High-throughput fitness experiments reveal specific vulnerabilities of human-adapted Salmonella during stress and infection.

Salmonella enterica is comprised of genetically distinct 'serovars' that together provide an intriguing model for exploring the genetic basis of pathogen evolution. Although the genomes of numerous Salmonella isolates with broad variations in host range and human disease manifestations have been sequenced, the functional links between genetic and phenotypic differences among these serovars remain poorly understood. Here, we conduct high-throughput functional genomics on both generalist (Typhimurium) and human-restricted (Typhi and Paratyphi A) Salmonella at unprecedented scale in the study of this enteric pathogen. Using a comprehensive systems biology approach, we identify gene networks with serovar-specific fitness effects across 25 host-associated stresses encountered at key stages of human infection. By experimentally perturbing these networks, we characterize previously undescribed pseudogenes in human-adapted Salmonella. Overall, this work highlights specific vulnerabilities encoded within human-restricted Salmonella that are linked to the degradation of their genomes, shedding light into the evolution of this enteric pathogen.

Salmonella enterica serovar Typhimurium ST313 sublineage 2.2 has emerged in Malawi with a characteristic gene expression signature and a fitness advantage.

Invasive non-typhoidal Salmonella (iNTS) disease is a serious bloodstream infection that targets immune-compromised individuals, and causes significant mortality in sub-Saharan Africa. Salmonella enterica serovar Typhimurium ST313 causes the majority of iNTS in Malawi. We performed an intensive comparative genomic analysis of 608 S. Typhimurium ST313 isolates dating between 1996 and 2018 from Blantyre, Malawi. We discovered that following the arrival of the well-characterized S. Typhimurium ST313 lineage 2 in 1999, two multidrug-resistant variants emerged in Malawi in 2006 and 2008, designated sublineages 2.2 and 2.3, respectively. The majority of S. Typhimurium isolates from human bloodstream infections in Malawi now belong to sublineages 2.2 or 2.3. To understand the emergence of the prevalent ST313 sublineage 2.2, we studied two representative strains, D23580 (lineage 2) and D37712 (sublineage 2.2). The chromosome of ST313 lineage 2 and sublineage 2.2 only differed by 29 SNPs/small indels and a 3 kb deletion of a Gifsy-2 prophage region including the sseI pseudogene. Lineage 2 and sublineage 2.2 had distinctive plasmid profiles. The transcriptome was investigated in 15 infection-relevant in vitro conditions and within macrophages. During growth in physiological conditions that do not usually trigger S. Typhimurium SPI2 gene expression, the SPI2 genes of D37712 were transcriptionally active. We identified down-regulation of flagellar genes in D37712 compared with D23580. Following phenotypic confirmation of transcriptomic differences, we discovered that sublineage 2.2 had increased fitness compared with lineage 2 during mixed growth in minimal media. We speculate that this competitive advantage is contributing to the emergence of sublineage 2.2 in Malawi.

Succinate utilisation by Salmonella is inhibited by multiple regulatory systems.

Succinate is a potent immune signalling molecule that is present in the mammalian gut and within macrophages. Both of these infection niches are colonised by the pathogenic bacterium Salmonella enterica serovar Typhimurium during infection. Succinate is a C4-dicarboyxlate that can serve as a source of carbon for bacteria. When succinate is provided as the sole carbon source for in vitro cultivation, Salmonella and other enteric bacteria exhibit a slow growth rate and a long lag phase. This growth inhibition phenomenon was known to involve the sigma factor RpoS, but the genetic basis of the repression of bacterial succinate utilisation was poorly understood. Here, we use an experimental evolution approach to isolate fast-growing mutants during growth of S. Typhimurium on succinate containing minimal medium. Our approach reveals novel RpoS-independent systems that inhibit succinate utilisation. The CspC RNA binding protein restricts succinate utilisation, an inhibition that is antagonised by high levels of the small regulatory RNA (sRNA) OxyS. We discovered that the Fe-S cluster regulatory protein IscR inhibits succinate utilisation by repressing the C4-dicarboyxlate transporter DctA. Furthermore, the ribose operon repressor RbsR is required for the complete RpoS-driven repression of succinate utilisation, suggesting a novel mechanism of RpoS regulation. Our discoveries shed light on the redundant regulatory systems that tightly regulate the utilisation of succinate. We speculate that the control of central carbon metabolism by multiple regulatory systems in Salmonella governs the infection niche-specific utilisation of succinate.

