Biochemistry and Pharmacology - Research Publications
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ItemKlebsiella pneumoniae with capsule type K64 is overrepresented among invasive disease in Vietnam(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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ItemRapid Whole Genome Sequencing of Serotype K1 Hypervirulent Klebsiella pneumoniae from an Undocumented Chinese Migrant(HINDAWI LTD, 2021-04-28)BACKGROUND: Hypervirulent Klebsiella pneumoniae causes severe disseminated infections, typically with hepatic and central nervous system involvement including endophthalmitis. Case Presentation. We report a fatal case of an undocumented Chinese migrant in her 60s who presented to the emergency department with abdominal pain, lethargy, and headache over the preceding two weeks. She had a new diagnosis of diabetes mellitus on admission. Computed tomography scan of the thorax, abdomen, and pelvis showed bilateral pneumonia with liver abscesses. The patient was treated with empirical broad-spectrum antibiotics before K. pneumoniae was isolated from cerebrospinal fluid and blood cultures. The isolate was further characterised as a ST23 (ST: sequence type), serotype K1 hypervirulent strain using Nanopore sequencing. Despite admission to the intensive care unit, the patient died within 48 hrs of admission. CONCLUSIONS: This case demonstrates the need for increased awareness of hypervirulent K. pneumoniae, even in countries where it occurs infrequently. Novel, rapid, sequencing technologies can support diagnosis in unusual presentations.
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ItemContext-aware genomic surveillance reveals hidden transmission of a carbapenemase-producing Klebsiella pneumoniae.(Microbiology Society, 2021-12)Genomic surveillance can inform effective public health responses to pathogen outbreaks. However, integration of non-local data is rarely done. We investigate two large hospital outbreaks of a carbapenemase-carrying Klebsiella pneumoniae strain in Germany and show the value of contextual data. By screening about 10 000 genomes, over 400 000 metagenomes and two culture collections using in silico and in vitro methods, we identify a total of 415 closely related genomes reported in 28 studies. We identify the relationship between the two outbreaks through time-dated phylogeny, including their respective origin. One of the outbreaks presents extensive hidden transmission, with descendant isolates only identified in other studies. We then leverage the genome collection from this meta-analysis to identify genes under positive selection. We thereby identify an inner membrane transporter (ynjC) with a putative role in colistin resistance. Contextual data from other sources can thus enhance local genomic surveillance at multiple levels and should be integrated by default when available.
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ItemSNPPar: identifying convergent evolution and other homoplasies from microbial whole-genome alignments.(Microbiology Society, 2021-12)Homoplasic SNPs are considered important signatures of strong (positive) selective pressure, and hence of adaptive evolution for clinically relevant traits such as antibiotic resistance and virulence. Here we present a new tool, SNPPar, for efficient detection and analysis of homoplasic SNPs from large whole genome sequencing datasets (>1000 isolates and/or >100 000 SNPs). SNPPar takes as input an SNP alignment, tree and annotated reference genome, and uses a combination of simple monophyly tests and ancestral state reconstruction (ASR, via TreeTime) to assign mutation events to branches and identify homoplasies. Mutations are annotated at the level of codon and gene, to facilitate analysis of convergent evolution. Testing on simulated data (120 Mycobacterium tuberculosis alignments representing local and global samples) showed SNPPar can detect homoplasic SNPs with very high specificity (zero false-positives in all tests) and high sensitivity (zero false-negatives in 89 % of tests). SNPPar analysis of three empirically sampled datasets (Elizabethkingia anophelis, Burkholderia dolosa and M. tuberculosis) produced results that were in concordance with previous studies, in terms of both individual homoplasies and evidence of convergence at the codon and gene levels. SNPPar analysis of a simulated alignment of ~64 000 genome-wide SNPs from 2000 M. tuberculosis genomes took ~23 min and ~2.6 GB of RAM to generate complete annotated results on a laptop. This analysis required ASR be conducted for only 1.25 % of SNPs, and the ASR step took ~23 s and 0.4 GB of RAM. SNPPar automates the detection and annotation of homoplasic SNPs efficiently and accurately from large SNP alignments. As demonstrated by the examples included here, this information can be readily used to explore the role of homoplasy in parallel and/or convergent evolution at the level of nucleotide, codon and/or gene.
