Publications by authors named "Robert A Kingsley"

80 Publications

Enhanced biofilm and extracellular matrix production by chronic carriage versus acute isolates of Salmonella Typhi.

PLoS Pathog 2021 Jan 19;17(1):e1009209. Epub 2021 Jan 19.

Center for Microbial Pathogenesis, Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States of America.

Salmonella Typhi is the primary causative agent of typhoid fever; an acute systemic infection that leads to chronic carriage in 3-5% of individuals. Chronic carriers are asymptomatic, difficult to treat and serve as reservoirs for typhoid outbreaks. Understanding the factors that contribute to chronic carriage is key to development of novel therapies to effectively resolve typhoid fever. Herein, although we observed no distinct clustering of chronic carriage isolates via phylogenetic analysis, we demonstrated that chronic isolates were phenotypically distinct from acute infection isolates. Chronic carriage isolates formed significantly thicker biofilms with greater biomass that correlated with significantly higher relative levels of extracellular DNA (eDNA) and DNABII proteins than biofilms formed by acute infection isolates. Importantly, extracellular DNABII proteins include integration host factor (IHF) and histone-like protein (HU) that are critical to the structural integrity of bacterial biofilms. In this study, we demonstrated that the biofilm formed by a chronic carriage isolate in vitro, was susceptible to disruption by a specific antibody against DNABII proteins, a successful first step in the development of a therapeutic to resolve chronic carriage.
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http://dx.doi.org/10.1371/journal.ppat.1009209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815147PMC
January 2021

Salmonella enterica serovar Typhi H58 clone has been endemic in Zimbabwe from 2012 to 2019.

J Antimicrob Chemother 2020 Dec 21. Epub 2020 Dec 21.

Department of Medical Microbiology, University of Pretoria, Pretoria, South Africa.

Background: Typhoid fever, caused by S. enterica ser. Typhi, continues to be a substantial health burden in developing countries. Little is known of the genotypic diversity of S. enterica ser. Typhi in Zimbabwe, but this is key for understanding the emergence and spread of this pathogen and devising interventions for its control.

Objectives: To report the molecular epidemiology of S. enterica ser. Typhi outbreak strains circulating from 2012 to 2019 in Zimbabwe, using comparative genomics.

Methods: : A review of typhoid cases records from 2012 to 2019 in Zimbabwe was performed. The phylogenetic relationship of outbreak isolates from 2012 to 2019 and emergence of antibiotic resistance was investigated by whole-genome sequence analysis.

Results: A total 22 479 suspected typhoid cases, 760 confirmed cases were reported from 2012 to 2019 and 29 isolates were sequenced. The majority of the sequenced isolates were predicted to confer resistance to aminoglycosides, β-lactams, phenicols, sulphonamides, tetracycline and fluoroquinolones (including qnrS detection). The qnrS1 gene was associated with an IncN (subtype PST3) plasmid in 79% of the isolates. Whole-genome SNP analysis, SNP-based haplotyping and resistance determinant analysis showed that 93% of the isolates belonged to a single clade represented by multidrug-resistant H58 lineage I (4.3.1.1), with a maximum pair-wise distance of 22 SNPs.

Conclusions: This study has provided detailed genotypic characterization of the outbreak strain, identified as S. Typhi 4.3.1.1 (H58). The strain has reduced susceptibility to ciprofloxacin due to qnrS carried by an IncN (subtype PST3) plasmid resulting from ongoing evolution to full resistance.
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http://dx.doi.org/10.1093/jac/dkaa519DOI Listing
December 2020

Mutation of hilD in a Salmonella Derby lineage linked to swine adaptation and reduced risk to human health.

Sci Rep 2020 12 9;10(1):21539. Epub 2020 Dec 9.

Risk Analysis and Genomic Epidemiology Unit, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Parma, Italy.

Salmonella enterica variants exhibit diverse host adaptation, outcome of infection, and associated risk to food safety. Analysis of the distribution of Salmonella enterica serovar Derby (S. Derby) subtypes in human and swine identified isolates with a distinct PFGE profile that were significantly under-represented in human infections, consistent with further host adaptation to swine. Here we show that isolates with this PFGE profile form a distinct phylogenetic sub-clade within S. Derby and exhibit a profound reduction in invasion of human epithelial cells, and a relatively small reduction in swine epithelial cells. A single missense mutation in hilD, that encodes the master-regulator of the Salmonella Pathogenicity Island 1 (SPI-1), was present in the adapted lineage. The missense mutation resulted in a loss of function of HilD that accounted for reduced invasion in human epithelial cells. The relatively small impact of the mutation on interaction with swine cells was consistent with an alternative mechanism of invasion in this pathogen-host combination.
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http://dx.doi.org/10.1038/s41598-020-78443-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726570PMC
December 2020

Whole-genome epidemiology links phage-mediated acquisition of a virulence gene to the clonal expansion of a pandemic serovar Typhimurium clone.

Microb Genom 2020 11;6(11)

Quadram Institute Bioscience, Norwich Research Park, Norwich, UK.

