Intracellular Salmonella Paratyphi A is motile and differs in the expression of flagella-chemotaxis, SPI-1 and carbon utilization pathways in comparison to intracellular S. Typhimurium.

Although Salmonella Typhimurium (STM) and Salmonella Paratyphi A (SPA) belong to the same phylogenetic species, share large portions of their genome and express many common virulence factors, they differ vastly in their host specificity, the immune response they elicit, and the clinical manifestations they cause. In this work, we compared their intracellular transcriptomic architecture and cellular phenotypes during human epithelial cell infection. While transcription induction of many metal transport systems, purines, biotin, PhoPQ and SPI-2 regulons was similar in both intracellular SPA and STM, we identified 234 differentially expressed genes that showed distinct expression patterns in intracellular SPA vs. STM. Surprisingly, clear expression differences were found in SPI-1, motility and chemotaxis, and carbon (mainly citrate, galactonate and ethanolamine) utilization pathways, indicating that these pathways are regulated differently during their intracellular phase. Concurring, on the cellular level, we show that while the majority of STM are non-motile and reside within Salmonella-Containing Vacuoles (SCV), a significant proportion of intracellular SPA cells are motile and compartmentalized in the cytosol. Moreover, we found that the elevated expression of SPI-1 and motility genes by intracellular SPA results in increased invasiveness of SPA, following exit from host cells. These findings demonstrate unexpected flagellum-dependent intracellular motility of a typhoidal Salmonella serovar and intriguing differences in intracellular localization between typhoidal and non-typhoidal salmonellae. We propose that these differences facilitate new cycles of host cell infection by SPA and may contribute to the ability of SPA to disseminate beyond the intestinal lamina propria of the human host during enteric fever.

Pathoadaptation of the passerine-associated Salmonella enterica serovar Typhimurium lineage to the avian host.

Salmonella enterica is a diverse bacterial pathogen and a primary cause of human and animal infections. While many S. enterica serovars present a broad host-specificity, several specialized pathotypes have been adapted to colonize and cause disease in one or limited numbers of host species. The underlying mechanisms defining Salmonella host-specificity are far from understood. Here, we present genetic analysis, phenotypic characterization and virulence profiling of a monophasic S. enterica serovar Typhimurium strain that was isolated from several wild sparrows in Israel. Whole genome sequencing and complete assembly of its genome demonstrate a unique genetic signature that includes the integration of the BTP1 prophage, loss of the virulence plasmid, pSLT and pseudogene accumulation in multiple T3SS-2 effectors (sseJ, steC, gogB, sseK2, and sseK3), catalase (katE), tetrathionate respiration (ttrB) and several adhesion/ colonization factors (lpfD, fimH, bigA, ratB, siiC and siiE) encoded genes. Correspondingly, this strain demonstrates impaired biofilm formation, intolerance to oxidative stress and compromised intracellular replication within non-phagocytic host cells. Moreover, while this strain showed attenuated pathogenicity in the mouse, it was highly virulent and caused an inflammatory disease in an avian host. Overall, our findings demonstrate a unique phenotypic profile and genetic makeup of an overlooked S. Typhimurium sparrow-associated lineage and present distinct genetic signatures that are likely to contribute to its pathoadaptation to passerine birds.

Std fimbriae-fucose interaction increases Salmonella-induced intestinal inflammation and prolongs colonization.

Expression of ABO and Lewis histo-blood group antigens by the gastrointestinal epithelium is governed by an α-1,2-fucosyltransferase enzyme encoded by the Fut2 gene. Alterations in mucin glycosylation have been associated with susceptibility to various bacterial and viral infections. Salmonella enterica serovar Typhimurium is a food-borne pathogen and a major cause of gastroenteritis. In order to determine the role of Fut2-dependent glycans in Salmonella-triggered intestinal inflammation, Fut2+/+ and Fut2-/- mice were orally infected with S. Typhimurium and bacterial colonization and intestinal inflammation were analyzed. Bacterial load in the intestine of Fut2-/- mice was significantly lower compared to Fut2+/+ mice. Analysis of histopathological changes revealed significantly lower levels of intestinal inflammation in Fut2-/- mice compared to Fut2+/+ mice and measurement of lipocalin-2 level in feces corroborated histopathological findings. Salmonella express fimbriae that assist in adherence of bacteria to host cells thereby facilitating their invasion. The std fimbrial operon of S. Typhimurium encodes the π-class Std fimbriae which bind terminal α(1,2)-fucose residues. An isogenic mutant of S. Typhimurium lacking Std fimbriae colonized Fut2+/+ and Fut2-/- mice to similar levels and resulted in similar intestinal inflammation. In vitro adhesion assays revealed that bacteria possessing Std fimbriae adhered significantly more to fucosylated cell lines or primary epithelial cells in comparison to cells lacking α(1,2)-fucose. Overall, these results indicate that Salmonella-triggered intestinal inflammation and colonization are dependent on Std-fucose interaction.

