Genomic analysis of clinical Aeromonas isolates reveals genetic diversity but little evidence of genetic determinants for diarrhoeal disease.

Aeromonas spp. are associated with a number of infectious syndromes in humans including gastroenteritis and dysentery. Our understanding of the genetic diversity, population structure, virulence determinants and antimicrobial resistance of the genus has been limited by a lack of sequenced genomes linked to metadata. We performed a comprehensive analysis of the whole genome sequences of 447 Aeromonas isolates from children in Karachi, Pakistan, with moderate-to-severe diarrhoea (MSD) and from matched controls without diarrhoea that were collected as part of the Global Enteric Multicenter Study (GEMS). Human-associated Aeromonas isolates exhibited high species diversity and extensive antimicrobial and virulence gene content. Aeromonas caviae, A. dhankensis, A. veronii and A. enteropelogenes were all significantly associated with MSD in at least one cohort group. The maf2 and lafT genes that encode components of polar and lateral flagella, respectively, exhibited a weak association with isolates originating from cases of gastroenteritis.

Determining the effects of AR/VR HMD design parameters (mass and inertia) on cervical spine joint torques.

This study aimed to determine gravitational and dynamic torques and muscle activity of the neck across a series of design parameters of head mounted displays (mass, center of mass, and counterweights) associated with virtual and augmented reality (VR/AR). Twenty young adult participants completed five movement types (Slow and Fast Flexion/Extension and Rotation, and Search) while wearing a custom-designed prototype headset that varied the three design parameters: display mass (0, 200, 500, and 750 g), distance of the display's center of mass in front of the eyes (approximately 1, 3, and 5 cm anteriorly), and counterweights of 0, 166, 332, and 500 g to balance the display mass of 500 g at 7 cm. Inverse dynamics of a link segment model of the head and headset provided estimates of the torques about the joint between the skull and the occiput-first cervical vertebrae (OC1) and joint between the C7 and T1 vertebrae (C7). Surface electromyography (EMG) measured bilateral muscle activity of the splenius and upper trapezius muscles. Adding 750 g of display mass nearly doubled root mean square joint torques across all movement types. Increasing the distance of the display mass in front of the eyes by 4 cm increased torques about OC1 for the Slow and Fast Rotation and Search movements by approximately 20%. Adding a counterweight decreased torques about OC1 during the rotation and search tasks but did not decrease the torques experienced in the lower cervical spine (C7). For the flexion/extension axis, the magnitude of the dynamic torque component was 20% or less of the total torque experienced whereas for the rotation axis the magnitude of the dynamic torque component was greater than 50% of the total torque. Surface EMG root mean square values significantly varied across movement types with the fast rotation having the largest values; however, they did not vary significantly across the headset configurations.

Genomic epidemiology of Salmonella Typhi in Central Division, Fiji, 2012 to 2016.

Background: Typhoid fever is endemic in some Pacific Island Countries including Fiji and Samoa yet genomic surveillance is not routine in such settings. Previous studies suggested imports of the global H58 clade of Salmonella enterica var Typhi (Salmonella Typhi) contribute to disease in these countries which, given the MDR potential of H58, does not auger well for treatment. The objective of the study was to define the genomic epidemiology of Salmonella Typhi in Fiji. Methods: Genomic sequencing approaches were implemented to study the distribution of 255 Salmonella Typhi isolates from the Central Division of Fiji. We augmented epidemiological surveillance and Bayesian phylogenomic approaches with a multi-year typhoid case-control study to define geospatial patterns among typhoid cases. Findings: Genomic analyses showed Salmonella Typhi from Fiji resolved into 2 non-H58 genotypes with isolates from the two dominant ethnic groups, the Indigenous (iTaukei) and non-iTaukei genetically indistinguishable. Low rates of international importation of clones was observed and overall, there were very low levels an antibiotic resistance within the endemic Fijian typhoid genotypes. Genomic epidemiological investigations were able to identify previously unlinked case clusters. Bayesian phylodynamic analyses suggested that genomic variation within the larger endemic Salmonella Typhi genotype expanded at discreet times, then contracted. Interpretation: Cyclones and flooding drove 'waves' of typhoid outbreaks in Fiji which, through population aggregation, poor sanitation and water safety, and then mobility of the population, spread clones more widely. Minimal international importations of new typhoid clones suggest that targeted local intervention strategies may be useful in controlling endemic typhoid infection. These findings add to our understanding of typhoid transmission networks in an endemic island country with broad implications, particularly across Pacific Island Countries. Funding: This work was supported by the Coalition Against Typhoid through the Bill and Melinda Gates Foundation [grant number OPP1017518], the Victorian Government, the National Health and Medical Research Council Australia, the Australian Research Council, and the Fiji Ministry of Health and Medical Services.

