Presence of phage-plasmids in multiple serovars of Salmonella enterica.

Evidence is accumulating in the literature that the horizontal spread of antimicrobial resistance (AMR) genes mediated by bacteriophages and bacteriophage-like plasmid (phage-plasmid) elements is much more common than previously envisioned. For instance, we recently identified and characterized a circular P1-like phage-plasmid harbouring a bla CTX-M-15 gene conferring extended-spectrum beta-lactamase (ESBL) resistance in Salmonella enterica serovar Typhi. As the prevalence and epidemiological relevance of such mechanisms has never been systematically assessed in Enterobacterales, in this study we carried out a follow-up retrospective analysis of UK Salmonella isolates previously sequenced as part of routine surveillance protocols between 2016 and 2021. Using a high-throughput bioinformatics pipeline we screened 47 784 isolates for the presence of the P1 lytic replication gene repL, identifying 226 positive isolates from 25 serovars and demonstrating that phage-plasmid elements are more frequent than previously thought. The affinity for phage-plasmids appears highly serovar-dependent, with several serovars being more likely hosts than others; most of the positive isolates (170/226) belonged to S. Typhimurium ST34 and ST19. The phage-plasmids ranged between 85.8 and 98.2 kb in size, with an average length of 92.1 kb; detailed analysis indicated a high amount of diversity in gene content and genomic architecture. In total, 132 phage-plasmids had the p0111 plasmid replication type, and 94 the IncY type; phylogenetic analysis indicated that both horizontal and vertical gene transmission mechanisms are likely to be involved in phage-plasmid propagation. Finally, phage-plasmids were present in isolates that were resistant and non-resistant to antimicrobials. In addition to providing a first comprehensive view of the presence of phage-plasmids in Salmonella, our work highlights the need for a better surveillance and understanding of phage-plasmids as AMR carriers, especially through their characterization with long-read sequencing.

Particular genomic and virulence traits associated with preterm infant-derived toxigenic Clostridium perfringens strains.

Clostridium perfringens is an anaerobic toxin-producing bacterium associated with intestinal diseases, particularly in neonatal humans and animals. Infant gut microbiome studies have recently indicated a link between C. perfringens and the preterm infant disease necrotizing enterocolitis (NEC), with specific NEC cases associated with overabundant C. perfringens termed C. perfringens-associated NEC (CPA-NEC). In the present study, we carried out whole-genome sequencing of 272 C. perfringens isolates from 70 infants across 5 hospitals in the United Kingdom. In this retrospective analysis, we performed in-depth genomic analyses (virulence profiling, strain tracking and plasmid analysis) and experimentally characterized pathogenic traits of 31 strains, including 4 from CPA-NEC patients. We found that the gene encoding toxin perfringolysin O, pfoA, was largely deficient in a human-derived hypovirulent lineage, as well as certain colonization factors, in contrast to typical pfoA-encoding virulent lineages. We determined that infant-associated pfoA+ strains caused significantly more cellular damage than pfoA- strains in vitro, and further confirmed this virulence trait in vivo using an oral-challenge C57BL/6 murine model. These findings suggest both the importance of pfoA+ C. perfringens as a gut pathogen in preterm infants and areas for further investigation, including potential intervention and therapeutic strategies.

Epitopes in the capsular polysaccharide and the porin OmpK36 receptors are required for bacteriophage infection of Klebsiella pneumoniae.

To kill bacteria, bacteriophages (phages) must first bind to a receptor, triggering the release of the phage DNA into the bacterial cell. Many bacteria secrete polysaccharides that had been thought to shield bacterial cells from phage attack. We use a comprehensive genetic screen to distinguish that the capsule is not a shield but is instead a primary receptor enabling phage predation. Screening of a transposon library to select phage-resistant Klebsiella shows that the first receptor-binding event docks to saccharide epitopes in the capsule. We discover a second step of receptor binding, dictated by specific epitopes in an outer membrane protein. This additional and necessary event precedes phage DNA release to establish a productive infection. That such discrete epitopes dictate two essential binding events for phages has profound implications for understanding the evolution of phage resistance and what dictates host range, two issues critically important to translating knowledge of phage biology into phage therapies.

First report and whole-genome sequencing of Pseudochrobactrum saccharolyticum in Latin America.

