Poultry Body Temperature Contributes to Invasion Control through Reduced Expression of Salmonella Pathogenicity Island 1 Genes in Salmonella enterica Serovars Typhimurium and Enteritidis

Salmonella enterica serovars Typhimurium (S. Typhimurium) and Enteritidis (S. Enteritidis) are foodborne pathogens, and outbreaks are often associated with poultry products. Chickens are typically asymptomatic when colonized by these serovars; however, the factors contributing to this observation are uncharacterized. Whereas symptomatic mammals have a body temperature between 37°C and 39°C, chickens have a body temperature of 41°C to 42°C. Here, in vivo experiments using chicks demonstrated that numbers of viable S. Typhimurium or S. Enteritidis bacteria within the liver and spleen organ sites were ≥4 orders of magnitude lower than those within the ceca. When similar doses of S. Typhimurium or S. Enteritidis were given to C3H/HeN mice, the ratio of the intestinal concentration to the liver/spleen concentration was 1:1. In the avian host, this suggested poor survival within these tissues or a reduced capacity to traverse the host epithelial layer and reach liver/spleen sites or both. Salmonella pathogenicity island 1 (SPI-1) promotes localization to liver/spleen tissues through invasion of the epithelial cell layer. Following in vitro growth at 42°C, SPI-1 genes sipC, invF, and hilA and the SPI-1 rtsA activator were downregulated compared to expression at 37°C. Overexpression of the hilA activators fur, fliZ, and hilD was capable of inducing hilA-lacZ at 37°C but not at 42°C despite the presence of similar levels of protein at the two temperatures. In contrast, overexpression of either hilC or rtsA was capable of inducing hilA and sipC at 42°C. These data indicate that physiological parameters of the poultry host, such as body temperature, have a role in modulating expression of virulence.     

A plasmid locus associated with Klebsiella clinical infections encodes a microbiome-dependent gut fitness factor

Klebsiella pneumoniae (Kp) is an important cause of healthcare-associated infections, which increases patient morbidity, mortality, and hospitalization costs. Gut colonization by Kp is consistently associated with subsequent Kp disease, and patients are predominantly infected with their colonizing strain. Our previous comparative genomics study, between disease-causing and asymptomatically colonizing Kp isolates, identified a plasmid-encoded tellurite (TeO₃^-2)-resistance (ter) operon as strongly associated with infection. However, TeO₃^-2 is extremely rare and toxic to humans. Thus, we used a multidisciplinary approach to determine the biological link between ter and Kp infection. First, we used a genomic and bioinformatic approach to extensively characterize Kp plasmids encoding the ter locus. These plasmids displayed substantial variation in plasmid incompatibility type and gene content. Moreover, the ter operon was genetically independent of other plasmid-encoded virulence and antibiotic resistance loci, both in our original patient cohort and in a large set (n = 88) of publicly available ter operon-encoding Kp plasmids, indicating that the ter operon is likely playing a direct, but yet undescribed role in Kp disease. Next, we employed multiple mouse models of infection and colonization to show that 1) the ter operon is dispensable during bacteremia, 2) the ter operon enhances fitness in the gut, 3) this phenotype is dependent on the colony of origin of mice, and 4) antibiotic disruption of the gut microbiota eliminates the requirement for ter. Furthermore, using 16S rRNA gene sequencing, we show that the ter operon enhances Kp fitness in the gut in the presence of specific indigenous microbiota, including those predicted to produce short chain fatty acids. Finally, administration of exogenous short-chain fatty acids in our mouse model of colonization was sufficient to reduce fitness of a ter mutant. These findings indicate that the ter operon, strongly associated with human infection, encodes factors that resist stress induced by the indigenous gut microbiota during colonization. This work represents a substantial advancement in our molecular understanding of Kp pathogenesis and gut colonization, directly relevant to Kp disease in healthcare settings.     

Seasonality affects leaf nutrient and condensed tannin concentration in southern African savannah browse

Trees that maintain some leaves throughout dry seasons become important ruminant browse depending on nutritive and antinutritive values. Leaves from seven tree species that maintained some leaves during the dry season were collected during dry and wet seasons and analysed for nutritive and antinutritive values. Neutral detergent fibre of leaves was either not different or less (P ≤ 0.05) during the dry season as compared to the wet season depending on species. Acid detergent fibre was either not different or greater (P ≤ 0.05) during the dry season as compared to the wet season. Crude protein and condensed tannins (CT) were either not different or less (P ≤ 0.05) during the dry season than during the wet season for the seven species. The biological activity (protein‐binding ability; PB) of the CT was highly species specific and was either not different between seasons, more bioactive during the wet season, or more bioactive during the dry season depending on the species. Based on combinations of low fibre, high protein and potentially beneficial levels of bioactive CT, Senegalia caffra, Vachellia karoo and Searsia lancea may be the most promising dry‐season browse of the species studied.     

