CpGB DNA activates dermal macrophages and specifically recruits inflammatory monocytes into the skin.

Toll-like receptor 9 (TLR9) drives innate immune responses after recognition of foreign or endogenous DNA containing unmethylated CpG motifs. DNA-mediated TLR9 activation is highly implicated in the pathogenesis of several autoimmune skin diseases, yet its contribution to the inflammation seen in these diseases remains unclear. In this study, TLR9 ligand, CpGB DNA, was administered to mice via a subcutaneous osmotic pump with treatment lasting 1 or 4 weeks. Gene expression and immunofluorescence analyses were used to determine chemokine expression and cell recruitment in the skin surrounding the pump outlet. CpGB DNA skin treatment dramatically induced a marked influx of CD11b+ F4/80+ macrophages, increasing over 4 weeks of treatment, and induction of IFNγ and TNFα expression. Chemokines, CCL2, CCL4, CCL5, CXCL9 and CXCL10, were highly induced in CpGB DNA-treated skin, although abrogation of these signalling pathways individually did not alter macrophage accumulation. Flow cytometry analysis showed that TLR9 activation in the skin increased circulating CD11b+ CD115+ Ly6C(hi) inflammatory monocytes following 1 week of CpGB DNA treatment. Additionally, skin-resident CD11b+ cells were found to initially take up subcutaneous CpGB DNA and propagate the subsequent immune response. Using diphtheria toxin-induced monocyte depletion mouse model, gene expression analysis demonstrated that CD11b+ cells are responsible for the CpGB DNA-induced cytokine and chemokine response. Overall, these data demonstrate that chronic TLR9 activation induces a specific inflammatory response, ultimately leading to a striking and selective accumulation of macrophages in the skin.

Antibiotic Resistance in Salmonella enterica Serovar Typhimurium Associates with CRISPR Sequence Type.

Salmonella enterica subsp. enterica serovar Typhimurium is a leading cause of food-borne salmonellosis in the United States. The number of antibiotic-resistant isolates identified in humans is steadily increasing, suggesting that the spread of antibiotic-resistant strains is a major threat to public health. S Typhimurium is commonly identified in a wide range of animal hosts, food sources, and environments, but little is known about the factors mediating the spread of antibiotic resistance in this ecologically complex serovar. Previously, we developed a subtyping method, CRISPR-multi-virulence-locus sequence typing (MVLST), which discriminates among strains of several common S. enterica serovars. Here, CRISPR-MVLST identified 22 sequence types within a collection of 76 S Typhimurium isolates from a variety of animal sources throughout central Pennsylvania. Six of the sequence types were identified in more than one isolate, and we observed statistically significant differences in resistance among these sequence types to 7 antibiotics commonly used in veterinary and human medicine, such as ceftiofur and ampicillin (P < 0.05). Importantly, five of these sequence types were subsequently identified in human clinical isolates, and a subset of these isolates had identical antibiotic resistance patterns, suggesting that these subpopulations are being transmitted through the food system. Therefore, CRISPR-MVLST is a promising subtyping method for monitoring the farm-to-fork spread of antibiotic resistance in S Typhimurium.

CRISPR-MVLST subtyping of Salmonella enterica subsp. enterica serovars Typhimurium and Heidelberg and application in identifying outbreak isolates.

BACKGROUND: Salmonella enterica subsp. enterica serovars Typhimurium (S. Typhimurium) and Heidelberg (S. Heidelberg) are major causes of foodborne salmonellosis, accounting for a fifth of all annual salmonellosis cases in the United States. Rapid, efficient and accurate methods for identification are required for routine surveillance and to track specific strains during outbreaks. We used Pulsed-field Gel Electrophoresis (PFGE) and a recently developed molecular subtyping approach termed CRISPR-MVLST that exploits the hypervariable nature of virulence genes and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) to subtype clinical S. Typhimurium and S. Heidelberg isolates. RESULTS: We analyzed a broad set of 175 S. Heidelberg and S. Typhimurium isolates collected over a five-year period. We identified 21 Heidelberg Sequence Types (HSTs) and 37 Typhimurium STs (TSTs) that were represented by 27 and 45 PFGE pulsotypes, respectively, and determined the discriminatory power of each method. CONCLUSIONS: For S. Heidelberg, our data shows that combined typing by both CRISPR-MVLST and PFGE provided a discriminatory power of 0.9213. Importantly, CRISPR-MVLST was able to separate common PFGE patterns such as JF6X01.0022 into distinct STs, thus providing significantly greater discriminatory power. Conversely, we show that subtyping by either CRISPR-MVLST or PFGE independently provides a sufficient discriminatory power (0.9345 and 0.9456, respectively) for S. Typhimurium. Additionally, using isolates from two S. Typhimurium outbreaks, we demonstrate that CRISPR-MVLST provides excellent epidemiologic concordance.