Epidemic and pandemic clones of bacterial pathogens with distinct characteristics continually emerge, replacing those previously dominant through mechanisms that remain poorly characterized. Here, whole-genome-sequencing-powered epidemiology linked horizontal transfer of a virulence gene, , to the emergence and clonal expansion of a new epidemic serovar Typhimurium (. Typhimurium) clone. The gene is sporadically distributed within the genus and rare in . Typhimurium lineages, but was acquired multiple times during clonal expansion of the currently dominant pandemic monophasic . Typhimurium sequence type (ST) 34 clone. Ancestral state reconstruction and time-scaled phylogenetic analysis indicated that was not present in the common ancestor of the epidemic clade, but later acquisition resulted in increased clonal expansion of -containing clones that was temporally associated with emergence of the epidemic, consistent with increased fitness. The gene was mainly associated with a temperate bacteriophage mTmV, but recombination with other bacteriophage and apparent horizontal gene transfer of the gene cassette resulted in distribution among at least four mobile genetic elements within the monophasic . Typhimurium ST34 epidemic clade. The mTmV prophage lysogenic transfer to other serovars was limited, but included the common pig-associated . Derby (. Derby). This may explain mTmV in . Derby co-circulating on farms with monophasic . Typhimurium ST34, highlighting the potential for further transfer of the virulence gene in nature. We conclude that whole-genome epidemiology pinpoints potential drivers of evolutionary and epidemiological dynamics during pathogen emergence, and identifies targets for subsequent research in epidemiology and bacterial pathogenesis.
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http://dx.doi.org/10.1099/mgen.0.000456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725340PMC
November 2020

Evolution of Salmonella enterica serotype Typhimurium driven by anthropogenic selection and niche adaptation.

PLoS Genet 2020 06 8;16(6):e1008850. Epub 2020 Jun 8.

Quadram Institute Biosciences, Norwich Research Park, Norwich, United Kingdom.

Salmonella enterica serotype Typhimurium (S. Typhimurium) is a leading cause of gastroenteritis and bacteraemia worldwide, and a model organism for the study of host-pathogen interactions. Two S. Typhimurium strains (SL1344 and ATCC14028) are widely used to study host-pathogen interactions, yet genotypic variation results in strains with diverse host range, pathogenicity and risk to food safety. The population structure of diverse strains of S. Typhimurium revealed a major phylogroup of predominantly sequence type 19 (ST19) and a minor phylogroup of ST36. The major phylogroup had a population structure with two high order clades (α and β) and multiple subclades on extended internal branches, that exhibited distinct signatures of host adaptation and anthropogenic selection. Clade α contained a number of subclades composed of strains from well characterized epidemics in domesticated animals, while clade β contained multiple subclades associated with wild avian species. The contrasting epidemiology of strains in clade α and β was reflected by the distinct distribution of antimicrobial resistance (AMR) genes, accumulation of hypothetically disrupted coding sequences (HDCS), and signatures of functional diversification. These observations were consistent with elevated anthropogenic selection of clade α lineages from adaptation to circulation in populations of domesticated livestock, and the predisposition of clade β lineages to undergo adaptation to an invasive lifestyle by a process of convergent evolution with of host adapted Salmonella serotypes. Gene flux was predominantly driven by acquisition and recombination of prophage and associated cargo genes, with only occasional loss of these elements. The acquisition of large chromosomally-encoded genetic islands was limited, but notably, a feature of two recent pandemic clones (DT104 and monophasic S. Typhimurium ST34) of clade α (SGI-1 and SGI-4).
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http://dx.doi.org/10.1371/journal.pgen.1008850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302871PMC
June 2020

Salmonella intracellular adaptation is key to understand cephalosporin treatment relapse.

EBioMedicine 2020 Jun 23;56:102802. Epub 2020 May 23.

Quadram Institute Bioscience, Norwich, United Kingdom; University of East Anglia, Norwich, United Kingdom. Electronic address:

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http://dx.doi.org/10.1016/j.ebiom.2020.102802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7248422PMC
June 2020

Emergence of NDM-producing Klebsiella pneumoniae and Escherichia coli in Spain: phylogeny, resistome, virulence and plasmids encoding blaNDM-like genes as determined by WGS.

J Antimicrob Chemother 2019 12;74(12):3489-3496

Laboratorio de Referencia e Investigación en Resistencia a Antibióticos e Infecciones Relacionadas con la Asistencia Sanitaria, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.

Objectives: NDM carbapenemases have spread worldwide. However, little information exists about the impact of NDM-producing Enterobacteriaceae in Spain. By WGS, we sought to elucidate the population structure of NDM-like-producing Klebsiella pneumoniae and Escherichia coli in Spain and to determine the plasmids harbouring blaNDM-like genes.

Methods: High-resolution SNP typing, core-genome MLST and plasmid reconstruction (PlasmidID) were performed on 59 NDM-like-producing K. pneumoniae and 8 NDM-like-producing E. coli isolated over an 8 year period in Spain.

Results: Five major epidemic clones of NDM-producing K. pneumoniae caused five important nationwide outbreaks: ST437/NDM-7, ST437/NDM-1, ST147/NDM-1, ST11/NDM-1 and ST101/NDM-1; in contrast, the spread of NDM-producing E. coli was polyclonal. Three blaNDM types were identified: blaNDM-1, 61.2%; blaNDM-7, 32.8%; and blaNDM-5, 6%. Five K. pneumoniae isolates co-produced other carbapenemases (three blaOXA-48 and two blaVIM-1). The average number of acquired resistance genes was higher in K. pneumoniae than in E. coli. The plasmids encoding blaNDM-like genes belonged to IncFII, IncFIB, IncX3, IncR, IncN and IncC types, of which IncF, IncR and IncC were associated with MDR. The genetic surroundings of blaNDM-like genes showed a highly variable region upstream of ISAba125.