The Brucellaceae family comprises microorganisms similar both phenotypically and genotypically, making it difficult to identify the etiological agent of these infections. This study reports the first isolation, identification, and characterization of Pseudochrobactrum saccharolyticum (strain 115) from Latin America. Strain 115 was isolated in 2007 from a bovine in Brazil and was initially classified as Brucella spp. by classical microbiological tests and bcsp31 PCR. The antimicrobial susceptibility of strain 115 was tested against drugs used to treat human brucellosis by minimal inhibitory concentration test. Subsequently, the whole genome of the strain was sequenced, assembled, and characterized. Phylogenetic trees built from 16S rRNA and recA gene sequences enabled the classification of strain 115 as Pseudochrobactrum spp. Phylogenomic analysis using Single Nucleotide Polymorphisms and Average Nucleotide Identity allowed the classification of the strain as P. saccharolyticum. Additionally, a Tetra Correlation Search identified one related genome from the same species, which was compared with strain 115 by analyzing genomic islands. This is the first identification and whole-genome sequence of P. saccharolyticum in Latin America and highlights a challenge in the diagnosis of bovine brucellosis, which could be solved by including the sequencing of 16S rRNA and recA genes in routine diagnostics.

Multiplex PCR assay to detect high risk lineages of Salmonella Typhi and Paratyphi A.

Enteric fever infections remain a significant public health issue, with up to 20 million infections per year. Increasing rates of antibiotic resistant strains have rendered many first-line antibiotics potentially ineffective. Genotype 4.3.1 (H58) is the main circulating lineage of S. Typhi in many South Asian countries and is associated with high levels of antibiotic resistance. The emergence and spread of extensively drug resistant (XDR) typhoid strains has increased the need for a rapid molecular test to identify and track these high-risk lineages for surveillance and vaccine prioritisation. Current methods require samples to be cultured for several days, followed by DNA extraction and sequencing to determine the specific lineage. We designed and evaluated the performance of a new multiplex PCR assay, targeting S. Paratyphi A as well as the H58 and XDR lineages of S. Typhi on a collection of bacterial strains. Our assay was 100% specific for the identification of lineage specific S. Typhi and S. Paratyphi A, when tested with a mix of non-Typhi Salmonella and non-Salmonella strains. With additional testing on clinical and environmental samples, this assay will allow rapid lineage level detection of typhoid of clinical significance, at a significantly lower cost to whole-genome sequencing. To our knowledge, this is the first report of a SNP-based multiplex PCR assay for the detection of lineage specific serovars of Salmonella Typhi.

Genome placement of alpha-haemolysin cluster is associated with alpha-haemolysin sequence variation, adhesin and iron acquisition factor profile of Escherichia coli.

Since the discovery of haemolysis, many studies focused on a deeper understanding of this phenotype in Escherichia coli and its association with other virulence genes, diseases and pathogenic attributes/functions in the host. Our virulence-associated factor profiling and genome-wide association analysis of genomes of haemolytic and nonhaemolytic E. coli unveiled high prevalence of adhesins, iron acquisition genes and toxins in haemolytic bacteria. In the case of fimbriae with high prevalence, we analysed sequence variation of FimH, EcpD and CsgA, and showed that different adhesin variants were present in the analysed groups, indicating altered adhesive capabilities of haemolytic and nonhaemolytic E. coli. Analysis of over 1000 haemolytic E. coli genomes revealed that they are pathotypically, genetically and antigenically diverse, but their adhesin and iron acquisition repertoire is associated with genome placement of hlyCABD cluster. Haemolytic E. coli with chromosome-encoded alpha-haemolysin had high frequency of P, S, Auf fimbriae and multiple iron acquisition systems such as aerobactin, yersiniabactin, salmochelin, Fec, Sit, Bfd and hemin uptake systems. Haemolytic E. coli with plasmid-encoded alpha-haemolysin had similar adhesin profile to nonpathogenic E. coli, with high prevalence of Stg, Yra, Ygi, Ycb, Ybg, Ycf, Sfm, F9 fimbriae, Paa, Lda, intimin and type 3 secretion system encoding genes. Analysis of HlyCABD sequence variation revealed presence of variants associated with genome placement and pathotype.

Mechanistic Insights into the Capsule-Targeting Depolymerase from a Klebsiella pneumoniae Bacteriophage.