Application of the Child Health and Nutrition Research Initiative (CHNRI) methodology to prioritize research to enable the implementation of Ending Cholera: A global roadmap to 2030.

BACKGROUND: The "Ending Cholera: A Global Roadmap to 2030" (Roadmap) was launched in October 2017. Following its launch, it became clear that additional evidence is needed to assist countries in controlling cholera and that a prioritized list of research questions is required to focus the limited resources to address the issues most relevant to the implementation of the Roadmap. METHODS: A comprehensive list of research questions was developed based on inputs from the Working Groups of the Global Taskforce for Cholera Control and other experts. The Child Health and Nutrition Research Initiative methodology was adapted to identify the relevant assessment criteria and assign weights to each criterion. The assessment criteria were applied to each research question by cholera experts to derive a score based on which they were prioritized. FINDINGS: The consultation process involved 177 experts and stakeholders representing different constituencies and geographies with research priority scores ranging from 88·8 to 65·7% and resulted in the prioritization of the top 20 research questions across all Roadmap pillars, the top five research questions for each Roadmap pillar, and three discovery research questions. This resulted in 32 non-duplicative research questions that considers both immediate and long-term Roadmap goals. INTERPRETATION: The transparent, inclusive, and rigorous process to develop a Research Agenda is aimed to secure broad buy-in and serve as a guide for funding agencies and researchers to focus their efforts to fill the evidence gaps plaguing cholera-endemic countries.

Innovative vaccine approaches-a Keystone Symposia report.

The rapid development of COVID-19 vaccines was the result of decades of research to establish flexible vaccine platforms and understand pathogens with pandemic potential, as well as several novel changes to the vaccine discovery and development processes that partnered industry and governments. And while vaccines offer the potential to drastically improve global health, low-and-middle-income countries around the world often experience reduced access to vaccines and reduced vaccine efficacy. Addressing these issues will require novel vaccine approaches and platforms, deeper insight how vaccines mediate protection, and innovative trial designs and models. On June 28-30, 2021, experts in vaccine research, development, manufacturing, and deployment met virtually for the Keystone eSymposium "Innovative Vaccine Approaches" to discuss advances in vaccine research and development.

Population structure and antimicrobial resistance patterns of Salmonella Typhi isolates in urban Dhaka, Bangladesh from 2004 to 2016.

BACKGROUND: Multi-drug resistant typhoid fever remains an enormous public health threat in low and middle-income countries. However, we still lack a detailed understanding of the epidemiology and genomics of S. Typhi in many regions. Here we have undertaken a detailed genomic analysis of typhoid in urban Dhaka, Bangladesh to unravel the population structure and antimicrobial resistance patterns in S. Typhi isolated between 2004-2016. PRINCIPAL FINDINGS: Whole genome sequencing of 202 S. Typhi isolates obtained from three study locations in urban Dhaka revealed a diverse range of S. Typhi genotypes and AMR profiles. The bacterial population within Dhaka were relatively homogenous with little stratification between different healthcare facilities or age groups. We also observed evidence of exchange of Bangladeshi genotypes with neighboring South Asian countries (India, Pakistan and Nepal) suggesting these are circulating throughout the region. This analysis revealed a decline in H58 (genotype 4.3.1) isolates from 2011 onwards, coinciding with a rise in a diverse range of non-H58 genotypes and a simultaneous rise in isolates with reduced susceptibility to fluoroquinolones, potentially reflecting a change in treatment practices. We identified a novel S. Typhi genotype, subclade 3.3.2 (previously defined only to clade level, 3.3), which formed two localized clusters (3.3.2.Bd1 and 3.3.2.Bd2) associated with different mutations in the Quinolone Resistance Determining Region (QRDR) of gene gyrA. SIGNIFICANCE: Our analysis of S. Typhi isolates from urban Dhaka, Bangladesh isolated over a twelve year period identified a diverse range of AMR profiles and genotypes. The observed increase in non-H58 genotypes associated with reduced fluoroquinolone susceptibility may reflect a change in treatment practice in this region and highlights the importance of continued molecular surveillance to monitor the ongoing evolution of AMR in Dhaka. We have defined new genotypes and lineages of Bangladeshi S. Typhi which will facilitate the identification of these emerging AMR clones in future surveillance efforts.

