Biochemistry and Pharmacology - Research Publications

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    Detection of Klebsiella pneumoniae human gut carriage: a comparison of culture, qPCR, and whole metagenomic sequencing methods
    Lindstedt, K ; Buczek, D ; Pedersen, T ; Hjerde, E ; Raffelsberger, N ; Suzuki, Y ; Brisse, S ; Holt, K ; Samuelsen, O ; Sundsfjord, A (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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    Genomic population structures of microbial pathogens
    Holt, KE ; Aanensen, DM ; Achtman, M (ROYAL SOC, 2022-10-10)
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    ESBL plasmids in Klebsiella pneumoniae: diversity, transmission and contribution to infection burden in the hospital setting.
    Hawkey, J ; Wyres, KL ; Judd, LM ; Harshegyi, T ; Blakeway, L ; Wick, RR ; Jenney, AWJ ; Holt, KE (Springer Science and Business Media LLC, 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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    Epidemiology and genomic analysis of Klebsiella oxytoca from a single hospital network in Australia
    Stewart, J ; Judd, LM ; Jenney, A ; Holt, KE ; Wyres, KL ; Hawkey, J (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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    Genomic dissection of Klebsiella pneumoniae infections in hospital patients reveals insights into an opportunistic pathogen
    Gorrie, CL ; Mirceta, M ; Wick, RR ; Judd, LM ; Lam, MMC ; Gomi, R ; Abbott, IJ ; Thomson, NR ; Strugnell, RA ; Pratt, NF ; Garlick, JS ; Watson, KM ; Hunter, PC ; Pilcher, DV ; McGloughlin, SA ; Spelman, DW ; Wyres, KL ; Jenney, AWJ ; Holt, KE (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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    Kaptive 2.0: updated capsule and lipopolysaccharide locus typing for the Klebsiella pneumoniae species complex
    Lam, MMC ; Wick, RR ; Judd, LM ; Holt, KE ; Wyres, KL (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.
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    Linear plasmids in Klebsiella and other Enterobacteriaceae
    Hawkey, J ; Cottingham, H ; Tokolyi, A ; Wick, RR ; Judd, LM ; Cerdeira, L ; Garcia, DDO ; Wyres, KL ; Holt, KE (MICROBIOLOGY SOC, 2022-04-01)
    Linear plasmids are extrachromosomal DNA elements that have been found in a small number of bacterial species. To date, the only linear plasmids described in the family Enterobacteriaceae belong to Salmonella, first found in Salmonella enterica Typhi. Here, we describe a collection of 12 isolates of the Klebsiella pneumoniae species complex in which we identified linear plasmids. Screening of assembly graphs assembled from public read sets identified linear plasmid structures in a further 13 K. pneumoniae species complex genomes. We used these 25 linear plasmid sequences to query all bacterial genome assemblies in the National Center for Biotechnology Information database, and discovered an additional 61 linear plasmid sequences in a variety of Enterobacteriaceae species. Gene content analysis divided these plasmids into five distinct phylogroups, with very few genes shared across more than two phylogroups. The majority of linear plasmid-encoded genes are of unknown function; however, each phylogroup carried its own unique toxin-antitoxin system and genes with homology to those encoding the ParAB plasmid stability system. Passage in vitro of the 12 linear plasmid-carrying Klebsiella isolates in our collection (which include representatives of all five phylogroups) indicated that these linear plasmids can be stably maintained, and our data suggest they can transmit between K. pneumoniae strains (including members of globally disseminated multidrug-resistant clones) and also between diverse Enterobacteriaceae species. The linear plasmid sequences, and representative isolates harbouring them, are made available as a resource to facilitate future studies on the evolution and function of these novel plasmids.
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    A curated collection of Klebsiella metabolic models reveals variable substrate usage and gene essentiality
    Hawkey, J ; Vezina, B ; Monk, JM ; Judd, LM ; Harshegyi, T ; Lopez-Fernandez, S ; Rodrigues, C ; Brisse, S ; Holt, KE ; Wyres, KL (COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2022-05-01)
    The Klebsiella pneumoniae species complex (KpSC) is a set of seven Klebsiella taxa that are found in a variety of niches and are an important cause of opportunistic health care-associated infections in humans. Because of increasing rates of multi-drug resistance within the KpSC, there is a growing interest in better understanding the biology and metabolism of these organisms to inform novel control strategies. We collated 37 sequenced KpSC isolates isolated from a variety of niches, representing all seven taxa. We generated strain-specific genome-scale metabolic models (GEMs) for all 37 isolates and simulated growth phenotypes on 511 distinct carbon, nitrogen, sulfur, and phosphorus substrates. Models were curated and their accuracy was assessed using matched phenotypic growth data for 94 substrates (median accuracy of 96%). We explored species-specific growth capabilities and examined the impact of all possible single gene deletions using growth simulations in 145 core carbon substrates. These analyses revealed multiple strain-specific differences, within and between species, and highlight the importance of selecting a diverse range of strains when exploring KpSC metabolism. This diverse set of highly accurate GEMs could be used to inform novel drug design, enhance genomic analyses, and identify novel virulence and resistance determinants. We envisage that these 37 curated strain-specific GEMs, covering all seven taxa of the KpSC, provide a valuable resource to the Klebsiella research community.
