Salmonella Typhimurium and Vibrio cholerae can be transferred from plastic mulch to basil and spinach salad leaves.

Plastic pollution is increasingly found in agricultural environments, where it contaminates soil and crops. Microbial biofilms rapidly colonise environmental plastics, such as the plastic mulches used in agricultural systems, which provide a unique environment for microbial plastisphere communities. Human pathogens can also persist in the plastisphere, and enter agricultural environments via flooding or irrigation with contaminated water. In this study we examined whether Salmonella Typhimurium and Vibrio cholerae can be transferred from the plastisphere on plastic mulch to the surface of ready-to-eat crop plants, and subsequently persist on the leaf surface. Both S. Typhimurium and V. cholerae were able to persist for 14 days on fragments of plastic mulch adhering to the surface of leaves of both basil and spinach. Importantly, within 24 h both pathogens were capable of dissociating from the surface of the plastic and were transferred onto the surface of both basil and spinach leaves. This poses a further risk to food safety and human health, as even removal of adhering plastics and washing of these ready-to-eat crops would not completely remove these pathogens. As the need for more intensive food production increases, so too does the use of plastic mulches in agronomic systems. Therefore, there is now an urgent need to understand the unquantified co-pollutant pathogen risk of contaminating agricultural and food production systems with plastic pollution.

Can plastic pollution drive the emergence and dissemination of novel zoonotic diseases?

As the volume of plastic in the environment increases, so too does human interactions with plastic pollution. Similarly, domestic, feral, and wild animals are increasingly interacting with plastic pollution, highlighting the potential for contamination of plastic wastes with animal faeces, urine, saliva, and blood. Substantial evidence indicates that once in the environment, plastics rapidly become colonised by microbial biofilm (the so-called 'plastisphere), which often includes potentially harmful microbial pathogens (including pathogens that are zoonotic in nature). Climate change, increased urbanisation, and the intensification of agriculture, mean that the three-way interactions between humans, animals, and plastic pollution are becoming more frequent, which is significant as almost 60% of emerging human infectious diseases during the last century have been zoonotic. Here, we critically review the potential for contaminated environmental plastics to facilitate the evolution of novel pathogenic strains of microorganisms, and the subsequent role of plastic pollution in the cyclical dissemination of zoonotic pathogens. As the interactions between humans, animals, and plastic pollution continues to grow, and the volume of plastics entering the environment increases, there is clearly an urgent need to better understand the role of plastic waste in facilitating zoonotic pathogen evolution and dissemination, and the effect this can have on environmental and human health.

An intact S-layer is advantageous to Clostridioides difficile within the host.

Clostridioides difficile is responsible for substantial morbidity and mortality in antibiotically-treated, hospitalised, elderly patients, in which toxin production correlates with diarrhoeal disease. While the function of these toxins has been studied in detail, the contribution of other factors, including the paracrystalline surface layer (S-layer), to disease is less well understood. Here, we highlight the essentiality of the S-layer in vivo by reporting the recovery of S-layer variants, following infection with the S-layer-null strain, FM2.5. These variants carry either correction of the original point mutation, or sequence modifications which restored the reading frame, and translation of slpA. Selection of these variant clones was rapid in vivo, and independent of toxin production, with up to 90% of the recovered C. difficile population encoding modified slpA sequence within 24 h post infection. Two variants, subsequently named FM2.5varA and FM2.5varB, were selected for study in greater detail. Structural determination of SlpA from FM2.5varB indicated an alteration in the orientation of protein domains, resulting in a reorganisation of the lattice assembly, and changes in interacting interfaces, which might alter function. Interestingly, variant FM2.5varB displayed an attenuated, FM2.5-like phenotype in vivo compared to FM2.5varA, which caused disease severity more comparable to that of R20291. Comparative RNA sequencing (RNA-Seq) analysis of in vitro grown isolates revealed large changes in gene expression between R20291 and FM2.5. Downregulation of tcdA/tcdB and several genes associated with sporulation and cell wall integrity may account for the reported attenuated phenotype of FM2.5 in vivo. RNA-seq data correlated well with disease severity with the more virulent variant, FM2.5varA, showing s similar profile of gene expression to R20291 in vitro, while the attenuated FM2.5varB showed downregulation of many of the same virulence associated traits as FM2.5. Cumulatively, these data add to a growing body of evidence that the S-layer contributes to C. difficile pathogenesis and disease severity.

