Biochemistry and Pharmacology - Research Publications

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    Multidrug-Resistant Salmonella enterica, Serotype Typhi, Gulf of Guinea Region, Africa
    Baltazar, M ; Ngandjio, A ; Holt, KE ; Lepillet, E ; Pardos de la Gandara, M ; Collard, J-M ; Bercion, R ; Nzouankeu, A ; Le Hello, S ; Dougan, G ; Fonkoua, M-C ; Weill, F-X (CENTERS DISEASE CONTROL, 2015-04)
    We identified 3 lineages among multidrug-resistant (MDR) Salmonella enterica serotype Typhi isolates in the Gulf of Guinea region in Africa during the 2000s. However, the MDR H58 haplotype, which predominates in southern Asia and Kenya, was not identified. MDR quinolone-susceptible isolates contained a 190-kb incHI1 pST2 plasmid or a 50-kb incN pST3 plasmid.
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    Shigella sonnei genome sequencing and phylogenetic analysis indicate recent global dissemination from Europe
    Holt, KE ; Baker, S ; Weill, F-X ; Holmes, EC ; Kitchen, A ; Yu, J ; Sangal, V ; Brown, DJ ; Coia, JE ; Kim, DW ; Choi, SY ; Kim, SH ; da Silveira, WD ; Pickard, DJ ; Farrar, JJ ; Parkhill, J ; Dougan, G ; Thomson, NR (NATURE PORTFOLIO, 2012-09)
    Shigella are human-adapted Escherichia coli that have gained the ability to invade the human gut mucosa and cause dysentery(1,2), spreading efficiently via low-dose fecal-oral transmission(3,4). Historically, S. sonnei has been predominantly responsible for dysentery in developed countries but is now emerging as a problem in the developing world, seeming to replace the more diverse Shigella flexneri in areas undergoing economic development and improvements in water quality(4-6). Classical approaches have shown that S. sonnei is genetically conserved and clonal(7). We report here whole-genome sequencing of 132 globally distributed isolates. Our phylogenetic analysis shows that the current S. sonnei population descends from a common ancestor that existed less than 500 years ago and that diversified into several distinct lineages with unique characteristics. Our analysis suggests that the majority of this diversification occurred in Europe and was followed by more recent establishment of local pathogen populations on other continents, predominantly due to the pandemic spread of a single, rapidly evolving, multidrug-resistant lineage.
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    Identification of a marker for two lineages within the GC1 clone of Acinetobacter baumannii
    Hamidian, M ; Wynn, M ; Holt, KE ; Pickard, D ; Dougan, G ; Hall, RM (OXFORD UNIV PRESS, 2014-02)
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    Characterization of the yehUT Two-Component Regulatory System of Salmonella enterica Serovar Typhi and Typhimurium
    Wong, VK ; Pickard, DJ ; Barquist, L ; Sivaraman, K ; Page, AJ ; Hart, PJ ; Arends, MJ ; Holt, KE ; Kane, L ; Mottram, LF ; Ellison, L ; Bautista, R ; McGee, CJ ; Kay, SJ ; Wileman, TM ; Kenney, LJ ; MacLennan, CA ; Kingsley, RA ; Dougan, G ; Cloeckaert, A (PUBLIC LIBRARY SCIENCE, 2013-12-30)
    Proteins exhibiting hyper-variable sequences within a bacterial pathogen may be associated with host adaptation. Several lineages of the monophyletic pathogen Salmonella enterica serovar Typhi (S. Typhi) have accumulated non-synonymous mutations in the putative two-component regulatory system yehUT. Consequently we evaluated the function of yehUT in S. Typhi BRD948 and S. Typhimurium ST4/74. Transcriptome analysis identified the cstA gene, encoding a carbon starvation protein as the predominantly yehUT regulated gene in both these serovars. Deletion of yehUT had no detectable effect on the ability of these mutant Salmonella to invade cultured epithelial cells (S. Typhi and S. Typhimurium) or induce colitis in a murine model (S. Typhimurium only). Growth, metabolic and antimicrobial susceptibility tests identified no obvious influences of yehUT on these phenotypes.