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    Klebsiella pneumoniae with capsule type K64 is overrepresented among invasive disease in Vietnam
    Vu Thi Ngoc, B ; Brisse, S ; Dao Tuyet, T ; Vu Tien Viet, D ; Holt, KE ; Nguyen Vu, T ; Tran Thi Kieu, H ; Nguyen Thi Ngoc, D ; van Doorn, HR ; Wertheim, HFL (F1000 Research Ltd, 2021-06-08)
    Introduction: Recent reports indicate the emergence of community-acquired pneumonia associated with K64-Klebsiella pneumoniae. Here, we identify the capsular types and sequence type of invasive and commensal K. pneumoniae isolates from Vietnam. Methods: We included 93 K. pneumoniae isolates from patients hospitalized at the National Hospital for Tropical Diseases, Hanoi between 2007 and 2011; and 110 commensal isolates from throat swabs from healthy volunteers living in rural and urban Hanoi in 2012. We determined sequence types (STs) by multi-locus sequence typing (MLST) and capsule typing for seven K types by PCR. Antibiotic susceptibility testing was performed using disk diffusion. Results: The most common detected capsule types were K1 (39/203, 19.2%, mainly ST23) and K2 (31/203, 15.3%, multiple STs: ST65, ST86, ST380). We found significantly more K2 isolates among invasive in comparison to commensal isolates (22.6% vs 9%, p = 0.01) but no significant difference was observed between invasive and commensal K1 isolates (14.5% vs 24.7%, p = 0.075). K64 with varying sequence types were predominantly seen among invasive K. pneumoniae (8 vs. 3) and were isolated from sepsis and meningitis patients. Among K64 isolates, one was carbapenem-resistant with ST799. Conclusion: Our study confirms that capsule type K64 K. pneumoniae is associated with community-acquired invasive infections in Vietnam. Research is needed to unravel the mechanisms of virulence of capsule type K64 in both community and hospital settings.
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    Whole genome sequence analysis of Salmonella Typhi in Papua New Guinea reveals an established population of genotype 2.1.7 sensitive to antimicrobials
    Dyson, ZA ; Malau, E ; Horwood, PF ; Ford, R ; Siba, V ; Yoannes, M ; Pomat, W ; Passey, M ; Judd, LM ; Ingle, DJ ; Williamson, DA ; Dougan, G ; Greenhill, AR ; Holt, KE ; Senok, A (PUBLIC LIBRARY SCIENCE, 2022-03-01)
    BACKGROUND: Typhoid fever, a systemic infection caused by Salmonella enterica serovar Typhi, remains a considerable public health threat in impoverished regions within many low- and middle-income settings. However, we still lack a detailed understanding of the emergence, population structure, molecular mechanisms of antimicrobial resistance (AMR), and transmission dynamics of S. Typhi across many settings, particularly throughout the Asia-Pacific islands. Here we present a comprehensive whole genome sequence (WGS) based overview of S. Typhi populations circulating in Papua New Guinea (PNG) over 30 years. PRINCIPLE FINDINGS: Bioinformatic analysis of 86 S. Typhi isolates collected between 1980-2010 demonstrated that the population structure of PNG is dominated by a single genotype (2.1.7) that appears to have emerged in the Indonesian archipelago in the mid-twentieth century with minimal evidence of inter-country transmission. Genotypic and phenotypic data demonstrated that the PNG S. Typhi population appears to be susceptible to former first line drugs for treating typhoid fever (chloramphenicol, ampicillin and co-trimoxazole), as well as fluoroquinolones, third generation cephalosporins, and macrolides. PNG genotype 2.1.7 was genetically conserved, with very few deletions, and no evidence of plasmid or prophage acquisition. Genetic variation among this population was attributed to either single point mutations, or homologous recombination adjacent to repetitive ribosomal RNA operons. SIGNIFICANCE: Antimicrobials remain an effective option for the treatment of typhoid fever in PNG, along with other intervention strategies including improvements to water, sanitation and hygiene (WaSH) related infrastructure and potentially the introduction of Vi-conjugate vaccines. However, continued genomic surveillance is warranted to monitor for the emergence of AMR within local populations, or the introduction of AMR associated genotypes of S. Typhi in this setting.