PF-431396 hydrate inhibition of kinase phosphorylation during adherent-invasive Escherichia coli infection inhibits intra-macrophage replication and inflammatory cytokine release.

Adherent-invasive Escherichia coli (AIEC) have been implicated in the aetiology of Crohn's disease (CD). They are characterized by an ability to adhere to and invade intestinal epithelial cells, and to replicate intracellularly in macrophages resulting in inflammation. Proline-rich tyrosine kinase 2 (PYK2) has previously been identified as a risk locus for inflammatory bowel disease and a regulator of intestinal inflammation. It is overexpressed in patients with colorectal cancer, a major long-term complication of CD. Here we show that Pyk2 levels are significantly increased during AIEC infection of murine macrophages while the inhibitor PF-431396 hydrate, which blocks Pyk2 activation, significantly decreased intramacrophage AIEC numbers. Imaging flow cytometry indicated that Pyk2 inhibition blocked intramacrophage replication of AIEC with no change in the overall number of infected cells, but a significant reduction in bacterial burden per cell. This reduction in intracellular bacteria resulted in a 20-fold decrease in tumour necrosis factor α secretion by cells post-AIEC infection. These data demonstrate a key role for Pyk2 in modulating AIEC intracellular replication and associated inflammation and may provide a new avenue for future therapeutic intervention in CD.

Open defaecation by proxy: Tackling the increase of disposable diapers in waste piles in informal settlements.

Disposable diapers are becoming increasingly popular and present an emerging challenge for global waste management, particularly within LMICs. They offer a cheap and convenient way for caregivers to manage child excreta; however, insufficient understanding of safe disposal methods, combined with limited access to waste management services results in hazardous disposal. Used diapers are being increasingly found dumped in the open environment, including in water bodies and in open fields, leading to faecal contamination of the environment and an enhanced risk of transmission of faecal-oral diseases such as cholera and typhoid. United Nations SDG 6 aims to end open defaecation globally by 2030; however, improper disposal of used diapers will hamper progress towards reaching this goal. In this review, we identify current trends in use and subsequent disposal of single use disposable diapers in LMICs, and critically discuss the environmental and public health impacts of current practices, and potential solutions to address these challenges. Contemporary methods for managing the disposal of single use diapers for communities in LMICs tend to be cost prohibitive with few alternative options other than dumping in the environment. Modern cloth diapers offer a low waste alternative to disposable diapers but often carry an unaffordable high upfront cost. Here, in addition to advocating improved efforts by governments to upgrade access and quality of waste management services, we recommend the design and implementation of intervention schemes aimed to increase awareness of safe and hygienic disposal practices for disposable diapers.

Plastic pollution and fungal, protozoan, and helminth pathogens - A neglected environmental and public health issue?

Plastic waste is ubiquitous in the environment and can become colonised by distinct microbial biofilm communities, known collectively as the 'plastisphere.' The plastisphere can facilitate the increased survival and dissemination of human pathogenic prokaryotes (e.g., bacteria); however, our understanding of the potential for plastics to harbour and disseminate eukaryotic pathogens is lacking. Eukaryotic microorganisms are abundant in natural environments and represent some of the most important disease-causing agents, collectively responsible for tens of millions of infections, and millions of deaths worldwide. While prokaryotic plastisphere communities in terrestrial, freshwater, and marine environments are relatively well characterised, such biofilms will also contain eukaryotic species. Here, we critically review the potential for fungal, protozoan, and helminth pathogens to associate with the plastisphere, and consider the regulation and mechanisms of this interaction. As the volume of plastics in the environment continues to rise there is an urgent need to understand the role of the plastisphere for the survival, virulence, dissemination, and transfer of eukaryotic pathogens, and the effect this can have on environmental and human health.