Mathematical modelling for antibiotic resistance control policy: do we know enough?

BACKGROUND: Antibiotics remain the cornerstone of modern medicine. Yet there exists an inherent dilemma in their use: we are able to prevent harm by administering antibiotic treatment as necessary to both humans and animals, but we must be mindful of limiting the spread of resistance and safeguarding the efficacy of antibiotics for current and future generations. Policies that strike the right balance must be informed by a transparent rationale that relies on a robust evidence base. MAIN TEXT: One way to generate the evidence base needed to inform policies for managing antibiotic resistance is by using mathematical models. These models can distil the key drivers of the dynamics of resistance transmission from complex infection and evolutionary processes, as well as predict likely responses to policy change in silico. Here, we ask whether we know enough about antibiotic resistance for mathematical modelling to robustly and effectively inform policy. We consider in turn the challenges associated with capturing antibiotic resistance evolution using mathematical models, and with translating mathematical modelling evidence into policy. CONCLUSIONS: We suggest that in spite of promising advances, we lack a complete understanding of key principles. From this we advocate for priority areas of future empirical and theoretical research.

An African Salmonella Typhimurium ST313 sublineage with extensive drug-resistance and signatures of host adaptation.

Bloodstream infections by Salmonella enterica serovar Typhimurium constitute a major health burden in sub-Saharan Africa (SSA). These invasive non-typhoidal (iNTS) infections are dominated by isolates of the antibiotic resistance-associated sequence type (ST) 313. Here, we report emergence of ST313 sublineage II.1 in the Democratic Republic of the Congo. Sublineage II.1 exhibits extensive drug resistance, involving a combination of multidrug resistance, extended spectrum ß-lactamase production and azithromycin resistance. ST313 lineage II.1 isolates harbour an IncHI2 plasmid we name pSTm-ST313-II.1, with one isolate also exhibiting decreased ciprofloxacin susceptibility. Whole genome sequencing reveals that ST313 II.1 isolates have accumulated genetic signatures potentially associated with altered pathogenicity and host adaptation, related to changes observed in biofilm formation and metabolic capacity. Sublineage II.1 emerged at the beginning of the 21st century and is involved in on-going outbreaks. Our data provide evidence of further evolution within the ST313 clade associated with iNTS in SSA.

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

Background: Salmonella 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 Salmonellae 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 S. Typhimurium ST313. Methods: Six S. 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.   Results: 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 wca locus genes, involved in the biosynthesis of colanic acid exopolysaccharide, in susceptible strains and increased expression of fepE, a regulator of very long-chain lipopolysaccharide in resistant strains. Conclusion: Clinical isolates of S. Typhimurium ST313 exhibit distinct antibody susceptibility phenotypes that may be associated with changes in gene expression on exposure to serum.

Antibiotic Resistance and Typhoid.