Protocol for the challenge non-typhoidal Salmonella (CHANTS) study: a first-in-human, in-patient, double-blind, randomised, safety and dose-escalation controlled human infection model in the UK.

INTRODUCTION: Invasive non-typhoidal Salmonella (iNTS) serovars are a major cause of community-acquired bloodstream infections in sub-Saharan Africa (SSA). In this setting, Salmonella enterica serovar Typhimurium accounts for two-thirds of infections and is associated with an estimated case fatality rate of 15%-20%. Several iNTS vaccine candidates are in early-stage assessment which-if found effective-would provide a valuable public health tool to reduce iNTS disease burden. The CHANTS study aims to develop a first-in-human Salmonella Typhimurium controlled human infection model, which can act as a platform for future vaccine evaluation, in addition to providing novel insights into iNTS disease pathogenesis. METHODS AND ANALYSIS: This double-blind, safety and dose-escalation study will randomise 40-80 healthy UK participants aged 18-50 to receive oral challenge with one of two strains of S. Typhimurium belonging to the ST19 (strain 4/74) or ST313 (strain D23580) lineages. 4/74 is a global strain often associated with diarrhoeal illness predominantly in high-income settings, while D23580 is an archetypal strain representing invasive disease-causing isolates found in SSA. The primary objective is to determine the minimum infectious dose (colony-forming unit) required for 60%-75% of participants to develop clinical or microbiological features of systemic salmonellosis. Secondary endpoints are to describe and compare the clinical, microbiological and immunological responses following challenge. Dose escalation or de-escalation will be undertaken by continual-reassessment methodology and limited within prespecified safety thresholds. Exploratory objectives are to describe mechanisms of iNTS virulence, identify putative immune correlates of protection and describe host-pathogen interactions in response to infection. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the NHS Health Research Authority (London-Fulham Research Ethics Committee 21/PR/0051; IRAS Project ID 301659). The study findings will be disseminated in international peer-reviewed journals and presented at national/international stakeholder meetings. Study outcome summaries will be provided to both funders and participants. TRIAL REGISTRATION NUMBER: NCT05870150.

The Challenge Non-Typhoidal Salmonella (CHANTS) Consortium: Development of a non-typhoidal Salmonella controlled human infection model: Report from a consultation group workshop, 05 July 2022, London, UK.

Invasive non-typhoidal Salmonella disease (iNTS) is a major cause of morbidity and mortality globally, particularly as a cause of bloodstream infection in children and immunocompromised adults in sub-Saharan Africa. Vaccines to prevent non-typhoidal Salmonella (NTS) would represent a valuable public health tool in this setting to avert cases and prevent expansion of antimicrobial resistance. Several NTS and combination typhoidal-NTS vaccine candidates are in early-stage development, although the pathway to licensure is unclear due to challenges in conducting large phase III field trials. Controlled human infection models (CHIM) present an opportunity to accelerate vaccine development for a range of enteric pathogens. Several recent typhoidal Salmonella CHIMs have been conducted safely and have played pivotal roles in progressing vaccine candidates to pre-qualification and licensure. The Challenge Non-Typhoidal Salmonella (CHANTS) consortium has been formed with funding from the Wellcome Trust, to deliver the first NTS CHIM, which can act as a platform for future vaccine evaluation. This paper reports the conclusions of a consultation group workshop convened with key stakeholders. The aims of this meeting were to: (1) define the rationale for an NTS CHIM (2) map the NTS vaccine pipeline (3) refine study design and (4) establish potential future use cases.

Diverse Durham collection phages demonstrate complex BREX defense responses.

Bacteriophages (phages) outnumber bacteria ten-to-one and cause infections at a rate of 1025 per second. The ability of phages to reduce bacterial populations makes them attractive alternative antibacterials for use in combating the rise in antimicrobial resistance. This effort may be hindered due to bacterial defenses such as Bacteriophage Exclusion (BREX) that have arisen from the constant evolutionary battle between bacteria and phages. For phages to be widely accepted as therapeutics in Western medicine, more must be understood about bacteria-phage interactions and the outcomes of bacterial phage defense. Here, we present the annotated genomes of 12 novel bacteriophage species isolated from water sources in Durham, UK, during undergraduate practical classes. The collection includes diverse species from across known phylogenetic groups. Comparative analyses of two novel phages from the collection suggest they may be founding members of a new genus. Using this Durham phage collection, we determined that particular BREX defense systems were likely to confer a varied degree of resistance against an invading phage. We concluded that the number of BREX target motifs encoded in the phage genome was not proportional to the degree of susceptibility. IMPORTANCE Bacteriophages have long been the source of tools for biotechnology that are in everyday use in molecular biology research laboratories worldwide. Phages make attractive new targets for the development of novel antimicrobials. While the number of phage genome depositions has increased in recent years, the expected bacteriophage diversity remains underrepresented. Here we demonstrate how undergraduates can contribute to the identification of novel phages and that a single City in England can provide ample phage diversity and the opportunity to find novel technologies. Moreover, we demonstrate that the interactions and intricacies of the interplay between bacterial phage defense systems such as Bacteriophage Exclusion (BREX) and phages are more complex than originally thought. Further work will be required in the field before the dynamic interactions between phages and bacterial defense systems are fully understood and integrated with novel phage therapies.