The combination of CRISPR-MVLST and PFGE provides increased discriminatory power for differentiating human clinical isolates of Salmonella enterica subsp. enterica serovar Enteritidis.

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is a major cause of foodborne salmonellosis. Rapid, efficient and accurate methods for identification are required to track specific strains of S. Enteritidis during outbreaks of human salmonellosis. By exploiting the hypervariable nature of virulence genes and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), we previously developed a powerful sequence-based subtyping approach, designated CRISPR-MVLST. To substantiate the applicability of CRISPR-MVLST, we analyzed a broad set of S. Enteritidis isolates collected over a six-year period. Among 141 isolates we defined 22 Enteritidis Sequence Types (ESTs), the majority of which were novel. Notably, strains exhibiting the common PFGE pattern, JEGX01.0004 (characteristic of ∼40% of S. Enteritidis isolates in the United States), were separated into twelve distinct sequence types. Conversely, isolates of EST4, the most predominant EST we observed, comprised eight different PFGE patterns. Importantly, we showed that some genotypes that were previously associated with the food supply chain at the farm level have now been identified in clinical samples. CRISPR sequence data shows subtle but distinct differences among different alleles of S. Enteritidis, suggesting that evolution of these loci occurs vertically, as opposed to previously reported evolution by spacer acquisition in other bacteria.

Identification of a mouse Lactobacillus johnsonii strain with deconjugase activity against the FXR antagonist T-ß-MCA.

Bile salt hydrolase (BSH) activity against the bile acid tauro-beta-muricholic acid (T-ß-MCA) was recently reported to mediate host bile acid, glucose, and lipid homeostasis via the farnesoid X receptor (FXR) signaling pathway. An earlier study correlated decreased Lactobacillus abundance in the cecum with increased concentrations of intestinal T-ß-MCA, an FXR antagonist. While several studies have characterized BSHs in lactobacilli, deconjugation of T-ß-MCA remains poorly characterized among members of this genus, and therefore it was unclear what strain(s) were responsible for this activity. Here, a strain of L. johnsonii with robust BSH activity against T-ß-MCA in vitro was isolated from the cecum of a C57BL/6J mouse. A screening assay performed on a collection of 14 Lactobacillus strains from nine different species identified BSH substrate specificity for T-ß-MCA only in two of three L. johnsonii strains. Genomic analysis of the two strains with this BSH activity revealed the presence of three bsh genes that are homologous to bsh genes in the previously sequenced human-associated strain L. johnsonii NCC533. Heterologous expression of several bsh genes in E. coli followed by enzymatic assays revealed broad differences in substrate specificity even among closely related bsh homologs, and suggests that the phylogeny of these enzymes does not closely correlate with substrate specificity. Predictive modeling allowed us to propose a potential mechanism driving differences in BSH activity for T-ß-MCA in these homologs. Our data suggests that L. johnsonii regulates T-ß-MCA levels in the mouse intestinal environment, and that this species may play a central role in FXR signaling in the mouse.

Prevalence and Phylogenetic Characterization of Escherichia coli and Hygiene Indicator Bacteria Isolated from Leafy Green Produce, Beef, and Pork Obtained from Farmers' Markets in Pennsylvania.

