Salvatore Rubino

Epitope tagging of chromosomal genes in Salmonella

We have developed a simple and efficient procedure for adding an epitope-encoding tail to one or more genes of interest in the bacterial chromosome. The procedure is a modification of the gene replacement method of Datsenko and Wanner [Datsenko, K. A. & Wanner, B. L. (2000) Proc. Natl. Acad. Sci. USA 97, 6640–6645]. A DNA module that begins with the epitope-encoding sequence and includes a selectable marker is amplified by PCR with primers that carry extensions (as short as 36 nt) homologous to the last portion of the targeted gene and to a region downstream from it. Transformation of a strain expressing bacteriophage λ red functions yields recombinants carrying the targeted gene fused to the epitope-encoding sequence. The resulting C-terminal-tagged protein can be identified by standard immuno-detection techniques. In an initial application of the method, we have added the sequences encoding the FLAG and 3xFLAG and influenza virus hemagglutinin epitopes to various genes of Salmonella enterica serovar Typhimurium, including putative and established pathogenic determinants present in prophage genomes. Epitope fusion proteins were detected in bacteria growing in vitro, tissue culture cells, and infected mouse tissues. This work identified a prophage locus specifically expressed in bacteria growing intracellularly. The procedure described here should be applicable to a wide variety of Gram-negative bacteria and is particularly suited for the study of intracellular pathogens.

Host adapted serotypes of Salmonella enterica.

Salmonella constitutes a genus of zoonotic bacteria of worldwide economic and health importance. The current view of salmonella taxonomy assigns the members of this genus to two species: S. enterica and S. bongori. S. enterica itself is divided into six subspecies, enterica, salamae, arizonae, diarizonae, indica, and houtenae, also known as subspecies I, II, IIIa, IIIb, IV, and VI, respectively [1]. Members of Salmonella enterica subspecies enterica are mainly associated with warm-blooded vertebrates and are usually transmitted by ingestion of food or water contaminated by infected faeces. The pathogenicity of most of the distinct serotypes remains undefined, and even within the most common serotypes, many questions remain to be answered regarding the interactions between the organism and the infected host.Salmonellosis manifests itself in three major forms: enteritis, septicaemia, and abortion, each of which may be present singly or in combination, depending on both the serotype and the host involved. Although currently over 2300 serovars of Salmonella are recognized, only about 50 serotypes are isolated in any significant numbers as human or animal pathogens [2, 3] and they all belong to subspecies enterica. Of these, most cause acute gastroenteritis characterized by a short incubation period and a severe systemic disease in man or animals, characterized by septicaemia, fever and/or abortion, and such serotypes are often associated with one or few host species [4–6].It is the intention of this review to present a summary of current knowledge of these host-adapted serotypes of S. enterica. The taxonomic relationships between the serotypes will be discussed together with a comparison of the pathology and pathogenesis of the disease that they cause in their natural host(s). Since much of our knowledge on salmonellosis is based on the results of work on Typhimurium, this serotype will often be used as the baseline in discussion. It is hoped that an appreciation of the differences that exist in the way these serotypes interact with the host will lead to a greater understanding of the complex host–parasite relationship that characterizes salmonella infections.

MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island

Short-read, high-throughput sequencing technology cannot identify the chromosomal position of repetitive insertion sequences that typically flank horizontally acquired genes such as bacterial virulence genes and antibiotic resistance genes. The MinION nanopore sequencer can produce long sequencing reads on a device similar in size to a USB memory stick. Here we apply a MinION sequencer to resolve the structure and chromosomal insertion site of a composite antibiotic resistance island in Salmonella Typhi Haplotype 58. Nanopore sequencing data from a single 18-h run was used to create a scaffold for an assembly generated from short-read Illumina data. Our results demonstrate the potential of the MinION device in clinical laboratories to fully characterize the epidemic spread of bacterial pathogens.