The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K. pneumoniae strains that produce the widespread, hypervirulence-associated K2-type capsular polysaccharide. Using transposon-directed insertion sequencing, we have shown that the production of capsule is an absolute requirement for efficient RAD2 infection by serving as a first-stage receptor. We have identified the depolymerase responsible for recognition and degradation of the capsule, determined that the depolymerase forms globular appendages on the phage virion tail tip, and present the cryo-electron microscopy structure of the RAD2 capsule depolymerase at 2.7-Å resolution. A putative active site for the enzyme was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides. Enzymatic assays coupled with mass spectrometric analyses of digested oligosaccharide products provided further mechanistic insight into the hydrolase activity of the enzyme, which, when incubated with K. pneumoniae, removes the capsule and sensitizes the cells to serum-induced killing. Overall, these findings expand our understanding of how phages target the Klebsiella capsule for infection, providing a framework for the use of depolymerases as antivirulence agents against this medically important pathogen. IMPORTANCE Klebsiella pneumoniae is a medically important pathogen that produces a thick protective capsule that is essential for pathogenicity. Phages are natural predators of bacteria, and many encode diverse "capsule depolymerases" which specifically degrade the capsule of their hosts, an exploitable trait for potential therapies. We have determined the first structure of a depolymerase that targets the clinically relevant K2 capsule and have identified its putative active site, providing hints to its mechanism of action. We also show that Klebsiella cells treated with a recombinant form of the depolymerase are stripped of capsule, inhibiting their ability to grow in the presence of serum, demonstrating the anti-infective potential of these robust and readily producible enzymes against encapsulated bacterial pathogens such as K. pneumoniae.

Genomic insights into the diversity, virulence and resistance of Klebsiella pneumoniae extensively drug resistant clinical isolates.

Klebsiella pneumoniae has been implicated in wide-ranging nosocomial outbreaks, causing severe infections without effective treatments due to antibiotic resistance. Here, we performed genome sequencing of 70 extensively drug resistant clinical isolates, collected from Brasília's hospitals (Brazil) between 2010 and 2014. The majority of strains (60 out of 70) belonged to a single clonal complex (CC), CC258, which has become distributed worldwide in the last two decades. Of these CC258 strains, 44 strains were classified as sequence type 11 (ST11) and fell into two distinct clades, but no ST258 strains were found. These 70 strains had a pan-genome size of 10 366 genes, with a core-genome size of ~4476 genes found in 95 % of isolates. Analysis of sequences revealed diverse mechanisms of resistance, including production of multidrug efflux pumps, enzymes with the same target function but with reduced or no affinity to the drug, and proteins that protected the drug target or inactivated the drug. ß-Lactamase production provided the most notable mechanism associated with K. pneumoniae. Each strain presented two or three different ß-lactamase enzymes, including class A (SHV, CTX-M and KPC), class B and class C AmpC enzymes, although no class D ß-lactamase was identified. Strains carrying the NDM enzyme involved three different ST types, suggesting that there was no common genetic origin.

Characterisation of Bacteriophage-Encoded Depolymerases Selective for Key Klebsiella pneumoniae Capsular Exopolysaccharides.

Capsular polysaccharides enable clinically important clones of Klebsiella pneumoniae to cause severe systemic infections in susceptible hosts. Phage-encoded capsule depolymerases have the potential to provide an alternative treatment paradigm in patients when multiple drug resistance has eroded the efficacy of conventional antibiotic chemotherapy. An investigation of 164 K. pneumoniae from intensive care patients in Thailand revealed a large number of distinct K types in low abundance but four (K2, K51, K1, K10) with a frequency of at least 5%. To identify depolymerases with the capacity to degrade capsules associated with these common K-types, 62 lytic phage were isolated from Thai hospital sewage water using K1, K2 and K51 isolates as hosts; phage plaques, without exception, displayed halos indicative of the presence of capsule-degrading enzymes. Phage genomes ranged in size from 41-348 kb with between 50 and 535 predicted coding sequences (CDSs). Using a custom phage protein database we were successful in applying annotation to 30 - 70% (mean = 58%) of these CDSs. The largest genomes, of so-called jumbo phage, carried multiple tRNAs as well as CRISPR repeat and spacer sequences. One of the smaller phage genomes was found to contain a putative Cas type 1E gene, indicating a history of host DNA acquisition in these obligate lytic phage. Whole-genome sequencing (WGS) indicated that some phage displayed an extended host range due to the presence of multiple depolymerase genes; in total, 42 candidate depolymerase genes were identified with up to eight in a single genome. Seven distinct virions were selected for further investigation on the basis of host range, phage morphology and WGS. Candidate genes for K1, K2 and K51 depolymerases were expressed and purified as his6-tagged soluble protein and enzymatic activity demonstrated against K. pneumoniae capsular polysaccharides by gel electrophoresis and Anton-Paar rolling ball viscometry. Depolymerases completely removed the capsule in K-type-specific fashion from K. pneumoniae cells. We conclude that broad-host range phage carry multiple enzymes, each with the capacity to degrade a single K-type, and any future use of these enzymes as therapeutic agents will require enzyme cocktails for utility against a range of K. pneumoniae infections.

