Thermal inactivation kinetics of Salmonella and Campylobacter in chicken livers.

Salmonella and Campylobacter are major foodborne pathogens that cause outbreaks associated with contaminated chicken liver. Proper cooking is necessary to avoid the risk of illness to consumers. This study tested the thermal inactivation of a 4-strain Salmonella cocktail and a 3-strain Campylobacter cocktail in chicken livers separately at temperatures ranging from 55.0 to 62.5°C. Inoculated livers were sealed in aluminum cells and immersed in a water bath. The decimal reduction time (D-values) of Salmonella in chicken livers were 9.01, 2.36, 0.82, and 0.23 min at 55.0, 57.5, 60.0, and 62.5°C, respectively. The D-values of Campylobacter ranged from 2.22 min at 55.0°C to 0.19 min at 60.0°C. Salmonella and Campylobacter had similar z-values in chicken livers of 4.8 and 4.6°C, respectively. Chicken livers can be heated to internal temperatures of 70.0 to 73.9°C for at least 1.6 to 0.2 s to achieve a 7-log reduction of Salmonella. Validation tests demonstrated that heating chicken livers to internal temperatures of 70.0 to 73.9°C for 2 to 0 s resulted in a reduction of Salmonella exceeding 7 logs. Collectively, these data show that Salmonella exhibits higher heat resistance than Campylobacter in chicken livers. Therefore, Salmonella could be considered as the target pathogen when designing thermal treatments or cooking instructions for liver products. These findings will aid in designing effective thermal processing for both industrial and home cooking to eliminate Salmonella and Campylobacter, ensuring consumer safety when consuming chicken liver products.

Discovery of novel treponemes associated with pododermatitis in elk (Cervus canadensis).

Pododermatitis, also known as treponeme-associated hoof disease (TAHD), presents a significant challenge to elk (Cervus canadensis) populations in the northwestern USA, with Treponema spp. consistently implicated in the lesion development. However, identifying species-specific Treponema strains from these lesions is hindered by its culture recalcitrance and limited genomic information. This study utilized shotgun sequencing, in silico genome reconstruction, and comparative genomics as a culture-independent approach to identify metagenome-assembled Treponema genomes (MATGs) from skin scraping samples collected from captive elk experimentally challenged with TAHD. The genomic analysis revealed 10 new MATGs, with 6 representing novel genomospecies associated with pododermatitis in elk and 4 corresponding to previously identified species-Treponema pedis and Treponema phagedenis. Importantly, genomic signatures of novel genomospecies identified in this study were consistently detected in biopsy samples of free-ranging elk diagnosed with TAHD, indicating a potential etiologic association. Comparative metabolic profiling of the MATGs against other Treponema genomes showed a distinct metabolic profile, suggesting potential host adaptation or geographic uniqueness of these newly identified genomospecies. The discovery of novel Treponema genomospecies enhances our understanding of the pathogenesis of pododermatitis and lays the foundation for the development of improved molecular surveillance tools to monitor and manage the disease in free-ranging elk.IMPORTANCETreponema spp. play an important role in the development of pododermatitis in free-ranging elk; however, the species-specific detection of Treponema from pododermatitis lesions is challenging due to culture recalcitrance and limited genomic information. The study utilized shotgun sequencing and in silico genome reconstruction to identify novel Treponema genomospecies from elk with pododermatitis. The discovery of the novel Treponema species opens new avenues to develop molecular diagnostic and epidemiologic tools for the surveillance of pododermatitis in elk. These findings significantly enhance our understanding of the genomic landscape of the Treponemataceae consortium while offering valuable insights into the etiology and pathogenesis of emerging pododermatitis in elk populations.

Transmission and lesion progression of treponeme-associated hoof disease in captive elk (Cervus canadensis).

Treponeme-associated hoof disease (TAHD) is a debilitating disease of free-ranging elk (Cervus canadensis) in the northwestern U.S. While treponemes are associated with lesions, the etiology and transmissibility between elk are unknown. Our objective was to determine whether the disease can be environmentally transmitted to captive elk. Four individually housed treatment elk and 2 control elk were challenged with soil mixed with inoculum prepared from free-ranging elk hooves from TAHD-positive elk or autoclaved hooves from normal elk, respectively. The inoculum for each group was applied to the interdigital space and added to pre-existing soil in each pen. Eight challenges were conducted at 1-4-week intervals and lesion development was assessed during a 138-day challenge period that was followed by a 170-day monitoring period to document lesion progression. All treatment elk, but no control elk, developed gross and histologic lesions consistent with TAHD. Treponema phylotypes similar to those in bovine digital dermatitis in cattle were detected using 16S rRNA gene amplicon sequencing from lesions in all treatment elk, but no control elk, during the challenge period. Lesions progressed from ulcerations in the interdigital space to extensive ulceration and underrunning of the hoof capsule by 35 and 173 days following the initial inoculation, respectively. Lameness in treatment elk was correlated with lesion development (R = 0.702, p≤0.001), and activity of infected elk was reduced during the challenge (p≤0.001) and monitoring periods (p = 0.004). Body condition was significantly lower in treatment than control elk 168 days following the initial inoculation (p = 0.05) and at each individual elk's study endpoint (p = 0.006). Three of 4 treatment elk were euthanized when they reached humane endpoints, and one elk recovered. These results provide direct evidence that TAHD is a transmissible infectious disease in elk. As such, actions that reduce transmission risk can support disease management and prevention.