Multiple drug (antibiotic) resistance (MDR) has become a major threat to the treatment of typhoid and other infectious diseases. Since the 1970s, this threat has increased in Salmonella enterica serovar Typhi, driven in part by the emergence of successful genetic clades, such as haplotype H58, associated with the MDR phenotype. H58 S. Typhi can express multiple antibiotic resistance determinants while retaining the ability to efficiently transmit and persist within the human population. The recent identification of extensively drug resistant S. Typhi only highlights the dangers of ignoring this threat. Here we discuss the evolution of the S. Typhi MDR phenotype and consider options for management.

Emergence of dominant multidrug-resistant bacterial clades: Lessons from history and whole-genome sequencing.

Antibiotic resistance in bacteria has emerged as a global challenge over the past 90 years, compromising our ability to effectively treat infections. There has been a dramatic increase in antibiotic resistance-associated determinants in bacterial populations, driven by the mobility and infectious nature of such determinants. Bacterial genome flexibility and antibiotic-driven selection are at the root of the problem. Genome evolution and the emergence of highly successful multidrug-resistant clades in different pathogens have made this a global challenge. Here, we describe some of the factors driving the origin, evolution, and spread of the antibiotic resistance genotype.

Fluoroquinolone resistance in Salmonella: insights by whole-genome sequencing.

Fluoroquinolone (FQ)-resistant Salmonella spp. were listed by the WHO in 2017 as priority pathogens for which new antibiotics were urgently needed. The overall global burden of Salmonella infections is high, but differs per region. Whereas typhoid fever is most prevalent in South and South-East Asia, non-typhoidal salmonellosis is prevalent across the globe and associated with a mild gastroenteritis. By contrast, invasive non-typhoidal Salmonella cause bloodstream infections associated with high mortality, particularly in sub-Saharan Africa. Most Salmonella strains from clinical sources are resistant to first-line antibiotics, with FQs now being the antibiotic of choice for treatment of invasive Salmonella infections. However, FQ resistance is increasingly being reported in Salmonella, and multiple molecular mechanisms are already described. Whole-genome sequencing (WGS) is becoming more frequently used to analyse bacterial genomes for antibiotic-resistance markers, and to understand the phylogeny of bacteria in relation to their antibiotic-resistance profiles. This mini-review provides an overview of FQ resistance in Salmonella, guided by WGS studies that demonstrate that WGS is a valuable tool for global surveillance.

Flaviviruses Exploit the Lipid Droplet Protein AUP1 to Trigger Lipophagy and Drive Virus Production.

Ubiquitylation is one of the most versatile protein post-translational modifications and is frequently altered during virus infections. Here we employed a functional proteomics screen to identify host proteins that are differentially ubiquitylated upon dengue virus (DENV) infection. Among the several differentially modified proteins identified in infected cells was AUP1, a lipid droplet-localized type-III membrane protein, which exists predominantly in the mono-ubiquitylated form. AUP1 associated with DENV NS4A and relocalized from lipid droplets to autophagosomes upon infection. Virus production was abolished in cells deleted for AUP1 or expressing an AUP1 acyltransferase domain mutant. Ubiquitylation disrupted the AUP1-NS4A interaction, resulting in inhibited acyltransferase activity, defective lipophagy, and attenuated virus production. Our results show that DENV-NS4A exploits the acyltransferase activity of AUP1 to trigger lipophagy, a process regulated by ubiquitylation. This mechanism appears to be a general phenomenon in biogenesis of flaviviruses and underscores the critical role of post-translational modifications in virus infections.

PlasmidTron: assembling the cause of phenotypes and genotypes from NGS data.