The Vi Capsular Antigen of Salmonella enterica Serotype Typhi Reduces Toll-Like Receptor-Dependent Interleukin-8 Expression in the Intestinal Mucosa

ABSTRACT Human infections with nontyphoidal Salmonella serotypes, such as S. enterica serotype Typhimurium, are characterized by a massive neutrophil influx in the colon and terminal ileum. In contrast, neutrophils are scarce in intestinal infiltrates of typhoid fever patients. Here, we show that in S. enterica serotype Typhi, the causative agent of typhoid fever, expression of the Vi capsular antigen reduced expression of the neutrophil chemoattractant interleukin-8 (IL-8) in host cells. Capsulated bacteria elicited IL-8 expression in polarized human epithelial cells (T84) and human macrophage-like cells (THP-1) in vitro at significantly reduced levels compared to noncapsulated bacteria. Experiments with a human cell line (HEK293) transfected with human Toll-like receptors (TLRs) demonstrated that in the presence of TLR5 or TLR4/MD2/CD14, a noncapsulated serotype Typhi mutant was able to induce the expression of IL-8, while this host response was significantly reduced when cells were infected with the capsulated serotype Typhi wild type. The relevance of these in vitro observations for the interaction of serotype Typhi with its human host was further studied ex vivo using human colonic tissue explants. Expression of IL-8 was detected in human colonic tissue explants infected with serotype Typhimurium or a noncapsulated serotype Typhi mutant. In contrast, infection with the serotype Typhi wild type did not elicit IL-8 expression in colonic tissue explants. Collectively, these data suggest that the scarcity of neutrophils in intestinal infiltrates of typhoid fever patients is due to a capsule-mediated reduction of TLR-dependent IL-8 production in the intestinal mucosa.

Inflammatory Cytokine Expression Is Associated with Chikungunya Virus Resolution and Symptom Severity

The Chikungunya virus infection zones have now quickly spread from Africa to parts of Asia, North America and Europe. Originally thought to trigger a disease of only mild symptoms, recently Chikungunya virus caused large-scale fatalities and widespread economic loss that was linked to recent virus genetic mutation and evolution. Due to the paucity of information on Chikungunya immunological progression, we investigated the serum levels of 13 cytokines/chemokines during the acute phase of Chikungunya disease and 6- and 12-month post-infection follow-up from patients of the Italian outbreak. We found that CXCL9/MIG, CCL2/MCP-1, IL-6 and CXCL10/IP-10 were significantly raised in the acute phase compared to follow-up samples. Furthermore, IL-1β, TNF-α, Il-12, IL-10, IFN-γ and IL-5 had low initial acute phase levels that significantly increased at later time points. Analysis of symptom severity showed association with CXCL9/MIG, CXCL10/IP-10 and IgG levels. These data give insight into Chikungunya disease establishment and subsequent convalescence, which is imperative to the treatment and containment of this quickly evolving and frequently re-emerging disease.

Interleukin-6 Is a Potential Biomarker for Severe Pandemic H1N1 Influenza A Infection

Pandemic H1N1 influenza A (H1N1pdm) is currently a dominant circulating influenza strain worldwide. Severe cases of H1N1pdm infection are characterized by prolonged activation of the immune response, yet the specific role of inflammatory mediators in disease is poorly understood. The inflammatory cytokine IL-6 has been implicated in both seasonal and severe pandemic H1N1 influenza A (H1N1pdm) infection. Here, we investigated the role of IL-6 in severe H1N1pdm infection. We found IL-6 to be an important feature of the host response in both humans and mice infected with H1N1pdm. Elevated levels of IL-6 were associated with severe disease in patients hospitalized with H1N1pdm infection. Notably, serum IL-6 levels associated strongly with the requirement of critical care admission and were predictive of fatal outcome. In C57BL/6J, BALB/cJ, and B6129SF2/J mice, infection with A/Mexico/4108/2009 (H1N1pdm) consistently triggered severe disease and increased IL-6 levels in both lung and serum. Furthermore, in our lethal C57BL/6J mouse model of H1N1pdm infection, global gene expression analysis indicated a pronounced IL-6 associated inflammatory response. Subsequently, we examined disease and outcome in IL-6 deficient mice infected with H1N1pdm. No significant differences in survival, weight loss, viral load, or pathology were observed between IL-6 deficient and wild-type mice following infection. Taken together, our findings suggest IL-6 may be a potential disease severity biomarker, but may not be a suitable therapeutic target in cases of severe H1N1pdm infection due to our mouse data.