Inhibitory Concentrations of Ciprofloxacin Induce an Adaptive Response Promoting the Intracellular Survival of Salmonella enterica Serovar Typhimurium.

Antimicrobial resistance (AMR) is a pressing global health crisis, which has been fueled by the sustained use of certain classes of antimicrobials, including fluoroquinolones. While the genetic mutations responsible for decreased fluoroquinolone (ciprofloxacin) susceptibility are known, the implications of ciprofloxacin exposure on bacterial growth, survival, and interactions with host cells are not well described. Aiming to understand the influence of inhibitory concentrations of ciprofloxacin in vitro, we subjected three clinical isolates of Salmonella enterica serovar Typhimurium to differing concentrations of ciprofloxacin, dependent on their MICs, and assessed the impact on bacterial growth, morphology, and transcription. We further investigated the differential morphology and transcription that occurred following ciprofloxacin exposure and measured the ability of ciprofloxacin-treated bacteria to invade and replicate in host cells. We found that ciprofloxacin-exposed S. Typhimurium is able to recover from inhibitory concentrations of ciprofloxacin and that the drug induces specific morphological and transcriptional signatures associated with the bacterial SOS response, DNA repair, and intracellular survival. In addition, ciprofloxacin-treated S. Typhimurium has increased capacity for intracellular replication in comparison to that of untreated organisms. These data suggest that S. Typhimurium undergoes an adaptive response under ciprofloxacin perturbation that promotes cellular survival, a consequence that may justify more measured use of ciprofloxacin for Salmonella infections. The combination of multiple experimental approaches provides new insights into the collateral effects that ciprofloxacin and other antimicrobials have on invasive bacterial pathogens. IMPORTANCE Antimicrobial resistance is a critical concern in global health. In particular, there is rising resistance to fluoroquinolones, such as ciprofloxacin, a first-line antimicrobial for many Gram-negative pathogens. We investigated the adaptive response of clinical isolates of Salmonella enterica serovar Typhimurium to ciprofloxacin, finding that the bacteria adapt in short timespans to high concentrations of ciprofloxacin in a way that promotes intracellular survival during early infection. Importantly, by studying three clinically relevant isolates, we were able to show that individual isolates respond differently to ciprofloxacin and that for each isolate, there was a heterogeneous response under ciprofloxacin treatment. The heterogeneity that arises from ciprofloxacin exposure may drive survival and proliferation of Salmonella during treatment and lead to drug resistance.

Functional analysis of colonization factor antigen I positive enterotoxigenic Escherichia coli identifies genes implicated in survival in water and host colonization.

Enterotoxigenic Escherichia coli (ETEC) expressing the colonization pili CFA/I are common causes of diarrhoeal infections in humans. Here, we use a combination of transposon mutagenesis and transcriptomic analysis to identify genes and pathways that contribute to ETEC persistence in water environments and colonization of a mammalian host. ETEC persisting in water exhibit a distinct RNA expression profile from those growing in richer media. Multiple pathways were identified that contribute to water survival, including lipopolysaccharide biosynthesis and stress response regulons. The analysis also indicated that ETEC growing in vivo in mice encounter a bottleneck driving down the diversity of colonizing ETEC populations.

Long-read-sequenced reference genomes of the seven major lineages of enterotoxigenic Escherichia coli (ETEC) circulating in modern time.