Clinically important E. coli strains can persist, and retain their pathogenicity, on environmental plastic and fabric waste.

Plastic waste is ubiquitous in the environment and there are increasing reports of such waste being colonised by human pathogens. However, the ability of pathogens to persist on plastics for long periods, and the risk that they pose to human health, is unknown. Here, under simulated environmental conditions, we aimed to determine if pathogenic bacteria can retain their virulence following a prolonged period on plastic. Using antibiotic selection and luciferase expression for quantification, we show that clinically important strains of E. coli can survive on plastic for at least 28-days. Importantly, these pathogens also retained their virulence (determined by using a Galleria mellonella model as a surrogate for human infection) and in some cases, had enhanced virulence following their recovery from the plastisphere. This indicates that plastics in the environment can act as reservoirs for human pathogens and could facilitate their persistence for extended periods of time. Most importantly human pathogens in the plastisphere are capable of retaining their pathogenicity. Pathogens colonising environmental plastic waste therefore pose a heightened public health risk, particularly in areas where people are exposed to pollution.

From wastewater discharge to the beach: Survival of human pathogens bound to microplastics during transfer through the freshwater-marine continuum.

Large quantities of microplastics are regularly discharged from wastewater treatment plants (WWTPs) into the aquatic environment. Once released, these plastics can rapidly become colonised by microbial biofilm, forming distinct plastisphere communities which may include potential pathogens. We hypothesised that the protective environment afforded by the plastisphere would facilitate the survival of potential pathogens during transitions between downstream environmental matrices and thus increase persistence and the potential for environmental dissemination of pathogens. The survival of Escherichia coli, Enterococcus faecalis and Pseudomonas aeruginosa colonising polyethylene or glass particles has been quantified in mesocosm incubation experiments designed to simulate, (1) the direct release of microplastics from WWTPs into freshwater and seawater environments; and (2) the movement of microplastics downstream following discharge from the WWTP through the river-estuary-marine-beach continuum. Culturable E. coli, E. faecalis and P. aeruginosa were successfully able to survive and persist on particles whether they remained in one environmental matrix or transitioned between different environmental matrices. All three bacteria were still detectable on both microplastic and glass particles after 25 days, with higher concentrations on microplastic compared to glass particles; however, there were no differences in bacterial die-off rates between the two materials. This potential for environmental survival of pathogens in the plastisphere could facilitate their transition into places where human exposure is greater (e.g., bathing waters and beach environments). Therefore, risks associated with pathogen-microplastic co-pollutants in the environment, emphasises the urgency for updated regulations on wastewater discharge and the management of microplastic generation and release.

Sewage-associated plastic waste washed up on beaches can act as a reservoir for faecal bacteria, potential human pathogens, and genes for antimicrobial resistance.

Sewage-associated plastic wastes, such as wet wipes and cotton bud sticks, commonly wash up on beaches; however, it is unclear whether this represents a public health risk. In this study, sewage-associated plastic waste, and naturally occurring substrates (seaweed and sand), were collected from ten beaches along the Firth of Forth estuary (Scotland, UK) and analysed using selective media for the faecal indicator organisms (FIOs) E. coli and intestinal enterococci (IE), and potential human pathogens (Vibrio spp.). Minimum inhibitory concentration (MIC) analysis was used to determine antibiotic resistance in selected strains. FIOs and Vibrio were more often associated with wet wipes and cotton bud sticks than with seaweed, and there was evidence of resistance to several antibiotics. This work demonstrates that plastics associated with sewage pollution can facilitate the survival and dissemination of FIOs and Vibrio and thus, could present an as yet unquantified potential risk to human health at the beach.

Monocytes mediate Salmonella Typhimurium-induced tumor growth inhibition in a mouse melanoma model.