Class 1 integrons in clinical and swine industry isolates of Salmonella Typhimurium from Colombia, dating 1997 to 2017.

Background. Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) has been linked to outbreaks of foodborne gastroenteritis disease, and the emergence of antimicrobial-resistant clones. In Colombia, laboratory surveillance of Salmonella spp. between 1997-2018 revealed that S. Typhimurium was the most ubiquitous serovar (27.6 % of all Salmonella isolates), with increasing levels of resistance to several families of antibiotics.Hypothesis. Resistant isolates of S. Typhimurium recovered from human clinical, food and swine samples carry class 1 integrons that are linked to antimicrobial resistance genes.Aim. Identify class 1 integrons, and investigate their association with other mobile genetic elements, and their relationship to the antimicrobial resistance of Colombian S. Typhimurium isolates.Methods. In this study, 442 isolates of S. Typhimurium were analysed, of which 237 were obtained from blood culture, 151 from other clinical sources, 4 from non-clinical sources and 50 from swine samples. Class 1 integrons and plasmid incompatibility groups were analysed by PCR and whole-genome sequencing (WGS), and regions flanking integrons were identified by WGS. The phylogenetic relationship was established by multilocus sequence typing (MLST) and single-nucleotide polymorphism (SNP) distances for 30 clinical isolates.Results . Overall, 39 % (153/392) of the human clinical isolates and 22 % (11/50) of the swine S. Typhimurium isolates carried complete class 1 integrons. Twelve types of gene cassette arrays were identified, including dfr7-aac-bla OXA-2 (Int1-Col1), which was the most common one in human clinical isolates (75.2 %, 115/153). Human clinical and swine isolates that carried class 1 integrons were resistant to up to five and up to three antimicrobial families, respectively. The Int1-Col1 integron was most prevalent in stool isolates and was associated with Tn21. The most common plasmid incompatibility group was IncA/C.Conclusions. The widespread presence of the IntI1-Col1 integron in Colombia since 1997 was striking. A possible relationship between integrons, source and mobile elements that favour the spread of antimicrobial resistance determinants in Colombian S. Typhimurium was identified.

Case-control investigation of invasive Salmonella disease in Malawi reveals no evidence of environmental or animal transmission of invasive strains, and supports human to human transmission.

BACKGROUND: Invasive Salmonella infections cause significant morbidity and mortality in Sub-Saharan Africa. However, the routes of transmission are uncertain. We conducted a case-control study of index-case and geographically-matched control households in Blantyre, Malawi, sampling Salmonella isolates from index cases, healthy people, animals, and the household environment. METHODOLOGY: Sixty index cases of human invasive Salmonella infection were recruited (March 2015-Oct 2016). Twenty-eight invasive Non-Typhoidal Salmonella (iNTS) disease and 32 typhoid patients consented to household sampling. Each index-case household was geographically matched to a control household. Extensive microbiological sampling included stool sampling from healthy household members, stool or rectal swabs from household-associated animals and boot-sock sampling of the household environment. FINDINGS: 1203 samples from 120 households, yielded 43 non-Typhoidal Salmonella (NTS) isolates from 25 households (overall sample positivity 3.6%). In the 28 iNTS patients, disease was caused by 3 STs of Salmonella Typhimurium, mainly ST313. In contrast, the isolates from households spanned 15 sequence types (STs). Two S. Typhimurium isolates from index cases closely matched isolates from their respective asymptomatic household members (2 and 3 SNP differences respectively). Despite the recovery of a diverse range of NTS, there was no overlap between the STs causing iNTS disease with any environmental or animal isolates. CONCLUSIONS: The finding of NTS strains from index cases that matched household members, coupled with lack of related animal or environmental isolates, supports a hypothesis of human to human transmission of iNTS infections in the household. The breadth of NTS strains found in animals and the household environment demonstrated the robustness of NTS sampling and culture methodology, and suggests a diverse ecology of Salmonella in this setting. Healthy typhoid (S. Typhi) carrier state was not detected. The lack of S. Typhi isolates from the household environment suggests that further methodological development is needed to culture S. Typhi from the environment.