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    Temporal Fluctuation of Multidrug Resistant Salmonella Typhi Haplotypes in the Mekong River Delta Region of Vietnam
    Holt, KE ; Dolecek, C ; Tran, TC ; Pham, TD ; Tran, TPL ; Nguyen, VMH ; Tran, VTN ; Campbell, JI ; Bui, HM ; Nguyen, VVC ; Tran, TH ; Farrar, J ; Dougan, G ; Baker, S ; Diemert, DJ (PUBLIC LIBRARY SCIENCE, 2011-01)
    BACKGROUND: typhoid fever remains a public health problem in Vietnam, with a significant burden in the Mekong River delta region. Typhoid fever is caused by the bacterial pathogen Salmonella enterica serovar Typhi (S. Typhi), which is frequently multidrug resistant with reduced susceptibility to fluoroquinolone-based drugs, the first choice for the treatment of typhoid fever. We used a GoldenGate (Illumina) assay to type 1,500 single nucleotide polymorphisms (SNPs) and analyse the genetic variation of S. Typhi isolated from 267 typhoid fever patients in the Mekong delta region participating in a randomized trial conducted between 2004 and 2005. PRINCIPAL FINDINGS: the population of S. Typhi circulating during the study was highly clonal, with 91% of isolates belonging to a single clonal complex of the S. Typhi H58 haplogroup. The patterns of disease were consistent with the presence of an endemic haplotype H58-C and a localised outbreak of S. Typhi haplotype H58-E2 in 2004. H58-E2-associated typhoid fever cases exhibited evidence of significant geo-spatial clustering along the Sông H u branch of the Mekong River. Multidrug resistance was common in the established clone H58-C but not in the outbreak clone H58-E2, however all H58 S. Typhi were nalidixic acid resistant and carried a Ser83Phe amino acid substitution in the gyrA gene. SIGNIFICANCE: the H58 haplogroup dominates S. Typhi populations in other endemic areas, but the population described here was more homogeneous than previously examined populations, and the dominant clonal complex (H58-C, -E1, -E2) observed in this study has not been detected outside Vietnam. IncHI1 plasmid-bearing S. Typhi H58-C was endemic during the study period whilst H58-E2, which rarely carried the plasmid, was only transient, suggesting a selective advantage for the plasmid. These data add insight into the outbreak dynamics and local molecular epidemiology of S. Typhi in southern Vietnam.
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    Combined high-resolution genotyping and geospatial analysis reveals modes of endemic urban typhoid fever transmission
    Baker, S ; Holt, KE ; Clements, ACA ; Karkey, A ; Arjyal, A ; Boni, MF ; Dongol, S ; Hammond, N ; Koirala, S ; Pham, TD ; Tran, VTN ; Campbell, JI ; Dolecek, C ; Basnyat, B ; Dougan, G ; Farrar, JJ (ROYAL SOC, 2011-10)
    Typhoid is a systemic infection caused by Salmonella Typhi and Salmonella Paratyphi A, human-restricted bacteria that are transmitted faeco-orally. Salmonella Typhi and S. Paratyphi A are clonal, and their limited genetic diversity has precluded the identification of long-term transmission networks in areas with a high disease burden. To improve our understanding of typhoid transmission we have taken a novel approach, performing a longitudinal spatial case-control study for typhoid in Nepal, combining single-nucleotide polymorphism genotyping and case localization via global positioning. We show extensive clustering of typhoid occurring independent of population size and density. For the first time, we demonstrate an extensive range of genotypes existing within typhoid clusters, and even within individual households, including some resulting from clonal expansion. Furthermore, although the data provide evidence for direct human-to-human transmission, we demonstrate an overwhelming contribution of indirect transmission, potentially via contaminated water. Consistent with this, we detected S. Typhi and S. Paratyphi A in water supplies and found that typhoid was spatially associated with public water sources and low elevation. These findings have implications for typhoid-control strategies, and our innovative approach may be applied to other diseases caused by other monophyletic or emerging pathogens.