Conclusions: In recent years NDM-producing K. pneumoniae and E. coli have emerged in Spain; the spread of a few high-risk K. pneumoniae clones such as ST437/NDM-7, ST437/NDM-1, ST147/NDM-1, ST11/NDM-1 and ST101/NDM-1 have caused several interregional outbreaks. In contrast, the spread of NDM-producing E. coli has been polyclonal. Plasmid types IncFII, IncFIB, IncX3, IncR, IncN and IncC carried blaNDM, and the same IncX3 plasmid was detected in K. pneumoniae and E. coli.
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http://dx.doi.org/10.1093/jac/dkz366DOI Listing
December 2019

Genome-wide Analysis of Salmonella enterica serovar Typhi in Humanized Mice Reveals Key Virulence Features.

Cell Host Microbe 2019 09 22;26(3):426-434.e6. Epub 2019 Aug 22.

Department of Microbiology, University of Washington, Seattle, WA 98195, USA; Department of Global Health, University of Washington, Seattle, WA 98195, USA; Department of Laboratory Medicine, University of Washington, Seattle, WA 98195, USA. Electronic address:

Salmonella enterica serovar Typhi causes typhoid fever only in humans. Murine infection with S. Typhimurium is used as a typhoid model, but its relevance to human typhoid is limited. Non-obese diabetic-scid IL2rγnull mice engrafted with human hematopoietic stem cells (hu-SRC-SCID) are susceptible to lethal S. Typhi infection. In this study, we use a high-density S. Typhi transposon library in hu-SRC-SCID mice to identify virulence loci using transposon-directed insertion site sequencing (TraDIS). Vi capsule, lipopolysaccharide (LPS), and aromatic amino acid biosynthesis were essential for virulence, along with the siderophore salmochelin. However, in contrast to the murine S. Typhimurium model, neither the PhoPQ two-component system nor the SPI-2 pathogenicity island was required for lethal S. Typhi infection, nor was the CdtB typhoid toxin. These observations highlight major differences in the pathogenesis of typhoid and non-typhoidal Salmonella infections and demonstrate the utility of humanized mice for understanding the pathogenesis of a human-specific pathogen.
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http://dx.doi.org/10.1016/j.chom.2019.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742556PMC
September 2019

Rapid transcriptional responses to serum exposure are associated with sensitivity and resistance to antibody-mediated complement killing in invasive Typhimurium ST313.

Wellcome Open Res 2019 25;4:74. Epub 2019 Apr 25.

Institute of Immunology and Immuotherapy, University of Birmingham, Birmingham, UK.

: Typhimurium ST313 exhibits signatures of adaptation to invasive human infection, including higher resistance to humoral immune responses than gastrointestinal isolates. Full resistance to antibody-mediated complement killing (serum resistance) among nontyphoidal is uncommon, but selection of highly resistant strains could compromise vaccine-induced antibody immunity. Here, we address the hypothesis that serum resistance is due to a distinct genotype or transcriptome response in . Typhimurium ST313. : Six . Typhimurium ST313 bloodstream isolates, three of which were antibody resistant, were studied. Genomic content (single nucleotide polymorphisms and larger chromosomal modifications) of the strains was determined by Illumina and PACBIO sequencing, and functionally characterized using RNA-seq, transposon directed insertion site sequencing (TraDIS), targeted gene deletion and transfer of selected point mutations in an attempt to identify features associated with serum resistance.   : Sequence polymorphisms in genes from strains with atypical serum susceptibility when transferred from strains that were highly resistant or susceptible to a strain that exhibited intermediate susceptibility did not significantly alter serum killing phenotype. No large chromosomal modifications typified serum resistance or susceptibility. Genes required for resistance to serum identified by TraDIS and RNA-seq included those involved in exopolysaccharide synthesis, iron scavenging and metabolism. Most of the down-regulated genes were associated with membrane proteins. Resistant and susceptible strains had distinct transcriptional responses to serum, particularly related to genes responsible for polysaccharide biosynthesis. There was higher upregulation of locus genes, involved in the biosynthesis of colanic acid exopolysaccharide, in susceptible strains and increased expression of , a regulator of very long-chain lipopolysaccharide in resistant strains. : Clinical isolates of . Typhimurium ST313 exhibit distinct antibody susceptibility phenotypes that may be associated with changes in gene expression on exposure to serum.
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http://dx.doi.org/10.12688/wellcomeopenres.15059.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6560496PMC
April 2019

Microevolution of antimicrobial resistance and biofilm formation of Salmonella Typhimurium during persistence on pig farms.

Sci Rep 2019 06 20;9(1):8832. Epub 2019 Jun 20.

Quadram Institute Bioscience, Norwich Research Park, Norwich, UK.