SURVEILLANCE FOR AN EMERGENT HOOF DISEASE IN ELK (CERVUS ELAPHUS) IN THE US PACIFIC WEST SUPPLEMENTED BY 16S RRNA GENE AMPLICON SEQUENCING.

A novel hoof disease of elk (Cervus elaphus) was described in southwestern Washington, US, in 2008 and was subsequently diagnosed in an adjacent area in northwestern Oregon in 2014. The disease, currently referred to as treponeme-associated hoof disease (TAHD), is characterized by lesions ranging from mild erosions, to severe ulcers with underrunning of the hoof capsule and heel-sole junction, to overgrown and avulsed hoof capsules. Histologically, lesions exhibit epithelial erosion or ulceration, suppurative inflammation, and the presence of argyrophilic spirochetes. We used data collected by the Washington Department of Fish and Wildlife and Oregon Department of Fish and Wildlife from 2008 to 2017 as reference for disease distribution. We then conducted enhanced surveillance in 2018-20 by obtaining 164 submissions from four US Pacific West states. We detected TAHD for the first time in Idaho and northern California, as well as in multiple counties in Washington and Oregon where it had not been previously reported. Given the unexpectedly broad disease distribution, continued surveillance is warranted to determine the full geographic extent of TAHD. From samples of 22 elk, we investigated 16S rRNA gene amplicon sequencing as a technique that could be used to supplement TAHD surveillance. Operational taxonomic units of the family Spirochaetaceae were identified in 10 of 12 histologically diagnosed TAHD-positive cases and two of 10 TAHD-negative cases. Phyla Spirochaetae (P<0.008), Fusobacteria (P<0.006), and Tenericutes (P<0.01) were overrepresented in samples from TAHD-positive feet when compared with TAHD-negative elk. A unique spirochete, PT19, was detected in hooves of 11 elk and from at least one elk in each state. Results support the use of 16S rRNA gene amplicon sequencing as a reliable and informative tool to supplement investigations into distribution and etiology of this presumed polybacterial disease.

Water sorption characteristics of freeze-dried bacteria in low-moisture foods.

Water sorption isotherms of bacteria reflect the water activity with the change of moisture content of bacteria at a specific temperature. The temperature-dependency of water activity change can help to understand the thermal resistance of bacteria during a thermal process. Thermal resistance of bacteria in low-moisture foods may differ significantly depending on the physiological characteristics of microorganisms, including cell structure, existence of biofilms, and growth state. Previous studies demonstrated that the incremental change of aw in bacterial cells during thermal treatments resulted in changes in their thermotolerance. In this study, a pathogen associated with low-moisture foods outbreaks, Salmonella Enteritidis PT30 (in planktonic and biofilm forms), and its validated surrogate, Enterococcus faecium, were lyophilized and their water sorption isotherms (WSI) at 20, 40, and 60 °C were determined by using a vapor sorption analyzer and simulated by the Guggenheim, Anderson and De Boer model (GAB). The published thermal death times at 80 °C (D80 °C-values) of these bacteria in low-moisture environments were related with their WSI-derived aw changes. The results showed that planktonic E. faecium and biofilms of Salmonella, exhibiting higher thermal resistance compared to the planktonic cultures of Salmonella, had a smaller increase in aw when thermally treated from 20 to 60 °C in sealed test cells. The computational modeling also showed that when temperature increased from 20 to 60 °C, with an increase in relative humidity from 10% to 60%, freeze-dried planktonic E. faecium and Salmonella cells would equilibrate to their surrounding environments in 0.15 s and 0.25 s, respectively, suggesting a rapid equilibration of bacterial cells to their microenvironment. However, control of bacteria with different cell structure and growth state would require further attentions on process design adjustment because of their different water sorption characteristics.

AT Homopolymer Strings in Salmonella enterica Subspecies I Contribute to Speciation and Serovar Diversity.