Persistent Infection and Long-Term Carriage of Typhoidal and Nontyphoidal Salmonellae.

The ability of pathogenic bacteria to affect higher organisms and cause disease is one of the most dramatic properties of microorganisms. Some pathogens can establish transient colonization only, but others are capable of infecting their host for many years or even for a lifetime. Long-term infection is called persistence, and this phenotype is fundamental for the biology of important human pathogens, including Helicobacter pylori, Mycobacterium tuberculosis, and Salmonella enterica Both typhoidal and nontyphoidal serovars of the species Salmonella enterica can cause persistent infection in humans; however, as these two Salmonella groups cause clinically distinct diseases, the characteristics of their persistent infections in humans differ significantly. Here, following a general summary of Salmonella pathogenicity, host specificity, epidemiology, and laboratory diagnosis, I review the current knowledge about Salmonella persistence and discuss the relevant epidemiology of persistence (including carrier rate, duration of shedding, and host and pathogen risk factors), the host response to Salmonella persistence, Salmonella genes involved in this lifestyle, as well as genetic and phenotypic changes acquired during prolonged infection within the host. Additionally, I highlight differences between the persistence of typhoidal and nontyphoidal Salmonella strains in humans and summarize the current gaps and limitations in our understanding, diagnosis, and curing of persistent Salmonella infections.

Emergence of new variants of antibiotic resistance genomic islands among multidrug-resistant Salmonella enterica in poultry.

Non-typhoidal Salmonella enterica (NTS) are diverse and important bacterial pathogens consisting of more than 2600 different serovars, with varying host-specificity. Here, we characterized the poultry-associated serovars in Israel, analysed their resistome and illuminated the molecular mechanisms underlying common multidrug resistance (MDR) patterns. We show that at least four serovars including Infantis, Muenchen, Newport and Virchow present a strong epidemiological association between their temporal trends in poultry and humans. Worrisomely, 60% from all of the poultry isolates tested (n = 188) were multidrug resistant, mediated by chromosomal SNPs and different mobile genetics elements. A novel streptomycin-azithromycin resistance island and previously uncharacterized versions of the mobilized Salmonella genomic island 1 (SGI1) were identified and characterized in S. Blockley and S. Kentucky isolates respectively. Moreover, we demonstrate that the acquisition of SGI1 does not impose fitness cost during growth under nutrient-limited conditions or in the context of Salmonella infection in the mouse model. Overall, our data emphasize the role of the poultry production as a pool of specific epidemic MDR strains and autonomous genetic elements, which confer resistance to heavy metals and medically relevant antibiotics. These are likely to disseminate to humans via the food chain and fuel the increasing global antibiotic resistance crisis.

Differences in the expression of SPI-1 genes pathogenicity and epidemiology between the emerging Salmonella enterica serovar Infantis and the model Salmonella enterica serovar Typhimurium.

BACKGROUND: Salmonella enterica serovar Infantis (S. Infantis) is one of the ubiquitous serovars of the bacterial pathogen S. enterica and recently has been emerging in many countries worldwide. Nonetheless, not much is known about its epidemiology, host adaptation, and virulence. METHODS: Epidemiological and molecular approaches were used together with tissue-culture and mouse models to conduct phenotypic comparison with the model S. enterica serovar Typhimurium. RESULTS: We show that S. Infantis is more frequently associated with infections in infants <2 years old and prone to cause significantly less invasive infections than serovar Typhimurium. Moreover, although S. Infantis adheres better to host cells and highly colonizes mouse intestines soon after infection, it is significantly less invasive and induces much lower inflammation and disease in vivo than S. Typhimurium. These differences were associated with lower expression of Salmonella pathogenicity island (SPI) 1 genes in S. Infantis than in S. Typhimurium. CONCLUSIONS: Our results demonstrate previously unknown differences in the epidemiology, virulence pathway expression, and pathogenicity between two highly abundant Salmonella serovars and suggest that native variation in the expression of the SPI-1 regulon is likely to contribute to epidemiological and virulence variation between genetically similar nontyphoidal Salmonella serovars.