The use of bacteria as an alternative cancer therapy has been reinvestigated in recent years. SL7207: an auxotrophic Salmonella enterica serovar Typhimurium aroA mutant with immune-stimulatory potential has proven a promising strain for this purpose. Here, we show that systemic administration of SL7207 induces melanoma tumor growth arrest in vivo, with greater survival of the SL7207-treated group compared to control PBS-treated mice. Administration of SL7207 is accompanied by a change in the immune phenotype of the tumor-infiltrating cells toward pro-inflammatory, with expression of the TH 1 cytokines IFN-γ, TNF-α, and IL-12 significantly increased. Interestingly, Ly6C+ MHCII+ monocytes were recruited to the tumors following SL7207 treatment and were pro-inflammatory. Accordingly, the abrogation of these infiltrating monocytes using clodronate liposomes prevented SL7207-induced tumor growth inhibition. These data demonstrate a previously unappreciated role for infiltrating inflammatory monocytes underlying bacterial-mediated tumor growth inhibition. This information highlights a possible novel role for monocytes in controlling tumor growth, contributing to our understanding of the immune responses required for successful immunotherapy of cancer.

Diversification of OmpA and OmpF of Yersinia ruckeri is independent of the underlying species phylogeny and evidence of virulence-related selection.

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM) which causes economically significant losses in farmed salmonids, especially Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss, Walbaum). However, very little is known about the genetic relationships of disease-causing isolates in these two host species or about factors responsible for disease. Phylogenetic analyses of 16 representative isolates based on the nucleotide sequences of 19 housekeeping genes suggests that pathogenic Atlantic salmon and rainbow trout isolates represent distinct host-specific lineages. However, the apparent phylogenies of certain isolates has been influenced by horizontal gene transfer and recombinational exchange. Splits decomposition analysis demonstrated a net-like phylogeny based on the housekeeping genes, characteristic of recombination. Comparative analysis of the distribution of individual housekeeping gene alleles across the isolates demonstrated evidence of genomic mosaicism and recombinational exchange involving certain Atlantic salmon and rainbow trout isolates. Comparative nucleotide sequence analysis of the key outer membrane protein genes ompA and ompF revealed that the corresponding gene trees were both non-congruent with respect to the housekeeping gene phylogenies providing evidence that horizontal gene transfer has influenced the evolution of both these surface protein-encoding genes. Analysis of inferred amino acid sequence variation in OmpA identified a single variant, OmpA.1, that was present in serotype O1 and O8 isolates representing typical pathogenic strains in rainbow trout and Atlantic salmon, respectively. In particular, the sequence of surface-exposed loop 3 differed by seven amino acids to that of other Y. ruckeri isolates. These findings suggest that positive selection has likely influenced the presence of OmpA.1 in these isolates and that loop 3 may play an important role in virulence. Amino acid sequence variation of OmpF was greater than that of OmpA and was similarly restricted mainly to the surface-exposed loops. Two OmpF variants, OmpF.1 and OmpF.2, were associated with pathogenic rainbow trout and Atlantic salmon isolates, respectively. These OmpF proteins had very similar amino acid sequences suggesting that positive evolutionary pressure has also favoured the selection of these variants in pathogenic strains infecting both species.

Microbiome-derived carnitine mimics as previously unknown mediators of gut-brain axis communication.

Alterations to the gut microbiome are associated with various neurological diseases, yet evidence of causality and identity of microbiome-derived compounds that mediate gut-brain axis interaction remain elusive. Here, we identify two previously unknown bacterial metabolites 3-methyl-4-(trimethylammonio)butanoate and 4-(trimethylammonio)pentanoate, structural analogs of carnitine that are present in both gut and brain of specific pathogen-free mice but absent in germ-free mice. We demonstrate that these compounds are produced by anaerobic commensal bacteria from the family Lachnospiraceae (Clostridiales) family, colocalize with carnitine in brain white matter, and inhibit carnitine-mediated fatty acid oxidation in a murine cell culture model of central nervous system white matter. This is the first description of direct molecular inter-kingdom exchange between gut prokaryotes and mammalian brain cells, leading to inhibition of brain cell function.

