulators of the mucoid phenotype). Here, we found that temperature was important in the HMV phenotype of Kp, and the impact of temperature on HMV was not uniform among strains. We investigated the HMV phenotype at 37 °C and room temperature (20-25 °C) in 170 clinically isolated hypermucoviscous Kp strains in Japan and analysed the association between the HMV phenotype, virulence genes and antimicrobial resistance (AMR) genes. String length distribution at different temperatures was correlated with the genomic population of Kp. The strains carrying rmpA/rmpA2 frequently showed the HMV phenotype at 37 °C, while the strains negative for these genes tended to show the HMV phenotype at room temperature. Hypervirulent Kp clusters carrying rmpA/rmpA2 without extended-spectrum beta-lactamases (ESBL)/carbapenemases produced higher string lengths at 37 °C than at room temperature, and were mostly isolated from the respiratory tract. Other HMV strains showed distinct characteristics of not carrying rmpA/rmpA2 but were positive for ESBL/carbapenemases, with a higher string length at room temperature than at 37 °C, and were frequently isolated from bloodstream infections. In total, 21 (13.5 %) HMV isolates carried ESBL and carbapenemases, among which five isolates were carbapenem-resistant hypervirulent Kp with a pLVPK-like plasmid (an epidemic virulence plasmid) and a pKPI-6-like plasmid (an epidemic blaIMP-6-bearing plasmid in Japan), suggesting the convergence of worldwide hypervirulence and epidemic AMR in Japan.
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ItemTransmission of Klebsiella strains and plasmids within and between grey-headed flying fox colonies(WILEY, 2022-09)The grey-headed flying fox (Pteropus poliocephalus) is an endemic Australian fruit bat, known to carry zoonotic pathogens. We recently showed they harbour bacterial pathogen Klebsiella pneumoniae and closely related species in the K. pneumoniae species complex (KpSC); however, the dynamics of KpSC transmission and gene flow within flying fox colonies are poorly understood. High-resolution genome comparisons of 39 KpSC isolates from grey-headed flying foxes identified five putative strain transmission clusters (four intra- and one inter-colony). The instance of inter-colony strain transmission of K. africana was found between two flying fox populations within flying distance, indicating either direct or indirect transmission through a common food/water source. All 11 plasmids identified within the KpSC isolates showed 73% coverage (mean) and ≥95% identity to human-associated KpSC plasmids, indicating gene flow between human clinical and grey-headed flying fox isolates. Along with strain transmission, inter-species horizontal plasmid transmission between K. pneumoniae and Klebsiella africana was also identified within a flying fox colony. Finally, genome-scale metabolic models were generated to predict and compare substrate usage to previously published KpSC models, from human and environmental sources. These models indicated no distinction on the basis of metabolic capabilities. Instead, metabolic capabilities were consistent with population structure and ST/lineage.
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ItemA Shigella sonnei clone with extensive drug resistance associated with waterborne outbreaks in China.(Springer Science and Business Media LLC, 2022-11-30)Antimicrobial resistance of Shigella sonnei has become a global concern. Here, we report a phylogenetic group of S. sonnei with extensive drug resistance, including a combination of multidrug resistance, coresistance to ceftriaxone and azithromycin (cefRaziR), reduced susceptibility to fluoroquinolones, and even colistin resistance (colR). This distinct clone caused six waterborne shigellosis outbreaks in China from 2015 to 2020. We collect 155 outbreak isolates and 152 sporadic isolates. The cefRaziR isolates, including outbreak strains, are mainly distributed in a distinct clade located in global Lineage III. The outbreak strains form a recently derived monophyletic group that may have emerged circa 2010. The cefRaziR and colR phenotypes are attributed to the acquisition of different plasmids, particularly the IncB/O/K/Z plasmid coharboring the blaCTX-M-14, mphA, aac(3)-IId, dfrA17, aadA5, and sul1 genes and the IncI2 plasmid with an mcr-1 gene. Genetic analyses identify 92 accessory genes and 60 single-nucleotide polymorphisms associated with the cefRaziR phenotype. Surveillance of this clone is required to determine its dissemination and threat to global public health.