The plasmid-encoded Ipf and Klf fimbriae display different expression and varying roles in the virulence of Salmonella enterica serovar Infantis in mouse vs. avian hosts.

Salmonella enterica serovar Infantis is one of the prevalent Salmonella serovars worldwide. Different emergent clones of S. Infantis were shown to acquire the pESI virulence-resistance megaplasmid affecting its ecology and pathogenicity. Here, we studied two previously uncharacterized pESI-encoded chaperone-usher fimbriae, named Ipf and Klf. While Ipf homologs are rare and were found only in S. enterica subspecies diarizonae and subspecies VII, Klf is related to the known K88-Fae fimbria and klf clusters were identified in seven S. enterica subspecies I serovars, harboring interchanging alleles of the fimbria major subunit, KlfG. Regulation studies showed that the klf genes expression is negatively and positively controlled by the pESI-encoded regulators KlfL and KlfB, respectively, and are activated by the ancestral leucine-responsive regulator (Lrp). ipf genes are negatively regulated by Fur and activated by OmpR. Furthermore, induced expression of both klf and ipf clusters occurs under microaerobic conditions and at 41°C compared to 37°C, in-vitro. Consistent with these results, we demonstrate higher expression of ipf and klf in chicks compared to mice, characterized by physiological temperature of 41.2°C and 37°C, respectively. Interestingly, while Klf was dispensable for S. Infantis colonization in the mouse, Ipf was required for maximal colonization in the murine ileum. In contrast to these phenotypes in mice, both Klf and Ipf contributed to a restrained infection in chicks, where the absence of these fimbriae has led to moderately higher bacterial burden in the avian host. Taken together, these data suggest that physiological differences between host species, such as the body temperature, can confer differences in fimbriome expression, affecting Salmonella colonization and other host-pathogen interplays.

The Stringent Response Regulator DksA Is Required for Salmonella enterica Serovar Typhimurium Growth in Minimal Medium, Motility, Biofilm Formation, and Intestinal Colonization.

Salmonella enterica serovar Typhimurium is a facultative intracellular human and animal bacterial pathogen posing a major threat to public health worldwide. Salmonella pathogenicity requires complex coordination of multiple physiological and virulence pathways. DksA is a conserved Gram-negative regulator that belongs to a distinct group of transcription factors that bind directly to the RNA polymerase secondary channel, potentiating the effect of the signaling molecule ppGpp during a stringent response. Here, we established that in S. Typhimurium, dksA is induced during the logarithmic phase and DksA is essential for growth in minimal defined medium and plays an important role in motility and biofilm formation. Furthermore, we determined that DksA positively regulates the Salmonella pathogenicity island 1 and motility-chemotaxis genes and is necessary for S. Typhimurium invasion of human epithelial cells and uptake by macrophages. In contrast, DksA was found to be dispensable for S. Typhimurium host cell adhesion. Finally, using the colitis mouse model, we found that dksA is spatially induced at the midcecum during the early stage of the infection and required for gastrointestinal colonization and systemic infection in vivo. Taken together, these data indicate that the ancestral stringent response regulator DksA coordinates various physiological and virulence S. Typhimurium programs and therefore is a key virulence regulator of Salmonella.

Differences in Host Cell Invasion and Salmonella Pathogenicity Island 1 Expression between Salmonella enterica Serovar Paratyphi A and Nontyphoidal S. Typhimurium.