Salmonella Typhimurium and its monophasic variant S. 4,[5],12:i:- are the dominant serotypes associated with pigs in many countries. We investigated their population structure on nine farms using whole genome sequencing, and their genotypic and phenotypic variation. The population structure revealed the presence of phylogenetically distinct clades consisting of closely related clones of S. Typhimurium or S. 4,[5],12:i:- on each pig farm, that persisted between production cycles. All the S. 4,[5],12:i:- strains carried the Salmonella genomic island-4 (SGI-4), which confers resistance to heavy metals, and half of the strains contained the mTmV prophage, harbouring the sopE virulence gene. Most clonal groups were highly drug resistant due to the presence of multiple antimicrobial resistance (AMR) genes, and two clades exhibited evidence of recent on-farm plasmid-mediated acquisition of additional AMR genes, including an IncHI2 plasmid. Biofilm formation was highly variable but had a strong phylogenetic signature. Strains capable of forming biofilm with the greatest biomass were from the S. 4,[5],12:i:- and S. Typhimurium DT104 clades, the two dominant pandemic clones found over the last 25 years. On-farm microevolution resulted in enhanced biofilm formation in subsequent production cycle.
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http://dx.doi.org/10.1038/s41598-019-45216-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586642PMC
June 2019

SGI-4 in Monophasic Typhimurium ST34 Is a Novel ICE That Enhances Resistance to Copper.

Front Microbiol 2019 24;10:1118. Epub 2019 May 24.

Quadram Institute Bioscience, Norwich, United Kingdom.

A multi drug resistant 4,[5],12:i- of sequence type 34 (monophasic . Typhimurium ST34) is a current pandemic clone associated with livestock, particularly pigs, and numerous outbreaks in the human population. A large genomic island, termed SGI-4, is present in the monophasic Typhimurium ST34 clade and absent from other . Typhimurium strains. SGI-4 consists of 87 open reading frames including and genes previously implicated in resistance to copper (Cu) and silver, and multiple genes predicted to be involved in mobilization and transfer by conjugation. SGI-4 was excised from the chromosome, circularized, and transferred to recipient strains of . Typhimurium at a frequency influenced by stress induced by mitomycin C, and oxygen tension. The presence of SGI-4 was associated with increased resistance to Cu, particularly but not exclusively under anaerobic conditions. The presence of genes, predicted to encode an RND family efflux pump that transports Cu from the periplasm to the external milieu, was sufficient to impart the observed enhanced resistance to Cu, above that commonly associated with . Typhimurium isolates. The presence of these genes resulted in the absence of Cu-dependent induction of genes encoding multiple proteins linked to Cu resistance, also present on SGI-4, suggesting that the system effectively limits the Cu availability in the periplasm, but did not affect SodCI-dependent macrophage survival.
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http://dx.doi.org/10.3389/fmicb.2019.01118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6543542PMC
May 2019

Bacterial flagellin promotes viral entry via an NF-kB and Toll Like Receptor 5 dependent pathway.

Sci Rep 2019 05 27;9(1):7903. Epub 2019 May 27.

Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, UK.

Viruses and bacteria colonize hosts by invading epithelial barriers. Recent studies have shown that interactions between the microbiota, pathogens and the host can potentiate infection through poorly understood mechanisms. Here, we investigated whether diverse bacterial species could modulate virus internalization into host cells, often a rate-limiting step in establishing infections. Lentiviral pseudoviruses expressing influenza, measles, Ebola, Lassa or vesicular stomatitis virus envelope glycoproteins enabled us to study entry of viruses that exploit diverse internalization pathways. Salmonella Typhimurium, Escherichia coli and Pseudomonas aeruginosa significantly increased viral uptake, even at low bacterial frequencies. This did not require bacterial contact with or invasion of host cells. Studies determined that the bacterial antigen responsible for this pro-viral activity was the Toll-Like Receptor 5 (TLR5) agonist flagellin. Exposure to flagellin increased virus attachment to epithelial cells in a temperature-dependent manner via TLR5-dependent activation of NF-ΚB. Importantly, this phenotype was both long lasting and detectable at low multiplicities of infection. Flagellin is shed from bacteria and our studies uncover a new bystander role for this protein in regulating virus entry. This highlights a new aspect of viral-bacterial interplay with significant implications for our understanding of polymicrobial-associated pathogenesis.
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http://dx.doi.org/10.1038/s41598-019-44263-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6536546PMC
May 2019

Adding function to the genome of African Salmonella Typhimurium ST313 strain D23580.

PLoS Biol 2019 01 15;17(1):e3000059. Epub 2019 Jan 15.

Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.

Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants.
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http://dx.doi.org/10.1371/journal.pbio.3000059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333337PMC
January 2019

Network Biology Approaches to Identify Molecular and Systems-Level Differences Between Salmonella Pathovars.

Methods Mol Biol 2019 ;1918:265-273

Quadram Institute Bioscience, Norwich Research Park, Norwich, UK.

The field of systems biology endeavors to map, study, and simulate cellular systems and their underlying mechanisms. The internal mechanisms of biological systems can be represented with networks comprising nodes and edges. Nodes denote the constituents of the biological system whereas edges indicate the relationships among them. Likewise, every layer of cellular organization can be represented by networks. Multilayered networks capture interactions between various network types, such as transcriptional regulatory networks, protein-protein interaction networks, and metabolic networks from the same biological system. This property makes multilayered networks representative of the system while its internal mechanisms are investigated. However, there are not many multilayered networks containing integrated data for nonmodel organisms including the bacterial pathogens Salmonella. Here, we outline the steps to create such an integrated network database, through the example of SalmoNet, the first integrated multilayered data resource for multiple strains belonging to distinct Salmonella serovars.
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http://dx.doi.org/10.1007/978-1-4939-9000-9_21DOI Listing
June 2019

Functional analysis of Salmonella Typhi adaptation to survival in water.

Environ Microbiol 2018 11;20(11):4079-4090

The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.