Multiplex PCR Assay for Clade Typing of Salmonella enterica Serovar Enteritidis.

Salmonella enterica serovar Enteritidis is one of the most commonly reported serovars of nontyphoidal Salmonella causing human disease and is responsible for both gastroenteritis and invasive nontyphoidal Salmonella (iNTS) disease worldwide. Whole-genome sequence (WGS) comparison of Salmonella Enteritidis isolates from across the world has identified three distinct clades, global epidemic, Central/East African, and West African, all of which have been implicated in epidemics: the global epidemic clade was linked to poultry-associated gastroenteritis, while the two African clades were related to iNTS disease. However, the distribution and epidemiology of these clades across Africa are poorly understood because identification of these clades currently requires whole-genome sequencing capacity. Here, we report a sensitive, time- and cost-effective real-time PCR assay capable of differentiating between the Salmonella Enteritidis clades to facilitate surveillance and to inform public health responses. The assay described here is limited to previously confirmed S. Enteritidis isolates. IMPORTANCE Challenges in the diagnosis and treatment of invasive Salmonella Enteritidis bloodstream infections in sub-Saharan Africa are responsible for a case fatality rate of approximately 15%. It is important to identify distinct clades of S. Enteritidis in diagnostic laboratories in the African setting to determine the different health outcomes associated with particular outbreaks. Here, we describe the development of a high-quality molecular classification assay for clade typing of S. Enteritidis that is ideal for use in public health laboratories in resource-limited settings.

Greater Breast Support Alters Trunk and Knee Joint Biomechanics Commonly Associated With Anterior Cruciate Ligament Injury.

Objective: The female breast is a passive tissue with little intrinsic support. Therefore, women rely on external breast support (sports bras) to control breast motion during athletic tasks. Research has demonstrated that lower levels of breast support are associated with altered trunk and pelvis movement patterns during running, a common athletic task. However, no previous study has identified the effect of sports bra support on movement patterns during other athletic tasks including landing. Therefore, the purpose of this study was to examine the effects of breast support on trunk and knee joint biomechanics in female collegiate athletes during a double-leg landing task. Methods: Fourteen female collegiate athletes completed five double-leg landing trials in each of three different sports bra conditions: no support, low support, and high support. A 10-camera motion capture system (250 Hz, Qualisys, Goteburg, Sweden) and two force platforms (1,250 Hz, AMTI, Watertown, MA, USA) were used to collect three-dimensional kinematics and ground reaction forces simultaneously. Visual 3D was used to calculate trunk segment and knee joint angles and moments. Custom software (MATLAB 2021a) was used to determine discrete values of dependent variables including vertical breast displacement, knee joint and trunk segment angles at initial contact and 100 ms post-initial contact, and peak knee joint moments. A repeated measures analysis of covariance with post-hoc paired samples t-tests were used to evaluate the effect of breast support on landing biomechanics. Results: Increasing levels of breast support were associated with reductions in peak knee flexion (Right: p = 0.008; Left: p = 0.029) and peak knee valgus angles (Right: p = 0.011; Left: p = 0.003) as well as reductions in peak knee valgus moments (Right: p = 0.033; Left: p = 0.013). There were no changes in peak knee extension moments (Right: p = 0.216; Left: p = 0.261). Increasing levels of breast support were associated with greater trunk flexion angles at initial contact (p = 0.024) and greater peak trunk flexion angles (p = 0.002). Conclusions: Lower levels of breast support are associated with knee joint and trunk biomechanical profiles suggested to increase ACL injury risk.

A widespread family of WYL-domain transcriptional regulators co-localizes with diverse phage defence systems and islands.