Propionic Acid Promotes the Virulent Phenotype of Crohn's Disease-Associated Adherent-Invasive Escherichia coli.

Propionic acid (PA) is a bacterium-derived intestinal antimicrobial and immune modulator used widely in food production and agriculture. Passage of Crohn's disease-associated adherent-invasive Escherichia coli (AIEC) through a murine model, in which intestinal PA levels are increased to mimic the human intestine, leads to the recovery of AIEC with significantly increased virulence. Similar phenotypic changes are observed outside the murine model when AIEC is grown in culture with PA as the sole carbon source; such PA exposure also results in AIEC that persists at 20-fold higher levels in vivo. RNA sequencing identifies an upregulation of genes involved in biofilm formation, stress response, metabolism, membrane integrity, and alternative carbon source utilization. PA exposure also increases virulence in a number of E. coli isolates from Crohn's disease patients. Removal of PA is sufficient to reverse these phenotypic changes. Our data indicate that exposure to PA results in AIEC resistance and increased virulence in its presence.

Author Correction: Increasing the bactofection capacity of a mammalian expression vector by removal of the f1 ori.

This Article was originally published under Nature Research's License to Publish, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the Article have been modified accordingly.

Inflammation associated ethanolamine facilitates infection by Crohn's disease-linked adherent-invasive Escherichia coli.

BACKGROUND: The predominance of specific bacteria such as adherent-invasive Escherichia coli (AIEC) within the Crohn's disease (CD) intestine remains poorly understood with little evidence uncovered to support a selective pressure underlying their presence. Intestinal ethanolamine is however readily accessible during periods of intestinal inflammation, and enables pathogens to outcompete the host microbiota under such circumstances. METHODS: Quantitative RT-PCR (qRT-PCR) to determine expression of genes central to ethanolamine metabolism; transmission electron microscopy to detect presence of bacterial microcompartments (MCPs); in vitro infections of both murine and human macrophage cell lines examining intracellular replication of the AIEC-type strain LF82 and clinical E. coli isolates in the presence of ethanolamine; determination of E. coli ethanolamine utilization (eut) operon transcription in faecal samples from healthy patients, patients with active CD and the same patients in remission following treatment. RESULTS: Growth on the intestinal short chain fatty acid propionic acid (PA) stimulates significantly increased transcription of the eut operon (fold change relative to glucose: >16.9; p-value <.01). Additionally ethanolamine was accessible to intra-macrophage AIEC and stimulated significant increases in growth intracellularly when it was added extracellularly at concentrations comparable to those in the human intestine. Finally, qRT-PCR indicated that expression of the E. coli eut operon was increased in children with active CD compared to healthy controls (fold change increase: >4.72; P < .02). After clinical remission post-exclusive enteral nutrition treatment, the same CD patients exhibited significantly reduced eut expression (Pre vs Post fold change decrease: >15.64; P < .01). INTERPRETATION: Our data indicates a role for ethanolamine metabolism in selecting for AIEC that are consistently overrepresented in the CD intestine. The increased E. coli metabolism of ethanolamine seen in the intestine during active CD, and its decrease during remission, indicates ethanolamine use may be a key factor in shaping the intestinal microbiome in CD patients, particularly during times of inflammation. FUND: This work was funded by Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/K008005/1 & BB/P003281/1 to DMW; by a Tenovus Scotland grant to MJO; by Glasgow Children's Hospital Charity, Nestle Health Sciences, Engineering and Physical Sciences Research Council (EPSRC) and Catherine McEwan Foundation grants awarded to KG; and by a Natural Environment Research Council (NERC) fellowship (NE/L011956/1) to UZI. The IBD team at the Royal Hospital for Children, Glasgow are supported by the Catherine McEwan Foundation and Yorkhill IBD fund. RKR and RH are supported by NHS Research Scotland Senior fellowship awards.