Increasingly rich metadata are now being linked to samples that have been whole-genome sequenced. However, much of this information is ignored. This is because linking this metadata to genes, or regions of the genome, usually relies on knowing the gene sequence(s) responsible for the particular trait being measured and looking for its presence or absence in that genome. Examples of this would be the spread of antimicrobial resistance genes carried on mobile genetic elements (MGEs). However, although it is possible to routinely identify the resistance gene, identifying the unknown MGE upon which it is carried can be much more difficult if the starting point is short-read whole-genome sequence data. The reason for this is that MGEs are often full of repeats and so assemble poorly, leading to fragmented consensus sequences. Since mobile DNA, which can carry many clinically and ecologically important genes, has a different evolutionary history from the host, its distribution across the host population will, by definition, be independent of the host phylogeny. It is possible to use this phenomenon in a genome-wide association study to identify both the genes associated with the specific trait and also the DNA linked to that gene, for example the flanking sequence of the plasmid vector on which it is encoded, which follows the same patterns of distribution as the marker gene/sequence itself. We present PlasmidTron, which utilizes the phenotypic data normally available in bacterial population studies, such as antibiograms, virulence factors, or geographical information, to identify traits that are likely to be present on DNA that can randomly reassort across defined bacterial populations. It is also possible to use this methodology to associate unknown genes/sequences (e.g. plasmid backbones) with a specific molecular signature or marker (e.g. resistance gene presence or absence) using PlasmidTron. PlasmidTron uses a k-mer-based approach to identify reads associated with a phylogenetically unlinked phenotype. These reads are then assembled de novo to produce contigs in a fast and scalable-to-large manner. PlasmidTron is written in Python 3 and is available under the open source licence GNU GPL3 from https://github.com/sanger-pathogens/plasmidtron.

Emergence of an Extensively Drug-Resistant Salmonella enterica Serovar Typhi Clone Harboring a Promiscuous Plasmid Encoding Resistance to Fluoroquinolones and Third-Generation Cephalosporins.

Antibiotic resistance is a major problem in Salmonella enterica serovar Typhi, the causative agent of typhoid. Multidrug-resistant (MDR) isolates are prevalent in parts of Asia and Africa and are often associated with the dominant H58 haplotype. Reduced susceptibility to fluoroquinolones is also widespread, and sporadic cases of resistance to third-generation cephalosporins or azithromycin have also been reported. Here, we report the first large-scale emergence and spread of a novel S Typhi clone harboring resistance to three first-line drugs (chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole) as well as fluoroquinolones and third-generation cephalosporins in Sindh, Pakistan, which we classify as extensively drug resistant (XDR). Over 300 XDR typhoid cases have emerged in Sindh, Pakistan, since November 2016. Additionally, a single case of travel-associated XDR typhoid has recently been identified in the United Kingdom. Whole-genome sequencing of over 80 of the XDR isolates revealed remarkable genetic clonality and sequence conservation, identified a large number of resistance determinants, and showed that these isolates were of haplotype H58. The XDR S Typhi clone encodes a chromosomally located resistance region and harbors a plasmid encoding additional resistance elements, including the blaCTX-M-15 extended-spectrum ß-lactamase, and carrying the qnrS fluoroquinolone resistance gene. This antibiotic resistance-associated IncY plasmid exhibited high sequence identity to plasmids found in other enteric bacteria isolated from widely distributed geographic locations. This study highlights three concerning problems: the receding antibiotic arsenal for typhoid treatment, the ability of S Typhi to transform from MDR to XDR in a single step by acquisition of a plasmid, and the ability of XDR clones to spread globally.IMPORTANCE Typhoid fever is a severe disease caused by the Gram-negative bacterium Salmonella enterica serovar Typhi. Antibiotic-resistant S Typhi strains have become increasingly common. Here, we report the first large-scale emergence and spread of a novel extensively drug-resistant (XDR) S Typhi clone in Sindh, Pakistan. The XDR S Typhi is resistant to the majority of drugs available for the treatment of typhoid fever. This study highlights the evolving threat of antibiotic resistance in S Typhi and the value of antibiotic susceptibility testing and whole-genome sequencing in understanding emerging infectious diseases. We genetically characterized the XDR S Typhi to investigate the phylogenetic relationship between these isolates and a global collection of S Typhi isolates and to identify multiple genes linked to antibiotic resistance. This S Typhi clone harbored a promiscuous antibiotic resistance plasmid previously identified in other enteric bacteria. The increasing antibiotic resistance in S Typhi observed here adds urgency to the need for typhoid prevention measures.