Active invasion into nonphagocytic host cells is central to Salmonella enterica pathogenicity and dependent on multiple genes within Salmonella pathogenicity island 1 (SPI-1). Here, we explored the invasion phenotype and the expression of SPI-1 in the typhoidal serovarS Paratyphi A compared to that of the nontyphoidal serovarS Typhimurium. We demonstrate that while S. Typhimurium is equally invasive under both aerobic and microaerobic conditions, S. Paratyphi A invades only following growth under microaerobic conditions. Transcriptome sequencing (RNA-Seq), reverse transcription-PCR (RT-PCR), Western blot, and secretome analyses established that S. Paratyphi A expresses much lower levels of SPI-1 genes and secretes lesser amounts of SPI-1 effector proteins than S. Typhimurium, especially under aerobic growth. Bypassing the native SPI-1 regulation by inducible expression of the SPI-1 activator, HilA, considerably elevated SPI-1 gene expression, host cell invasion, disruption of epithelial integrity, and induction of proinflammatory cytokine secretion by S. Paratyphi A but not by S. Typhimurium, suggesting that SPI-1 expression is naturally downregulated inS Paratyphi A. Using streptomycin-treated mice, we were able to establish substantial intestinal colonization byS Paratyphi A and showed moderately higher pathology and intestinal inflammation in mice infected with S. Paratyphi A overexpressing hilA Collectively, our results reveal unexpected differences in SPI-1 expression between S. Paratyphi A andS Typhimurium, indicate that S. Paratyphi A host cell invasion is suppressed under aerobic conditions, and suggest that lower invasion in aerobic sites and suppressed expression of immunogenic SPI-1 components contributes to the restrained inflammatory infection elicited by S. Paratyphi A.

Persistent Infections by Nontyphoidal Salmonella in Humans: Epidemiology and Genetics.

BACKGROUND: Although chronic infections by typhoidal Salmonella are well-known, prolonged human infections by nontyphoidal Salmonella (NTS) are poorly characterized. METHODS: We retrospectively analyzed 48 345 culture-confirmed NTS infections that occurred in Israel 1995-2012. A case-control study was performed to identify risk factors associated with persistent infections. Whole-genome-sequencing, pulsed-field gel electrophoresis (PFGE), and a mouse infection model were used to study genetic and phenotypic differences between same-patient persistent, recurring isolates. RESULTS: In total, 1047 cases of persistent NTS infections, comprising 2.2% of all reported cases of salmonellosis, were identified. The persistence periods ranged between 30 days to 8.3 years. The majority (93%) of the persistently infected patients were immunocompetent, and 65% were symptomatic with relapsing diarrhea, indicating a distinct clinical manifestation from the asymptomatic carriage of typhoidal Salmonella. Four NTS serovars (Mbandaka, Bredeney, Infantis and Virchow) were found to be significantly more frequently associated with persistence than others. Comparative genomics between early and later isolates obtained from the same patients confirmed clonal infection and showed 0 to 10 SNPs between persistent isolates. A different composition of mobile genetic elements (plasmids and phages) or amino acid substitutions in global regulators was identified in multiple cases. These changes resulted in differences in phenotype and virulence between early and later same-patient isolates. CONCLUSIONS: These results illuminate the overlooked clinical manifestation of persistent salmonellosis that can serve as a human reservoir for NTS infections. Additionally, we demonstrate mechanisms of in-host microevolution and exhibit their potential to shape Salmonella pathogenicity, antimicrobial resistance and host-pathogen interactions.

Travel- and Community-Based Transmission of Multidrug-Resistant Shigella sonnei Lineage among International Orthodox Jewish Communities.

Shigellae are sensitive indicator species for studying trends in the international transmission of antimicrobial-resistant Enterobacteriaceae. Orthodox Jewish communities (OJCs) are a known risk group for shigellosis; Shigella sonnei is cyclically epidemic in OJCs in Israel, and sporadic outbreaks occur in OJCs elsewhere. We generated whole-genome sequences for 437 isolates of S. sonnei from OJCs and non-OJCs collected over 22 years in Europe (the United Kingdom, France, and Belgium), the United States, Canada, and Israel and analyzed these within a known global genomic context. Through phylogenetic and genomic analysis, we showed that strains from outbreaks in OJCs outside of Israel are distinct from strains in the general population and relate to a single multidrug-resistant sublineage of S. sonnei that prevails in Israel. Further Bayesian phylogenetic analysis showed that this strain emerged approximately 30 years ago, demonstrating the speed at which antimicrobial drug-resistant pathogens can spread widely through geographically dispersed, but internationally connected, communities.

Feverlike Temperature is a Virulence Regulatory Cue Controlling the Motility and Host Cell Entry of Typhoidal Salmonella.