Contaminated water is a major risk factor associated with the transmission of Salmonella enterica serovar Typhi (S. Typhi), the aetiological agent of human typhoid. However, little is known about how this pathogen adapts to living in the aqueous environment. We used transcriptome analysis (RNA-seq) and transposon mutagenesis (TraDIS) to characterize these adaptive changes and identify multiple genes that contribute to survival. Over half of the genes in the S. Typhi genome altered expression level within the first 24 h following transfer from broth culture to water, although relatively few did so in the first 30 min. Genes linked to central metabolism, stress associated with arrested proton motive force and respiratory chain factors changed expression levels. Additionally, motility and chemotaxis genes increased expression, consistent with a scavenging lifestyle. The viaB-associated gene tviC encoding a glcNAc epimerase that is required for Vi polysaccharide biosynthesis was, along with several other genes, shown to contribute to survival in water. Thus, we define regulatory adaptation operating in S. Typhi that facilitates survival in water.
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http://dx.doi.org/10.1111/1462-2920.14458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282856PMC
November 2018

Evolution of Salmonella within Hosts.

Trends Microbiol 2018 12 25;26(12):986-998. Epub 2018 Jun 25.

Quadram Institute Bioscience, Norwich Research Park, Colney, Norwich, UK. Electronic address:

Within-host evolution has resulted in thousands of variants of Salmonella that exhibit remarkable diversity in host range and disease outcome, from broad host range to exquisite host restriction, causing gastroenteritis to disseminated disease such as typhoid fever. Within-host evolution is a continuing process driven by genomic variation that occurs during each infection, potentiating adaptation to a new niche resulting from changes in animal husbandry, the use of antimicrobials, and emergence of immune compromised populations. We discuss key advances in our understanding of the evolution of Salmonella within the host, inferred from (i) the process of host adaptation of Salmonella pathovars in the past, and (ii) direct observation of the generation of variation and selection of beneficial traits during single infections.
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http://dx.doi.org/10.1016/j.tim.2018.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249985PMC
December 2018

Genome Variation and Molecular Epidemiology of Salmonella enterica Serovar Typhimurium Pathovariants.

Infect Immun 2018 08 23;86(8). Epub 2018 Jul 23.

Quadram Institute Bioscience, Norwich Research Park, Colney, Norwich, United Kingdom

serovar Typhimurium is one of approximately 2,500 distinct serovars of the genus but is exceptional in its wide distribution in the environment, livestock, and wild animals. Typhimurium causes a large proportion of nontyphoidal (NTS) infections, accounting for a quarter of infections, second only to serovar Enteritidis in incidence. Typhimurium was once considered the archetypal broad-host-range serovar due to its wide distribution in livestock and wild animals, and much of what we know of the interaction of with the host comes from research using a small number of laboratory strains of the serovar (LT2, SL1344, and ATCC 14028). But it has become clear that these strains do not reflect the genotypic or phenotypic diversity of Typhimurium. Here, we review the epidemiological record of Typhimurium and studies of the host-pathogen interactions of diverse strains of Typhimurium. We present the concept of distinct pathovariants of Typhimurium that exhibit diversity of host range, distribution in the environment, pathogenicity, and risk to food safety. We review recent evidence from whole-genome sequencing that has revealed the extent of genomic diversity of Typhimurium pathovariants, the genomic basis of differences in the level of risk to human and animal health, and the molecular epidemiology of prominent strains. An improved understanding of the impact of genome variation of bacterial pathogens on pathogen-host and pathogen-environment interactions has the potential to improve quantitative risk assessment and reveal how new pathogens evolve.
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http://dx.doi.org/10.1128/IAI.00079-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6056856PMC
August 2018

IgG1 Is Required for Optimal Protection after Immunization with the Purified Porin OmpD from Typhimurium.

J Immunol 2017 12 10;199(12):4103-4109. Epub 2017 Nov 10.

Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham B15 2TT, United Kingdom;

In mice, the IgG subclass induced after Ag encounter can reflect the nature of the Ag. Th2 Ags such as alum-precipitated proteins and helminths induce IgG1, whereas Th1 Ags, such as Typhimurium, predominantly induce IgG2a. The contribution of different IgG isotypes to protection against bacteria such as Typhimurium is unclear, although as IgG2a is induced by natural infection, it is assumed this isotype is important. Previously, we have shown that purified Typhimurium porins including outer membrane protein OmpD, which induce both IgG1 and IgG2a in mice, provide protection to Typhimurium infection via Ab. In this study we report the unexpected finding that mice lacking IgG1, but not IgG2a, are substantially less protected after porin immunization than wild-type controls. IgG1-deficient mice produce more porin-specific IgG2a, resulting in total IgG levels that are similar to wild-type mice. The decreased protection in IgG1-deficient mice correlates with less efficient bacterial opsonization and uptake by macrophages, and this reflects the low binding of outer membrane protein OmpD-specific IgG2a to the bacterial surface. Thus, the Th2-associated isotype IgG1 can play a role in protection against Th1-associated organisms such as Typhimurium. Therefore, individual IgG subclasses to a single Ag can provide different levels of protection and the IgG isotype induced may need to be a consideration when designing vaccines and immunization strategies.
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http://dx.doi.org/10.4049/jimmunol.1700952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5713499PMC
December 2017

Role of and in Susceptibility to Antibody-Mediated Complement-Dependent Killing and Virulence of Salmonella enterica Serovar Typhimurium.