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

The ST313 pathovar of Salmonella enterica 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 S 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 S Typhimurium to immune serum identified a repertoire of S 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 S Typhimurium SL1344 as the host. Based on their initial levels of enhanced resistance to killing, yfgA and sapA mutants were selected for further characterization. The S Typhimurium yfgA mutant lost the characteristic Salmonella 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 S Typhimurium sapA 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 in vivo 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.

An evaluation of purified Salmonella Typhi protein antigens for the serological diagnosis of acute typhoid fever.

OBJECTIVES: The diagnosis of typhoid fever is a challenge. Aiming to develop a typhoid diagnostic we measured antibody responses against Salmonella Typhi (S. Typhi) protein antigens and the Vi polysaccharide in a cohort of Bangladeshi febrile patients. METHODS: IgM against 12 purified antigens and the Vi polysaccharide was measured by ELISA in plasma from patients with confirmed typhoid fever (n = 32), other confirmed infections (n = 17), and healthy controls (n = 40). ELISAs with the most specific antigens were performed on plasma from 243 patients with undiagnosed febrile disease. RESULTS: IgM against the S. Typhi protein antigens correlated with each other (rho > 0.8), but not against Vi (rho < 0.6). Typhoid patients exhibited higher IgM against 11/12 protein antigens and Vi than healthy controls and those with other infections. Vi, PilL, and CdtB exhibited the greatest sensitivity and specificity. Specificity and sensitivity was improved when Vi was combined with a protein antigen, generating sensitivities and specificities of 0.80 and >0.85, respectively. Applying a dynamic cut-off to patients with undiagnosed febrile disease suggested that 34-58% had an IgM response indicative of typhoid. CONCLUSIONS: We evaluated the diagnostic potential of several S. Typhi antigens; our assays give good sensitivity and specificity, but require further assessment in differing patient populations.

The Type III Secretion System Effector SptP of Salmonella enterica Serovar Typhi.

Strains of the various Salmonella enterica serovars cause gastroenteritis or typhoid fever in humans, with virulence depending on the action of two type III secretion systems (Salmonella pathogenicity island 1 [SPI-1] and SPI-2). SptP is a Salmonella SPI-1 effector, involved in mediating recovery of the host cytoskeleton postinfection. SptP requires a chaperone, SicP, for stability and secretion. SptP has 94% identity between S. enterica serovar Typhimurium and S Typhi; direct comparison of the protein sequences revealed that S Typhi SptP has numerous amino acid changes within its chaperone-binding domain. Subsequent comparison of ΔsptP S Typhi and S. Typhimurium strains demonstrated that, unlike SptP in S. Typhimurium, SptP in S Typhi was not involved in invasion or cytoskeletal recovery postinfection. Investigation of whether the observed amino acid changes within SptP of S Typhi affected its function revealed that S Typhi SptP was unable to complement S. Typhimurium ΔsptP due to an absence of secretion. We further demonstrated that while S. Typhimurium SptP is stable intracellularly within S Typhi, S Typhi SptP is unstable, although stability could be recovered following replacement of the chaperone-binding domain with that of S. Typhimurium. Direct assessment of the strength of the interaction between SptP and SicP of both serovars via bacterial two-hybrid analysis demonstrated that S Typhi SptP has a significantly weaker interaction with SicP than the equivalent proteins in S. Typhimurium. Taken together, our results suggest that changes within the chaperone-binding domain of SptP in S Typhi hinder binding to its chaperone, resulting in instability, preventing translocation, and therefore restricting the intracellular activity of this effector. IMPORTANCE: Studies investigating Salmonella pathogenesis typically rely on Salmonella Typhimurium, even though Salmonella Typhi causes the more severe disease in humans. As such, an understanding of S. Typhi pathogenesis is lacking. Differences within the type III secretion system effector SptP between typhoidal and nontyphoidal serovars led us to characterize this effector within S Typhi. Our results suggest that SptP is not translocated from typhoidal serovars, even though the loss of sptP results in virulence defects in S. Typhimurium. Although SptP is just one effector, our results exemplify that the behavior of these serovars is significantly different and genes identified to be important for S. Typhimurium virulence may not translate to S Typhi.

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

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 ß1 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.