Human infection with typhoidal Salmonella serovars causes a febrile systemic disease, termed enteric fever. Here we establish that in response to a temperature equivalent to fever (39 °C-42 °C) Salmonella enterica serovars Typhi, Paratyphi A, and Sendai significantly attenuate their motility, epithelial cell invasion, and uptake by macrophages. Under these feverlike conditions, the residual epithelial cell invasion of S. Paratyphi A occurs in a type III secretion system (T3SS) 1-independent manner and results in restrained disruption of epithelium integrity. The impaired motility and invasion are associated with down-regulation of T3SS-1 genes and class II and III (but not I) of the flagella-chemotaxis regulon. In contrast, we demonstrate up-regulation of particular Salmonella pathogenicity island 2 genes (especially spiC) and increased intraepithelial growth in a T3SS-2-dependent manner. These results indicate that elevated physiological temperature is a novel cue controlling virulence phenotypes in typhoidal serovars, which is likely to play a role in the distinct clinical manifestations elicited by typhoidal and nontyphoidal salmonellae.

Flagellin Is Required for Host Cell Invasion and Normal Salmonella Pathogenicity Island 1 Expression by Salmonella enterica Serovar Paratyphi A.

Salmonella enterica serovar Paratyphi A is a human-specific serovar that, together with Salmonella enterica serovar Typhi and Salmonella enterica serovar Sendai, causes enteric fever. Unlike the nontyphoidal Salmonella enterica serovar Typhimurium, the genomes of S. Typhi and S. Paratyphi A are characterized by inactivation of multiple genes, including in the flagellum-chemotaxis pathway. Here, we explored the motility phenotype of S. Paratyphi A and the role of flagellin in key virulence-associated phenotypes. Motility studies established that the human-adapted typhoidal S. Typhi, S. Paratyphi A, and S. Sendai are all noticeably less motile than S. Typhimurium, and comparative transcriptome sequencing (RNA-Seq) showed that in S. Paratyphi A, the entire motility-chemotaxis regulon is expressed at significantly lowers levels than in S. Typhimurium. Nevertheless, S. Paratyphi A, like S. Typhimurium, requires a functional flagellum for epithelial cell invasion and macrophage uptake, probably in a motility-independent mechanism. In contrast, flagella were found to be dispensable for host cell adhesion. Moreover, we demonstrate that in S. Paratyphi A, but not in S. Typhimurium, the lack of flagellin results in increased transcription of the flagellar and the Salmonella pathogenicity island 1 (SPI-1) regulons in a FliZ-dependent manner and in oversecretion of SPI-1 effectors via type three secretion system 1. Collectively, these results suggest a novel regulatory linkage between flagellin and SPI-1 in S. Paratyphi A that does not occur in S. Typhimurium and demonstrate curious distinctions in motility and the expression of the flagellum-chemotaxis regulon between these clinically relevant pathogens.

A unique megaplasmid contributes to stress tolerance and pathogenicity of an emergent Salmonella enterica serovar Infantis strain.

Of all known Salmonella enterica serovars, S. Infantis is one of the most commonly isolated and has been recently emerging worldwide. To understand the recent emergence of S. Infantis in Israel, we performed extensive comparative analyses between pre-emergent and the clonal emergent S. Infantis populations. We demonstrate the fixation of adaptive mutations in the DNA gyrase (gyrA) and nitroreductase (nfsA) genes, conferring resistance to quinolones and nitrofurans, respectively, and the carriage of an emergent-specific plasmid, designated pESI. This self-transferred episome is a mosaic megaplasmid (∼280 kb), which increases bacterial tolerance to environmental mercury (mer operon) and oxidative stress, and provides further resistance to tetracycline, sulfamethoxazole and trimethoprim, most likely due to the presence of tetRA, sulI and dfrA genes respectively. Moreover, pESI carries the yersiniabactin siderophore system and two novel chaperone-usher fimbriae. In vitro studies established that pESI conjugation into a plasmidless S. Infantis strain results in superior biofilm formation, adhesion and invasion into avian and mammalian host cells. In vivo mouse infections demonstrated higher pathogenicity and increased intestinal inflammation caused by an S. Infantis strain harboring pESI compared with the plasmidless parental strain. Our results indicate that the presence of pESI that was found only in the emergent population of S. Infantis in Israel contributes significantly to antimicrobials tolerance and pathogenicity of its carrier. It is highly likely that pESI plays a key role in the successful spread of the emergent clone that replaced the local S. Infantis community in the short time of only 2-3 years.