Infect Immun 2017 09 18;85(9). Epub 2017 Aug 18.

Novartis Vaccines Institute for Global Health, Siena, Italy

The ST313 pathovar of serovar Typhimurium contributes to a high burden of invasive disease among African infants and HIV-infected adults. It is characterized by genome degradation (loss of coding capacity) and has increased resistance to antibody-dependent complement-mediated killing compared with enterocolitis-causing strains of Typhimurium. Vaccination is an attractive disease-prevention strategy, and leading candidates focus on the induction of bactericidal antibodies. Antibody-resistant strains arising through further gene deletion could compromise such a strategy. Exposing a saturating transposon insertion mutant library of Typhimurium to immune serum identified a repertoire of Typhimurium genes that, when interrupted, result in increased resistance to serum killing. These genes included several involved in bacterial envelope biogenesis, protein translocation, and metabolism. We generated defined mutant derivatives using Typhimurium SL1344 as the host. Based on their initial levels of enhanced resistance to killing, and mutants were selected for further characterization. The Typhimurium mutant lost the characteristic rod-shaped appearance, exhibited increased sensitivity to osmotic and detergent stress, lacked very long lipopolysaccharide, was unable to invade enterocytes, and demonstrated decreased ability to infect mice. In contrast, the Typhimurium mutants had similar sensitivity to osmotic and detergent stress and lipopolysaccharide profile and an increased ability to infect enterocytes compared with the wild type, but it had no increased ability to cause infection. These findings indicate that increased resistance to antibody-dependent complement-mediated killing secondary to genetic deletion is not necessarily accompanied by increased virulence and suggest the presence of different mechanisms of antibody resistance.
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http://dx.doi.org/10.1128/IAI.00419-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563563PMC
September 2017

Emergence of host-adapted Salmonella Enteritidis through rapid evolution in an immunocompromised host.

Nat Microbiol 2016 Jan 25;1:15023. Epub 2016 Jan 25.

The Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

Host adaptation is a key factor contributing to the emergence of new bacterial, viral and parasitic pathogens. Many pathogens are considered promiscuous because they cause disease across a range of host species, while others are host-adapted, infecting particular hosts(1). Host adaptation can potentially progress to host restriction, where the pathogen is strictly limited to a single host species and is frequently associated with more severe symptoms. Host-adapted and host-restricted bacterial clades evolve from within a broader host-promiscuous species and sometimes target different niches within their specialist hosts, such as adapting from a mucosal to a systemic lifestyle. Genome degradation, marked by gene inactivation and deletion, is a key feature of host adaptation, although the triggers initiating genome degradation are not well understood. Here, we show that a chronic systemic non-typhoidal Salmonella infection in an immunocompromised human patient resulted in genome degradation targeting genes that are expendable for a systemic lifestyle. We present a genome-based investigation of a recurrent blood-borne Salmonella enterica serotype Enteritidis (S. Enteritidis) infection covering 15 years in an interleukin-12 β1 receptor-deficient individual that developed into an asymptomatic chronic infection. The infecting S. Enteritidis harboured a mutation in the mismatch repair gene mutS that accelerated the genomic mutation rate. Phylogenetic analysis and phenotyping of multiple patient isolates provides evidence for a remarkable level of within-host evolution that parallels genome changes present in successful host-restricted bacterial pathogens but never before observed on this timescale. Our analysis identifies common pathways of host adaptation and demonstrates the role that immunocompromised individuals can play in this process.
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http://dx.doi.org/10.1038/nmicrobiol.2015.23DOI Listing
January 2016

Distinct Salmonella Enteritidis lineages associated with enterocolitis in high-income settings and invasive disease in low-income settings.

Nat Genet 2016 10 22;48(10):1211-1217. Epub 2016 Aug 22.

Wellcome Trust Sanger Institute, Cambridge, UK.

An epidemiological paradox surrounds Salmonella enterica serovar Enteritidis. In high-income settings, it has been responsible for an epidemic of poultry-associated, self-limiting enterocolitis, whereas in sub-Saharan Africa it is a major cause of invasive nontyphoidal Salmonella disease, associated with high case fatality. By whole-genome sequence analysis of 675 isolates of S. Enteritidis from 45 countries, we show the existence of a global epidemic clade and two new clades of S. Enteritidis that are geographically restricted to distinct regions of Africa. The African isolates display genomic degradation, a novel prophage repertoire, and an expanded multidrug resistance plasmid. S. Enteritidis is a further example of a Salmonella serotype that displays niche plasticity, with distinct clades that enable it to become a prominent cause of gastroenteritis in association with the industrial production of eggs and of multidrug-resistant, bloodstream-invasive infection in Africa.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5047355PMC
http://dx.doi.org/10.1038/ng.3644DOI Listing
October 2016

Using a Human Challenge Model of Infection to Measure Vaccine Efficacy: A Randomised, Controlled Trial Comparing the Typhoid Vaccines M01ZH09 with Placebo and Ty21a.

PLoS Negl Trop Dis 2016 08 17;10(8):e0004926. Epub 2016 Aug 17.

Oxford Vaccine Group, Department of Paediatrics, and the NIHR Oxford Biomedical Research Centre, University of Oxford, Oxford, United Kingdom.