Salmonella enterica Serovar-Host Specificity Does Not Correlate with the Magnitude of Intestinal Invasion in Sheep

ABSTRACT The colonization of intestinal and systemic tissues bySalmonella enterica serovars with different host specificities was determined 7 days after inoculation of 1 to 2-month-old lambs. Following oral inoculation, S. entericaserovars Abortusovis, Dublin, and Gallinarum were recovered in comparable numbers from the intestinal mucosa, but serovar Gallinarum was recovered in lower numbers than the other serovars from systemic sites. The pattern of bacterial recovery from systemic sites following intravenous inoculation was similar. The magnitude of intestinal invasion was evaluated in ovine ligated ileal loops in vivo. Serovars Dublin and Gallinarum and the broad-host-range Salmonellaserovar Typhimurium were recovered in comparable numbers from ileal mucosa 3 h after loop inoculation, whereas the recovery of serovar Abortusovis was approximately 10-fold lower. Microscopic analysis of intestinal mucosae infected with serovars Typhimurium and Dublin showed dramatic morphological changes and infiltration of inflammatory cells, whereas mucosae infected with serovars Abortusovis and Gallinarum were indistinguishable from uninfected mucosae. Together these data suggest that Salmonella serovar specificity in sheep correlates with bacterial persistence at systemic sites. Intestinal invasion and avoidance of the hosts intestinal inflammatory response may contribute to but do not determine the specificity of serovar Abortosovis for sheep. Intestinal invasion by serovar Abortusovis was significantly reduced after mutation of invH but was not reduced following curing of the virulence plasmid, suggesting that theSalmonella pathogenicity island 1 influences but the virulence plasmid genes do not influence the ability of serovar Abortusovis to invade the intestinal mucosa in sheep.

Effects of crp deletion in Salmonella enterica serotype Gallinarum

BackgroundSalmonella enterica serotype Gallinarum (S. Gallinarum) remains an important pathogen of poultry, especially in developing countries. There is a need to develop effective and safe vaccines. In the current study, the effect of crp deletion was investigated with respect to virulence and biochemical properties and the possible use of a deletion mutant as vaccine candidate was preliminarily tested.MethodsMutants were constructed in S. Gallinarum by P22 transduction from Salmonella Typhimurium (S. Typhimurium) with deletion of the crp gene. The effect was characterized by measuring biochemical properties and by testing of invasion in a chicken loop model and by challenge of six-day-old chickens. Further, birds were immunized with the deleted strain and challenged with the wild type isolate.ResultsThe crp deletions caused complete attenuation of S. Gallinarum. This was shown by ileal loop experiments not to be due to significantly reduced invasion. Strains with such deletions may have vaccine potential, since oral inoculatoin with S. Gallinarum Δcrp completely protected against challenge with the same dose of wild type S. Gallinarum ten days post immunization. Interestingly, the mutations did not cause the same biochemical and growth changes to the two biotypes of S. Gallinarum. All biochemical effects but not virulence could be complemented by providing an intact crp- gene from S. Typhimurium on the plasmid pSD110.ConclusionTransduction of a Tn10 disrupted crp gene from S. Typhimurium caused attenuation in S. Gallinarum and mutated strains are possible candidates for live vaccines against fowl typhoid.

Lethal and sublethal effects of Brevibacillus laterosporus on the housefly ( Musca domestica )

Sporulated cultures of an isolate of the entomopathogenic bacterium Brevibacillus laterosporus (Laubach) from soil in Sardinia (Italy) were highly toxic by ingestion to both adults and juveniles of the housefly, Musca domestica L. (Diptera: Muscidae). Toxicity was associated with spores as the culture supernatant was not toxic and no parasporal inclusions were observed. The level of toxicity varied with the concentration of spores, the time of exposure to the treated diet, and the stage of insect development. Comparative LC50 values were 0.72 × 108 and 1.75 × 108 spores per gram of diet for 1st and 2nd instar larvae, respectively, and 3.84 × 108 spores per gram for adults. A significant increase in larval development time and reductions in pupal weight and emergence rate were observed when larvae were fed on diets containing sublethal concentrations of spores. Adults surviving after feeding on a sublethal diet showed a significant reduction in fecundity and longevity. Similarly, adults from treated larvae exhibited a lower fecundity, although their longevity was not influenced. A relationship between pupal weight and adult fecundity was found. Pupal stage duration and egg eclosion were not significantly affected.

Using next generation sequencing to tackle non-typhoidal Salmonella infections.

The publication of studies using next generation sequencing to analyse large numbers of bacterial isolates from global epidemics is transforming microbiology, epidemiology and public health. The emergence of multidrug resistant Salmonella Typhimurium ST313 is one example. While the epidemiology in Africa appears to be human-to-human spread and the association with invasive disease almost absolute, more needs to be done to exclude the possibility of animal reservoirs and to transfer the ability to track all Salmonella infections to the laboratories in the front line. In this mini-review we summarise what is currently known about non-typhoidal Salmonella in sub-Saharan Africa and discuss some of the issues which remain.