Integrative analysis of Salmonellosis in Israel reveals association of Salmonella enterica Serovar 9,12:l,v:- with extraintestinal infections, dissemination of endemic S. enterica Serovar Typhimurium DT104 biotypes, and severe underreporting of outbreaks.

Salmonella enterica is the leading etiologic agent of bacterial food-borne outbreaks worldwide. This ubiquitous species contains more than 2,600 serovars that may differ in their host specificity, clinical manifestations, and epidemiology. To characterize salmonellosis epidemiology in Israel and to study the association of nontyphoidal Salmonella (NTS) serovars with invasive infections, 48,345 Salmonella cases reported and serotyped at the National Salmonella Reference Center between 1995 and 2012 were analyzed. A quasi-Poisson regression was used to identify irregular clusters of illness, and pulsed-field gel electrophoresis in conjunction with whole-genome sequencing was applied to molecularly characterize strains of interest. Three hundred twenty-nine human salmonellosis clusters were identified, representing an annual average of 23 (95% confidence interval [CI], 20 to 26) potential outbreaks. We show that the previously unsequenced S. enterica serovar 9,12:l,v:- belongs to the B clade of Salmonella enterica subspecies enterica, and we show its frequent association with extraintestinal infections, compared to other NTS serovars. Furthermore, we identified the dissemination of two prevalent Salmonella enterica serovar Typhimurium DT104 clones in Israel, which are genetically distinct from other global DT104 isolates. Accumulatively, these findings indicate a severe underreporting of Salmonella outbreaks in Israel and provide insights into the epidemiology and genomics of prevalent serovars, responsible for recurring illness.

The transcriptional regulation of the horizontally acquired iron uptake system, yersiniabactin and its contribution to oxidative stress tolerance and pathogenicity of globally emerging salmonella strains.

The bacterial species Salmonella enterica (S. enterica) is a highly diverse pathogen containing more than 2600 distinct serovars, which can infect a wide range of animal and human hosts. Recent global emergence of multidrug resistant strains, from serovars Infantis and Muenchen is associated with acquisition of the epidemic megaplasmid, pESI that augments antimicrobial resistance and pathogenicity. One of the main pESI's virulence factors is the potent iron uptake system, yersiniabactin encoded by fyuA, irp2-irp1-ybtUTE, ybtA, and ybtPQXS gene cluster. Here we show that yersiniabactin, has an underappreciated distribution among different S. enterica serovars and subspecies, integrated in their chromosome or carried by different conjugative plasmids, including pESI. While the genetic organization and the coding sequence of the yersiniabactin genes are generally conserved, a 201-bp insertion sequence upstream to ybtA, was identified in pESI. Despite this insertion, pESI-encoded yersiniabactin is regulated by YbtA and the ancestral Ferric Uptake Regulator (Fur), which binds directly to the ybtA and irp2 promoters. Furthermore, we show that yersiniabactin genes are specifically induced during the mid-late logarithmic growth phase and in response to iron-starvation or hydrogen peroxide. Concurring, yersiniabactin was found to play a previously unknown role in oxidative stress tolerance and to enhance intestinal colonization of S. Infantis in mice. These results indicate that yersiniabactin contributes to Salmonella fitness and pathogenicity in vivo and is likely to play a role in the rapid dissemination of pESI among globally emerging Salmonella lineages.

A new Salmonella enterica serovar that was isolated from a wild sparrow presents a distinct genetic, metabolic and virulence profile.

Salmonella enterica is a ubiquitous and clinically-important bacterial pathogen, able to infect and cause different diseases in a wide range of hosts. Here, we report the isolation and characterization of a new S. enterica serovar (13,23:i:-; S. Tirat-Zvi), belonging to the Havana supper-lineage that was isolated from a wild house sparrow (Passer domesticus) in Israel. Whole genome sequencing and complete assembly of its genome indicated a plasmid-free, 4.7 Mb genome that carries the Salmonella pathogenicity islands 1-6, 9, 19 and an integrative and conjugative element (ICE), encoding arsenic resistance genes. Phenotypically, S. Tirat-Zvi isolate TZ282 was motile, readily formed biofilm, more versatile in carbon source utilization than S. Typhimurium and highly tolerant to arsenic, but impaired in host cell invasion. In-vivo infection studies indicated that while S. Tirat-Zvi was able to infect and cause an acute inflammatory enterocolitis in young chicks, it was compromised in mice colonization and did not cause an inflammatory colitis in mice compared to S. Typhimurium. We suggest that these phenotypes reflect the distinctive ecological niche of this new serovar and its evolutionary adaptation to passerine birds, as a permissive host. Moreover, these results further illuminate the genetic, phenotypic and ecological diversity of S. enterica pathovars.