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    Emergence of a Globally Dominant IncHI1 Plasmid Type Associated with Multiple Drug Resistant Typhoid
    Holt, KE ; Minh, DP ; Baker, S ; Pham, TD ; Tran, VTN ; Nair, S ; Turner, AK ; Walsh, C ; Fanning, S ; Farrell-Ward, S ; Dutta, S ; Kariuki, S ; Weill, F-X ; Parkhill, J ; Dougan, G ; Wain, J ; Ryan, ET (PUBLIC LIBRARY SCIENCE, 2011-07)
    Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), remains a serious global health concern. Since their emergence in the mid-1970s multi-drug resistant (MDR) S. Typhi now dominate drug sensitive equivalents in many regions. MDR in S. Typhi is almost exclusively conferred by self-transmissible IncHI1 plasmids carrying a suite of antimicrobial resistance genes. We identified over 300 single nucleotide polymorphisms (SNPs) within conserved regions of the IncHI1 plasmid, and genotyped both plasmid and chromosomal SNPs in over 450 S. Typhi dating back to 1958. Prior to 1995, a variety of IncHI1 plasmid types were detected in distinct S. Typhi haplotypes. Highly similar plasmids were detected in co-circulating S. Typhi haplotypes, indicative of plasmid transfer. In contrast, from 1995 onwards, 98% of MDR S. Typhi were plasmid sequence type 6 (PST6) and S. Typhi haplotype H58, indicating recent global spread of a dominant MDR clone. To investigate whether PST6 conferred a selective advantage compared to other IncHI1 plasmids, we used a phenotyping array to compare the impact of IncHI1 PST6 and PST1 plasmids in a common S. Typhi host. The PST6 plasmid conferred the ability to grow in high salt medium (4.7% NaCl), which we demonstrate is due to the presence in PST6 of the Tn6062 transposon encoding BetU.
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    High-throughput bacterial SNP typing identifies distinct clusters of Salmonella Typhi causing typhoid in Nepalese children
    Holt, KE ; Baker, S ; Dongol, S ; Basnyat, B ; Adhikari, N ; Thorson, S ; Pulickal, AS ; Song, Y ; Parkhill, J ; Farrar, JJ ; Murdoch, DR ; Kelly, DF ; Pollard, AJ ; Dougan, G (BMC, 2010-05-31)
    BACKGROUND: Salmonella Typhi (S. Typhi) causes typhoid fever, which remains an important public health issue in many developing countries. Kathmandu, the capital of Nepal, is an area of high incidence and the pediatric population appears to be at high risk of exposure and infection. METHODS: We recently defined the population structure of S. Typhi, using new sequencing technologies to identify nearly 2,000 single nucleotide polymorphisms (SNPs) that can be used as unequivocal phylogenetic markers. Here we have used the GoldenGate (Illumina) platform to simultaneously type 1,500 of these SNPs in 62 S. Typhi isolates causing severe typhoid in children admitted to Patan Hospital in Kathmandu. RESULTS: Eight distinct S. Typhi haplotypes were identified during the 20-month study period, with 68% of isolates belonging to a subclone of the previously defined H58 S. Typhi. This subclone was closely associated with resistance to nalidixic acid, with all isolates from this group demonstrating a resistant phenotype and harbouring the same resistance-associated SNP in GyrA (Phe83). A secondary clone, comprising 19% of isolates, was observed only during the second half of the study. CONCLUSIONS: Our data demonstrate the utility of SNP typing for monitoring bacterial populations over a defined period in a single endemic setting. We provide evidence for genotype introduction and define a nalidixic acid resistant subclone of S. Typhi, which appears to be the dominant cause of severe pediatric typhoid in Kathmandu during the study period.