Bacteria are under constant assault by bacteriophages and other mobile genetic elements. As a result, bacteria have evolved a multitude of systems that protect from attack. Genes encoding bacterial defence mechanisms can be clustered into 'defence islands', providing a potentially synergistic level of protection against a wider range of assailants. However, there is a comparative paucity of information on how expression of these defence systems is controlled. Here, we functionally characterize a transcriptional regulator, BrxR, encoded within a recently described phage defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a combination of reporters and electrophoretic mobility shift assays, we discovered that BrxR acts as a repressor. We present the structure of BrxR to 2.15 Å, the first structure of this family of transcription factors, and pinpoint a likely binding site for ligands within the WYL-domain. Bioinformatic analyses demonstrated that BrxR-family homologues are widespread amongst bacteria. About half (48%) of identified BrxR homologues were co-localized with a diverse array of known phage defence systems, either alone or clustered into defence islands. BrxR is a novel regulator that reveals a common mechanism for controlling the expression of the bacterial phage defence arsenal.

Biogenesis of a bacterial metabolosome for propanediol utilization.

Bacterial metabolosomes are a family of protein organelles in bacteria. Elucidating how thousands of proteins self-assemble to form functional metabolosomes is essential for understanding their significance in cellular metabolism and pathogenesis. Here we investigate the de novo biogenesis of propanediol-utilization (Pdu) metabolosomes and characterize the roles of the key constituents in generation and intracellular positioning of functional metabolosomes. Our results demonstrate that the Pdu metabolosome undertakes both "Shell first" and "Cargo first" assembly pathways, unlike the ß-carboxysome structural analog which only involves the "Cargo first" strategy. Shell and cargo assemblies occur independently at the cell poles. The internal cargo core is formed through the ordered assembly of multiple enzyme complexes, and exhibits liquid-like properties within the metabolosome architecture. Our findings provide mechanistic insight into the molecular principles driving bacterial metabolosome assembly and expand our understanding of liquid-like organelle biogenesis.

Transcriptional Organization of the Salmonella Typhimurium Phage P22 pid ORFan Locus.

Many phage genes lack sequence similarity to any other open reading frame (ORF) in current databases. These enigmatic ORFan genes can have a tremendous impact on phage propagation and host interactions but often remain experimentally unexplored. We previously revealed a novel interaction between phage P22 and its Salmonella Typhimurium host, instigated by the ORFan gene pid (for phage P22 encoded instigator of dgo expression) and resulting in derepression of the host dgoRKAT operon. The pid gene is highly expressed in phage carrier cells that harbor a polarly located P22 episome that segregates asymmetrically among daughter cells. Here, we discovered that the pid locus is fitted with a weak promoter, has an exceptionally long 5' untranslated region that is instructive for a secondary pid mRNA species, and has a 3' Rho-independent termination loop that is responsible for stability of the pid transcript.

An accessible, efficient and global approach for the large-scale sequencing of bacterial genomes.

We have developed an efficient and inexpensive pipeline for streamlining large-scale collection and genome sequencing of bacterial isolates. Evaluation of this method involved a worldwide research collaboration focused on the model organism Salmonella enterica, the 10KSG consortium. Following the optimization of a logistics pipeline that involved shipping isolates as thermolysates in ambient conditions, the project assembled a diverse collection of 10,419 isolates from low- and middle-income countries. The genomes were sequenced using the LITE pipeline for library construction, with a total reagent cost of less than USD$10 per genome. Our method can be applied to other large bacterial collections to underpin global collaborations.

The phage defence island of a multidrug resistant plasmid uses both BREX and type IV restriction for complementary protection from viruses.

Bacteria have evolved a multitude of systems to prevent invasion by bacteriophages and other mobile genetic elements. Comparative genomics suggests that genes encoding bacterial defence mechanisms are often clustered in 'defence islands', providing a concerted level of protection against a wider range of attackers. However, there is a comparative paucity of information on functional interplay between multiple defence systems. Here, we have functionally characterised a defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a suite of thirty environmentally-isolated coliphages, we demonstrate multi-layered and robust phage protection provided by a plasmid-encoded defence island that expresses both a type I BREX system and the novel GmrSD-family type IV DNA modification-dependent restriction enzyme, BrxU. We present the structure of BrxU to 2.12 Å, the first structure of the GmrSD family of enzymes, and show that BrxU can utilise all common nucleotides and a wide selection of metals to cleave a range of modified DNAs. Additionally, BrxU undergoes a multi-step reaction cycle instigated by an unexpected ATP-dependent shift from an intertwined dimer to monomers. This direct evidence that bacterial defence islands can mediate complementary layers of phage protection enhances our understanding of the ever-expanding nature of phage-bacterial interactions.

Prophages encode phage-defense systems with cognate self-immunity.