Background: Typhoid persists as a major cause of global morbidity. While several licensed vaccines to prevent typhoid are available, they are of only moderate efficacy and unsuitable for use in children less than two years of age. Development of new efficacious vaccines is complicated by the human host-restriction of Salmonella enterica serovar Typhi (S. Typhi) and lack of clear correlates of protection. In this study, we aimed to evaluate the protective efficacy of a single dose of the oral vaccine candidate, M01ZH09, in susceptible volunteers by direct typhoid challenge.

Methods And Findings: We performed a randomised, double-blind, placebo-controlled trial in healthy adult participants at a single centre in Oxford (UK). Participants were allocated to receive one dose of double-blinded M01ZH09 or placebo or 3-doses of open-label Ty21a. Twenty-eight days after vaccination, participants were challenged with 104CFU S. Typhi Quailes strain. The efficacy of M01ZH09 compared with placebo (primary outcome) was assessed as the percentage of participants reaching pre-defined endpoints constituting typhoid diagnosis (fever and/or bacteraemia) during the 14 days after challenge. Ninety-nine participants were randomised to receive M01ZH09 (n = 33), placebo (n = 33) or 3-doses of Ty21a (n = 33). After challenge, typhoid was diagnosed in 18/31 (58.1% [95% CI 39.1 to 75.5]) M01ZH09, 20/30 (66.7% [47.2 to 87.2]) placebo, and 13/30 (43.3% [25.5 to 62.6]) Ty21a vaccine recipients. Vaccine efficacy (VE) for one dose of M01ZH09 was 13% [95% CI -29 to 41] and 35% [-5 to 60] for 3-doses of Ty21a. Retrospective multivariable analyses demonstrated that pre-existing anti-Vi antibody significantly reduced susceptibility to infection after challenge; a 1 log increase in anti-Vi IgG resulting in a 71% decrease in the hazard ratio of typhoid diagnosis ([95% CI 30 to 88%], p = 0.006) during the 14 day challenge period. Limitations to the study included the requirement to limit the challenge period prior to treatment to 2 weeks, the intensity of the study procedures and the high challenge dose used resulting in a stringent model.

Conclusions: Despite successfully demonstrating the use of a human challenge study to directly evaluate vaccine efficacy, a single-dose M01ZH09 failed to demonstrate significant protection after challenge with virulent Salmonella Typhi in this model. Anti-Vi antibody detected prior to vaccination played a major role in outcome after challenge.

Trial Registration: ClinicalTrials.gov (NCT01405521) and EudraCT (number 2011-000381-35).
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http://dx.doi.org/10.1371/journal.pntd.0004926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988630PMC
August 2016

A profile-based method for identifying functional divergence of orthologous genes in bacterial genomes.

Bioinformatics 2016 12 8;32(23):3566-3574. Epub 2016 Aug 8.

School of Biological Sciences, University of Canterbury, Christchurch, New Zealand.

Motivation: Next generation sequencing technologies have provided us with a wealth of information on genetic variation, but predicting the functional significance of this variation is a difficult task. While many comparative genomics studies have focused on gene flux and large scale changes, relatively little attention has been paid to quantifying the effects of single nucleotide polymorphisms and indels on protein function, particularly in bacterial genomics.

Results: We present a hidden Markov model based approach we call delta-bitscore (DBS) for identifying orthologous proteins that have diverged at the amino acid sequence level in a way that is likely to impact biological function. We benchmark this approach with several widely used datasets and apply it to a proof-of-concept study of orthologous proteomes in an investigation of host adaptation in Salmonella enterica We highlight the value of the method in identifying functional divergence of genes, and suggest that this tool may be a better approach than the commonly used dN/dS metric for identifying functionally significant genetic changes occurring in recently diverged organisms.

Availability And Implementation: A program implementing DBS for pairwise genome comparisons is freely available at: https://github.com/UCanCompBio/deltaBS CONTACT: nicole.wheeler@pg.canterbury.ac.nz or lars.barquist@uni-wuerzburg.deSupplementary information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btw518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5181535PMC
December 2016

Interferon-driven alterations of the host's amino acid metabolism in the pathogenesis of typhoid fever.

J Exp Med 2016 05 23;213(6):1061-77. Epub 2016 May 23.

Oxford Vaccine Group, Department of Paediatrics, University of Oxford and the NIHR Oxford Biomedical Research Centre, Oxford OX3 7LE, England, UK.

Enteric fever, caused by Salmonella enterica serovar Typhi, is an important public health problem in resource-limited settings and, despite decades of research, human responses to the infection are poorly understood. In 41 healthy adults experimentally infected with wild-type S. Typhi, we detected significant cytokine responses within 12 h of bacterial ingestion. These early responses did not correlate with subsequent clinical disease outcomes and likely indicate initial host-pathogen interactions in the gut mucosa. In participants developing enteric fever after oral infection, marked transcriptional and cytokine responses during acute disease reflected dominant type I/II interferon signatures, which were significantly associated with bacteremia. Using a murine and macrophage infection model, we validated the pivotal role of this response in the expression of proteins of the host tryptophan metabolism during Salmonella infection. Corresponding alterations in tryptophan catabolites with immunomodulatory properties in serum of participants with typhoid fever confirmed the activity of this pathway, and implicate a central role of host tryptophan metabolism in the pathogenesis of typhoid fever.
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http://dx.doi.org/10.1084/jem.20151025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4886356PMC
May 2016

Emergence of host-adapted Enteritidis through rapid evolution in an immunocompromised host.

Nat Microbiol 2016 03 25;1(3). Epub 2016 Jan 25.

The Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom.