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ItemFive Years of GenoTyphi: Updates to the Global Salmonella Typhi Genotyping Framework(OXFORD UNIV PRESS INC, 2021-12-15)In 2016, a whole-genome sequence (WGS)-based genotyping framework (GenoTyphi) was developed and provided a phylogenetically informative nomenclature for lineages of Salmonella Typhi, the etiological agent of typhoid fever. Subsequent surveillance studies have revealed additional epidemiologically important subpopulations, which require the definition of new genotypes and extension of associated software to facilitate the detection of antimicrobial resistance (AMR) mutations. Analysis of 4632 WGS provide an updated overview of the global S Typhi population structure and genotyping framework, revealing the widespread nature of haplotype 58 ([H58] 4.3.1) genotypes and the diverse range of genotypes carrying AMR mutations.
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ItemBurden of enteric fever at three urban sites in Africa and Asia: a multicentre population-based study(ELSEVIER SCI LTD, 2021-12)BACKGROUND: Enteric fever is a serious public health concern in many low-income and middle-income countries. Numerous data gaps exist concerning the epidemiology of Salmonella enterica serotype Typhi (S Typhi) and Salmonella enterica serotype Paratyphi (S Paratyphi), which are the causative agents of enteric fever. We aimed to determine the burden of enteric fever in three urban sites in Africa and Asia. METHODS: In this multicentre population-based study, we did a demographic census at three urban sites in Africa (Blantyre, Malawi) and Asia (Kathmandu, Nepal and Dhaka, Bangladesh) between June 1, 2016, and Sept 25, 2018. Households were selected randomly from the demographic census. Participants from within the geographical census area presenting to study health-care facilities were approached for recruitment if they had a history of fever for 72 h or more (later changed to >48 h) or temperature of 38·0°C or higher. Facility-based passive surveillance was done between Nov 11, 2016, and Dec 31, 2018, with blood-culture collection for febrile illness. We also did a community-based serological survey to obtain data on Vi-antibody defined infections. We calculated crude incidence for blood-culture-confirmed S Typhi and S Paratyphi infection, and calculated adjusted incidence and seroincidence of S Typhi blood-culture-confirmed infection. FINDINGS: 423 618 individuals were included in the demographic census, contributing 626 219 person-years of observation for febrile illness surveillance. 624 S Typhi and 108 S Paratyphi A isolates were collected from the blood of 12 082 febrile patients. Multidrug resistance was observed in 44% S Typhi isolates and fluoroquinolone resistance in 61% of S Typhi isolates. In Blantyre, the overall crude incidence of blood-culture confirmed S Typhi was 58 cases per 100 000 person-years of observation (95% CI 48-70); the adjusted incidence was 444 cases per 100 000 person-years of observation (95% credible interval [CrI] 347-717). The corresponding rates were 74 (95% CI 62-87) and 1062 (95% CrI 683-1839) in Kathmandu, and 161 (95% CI 145-179) and 1135 (95% CrI 898-1480) in Dhaka. S Paratyphi was not found in Blantyre; overall crude incidence of blood-culture-confirmed S Paratyphi A infection was 6 cases per 100 000 person-years of observation (95% CI 3-11) in Kathmandu and 42 (95% CI 34-52) in Dhaka. Seroconversion rates for S Typhi infection per 100 000 person-years estimated from anti-Vi seroconversion episodes in serological surveillance were 2505 episodes (95% CI 1605-3727) in Blantyre, 7631 (95% CI 5913-9691) in Kathmandu, and 3256 (95% CI 2432-4270) in Dhaka. INTERPRETATION: High disease incidence and rates of antimicrobial resistance were observed across three different transmission settings and thus necessitate multiple intervention strategies to achieve global control of these pathogens. FUNDING: Wellcome Trust and the Bill & Melinda Gates Foundation.
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ItemAddressing parachute research and removing barriers for LMIC researchers in Microbial Genomics.(Microbiology Society, 2021-10)
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ItemRapid Genomic Characterization and Global Surveillance of Klebsiella Using Pathogenwatch.(Oxford University Press (OUP), 2021-12-01)BACKGROUND: Klebsiella species, including the notable pathogen K. pneumoniae, are increasingly associated with antimicrobial resistance (AMR). Genome-based surveillance can inform interventions aimed at controlling AMR. However, its widespread implementation requires tools to streamline bioinformatic analyses and public health reporting. METHODS: We developed the web application Pathogenwatch, which implements analytics tailored to Klebsiella species for integration and visualization of genomic and epidemiological data. We populated Pathogenwatch with 16 537 public Klebsiella genomes to enable contextualization of user genomes. We demonstrated its features with 1636 genomes from 4 low- and middle-income countries (LMICs) participating in the NIHR Global Health Research Unit (GHRU) on AMR. RESULTS: Using Pathogenwatch, we found that GHRU genomes were dominated by a small number of epidemic drug-resistant clones of K. pneumoniae. However, differences in their distribution were observed (eg, ST258/512 dominated in Colombia, ST231 in India, ST307 in Nigeria, ST147 in the Philippines). Phylogenetic analyses including public genomes for contextualization enabled retrospective monitoring of their spread. In particular, we identified hospital outbreaks, detected introductions from abroad, and uncovered clonal expansions associated with resistance and virulence genes. Assessment of loci encoding O-antigens and capsule in K. pneumoniae, which represent possible vaccine candidates, showed that 3 O-types (O1-O3) represented 88.9% of all genomes, whereas capsule types were much more diverse. CONCLUSIONS: Pathogenwatch provides a free, accessible platform for real-time analysis of Klebsiella genomes to aid surveillance at local, national, and global levels. We have improved representation of genomes from GHRU participant countries, further facilitating ongoing surveillance.