Comparative bioinformatic and proteomic approaches to evaluate the outer membrane proteome of the fish pathogen Yersinia ruckeri.

Yersinia ruckeri is the aetiological agent of enteric redmouth (ERM) disease and is responsible for significant economic losses in farmed salmonids. Enteric redmouth disease is associated primarily with rainbow trout (Oncorhynchus mykiss, Walbaum) but its incidence in Atlantic salmon (Salmo salar) is increasing. Outer membrane proteins (OMPs) of Gram-negative bacteria are located at the host-pathogen interface and play important roles in virulence. The outer membrane of Y. ruckeri is poorly characterised and little is known about its composition and the roles of individual OMPs in virulence. Here, we employed a bioinformatic pipeline to first predict the OMP composition of Y. ruckeri. Comparative proteomic approaches were subsequently used to identify those proteins expressed in vitro in eight representative isolates recovered from Atlantic salmon and rainbow trout. One hundred and forty-one OMPs were predicted from four Y. ruckeri genomes and 77 of these were identified in three or more genomes and were considered as "core" proteins. Gel-free and gel-based proteomic approaches together identified 65 OMPs in a single reference isolate and subsequent gel-free analysis identified 64 OMPs in the eight Atlantic salmon and rainbow trout isolates. Together, our gel-free and gel-based proteomic analyses identified 84 unique OMPs in Y. ruckeri. SIGNIFICANCE: Yersinia ruckeri is an important pathogen of Atlantic salmon and rainbow trout and is of major economic significance to the aquaculture industry worldwide. Disease outbreaks are becoming more problematic in Atlantic salmon and there is an urgent need to investigate in further detail the cell-surface (outer membrane) composition of strains infecting each of these host species. Currently, the outer membrane of Y. ruckeri is poorly characterised and very little is known about the OMP composition of strains infecting each of these salmonid species. This study represents the most comprehensive comparative outer membrane proteomic analysis of Y. ruckeri to date, encompassing isolates of different biotypes, serotypes, OMP-types and hosts of origin and provides insights into the potential roles of these diverse proteins in host-pathogen interactions. The study has identified key OMPs likely to be involved in disease pathogenesis and makes a significant contribution to furthering our understanding of the cell-surface composition of this important fish pathogen that will be relevant to the development of improved vaccines and therapeutics.

Increasing the bactofection capacity of a mammalian expression vector by removal of the f1 ori.

Bacterial-mediated cancer therapy has shown great promise in in vivo tumour models with increased survival rates post-bacterial treatment. Improving efficiency of bacterial-mediated tumour regression has focused on controlling and exacerbating bacterial cytotoxicity towards tumours. One mechanism that has been used to carry this out is the process of bactofection where post-invasion, bacteria deliver plasmid-borne mammalian genes into target cells for expression. Here we utilised the cancer-targeting Salmonella Typhimurium strain, SL7207, to carry out bactofection into triple negative breast cancer MDA-MB-231 cells. However, we noted that post-transformation with the commonly used mammalian expression vector pEGFP, S. Typhimurium became filamentous, attenuated and unable to invade target cells efficiently. Filamentation did not occur in Escherichia coli-transformed with the same plasmid. Further investigation identified the region inducing S. Typhimurium filamentation as being the f1 origin of replication (f1 ori), an artefact of historic use of mammalian plasmids for single stranded DNA production. Other f1 ori-containing plasmids also induced the attenuated phenotype, while removal of the f1 ori from pEGFP restored S. Typhimurium virulence and increased the bactofection capacity. This work has implications for interpretation of prior bactofection studies employing f1 ori-containing plasmids in S. Typhimurium, while also indicating that future use of S. Typhimurium in targeting tumours should avoid the use of these plasmids.

Correction: Increasing the bactofection capacity of a mammalian expression vector by removal of the f1 ori.