Kinase domain mutants of Bcr-Abl exhibit altered transformation potency, kinase activity, and substrate utilization, irrespective of sensitivity to imatinib

Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired imatinib resistance in patients with Philadelphia chromosome-positive leukemia. Mutations of the ATP binding loop (p-loop) have been associated with a poor prognosis. We compared the transformation potency of five common KD mutants in various biological assays. Relative to unmutated (native) Bcr-Abl, the ATP binding loop mutants Y253F and E255K exhibited increased transformation potency, M351T and H396P were less potent, and the performance of T315I was assay dependent. The transformation potency of Y253F and M351T correlated with intrinsic Bcr-Abl kinase activity, whereas the kinase activity of E255K, H396P, and T315I did not correlate with transforming capabilities, suggesting that additional factors influence transformation potency. Analysis of the phosphotyrosine proteome by mass spectroscopy showed differential phosphorylation among the mutants, a finding consistent with altered substrate specificity and pathway activation. Mutations in the KD of Bcr-Abl influence kinase activity and signaling in a complex fashion, leading to gain- or loss-of-function variants. The drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors.     

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of Crohn’s Disease - Crohn’s Colitis. Acute colitis may present with abdominal pain and distention, malabsorption, diarrhea, hematochezia and mucus in the stool. We are beginning to understand the complex interactions between the environment, genetics, and epithelial barrier dysfunction in Inflammatory Bowel Disease and animal models of colitis have been essential in advancing our understanding of this disease. One popular model involves supplementing the drinking water of mice with low-molecular weight Dextran Sodium Sulfate (DSS), resulting in epithelial damage and a robust inflammatory response in the colon lasting several days ¹.Variations of this approach can be used to model acute injury, acute injury followed by repair, and repeated cycles of DSS interspersed with recovery modeling chronic inflammatory diseases ². After a single four-day treatment of 3% DSS in drinking water, mice show signs of acute colitis including weight loss, bloody stools, and diarrhea. Mice are euthanized at the conclusion of the treatment course and at necropsy dissected colons are processed and can be ''Swiss rolled" ³ to allow microscopic analysis of the entire colon or infused with formalin as "sausages" to allow macroscopic analysis. Tissue is then embedded in paraffin, sectioned, and stained for histologic review.Open in a separate windowClick here to view.^{(49M, flv)}     

Aspergillomarasmine A inhibits metallo-β-lactamases by selectively sequestering Zn2+

Class B metallo-β-lactamases (MBLs) are Zn²⁺-dependent enzymes that catalyze the hydrolysis of β-lactam antibiotics to confer resistance in bacteria. Several problematic groups of MBLs belong to subclass B1, including the binuclear New Delhi MBL (NDM), Verona integrin-encoded MBL, and imipenemase-type enzymes, which are responsible for widespread antibiotic resistance. Aspergillomarasmine A (AMA) is a natural aminopolycarboxylic acid that functions as an effective inhibitor of class B1 MBLs. The precise mechanism of action of AMA is not thoroughly understood, but it is known to inactivate MBLs by removing one catalytic Zn²⁺ cofactor. We investigated the kinetics of MBL inactivation in detail and report that AMA is a selective Zn²⁺ scavenger that indirectly inactivates NDM-1 by encouraging the dissociation of a metal cofactor. To further investigate the mechanism in living bacteria, we used an active site probe and showed that AMA causes the loss of a Zn²⁺ ion from a low-affinity binding site of NDM-1. Zn²⁺-depleted NDM-1 is rapidly degraded, contributing to the efficacy of AMA as a β-lactam potentiator. However, MBLs with higher metal affinity and stability such as NDM-6 and imipenemase-7 exhibit greater tolerance to AMA. These results indicate that the mechanism of AMA is broadly applicable to diverse Zn²⁺ chelators and highlight that leveraging Zn²⁺ availability can influence the survival of MBL-producing bacteria when they are exposed to β-lactam antibiotics.     

Total cost estimation for implementing genome-enabled selection in a multi-level swine production system

Background

Determining an animal’s genetic merit using genomic information can improve estimated breeding value (EBV) accuracy; however, the magnitude of the accuracy improvement must be large enough to recover the costs associated with implementing genome-enabled selection. One way to reduce costs is to genotype nucleus herd selection candidates using a low-density chip and to use high-density chip genotyping for animals that are used as parents in the nucleus breeding herd. The objective of this study was to develop a tool to estimate the cost structure associated with incorporating genome-enabled selection into multi-level commercial breeding programs.

Results

For the purpose of this deterministic study, it was assumed that a commercial pig is created from a terminal line sire and a dam that is a cross between two maternal lines. It was also assumed that all male and female selection candidates from the 1000 sow maternal line nucleus herds were genotyped at low density and all animals used for breeding at high density. With the assumptions used in this analysis, it was estimated that genome-enabled selection costs for a maternal line would be approximately US$0.082 per weaned pig in the commercial production system. A total of US$0.164 per weaned pig is needed to incorporate genome-enabled selection into the two maternal lines. Similarly, for a 600 sow terminal line nucleus herd and genotyping only male selection candidates with the low-density panel, the cost per weaned pig in the commercial herd was estimated to be US$0.044. This means that US$0.21 per weaned pig produced at the commercial level and sired by boars obtained from the nucleus herd breeding program needs to be added to the genetic merit value in order to break even on the additional cost required when genome-enabled selection is used in both maternal lines and the terminal line.

Conclusions

By modifying the input values, such as herd size and genotyping strategy, a flexible spreadsheet tool developed from this work can be used to estimate the additional costs associated with genome-enabled selection. This tool will aid breeders in estimating the economic viability of incorporating genome-enabled selection into their specific breeding program.