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ItemwMel Wolbachia genome remains stable after 7 years in Australian Aedes aegypti field populations(MICROBIOLOGY SOC, 2021-09)Infection of wMel Wolbachia in Aedes aegypti imparts two signature features that enable its application for biocontrol of dengue. First, the susceptibility of mosquitoes to viruses such as dengue and Zika is reduced. Second, a reproductive manipulation is caused that enables wMel introgression into wild-type mosquito populations. The long-term success of this method relies, in part, on evolution of the wMel genome not compromising the critical features that make it an attractive biocontrol tool. This study compared the wMel Wolbachia genome at the time of initial releases and 1-7 years post-release in Cairns, Australia. Our results show the wMel genome remains highly conserved up to 7 years post-release in gene sequence, content, synteny and structure. This work suggests the wMel genome is stable in its new mosquito host and, therefore, provides reassurance on the potential for wMel to deliver long-term public-health impacts.
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ItemNo Preview AvailableGenomic Diversity and Antimicrobial Resistance of Haemophilus Colonizing the Airways of Young Children with Cystic Fibrosis(AMER SOC MICROBIOLOGY, 2021-08-31)Respiratory infection during childhood is a key risk factor in early cystic fibrosis (CF) lung disease progression. Haemophilus influenzae and Haemophilus parainfluenzae are routinely isolated from the lungs of children with CF; however, little is known about the frequency and characteristics of Haemophilus colonization in this context. Here, we describe the detection, antimicrobial resistance (AMR), and genome sequencing of H. influenzae and H. parainfluenzae isolated from airway samples of 147 participants aged ≤12 years enrolled in the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) program, Melbourne, Australia. The frequency of colonization per visit was 4.6% for H. influenzae and 32.1% for H. parainfluenzae, 80.3% of participants had H. influenzae and/or H. parainfluenzae detected on at least one visit, and using genomic data, we estimate 15.6% of participants had persistent colonization with the same strain for at least two consecutive visits. Isolates were genetically diverse and AMR was common, with 52% of H. influenzae and 82% of H. parainfluenzae displaying resistance to at least one drug. The genetic basis for AMR could be identified in most cases; putative novel determinants include a new plasmid encoding blaTEM-1 (ampicillin resistance), a new inhibitor-resistant blaTEM allele (augmentin resistance), and previously unreported mutations in chromosomally carried genes (pbp3, ampicillin resistance; folA/folP, cotrimoxazole resistance; rpoB, rifampicin resistance). Acquired AMR genes were more common in H. parainfluenzae than H. influenzae (51% versus 21%, P = 0.0107) and were mostly associated with the ICEHin mobile element carrying blaTEM-1, resulting in more ampicillin resistance in H. parainfluenzae (73% versus 30%, P = 0.0004). Genomic data identified six potential instances of Haemophilus transmission between participants, of which three involved participants who shared clinic visit days. IMPORTANCE Cystic fibrosis (CF) lung disease begins during infancy, and acute respiratory infections increase the risk of early disease development and progression. Microbes involved in advanced stages of CF are well characterized, but less is known about early respiratory colonizers. We report the population dynamics and genomic determinants of AMR in two early colonizer species, namely, Haemophilus influenzae and Haemophilus parainfluenzae, collected from a pediatric CF cohort. This investigation also reveals that H. parainfluenzae has a high frequency of AMR carried on mobile elements that may act as a potential reservoir for the emergence and spread of AMR to H. influenzae, which has greater clinical significance as a respiratory pathogen in children. This study provides insight into the evolution of AMR and the colonization of H. influenzae and H. parainfluenzae in a pediatric CF cohort, which will help inform future treatment.