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ItemDetection of Klebsiella pneumoniae human gut carriage: a comparison of culture, qPCR, and whole metagenomic sequencing methods(TAYLOR & FRANCIS INC, 2022-12-31)Klebsiella pneumoniae is an important opportunistic healthcare-associated pathogen and major contributor to the global spread of antimicrobial resistance. Gastrointestinal colonization with K. pneumoniae is a major predisposing risk factor for infection and forms an important hub for the dispersal of resistance. Current culture-based detection methods are time consuming, give limited intra-sample abundance and strain diversity information, and have uncertain sensitivity. Here we investigated the presence and abundance of K. pneumoniae at the species and strain level within fecal samples from 103 community-based adults by qPCR and whole metagenomic sequencing (WMS) compared to culture-based detection. qPCR demonstrated the highest sensitivity, detecting K. pneumoniae in 61.2% and 75.8% of direct-fecal and culture-enriched sweep samples, respectively, including 52/52 culture-positive samples. WMS displayed lower sensitivity, detecting K. pneumoniae in 71.2% of culture-positive fecal samples at a 0.01% abundance cutoff, and was inclined to false positives in proportion to the relative abundance of other Enterobacterales present. qPCR accurately quantified K. pneumoniae to 16 genome copies/reaction while WMS could estimate relative abundance to at least 0.01%. Quantification by both methods correlated strongly with each other (Spearman's rho = 0.91). WMS also supported accurate intra-sample K. pneumoniae sequence type (ST)-level diversity detection from fecal microbiomes to 0.1% relative abundance, agreeing with the culture-based detected ST in 16/19 samples. Our results show that qPCR and WMS are sensitive and reliable tools for detection, quantification, and strain analysis of K. pneumoniae from fecal samples with potential to support infection control and enhance insights in K. pneumoniae gastrointestinal ecology.
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ItemGenomic population structures of microbial pathogens(ROYAL SOC, 2022-10-10)
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ItemEpidemiology and genomic analysis of Klebsiella oxytoca from a single hospital network in Australia(BMC, 2022-08-24)BACKGROUND: Infections caused by Klebsiella oxytoca are the second most common cause of Klebsiella infections in humans. Most studies have focused on K. oxytoca outbreaks and few have examined the broader clinical context of K. oxytoca. METHODS: Here, we collected all clinical isolates identified as K. oxytoca in a hospital microbiological diagnostic lab across a 15-month period (n = 239). Whole genome sequencing was performed on a subset of 92 isolates (all invasive, third-generation cephalosporin resistant (3GCR) and non-urinary isolates collected > 48 h after admission), including long-read sequencing on a further six isolates with extended-spectrum beta-lactamase or carbapenemase genes. RESULTS: The majority of isolates were sensitive to antimicrobials, however 22 isolates were 3GCR, of which five were also carbapenem resistant. Genomic analyses showed those identified as K. oxytoca by the clinical laboratory actually encompassed four distinct species (K. oxytoca, Klebsiella michiganensis, Klebsiella grimontii and Klebsiella pasteurii), referred to as the K. oxytoca species complex (KoSC). There was significant diversity within the population, with only 10/67 multi-locus sequence types (STs) represented by more than one isolate. Strain transmission was rare, with only one likely event identified. Six isolates had extended spectrum beta-lactamase (blaSHV-12 and/or blaCTX-M-9) or carbapenemase (blaIMP-4) genes. One pair of K. michiganensis and K. pasteurii genomes carried identical blaIMP-4 IncL/M plasmids, indicative of plasmid transmission. CONCLUSION: Whilst antimicrobial resistance was rare, the resistance plasmids were similar to those found in other Enterobacterales, demonstrating that KoSC has access to the same plasmid reservoir and thus there is potential for multi-drug resistance. Further genomic studies are required to improve our understanding of the KoSC population and facilitate investigation into the attributes of successful nosocomial isolates.