The popularity of farmers' markets in the United States has led to over 8,400 farmers' markets being in operation in 2015. As farmers' markets have increased in size and complexity in the kinds of foods sold at these venues, so have the potential food safety risks. Since 2008, seven major foodborne illness outbreaks and two recalls associated with food products from farmers' markets have occurred, causing 80 known reported illnesses and one death. Various researchers also have observed vendors performing high-risk food safety retail behaviors, and others have identified microbiological hazards in foods sold at farmers' markets. In this study, the presence of hygiene indicators (coliforms, fecal coliforms, Listeria spp., and Escherichia coli ) was assessed in select samples of leafy green produce and meat obtained from farmers' markets in Pennsylvania. E. coli isolates were further characterized by phylogenetic profile and virulence potential. E. coli was present in 40% (20 of 50) and 18% (9 of 50) of beef and pork samples, respectively, and in 28% (15 of 54), 29% (15 of 52), and 17% (8 of 46) of kale, lettuce, and spinach samples, respectively. Listeria spp. was found in 8% (4 of 50) of beef samples, 2% (1 of 54) of kale samples, 4% (2 of 52) of lettuce samples, and 7% (3 of 46) of spinach samples. Among the 10 Listeria spp. isolates, 3 were identified as L. monocytogenes . E. coli isolated from meat samples mainly clustered into phylogroup B1 (66%; 19 of 29), whereas produce isolates clustered into phylogroups B2 (36%; 14 of 39) and B1 (33%; 13 of 39). These E. coli isolates possessed the fimH, iroN, hlyD, and eae genes associated with extraintestinal pathogenic E. coli and Shiga toxin-producing E. coli . The high prevalence but low levels of E. coli and Listeria spp. found on both produce and meat products obtained from farmers' markets in this study strongly indicate that farmers' market vendors would benefit greatly from food safety training and increased public health oversight.

Modeling innate immunity in murine skin: utilization of subcutaneous osmotic pumps for inflammatory and fibrotic skin disease.

Current models for systemic sclerosis have many limitations for mimicking human disease. We have recently described a novel model based on chronic stimulation of skin by poly(I:C), a form of double-stranded RNA known to stimulate innate immune responses. This model shows inflammation and extracellular matrix deposition, and upregulated expression of genes associated with fibrosis and vascular injury. We detail here the methodology for this model, utilizing osmotic pumps to deliver innate immune stimuli to subcutaneous tissue. Subcutaneous osmotic pumps provide a flexible system for studying innate immunity in the skin as well as the affects of other ligands on skin inflammation and fibrosis.

Antagonistic effect of the matricellular signaling protein CCN3 on TGF-beta- and Wnt-mediated fibrillinogenesis in systemic sclerosis and Marfan syndrome.

Abnormal fibrillinogenesis is associated with connective tissue disorders (CTDs), including Marfan syndrome (MFS), systemic sclerosis (SSc) and Tight-skin (Tsk) mice. We have previously shown that TGF-beta and Wnt stimulate fibrillin-1 assembly and that fibrillin-1 and the developmental regulator CCN3 are both highly increased in Tsk skin. We investigated the role of CCN3 in abnormal fibrillinogenesis in Tsk mice, MFS, and SSc. Smad3 deletion in Tsk mice decreased CCN3 overexpression, suggesting that TGF-beta mediates at least part of the effect of Tsk fibrillin on CCN3 which is consistent with a synergistic effect of TGF-beta and Wnt in vitro on CCN3 expression. Disruption of fibrillin-1 assembly by MFS fibrillin decreased CCN3 expression and skin from patients with early diffuse SSc showed a strong correlation between increased CCN3 and fibrillin-1 expression, suggesting that CCN3 regulation by fibrillin-1 extends to these CTDs. Diffuse SSc skin and sera also showed evidence of increased Wnt activity, implicating a Wnt stimulus behind this correlation. CCN3 overexpression markedly repressed fibrillin-1 assembly and also blocked other TGFbeta- and Wnt-regulated profibrotic gene expression. Together, these data indicate that CCN3 counter-regulates positive signals from TGF-beta and Wnt for fibrillin fibrillogenesis and profibrotic gene expression.