Host adaptation is a key factor contributing to the emergence of new bacterial, viral and parasitic pathogens. Many pathogens are considered promiscuous because they cause disease across a range of host species, while others are host-adapted, infecting particular hosts. Host adaptation can potentially progress to host restriction where the pathogen is strictly limited to a single host species and is frequently associated with more severe symptoms. Host-adapted and host-restricted bacterial clades evolve from within a broader host-promiscuous species and sometimes target different niches within their specialist hosts, such as adapting from a mucosal to a systemic lifestyle. Genome degradation, marked by gene inactivation and deletion, is a key feature of host adaptation, although the triggers initiating genome degradation are not well understood. Here, we show that a chronic systemic non-typhoidal infection in an immunocompromised human patient resulted in genome degradation targeting genes that are expendable for a systemic lifestyle. We present a genome-based investigation of a recurrent blood-borne serotype Enteritidis (. Enteritidis) infection covering 15 years in an interleukin (IL)-12 β-1 receptor-deficient individual that developed into an asymptomatic chronic infection. The infecting Enteritidis harbored a mutation in the mismatch repair gene that accelerated the genomic mutation rate. Phylogenetic analysis and phenotyping of multiple patient isolates provides evidence for a remarkable level of within-host evolution that parallels genome changes present in successful host-restricted bacterial pathogens but never before observed on this timescale. Our analysis identifies common pathways of host adaptation and demonstrates the role that immunocompromised individuals can play in this process.
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http://dx.doi.org/10.1038/nmicrobiol.2015.23DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4843968PMC
March 2016

Microevolution of Monophasic Salmonella Typhimurium during Epidemic, United Kingdom, 2005-2010.

Emerg Infect Dis 2016 Apr;22(4):617-24

Microevolution associated with emergence and expansion of new epidemic clones of bacterial pathogens holds the key to epidemiologic success. To determine microevolution associated with monophasic Salmonella Typhimurium during an epidemic, we performed comparative whole-genome sequencing and phylogenomic analysis of isolates from the United Kingdom and Italy during 2005-2012. These isolates formed a single clade distinct from recent monophasic epidemic clones previously described from North America and Spain. The UK monophasic epidemic clones showed a novel genomic island encoding resistance to heavy metals and a composite transposon encoding antimicrobial drug resistance genes not present in other Salmonella Typhimurium isolates, which may have contributed to epidemiologic success. A remarkable amount of genotypic variation accumulated during clonal expansion that occurred during the epidemic, including multiple independent acquisitions of a novel prophage carrying the sopE gene and multiple deletion events affecting the phase II flagellin locus. This high level of microevolution may affect antigenicity, pathogenicity, and transmission.
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http://dx.doi.org/10.3201/eid2204.150531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806966PMC
April 2016

Loss of Multicellular Behavior in Epidemic African Nontyphoidal Salmonella enterica Serovar Typhimurium ST313 Strain D23580.

mBio 2016 Mar 1;7(2):e02265. Epub 2016 Mar 1.

Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington, USA Department of Laboratory Medicine, School of Medicine, University of Washington, Seattle, Washington, USA

Unlabelled: Nontyphoidal Salmonella enterica serovar Typhimurium is a frequent cause of bloodstream infections in children and HIV-infected adults in sub-Saharan Africa. Most isolates from African patients with bacteremia belong to a single sequence type, ST313, which is genetically distinct from gastroenteritis-associated ST19 strains, such as 14028s and SL1344. Some studies suggest that the rapid spread of ST313 across sub-Saharan Africa has been facilitated by anthroponotic (person-to-person) transmission, eliminating the need for Salmonella survival outside the host. While these studies have not ruled out zoonotic or other means of transmission, the anthroponotic hypothesis is supported by evidence of extensive genomic decay, a hallmark of host adaptation, in the sequenced ST313 strain D23580. We have identified and demonstrated 2 loss-of-function mutations in D23580, not present in the ST19 strain 14028s, that impair multicellular stress resistance associated with survival outside the host. These mutations result in inactivation of the KatE stationary-phase catalase that protects high-density bacterial communities from oxidative stress and the BcsG cellulose biosynthetic enzyme required for the RDAR (red, dry, and rough) colonial phenotype. However, we found that like 14028s, D23580 is able to elicit an acute inflammatory response and cause enteritis in mice and rhesus macaque monkeys. Collectively, these observations suggest that African S. Typhimurium ST313 strain D23580 is becoming adapted to an anthroponotic mode of transmission while retaining the ability to infect and cause enteritis in multiple host species.

Importance: The last 3 decades have witnessed an epidemic of invasive nontyphoidal Salmonella infections in sub-Saharan Africa. Genomic analysis and clinical observations suggest that the Salmonella strains responsible for these infections are evolving to become more typhoid-like with regard to patterns of transmission and virulence. This study shows that a prototypical African nontyphoidal Salmonella strain has lost traits required for environmental stress resistance, consistent with an adaptation to a human-to-human mode of transmission. However, in contrast to predictions, the strain remains capable of causing acute inflammation in the mammalian intestine. This suggests that the systemic clinical presentation of invasive nontyphoidal Salmonella infections in Africa reflects the immune status of infected hosts rather than intrinsic differences in the virulence of African Salmonella strains. Our study provides important new insights into the evolution of host adaptation in bacterial pathogens.
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http://dx.doi.org/10.1128/mBio.02265-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810497PMC
March 2016