Adenine and thymine homopolymer strings of at least 8 nucleotides (AT 8+mers) were characterized in Salmonella enterica subspecies I. The motif differed between other taxonomic classes but not between Salmonella enterica serovars. The motif in plasmids was possibly associated with serovar. Approximately 12.3% of the S. enterica motif loci had mutations. Mutability of AT 8+mers suggests that genomes undergo frequent repair to maintain optimal gene content, and that the motif facilitates self-recognition; in addition, serovar diversity is associated with plasmid content. A theory that genome regeneration accounts for both persistence of predominant Salmonella serovars and serovar diversity provides a new framework for investigating root causes of foodborne illness.

Occurrence of potentially zoonotic and cephalosporin resistant enteric bacteria among shelter dogs in the Central and South-Central Appalachia.

BACKGROUND: Antimicrobial resistance and presence of zoonotic enteropathogens in shelter dogs pose a public health risk to shelter workers and potential adopters alike. In this study we investigated the prevalence of zoonotic bacterial pathogens and cephalosporin resistant (CefR) enteric bacteria in the feces of apparently healthy shelter dogs in the Cumberland Gap Region (CGR) in the US states of Kentucky, Tennessee and Virginia. RESULTS: Fecal samples of 59 dogs from 10 shelters in the CGR of Central and South-Central Appalachia were screened for the presence of Campylobacter jejuni, Clostridium perfringens, Salmonella and CefR enteric bacteria. C. jejuni, C. perfringens were detected by PCR based assays. Culture and PCR were used for Salmonella detection. Of 59 dogs, fecal samples from 14 (23.7%) and 8 (13.6%) dogs tested positive for cpa and hipO genes of C. perfringens and C. jejuni, respectively. Salmonella was not detected in any of the tested samples by PCR or culture. CefR enteric bacteria were isolated on MacConkey agar supplemented with ceftiofur followed by identification using MALDI-TOF. Fecal samples from 16 dogs (27.1%) yielded a total of 18 CefR enteric bacteria. Majority of CefR isolates (14/18, 77.8%) were E. coli followed by, one isolate each of Enterococcus hirae, Acinetobacter baumannii, Acinetobacter pittii, and Pseudomonas aeruginosa. CefR enteric bacteria were tested for resistance against 19- or 24-antibiotic panels using broth microdilution method. Seventeen (94.4%) CefR bacteria were resistant to more than one antimicrobial agent, and 14 (77.8%) displayed multidrug resistance (MDR). CONCLUSIONS: This study shows that shelter dogs within the CGR not only carry zoonotic bacterial pathogens, but also shed multidrug resistant enteric bacteria in their feces that may pose public health risks.

Quality changes in chicken livers during cooking.

Raw chicken livers are often contaminated with Campylobacter and Salmonella. Cooking is considered the last defense of pathogen control for meals containing chicken livers. However, consumers' preference for pink color and a creamy texture as desired attributes in preparing liver pâté may lead to inadequate cooking, thereby increasing the risk of foodborne illness. This study aimed to investigate the effects of different cooking conditions (60-90°C, 0-65 min) on quality changes in frozen and fresh chicken livers and develop cooking recommendations to produce safe liver products with desired qualities. Frozen storage reduced the water holding capacity of raw chicken livers and led to more cooking loss (reduction in the weight of liver pieces during cooking) and area shrinkage after heating. The cooking loss and area shrinkage increased with increasing heating time and temperature, following the first-order fractional model. Compared with fresh livers, the shear resistance for cutting through the cooked livers increased after heating at 73.9°C to 90°C and decreased at 60°C, whereas the livers heated at 70°C had shear resistance (~4.5 N/g) similar to the fresh liver, regardless of the heating times used in this study. Heating resulted in color changes in livers, shifting from red hue (0°) toward yellow hue (90°), as characterized by the increased hue angles after heating. Cooking livers to an internal temperature of 70°C to 73.9°C and hold for 101 to 26 s is recommended for food processing plants or restaurants to prepare ready-to-eat meals containing chicken livers to achieve microbial safety with respect to Salmonella and provide cooked livers with desired texture and pink color.

Comparative efficacy of spray-dried plasma and bacitracin methylene disalicylate in reducing cecal colonization by Salmonella Enteritidis in broiler chickens.