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    The Burden and Characteristics of Enteric Fever at a Healthcare Facility in a Densely Populated Area of Kathmandu
    Karkey, A ; Arjyal, A ; Anders, KL ; Boni, MF ; Dongol, S ; Koirala, S ; Phan, VTM ; Tran, VTN ; Clements, ACA ; Holt, KE ; Pham, TD ; Day, JN ; Campbell, JI ; Dougan, G ; Dolecek, C ; Farrar, J ; Basnyat, B ; Baker, S ; von Seidlein, L (PUBLIC LIBRARY SCIENCE, 2010-11-15)
    Enteric fever, caused by Salmonella enterica serovars Typhi and Paratyphi A (S. Typhi and S. Paratyphi A) remains a major public health problem in many settings. The disease is limited to locations with poor sanitation which facilitates the transmission of the infecting organisms. Efficacious and inexpensive vaccines are available for S. Typhi, yet are not commonly deployed to control the disease. Lack of vaccination is due partly to uncertainty of the disease burden arising from a paucity of epidemiological information in key locations. We have collected and analyzed data from 3,898 cases of blood culture-confirmed enteric fever from Patan Hospital in Lalitpur Sub-Metropolitan City (LSMC), between June 2005 and May 2009. Demographic data was available for a subset of these patients (n = 527) that were resident in LSMC and who were enrolled in trials. We show a considerable burden of enteric fever caused by S. Typhi (2,672; 68.5%) and S. Paratyphi A (1,226; 31.5%) at this Hospital over a four year period, which correlate with seasonal fluctuations in rainfall. We found that local population density was not related to incidence and we identified a focus of infections in the east of LSMC. With data from patients resident in LSMC we found that the median age of those with S. Typhi (16 years) was significantly less than S. Paratyphi A (20 years) and that males aged 15 to 25 were disproportionately infected. Our findings provide a snapshot into the epidemiological patterns of enteric fever in Kathmandu. The uneven distribution of enteric fever patients within the population suggests local variation in risk factors, such as contaminated drinking water. These findings are important for initiating a vaccination scheme and improvements in sanitation. We suggest any such intervention should be implemented throughout the LSMC area.
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    A linear plasmid truncation induces unidirectional flagellar phase change in H:z66 positive Salmonella Typhi
    Baker, S ; Holt, K ; Whitehead, S ; Goodhead, I ; Perkins, T ; Stocker, B ; Hardy, J ; Dougan, G (WILEY, 2007-12)
    The process by which bacteria regulate flagellar expression is known as phase variation and in Salmonella enterica this process permits the expression of one of two flagellin genes, fliC or fljB, at any one time. Salmonella Typhi (S. Typhi) is normally not capable of phase variation of flagellar antigen expression as isolates only harbour the fliC gene (H:d) and lacks an equivalent fljB locus. However, some S. Typhi isolates, exclusively from Indonesia, harbour an fljB equivalent encoded on linear plasmid, pBSSB1 that drives the expression of a novel flagellin named H:z66. H:z66+S. Typhi isolates were stimulated to change flagellar phase and genetically analysed for the mechanism of variation. The phase change was demonstrated to be unidirectional, reverting to expression from the resident chromosomal fliC gene. DNA sequencing demonstrated that pBSSB1 linear DNA was still detectable but that these derivatives had undergone deletion and were lacking fljA(z66) (encoding a flagellar repressor) and fljB(z66). The deletion end-point was found to involve one of the plasmid termini and a palindromic repeat sequence within fljB(z66), distinct to that found at the terminus of pBSSB1. These data demonstrate that, like some Streptomyces linear elements, at least one of the terminal inverted repeats of pBSSB1 is non-essential, but that a palindromic repeat sequence may be necessary for replication.