Enterotoxigenic Escherichia coli (ETEC) is an enteric pathogen responsible for the majority of diarrheal cases worldwide. ETEC infections are estimated to cause 80,000 deaths annually, with the highest rates of burden, ca 75 million cases per year, amongst children under 5 years of age in resource-poor countries. It is also the leading cause of diarrhoea in travellers. Previous large-scale sequencing studies have found seven major ETEC lineages currently in circulation worldwide. We used PacBio long-read sequencing combined with Illumina sequencing to create high-quality complete reference genomes for each of the major lineages with manually curated chromosomes and plasmids. We confirm that the major ETEC lineages all harbour conserved plasmids that have been associated with their respective background genomes for decades, suggesting that the plasmids and chromosomes of ETEC are both crucial for ETEC virulence and success as pathogens. The in-depth analysis of gene content, synteny and correct annotations of plasmids will elucidate other plasmids with and without virulence factors in related bacterial species. These reference genomes allow for fast and accurate comparison between different ETEC strains, and these data will form the foundation of ETEC genomics research for years to come.

Identification of Natural Mutations Responsible for Altered Infection Phenotypes of Salmonella enterica Clinical Isolates by Using Cell Line Infection Screens.

The initial steps of Salmonella pathogenesis involve adhesion to and invasion into host epithelial cells. While well-studied for Salmonella enterica serovar Typhimurium, the factors contributing to this process in other, host-adapted serovars remains unexplored. Here, we screened clinical isolates of serovars Gallinarum, Dublin, Choleraesuis, Typhimurium, and Enteritidis for adhesion to and invasion into intestinal epithelial cell lines of human, porcine, and chicken origins. Thirty isolates with altered infectivity were used for genomic analyses, and 14 genes and novel mutations associated with high or low infectivity were identified. The functions of candidate genes included virulence gene expression regulation and cell wall or membrane synthesis and components. The role of several of these genes in Salmonella adhesion to and invasion into cells has not previously been investigated. The genes dksA (encoding a stringent response regulator) and sanA (encoding a vancomycin high-temperature exclusion protein) were selected for further analyses, and we confirmed their roles in adhesion to and invasion into host cells. Furthermore, transcriptomic analyses were performed for S Enteritidis and S Typhimurium, with two highly infective and two marginally infective isolates for each serovar. Expression profiles for the isolates with altered infection phenotypes revealed the importance of type 3 secretion system expression levels in the determination of an isolate's infection phenotype. Taken together, these data indicate a new role in cell host infection for genes or gene variants previously not associated with adhesion to and invasion into the epithelial cells.IMPORTANCESalmonella is a foodborne pathogen affecting over 200 million people and resulting in over 200,000 fatal cases per year. Its adhesion to and invasion into intestinal epithelial cells represent one of the first and key steps in the pathogenesis of salmonellosis. Still, around 35 to 40% of bacterial genes have no experimentally validated function, and their contribution to bacterial virulence, including adhesion and invasion, remains largely unknown. Therefore, the significance of this study is in the identification of new genes or gene allelic variants previously not associated with adhesion and invasion. It is well established that blocking adhesion and/or invasion would stop or hamper bacterial infection; therefore, the new findings from this study could be used in future developments of anti-Salmonella therapy targeting genes involved in these key processes. Such treatment could be a valuable alternative, as the prevalence of antibiotic-resistant bacteria is increasing very rapidly.

The architecture and stabilisation of flagellotropic tailed bacteriophages.

Flagellotropic bacteriophages engage flagella to reach the bacterial surface as an effective means to increase the capture radius for predation. Structural details of these viruses are of great interest given the substantial drag forces and torques they face when moving down the spinning flagellum. We show that the main capsid and auxiliary proteins form two nested chainmails that ensure the integrity of the bacteriophage head. Core stabilising structures are conserved in herpesviruses suggesting their ancestral origin. The structure of the tail also reveals a robust yet pliable assembly. Hexameric rings of the tail-tube protein are braced by the N-terminus and a ß-hairpin loop, and interconnected along the tail by the splayed ß-hairpins. By contrast, we show that the ß-hairpin has an inhibitory role in the tail-tube precursor, preventing uncontrolled self-assembly. Dyads of acidic residues inside the tail-tube present regularly-spaced motifs well suited to DNA translocation into bacteria through the tail.