Temperate phages are pervasive in bacterial genomes, existing as vertically inherited islands termed prophages. Prophages are vulnerable to predation of their host bacterium by exogenous phages. Here, we identify BstA, a family of prophage-encoded phage-defense proteins in diverse Gram-negative bacteria. BstA localizes to sites of exogenous phage DNA replication and mediates abortive infection, suppressing the competing phage epidemic. During lytic replication, the BstA-encoding prophage is not itself inhibited by BstA due to self-immunity conferred by the anti-BstA (aba) element, a short stretch of DNA within the bstA locus. Inhibition of phage replication by distinct BstA proteins from Salmonella, Klebsiella, and Escherichia prophages is generally interchangeable, but each possesses a cognate aba element. The specificity of the aba element ensures that immunity is exclusive to the replicating prophage, preventing exploitation by variant BstA-encoding phages. The BstA protein allows prophages to defend host cells against exogenous phage attack without sacrificing the ability to replicate lytically.

Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease.

The FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. While the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth suppression of Salmonella with succinate as the sole carbon source, an experimental evolution approach was devised to exploit this phenotype. By coupling mutational scanning with loss-of-function selection, we identified multiple ProQ residues in both the amino-terminal FinO domain and the variable carboxy-terminal region that are required for ProQ activity. Two carboxy-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. In contrast, several mutations in the FinO domain rendered ProQ both functionally inactive and unable to interact with target RNA in vivo. Alteration of the FinO domain stimulated the rapid turnover of ProQ by Lon-mediated proteolysis, suggesting a quality control mechanism that prevents the accumulation of nonfunctional ProQ molecules. We extend this observation to Hfq, the other major sRNA chaperone of enteric bacteria. The Hfq Y55A mutant protein, defective in RNA-binding and oligomerization, proved to be labile and susceptible to degradation by Lon. Taken together, our findings connect the major AAA+ family protease Lon with RNA-dependent quality control of Hfq and ProQ, the two major sRNA chaperones of Gram-negative bacteria.

Intracellular niche-specific profiling reveals transcriptional adaptations required for the cytosolic lifestyle of Salmonella enterica.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that causes diarrheal disease in humans and animals. During salmonellosis, S. Typhimurium colonizes epithelial cells lining the gastrointestinal tract. S. Typhimurium has an unusual lifestyle in epithelial cells that begins within an endocytic-derived Salmonella-containing vacuole (SCV), followed by escape into the cytosol, epithelial cell lysis and bacterial release. The cytosol is a more permissive environment than the SCV and supports rapid bacterial growth. The physicochemical conditions encountered by S. Typhimurium within the epithelial cytosol, and the bacterial genes required for cytosolic colonization, remain largely unknown. Here we have exploited the parallel colonization strategies of S. Typhimurium in epithelial cells to decipher the two niche-specific bacterial virulence programs. By combining a population-based RNA-seq approach with single-cell microscopic analysis, we identified bacterial genes with cytosol-induced or vacuole-induced expression signatures. Using these genes as environmental biosensors, we defined that Salmonella is exposed to oxidative stress and iron and manganese deprivation in the cytosol and zinc and magnesium deprivation in the SCV. Furthermore, iron availability was critical for optimal S. Typhimurium replication in the cytosol, as well as entC, fepB, soxS, mntH and sitA. Virulence genes that are typically associated with extracellular bacteria, namely Salmonella pathogenicity island 1 (SPI1) and SPI4, showed increased expression in the cytosol compared to vacuole. Our study reveals that the cytosolic and vacuolar S. Typhimurium virulence gene programs are unique to, and tailored for, residence within distinct intracellular compartments. This archetypical vacuole-adapted pathogen therefore requires extensive transcriptional reprogramming to successfully colonize the mammalian cytosol.

Isolation and Characterisation of Bacteriophages with Activity against Invasive Non-Typhoidal Salmonella Causing Bloodstream Infection in Malawi.

In recent years, novel lineages of invasive non-typhoidal Salmonella (iNTS) serovars Typhimurium and Enteritidis have been identified in patients with bloodstream infection in Sub-Saharan Africa. Here, we isolated and characterised 32 phages capable of infecting S. Typhimurium and S. Enteritidis, from water sources in Malawi and the UK. The phages were classified in three major phylogenetic clusters that were geographically distributed. In terms of host range, Cluster 1 phages were able to infect all bacterial hosts tested, whereas Clusters 2 and 3 had a more restricted profile. Cluster 3 contained two sub-clusters, and 3.b contained the most novel isolates. This study represents the first exploration of the potential for phages to target the lineages of Salmonella that are responsible for bloodstream infections in Sub-Saharan Africa.