Persistent Salmonella infections in humans are associated with mutations in the BarA/SirA regulatory pathway.

Several bacterial pathogens, including Salmonella enterica, can cause persistent infections in humans by mechanisms that are poorly understood. By comparing genomes of isolates longitudinally collected from 256 prolonged salmonellosis patients, we identified repeated mutations in global regulators, including the barA/sirA two-component regulatory system, across multiple patients and Salmonella serovars. Comparative RNA-seq analysis revealed that distinct mutations in barA/sirA led to diminished expression of Salmonella pathogenicity islands 1 and 4 genes, which are required for Salmonella invasion and enteritis. Moreover, barA/sirA mutants were attenuated in an acute salmonellosis mouse model and induced weaker transcription of host immune responses. In contrast, in a persistent infection mouse model, these mutants exhibited long-term colonization and prolonged shedding. Taken together, these findings suggest that selection of mutations in global virulence regulators facilitates persistent Salmonella infection in humans, by attenuating Salmonella virulence and inducing a weaker host inflammatory response.

The Effect of Outer Space and Other Environmental Cues on Bacterial Conjugation.

Bacterial conjugation is one of the most abundant horizontal gene transfer (HGT) mechanisms, playing a fundamental role in prokaryote evolution. A better understanding of bacterial conjugation and its cross talk with the environment is needed for a more complete understanding of HGT mechanisms and to fight the dissemination of malicious genes between bacteria. Here, we studied the effect of outer space, microgravity, and additional key environmental cues on transfer (tra) gene expression and conjugation efficiency, using the under studied broad-host range plasmid pN3, as a model. High resolution scanning electron microscopy revealed the morphology of the pN3 conjugative pili and mating pair formation during conjugation. Using a nanosatellite carrying a miniaturized lab, we studied pN3 conjugation in outer space, and used qRT-PCR, Western blotting and mating assays to determine the effect of ground physicochemical parameters on tra gene expression and conjugation. We showed for the first time that bacterial conjugation can occur in outer space and on the ground, under microgravity-simulated conditions. Furthermore, we demonstrated that microgravity, liquid media, elevated temperature, nutrient depletion, high osmolarity and low oxygen significantly reduce pN3 conjugation. Interestingly, under some of these conditions we observed an inverse correlation between tra gene transcription and conjugation frequency and found that induction of at least traK and traL can negatively affect pN3 conjugation frequency in a dose-dependent manner. Collectively, these results uncover pN3 regulation by various environmental cues and highlight the diversity of conjugation systems and the different ways in which they may be regulated in response to abiotic signals. IMPORTANCE Bacterial conjugation is a highly ubiquitous and promiscuous process, by which a donor bacterium transfers a large portion of genetic material to a recipient cell. This mechanism of horizontal gene transfer plays an important role in bacterial evolution and in the ability of bacteria to acquire resistance to antimicrobial drugs and disinfectants. Bacterial conjugation is a complex and energy-consuming process, that is tightly regulated and largely affected by various environmental signals sensed by the bacterial cell. Comprehensive knowledge about bacterial conjugation and the ways it is affected by environmental cues is required to better understand bacterial ecology and evolution and to find new effective ways to counteract the threating dissemination of antibiotic resistance genes between bacterial populations. Moreover, characterizing this process under stress or suboptimal growth conditions such as elevated temperatures, high salinity or in the outer space, may provide insights relevant to future habitat environmental conditions.

Profiling of Secreted Type 3 Secretion System Substrates by Salmonella enterica.

Many of Salmonella enterica virulence-associated phenotypes, including its ability to manipulate various host pathways are mediated by translocation of specific effector proteins via type 3 secretion systems (T3SSs) into the host cell. Culturing Salmonella under a defined set of stimulating conditions in vitro can mimic the physiological signals Salmonella senses during the infection and results in the secretion of these effectors into the growth medium. Here we describe a Salmonella secretion assay to identify and quantify protein substrates secreted by T3SS-1 and demonstrate how this method can be utilized to study the secretion of T3SS-1 effectors and flagellum components in different genetic backgrounds or under varying growth conditions.