This Article was originally published with one of the panels in Figure 5A inserted twice (SL-pEGFP). In Figure 5B there was also a typo. SL-LacZ should have read SL-pEGFP(-f1). Both 5A and 5B are corrected in both the PDF and HTML versions of the Article.

Multilocus Variable-Number Tandem-Repeat Analysis of Yersinia ruckeri Confirms the Existence of Host Specificity, Geographic Endemism, and Anthropogenic Dissemination of Virulent Clones.

A multilocus variable-number tandem-repeat analysis (MLVA) assay was developed for epizootiological study of the internationally significant fish pathogen Yersinia ruckeri, which causes yersiniosis in salmonids. The assay involves amplification of 10 variable-number tandem-repeat (VNTR) loci in two five-plex PCRs, followed by capillary electrophoresis. A collection of 484 Y. ruckeri isolates, originating from various biological sources and collected from four continents over 7 decades, was analyzed. Minimum-spanning-tree cluster analysis of MLVA profiles separated the studied population into nine major clonal complexes and a number of minor clusters and singletons. The major clonal complexes could be associated with host species, geographic origin, and serotype. A single large clonal complex of serotype O1 isolates dominating the yersiniosis situation in international rainbow trout farming suggests anthropogenic spread of this clone, possibly related to transport of fish. Moreover, subclustering within this clonal complex indicates putative transmission routes and multiple biotype shift events. In contrast to the situation in rainbow trout, Y. ruckeri strains associated with disease in Atlantic salmon appear as more or less geographically isolated clonal complexes. A single complex of serotype O1 exclusive to Norway was found to be responsible for almost all major yersiniosis outbreaks in modern Norwegian salmon farming, and site-specific subclustering further indicates persistent colonization of freshwater farms in Norway. Identification of genetically diverse Y. ruckeri isolates from clinically healthy fish and environmental sources also suggests the widespread existence of less-virulent or avirulent strains.IMPORTANCE This comprehensive population study substantially improves our understanding of the epizootiological history and nature of an internationally important fish-pathogenic bacterium. The MLVA assay developed and presented represents a high-resolution typing tool particularly well suited for Yersinia ruckeri infection tracing, selection of strains for vaccine inclusion, and risk assessment. The ability of the assay to separate isolates into geographically linked and/or possibly host-specific clusters reflects its potential utility for maintenance of national biosecurity. The MLVA is internationally applicable and robust, and it provides clear, unambiguous, and easily interpreted results. Typing is reasonably inexpensive, with a moderate technological requirement, and may be completed from a harvested colony within a single working day. As the resulting MLVA profiles are readily portable, any Y. ruckeri strain may rapidly be placed in a global epizootiological context.

SipA Activation of Caspase-3 Is a Decisive Mediator of Host Cell Survival at Early Stages of Salmonella enterica Serovar Typhimurium Infection.

Salmonella invasion protein A (SipA) is a dual-function effector protein that plays roles in both actin polymerization and caspase-3 activation in intestinal epithelial cells. To date its function in other cell types has remained largely unknown despite its expression in multiple cell types and its extracellular secretion during infection. Here we show that in macrophages SipA induces increased caspase-3 activation early in infection. This activation required a threshold level of SipA linked to multiplicity of infection and may be a limiting factor controlling bacterial numbers in infected macrophages. In polymorphonuclear leukocytes, SipA or other Salmonella pathogenicity island 1 effectors had no effect on induction of caspase-3 activation either alone or in the presence of whole bacteria. Tagging of SipA with the small fluorescent phiLOV tag, which can pass through the type three secretion system, allowed visualization and quantification of caspase-3 activation by SipA-phiLOV in macrophages. Additionally, SipA-phiLOV activation of caspase-3 could be tracked in the intestine through multiphoton laser scanning microscopy in an ex vivo intestinal model. This allowed visualization of areas where the intestinal epithelium had been compromised and demonstrated the potential use of this fluorescent tag for in vivo tracking of individual effectors.