Publisher Correction: Comparative genomics of Salmonella enterica serovar Enteritidis ST-11 isolated in Uruguay reveals lineages associated with particular epidemiological traits.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genomic surveillance of Escherichia coli ST131 identifies local expansion and serial replacement of subclones.

Escherichia coli sequence type 131 (ST131) is a pandemic clone that is evolving rapidly with increasing levels of antimicrobial resistance. Here, we investigated an outbreak of E. coli ST131 producing extended spectrum ß-lactamases (ESBLs) in a long-term care facility (LTCF) in Ireland by combining data from this LTCF (n=69) with other Irish (n=35) and global (n=690) ST131 genomes to reconstruct the evolutionary history and understand changes in population structure and genome architecture over time. This required a combination of short- and long-read genome sequencing, de novo assembly, read mapping, ESBL gene screening, plasmid alignment and temporal phylogenetics. We found that Clade C was the most prevalent (686 out of 794 isolates, 86 %) of the three major ST131 clades circulating worldwide (A with fimH41, B with fimH22, C with fimH30), and was associated with the presence of different ESBL alleles, diverse plasmids and transposable elements. Clade C was estimated to have emerged in c. 1985 and subsequently acquired different ESBL gene variants (blaCTX-M-14 vs blaCTX-M-15). An ISEcp1-mediated transposition of the blaCTX-M-15 gene further increased the diversity within Clade C. We discovered a local clonal expansion of a rare C2 lineage (C2_8) with a chromosomal insertion of blaCTX-M-15 at the mppA gene. This was acquired from an IncFIA plasmid. The C2_8 lineage clonally expanded in the Irish LTCF from 2006, displacing the existing C1 strain (C1_10), highlighting the potential for novel ESBL-producing ST131 with a distinct genetic profile to cause outbreaks strongly associated with specific healthcare environments.

Comparative genomics of Salmonella enterica serovar Enteritidis ST-11 isolated in Uruguay reveals lineages associated with particular epidemiological traits.

Salmonella enterica serovar Enteritidis is a major cause of foodborne disease in Uruguay since 1995. We used a genomic approach to study a set of isolates from different sources and years. Whole genome phylogeny showed that most of the strains are distributed in two major lineages (E1 and E2), both belonging to MLST sequence type 11 the major ST among serovar Enteritidis. Strikingly, E2 isolates are over-represented in periods of outbreak abundance in Uruguay, while E1 span all epidemic periods. Both lineages circulate in neighbor countries at the same timescale as in Uruguay, and are present in minor numbers in distant countries. We identified allelic variants associated with each lineage. Three genes, ycdX, pduD and hsdM, have distinctive variants in E1 that may result in defective products. Another four genes (ybiO, yiaN, aas, aceA) present variants specific for the E2 lineage. Overall this work shows that S. enterica serovar Enteritidis strains circulating in Uruguay have the same phylogenetic profile than strains circulating in the region, as well as in more distant countries. Based on these results we hypothesize that the E2 lineage, which is more prevalent during epidemics, exhibits a combination of allelic variants that could be associated with its epidemic ability.

Phylogenetic Analysis Indicates a Longer Term Presence of the Globally Distributed H58 Haplotype of Salmonella Typhi in Southern India.

BACKGROUND: Typhoid fever caused by Salmonella Typhi is a major public health concern in low-/middle-income countries. A recent study of 1900 global S. Typhi indicated that South Asia might be the site of the original emergence of the most successful and hypervirulent clone belonging to the 4.3.1 genotype. However, this study had limited samples from India. METHODS: We analyzed 194 clinical S. Typhi, temporal representatives from those isolated from blood and bone marrow cultures in southern India, over 26 years (1991-2016). Antimicrobial resistance (AMR) testing was performed for most common clinical agents. Whole-genome sequencing and SNP-level analysis was conducted. Comparative genomics of Vellore isolates was performed to infer transmission and AMR events. RESULTS: We identified multidrug-resistance (MDR)-associated clade 4.3.1 as the dominant genotype. We detected 4.3.1 S. Typhi as early as 1991, the earliest to be reported form India, and the majority were fluoroquinolone resistant and not MDR. MDR was not detected at all in other genotypes circulating in Vellore. Comparison with global S. Typhi showed 2 Vellore subgroups (I and II) that were phylogenetically highly related to previously described South Asia (subgroup I, II) and Southeast Asia (subgroup II) clades. CONCLUSIONS: 4.3.1 S. Typhi has dominated in Vellore for 2 decades. Our study would assist public health agencies in better tracking of transmission and persistence of this successful clade in India and globally. It informs clinicians of the AMR pattern of circulating clone, which would add confidence to their prophylactic/treatment decision making and facilitate efficient patient care.