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ItemESBL plasmids in Klebsiella pneumoniae: diversity, transmission and contribution to infection burden in the hospital setting(BMC, 2022-08-23)BACKGROUND: Resistance to third-generation cephalosporins, often mediated by extended-spectrum beta-lactamases (ESBLs), is a considerable issue in hospital-associated infections as few drugs remain for treatment. ESBL genes are often located on large plasmids that transfer horizontally between strains and species of Enterobacteriaceae and frequently confer resistance to additional drug classes. Whilst plasmid transmission is recognised to occur in the hospital setting, the frequency and impact of plasmid transmission on infection burden, compared to ESBL + strain transmission, is not well understood. METHODS: We sequenced the genomes of clinical and carriage isolates of Klebsiella pneumoniae species complex from a year-long hospital surveillance study to investigate ESBL burden and plasmid transmission in an Australian hospital. Long-term persistence of a key transmitted ESBL + plasmid was investigated via sequencing of ceftriaxone-resistant isolates during 4 years of follow-up, beginning 3 years after the initial study. RESULTS: We found 25 distinct ESBL plasmids. We identified one plasmid, which we called Plasmid A, that carried blaCTX-M-15 in an IncF backbone similar to pKPN-307. Plasmid A was transmitted at least four times into different Klebsiella species/lineages and was responsible for half of all ESBL episodes during the initial 1-year study period. Three of the Plasmid A-positive strains persisted locally 3-6 years later, and Plasmid A was detected in two additional strain backgrounds. Overall Plasmid A accounted for 21% of ESBL + infections in the follow-up period. CONCLUSIONS: Here, we systematically surveyed ESBL strain and plasmid transmission over 1 year in a single hospital network. Whilst ESBL plasmid transmission events were rare in this setting, they had a significant and sustained impact on the burden of ceftriaxone-resistant and multidrug-resistant infections. If onward transmission of Plasmid A-carrying strains could have been prevented, this may have reduced the number of opportunities for Plasmid A to transmit and create novel ESBL + strains, as well as reducing overall ESBL infection burden.
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ItemGenomic dissection of Klebsiella pneumoniae infections in hospital patients reveals insights into an opportunistic pathogen(NATURE PORTFOLIO, 2022-05-31)Klebsiella pneumoniae is a major cause of opportunistic healthcare-associated infections, which are increasingly complicated by the presence of extended-spectrum beta-lactamases (ESBLs) and carbapenem resistance. We conducted a year-long prospective surveillance study of K. pneumoniae clinical isolates in hospital patients. Whole-genome sequence (WGS) data reveals a diverse pathogen population, including other species within the K. pneumoniae species complex (18%). Several infections were caused by K. variicola/K. pneumoniae hybrids, one of which shows evidence of nosocomial transmission. A wide range of antimicrobial resistance (AMR) phenotypes are observed, and diverse genetic mechanisms identified (mainly plasmid-borne genes). ESBLs are correlated with presence of other acquired AMR genes (median n = 10). Bacterial genomic features associated with nosocomial onset are ESBLs (OR 2.34, p = 0.015) and rhamnose-positive capsules (OR 3.12, p < 0.001). Virulence plasmid-encoded features (aerobactin, hypermucoidy) are observed at low-prevalence (<3%), mostly in community-onset cases. WGS-confirmed nosocomial transmission is implicated in just 10% of cases, but strongly associated with ESBLs (OR 21, p < 1 × 10-11). We estimate 28% risk of onward nosocomial transmission for ESBL-positive strains vs 1.7% for ESBL-negative strains. These data indicate that K. pneumoniae infections in hospitalised patients are due largely to opportunistic infections with diverse strains, with an additional burden from nosocomially-transmitted AMR strains and community-acquired hypervirulent strains.
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ItemKaptive 2.0: updated capsule and lipopolysaccharide locus typing for the Klebsiella pneumoniae species complex(MICROBIOLOGY SOC, 2022-03-01)The outer polysaccharide capsule and lipopolysaccharide (LPS) antigens are key targets for novel control strategies targeting Klebsiella pneumoniae and related taxa from the K. pneumoniae species complex (KpSC), including vaccines, phage and monoclonal antibody therapies. Given the importance and growing interest in these highly diverse surface antigens, we had previously developed Kaptive, a tool for rapidly identifying and typing capsule (K) and outer LPS (O) loci from whole genome sequence data. Here, we report two significant updates, now freely available in Kaptive 2.0 (https://github.com/katholt/kaptive): (i) the addition of 16 novel K locus sequences to the K locus reference database following an extensive search of >17 000 KpSC genomes; and (ii) enhanced O locus typing to enable prediction of the clinically relevant O2 antigen (sub)types, for which the genetic determinants have been recently described. We applied Kaptive 2.0 to a curated dataset of >12 000 public KpSC genomes to explore for the first time, to the best of our knowledge, the distribution of predicted O (sub)types across species, sampling niches and clones, which highlighted key differences in the distributions that warrant further investigation. As the uptake of genomic surveillance approaches continues to expand globally, the application of Kaptive 2.0 will generate novel insights essential for the design of effective KpSC control strategies.