A Genome-Wide Knockout Screen in Human Macrophages Identified Host Factors Modulating Salmonella Infection.

A genome-scale CRISPR knockout library screen of THP-1 human macrophages was performed to identify loss-of-function mutations conferring resistance to Salmonella uptake. The screen identified 183 candidate genes, from which 14 representative genes involved in actin dynamics (ACTR3, ARPC4, CAPZB, TOR3A, CYFIP2, CTTN, and NHLRC2), glycosaminoglycan metabolism (B3GNT1), receptor signaling (PDGFB and CD27), lipid raft formation (CLTCL1), calcium transport (ATP2A2 and ITPR3), and cholesterol metabolism (HMGCR) were analyzed further. For some of these pathways, known chemical inhibitors could replicate the Salmonella resistance phenotype, indicating their potential as targets for host-directed therapy. The screen indicated a role for the relatively uncharacterized gene NHLRC2 in both Salmonella invasion and macrophage differentiation. Upon differentiation, NHLRC2 mutant macrophages were hyperinflammatory and did not exhibit characteristics typical of macrophages, including atypical morphology and inability to interact and phagocytose bacteria/particles. Immunoprecipitation confirmed an interaction of NHLRC2 with FRYL, EIF2AK2, and KLHL13.IMPORTANCESalmonella exploits macrophages to gain access to the lymphatic system and bloodstream to lead to local and potentially systemic infections. With an increasing number of antibiotic-resistant isolates identified in humans, Salmonella infections have become major threats to public health. Therefore, there is an urgent need to identify alternative approaches to anti-infective therapy, including host-directed therapies. In this study, we used a simple genome-wide screen to identify 183 candidate host factors in macrophages that can confer resistance to Salmonella infection. These factors may be potential therapeutic targets against Salmonella infections.

Phylogenomic analysis of gastroenteritis-associated Clostridium perfringens in England and Wales over a 7-year period indicates distribution of clonal toxigenic strains in multiple outbreaks and extensive involvement of enterotoxin-encoding (CPE) plasmids.

Clostridium perfringens is a major enteric pathogen known to cause gastroenteritis in human adults. Although major outbreak cases are frequently reported, only limited whole-genome sequencing (WGS) based studies have been performed to understand the genomic epidemiology and virulence gene content of outbreak-associated C. perfringens strains. We performed phylogenomic analysis on 109 C. perfringens isolates (human and food) obtained from disease cases in England and Wales between 2011 and 2017. Initial findings highlighted the enhanced discriminatory power of WGS in profiling outbreak C. perfringens strains, when compared to the current Public Health England referencing laboratory technique of fluorescent amplified fragment length polymorphism analysis. Further analysis identified that isogenic C. perfringens strains were associated with nine distinct care-home-associated outbreaks over the course of a 5-year interval, indicating a potential common source linked to these outbreaks or transmission over time and space. As expected, the enterotoxin cpe gene was encoded in all but 4 isolates (96.3 %; 105/109), with virulence plasmids encoding cpe (particularly pCPF5603 and pCPF4969 plasmids) extensively distributed (82.6 %; 90/109). Genes encoding accessory virulence factors, such as beta-2 toxin, were commonly detected (46.7 %; 51/109), and genes encoding phage proteins were also frequently identified. Overall, this large-scale genomic study of gastroenteritis-associated C. perfringens suggested that three major cpe-encoding (toxinotype F) genotypes underlie these outbreaks: strains carrying (1) pCPF5603 plasmid, (2) pCPF4969 plasmid and (3) chromosomal-cpe strains. Our findings substantially expanded our knowledge on type F C. perfringens involved in human-associated gastroenteritis, with further studies required to fully probe the dissemination and regional reservoirs of this enteric pathogen, which may help devise effective prevention strategies to reduce the food-poisoning disease burden in vulnerable patients, such as the elderly.