Spray-dried plasma (SDP) contains immunoglobulins and glycoproteins that possess antibacterial properties. Two floor-pen trials were conducted to determine the efficacy of dietary SDP and bacitracin methylene disalicylate (BMD) antibiotic in reducing intestinal colonization by Salmonella Enteritidis (SE) in broiler chickens. Experiment 1 was a 2-wk, 3 × 2 factorial design consisting of 6 treatments. Treatment CON consisted of chicks fed unmedicated corn-soybean meal (SBM) basal without SDP. Treatment BMD consisted of chicks given unmedicated corn-SBM basal into which BMD was added at 0.055g/kg diet. Treatment SDP consisted of chicks given unmedicated corn-SBM basal into which SDP was added at 30g/kg diet. Treatments CON-SE, BMD-SE, and SDP-SE consisted of chicks that were given diets similar to CON, BMD, and SDP, respectively, and were each inoculated with 7.46 × 108 CFU SE /mL at 1 day of age. Experiment 2 was a 42-day trial that was similar to Experiment 1 in design, except that chicks were placed on fresh clean litter. On d 3, 7, 14, and 28 post-challenge (PC), ceca SE concentration was enumerated on xylose lysine tergitol-4 (XLT4) agar. Body weight gain (BWG) and feed conversion ratio (FCR) were also recorded. Results for d 3 showed that BMD- and SDP-fed chicks had similar (P > 0.05) cecal SE (3.39 log 10 CFU / g and 3.58 log 10 CFU / g, respectively), but these levels were lower (P < 0.05) than that of CON-fed chicks (5.68 log 10 CFU / g). A similar trend was observed on d 7 and 14 PC. The BMD- and SDP-fed chicks also had higher BWG and FCR (P < 0.05) when compared with CON-fed chicks up to d 14. Thereafter, only BMD treatment sustained this growth-promoting effect till d 42 in SE-challenged birds. In conclusion, BMD and SDP showed similar efficacy in reducing cecal Salmonella and in mitigating consequent growth-depressing effect(s) in broiler chicks up to 2 wk of age.

Phenelzine and Amoxapine Inhibit Tyramine and d-Glucuronic Acid Catabolism in Clinically Significant Salmonella in A Serotype-Independent Manner.

Non-typhoidal Salmonella ingeniously scavenges energy for growth from tyramine (TYR) and d-glucuronic acid (DGA), both of which occur in the host as the metabolic byproducts of the gut microbial metabolism. A critical first step in energy scavenging from TYR and DGA in Salmonella involves TYR-oxidation via TYR-oxidoreductase and production of free-DGA via ß-glucuronidase (GUS)-mediated hydrolysis of d-glucuronides (conjugated form of DGA), respectively. Here, we report that Salmonella utilizes TYR and DGA as sole sources of energy in a serotype-independent manner. Using colorimetric and radiometric approaches, we report that genes SEN2971, SEN3065, and SEN2426 encode TYR-oxidoreductases. Some Salmonella serotypes produce GUS, thus can also scavenge energy from d-glucuronides. We repurposed phenelzine (monoaminoxidase-inhibitor) and amoxapine (GUS-inhibitor) to inhibit the TYR-oxidoreductases and GUS encoded by Salmonella, respectively. We show that phenelzine significantly inhibits the growth of Salmonella by inhibiting TYR-oxidoreductases SEN2971, SEN3065, and SEN2426. Similarly, amoxapine significantly inhibits the growth of Salmonella by inhibiting GUS-mediated hydrolysis of d-glucuronides. Because TYR and DGA serve as potential energy sources for Salmonella growth in vivo, the data and the novel approaches used here provides a better understanding of the role of TYR and DGA in Salmonella pathogenesis and nutritional virulence.

Global transcriptional profiling of tyramine and d-glucuronic acid catabolism in Salmonella.

Salmonella has evolved various metabolic pathways to scavenge energy from the metabolic byproducts of the host gut microbiota, however, the precise metabolic byproducts and pathways utilized by Salmonella remain elusive. Previously we reported that Salmonella can proliferate by deriving energy from two metabolites that naturally occur in the host as gut microbial metabolic byproducts, namely, tyramine (TYR, an aromatic amine) and d-glucuronic acid (DGA, a hexuronic acid). Salmonella Pathogenicity Island 13 (SPI-13) plays a critical role in the ability of Salmonella to derive energy from TYR and DGA, however the catabolic pathways of these two micronutrients in Salmonella are poorly defined. The objective of this study was to identify the specific genetic components and construct the regulatory circuits for the TYR and DGA catabolic pathways in Salmonella. To accomplish this, we employed TYR and DGA-induced global transcriptional profiling and gene functional network analysis approaches. We report that TYR induced differential expression of 319 genes (172 up-regulated and 157 down-regulated) when Salmonella was grown in the presence of TYR as a sole energy source. These included the genes originally predicted to be involved in the classical TYR catabolic pathway. TYR also induced expression of majority of genes involved in the acetaldehyde degradation pathway and aided identification of a few new genes that are likely involved in alternative pathway for TYR catabolism. In contrast, DGA induced differential expression of 71 genes (58 up-regulated and 13 down-regulated) when Salmonella was grown in the presence of DGA as a sole energy source. These included the genes originally predicted to be involved in the classical pathway and a few new genes likely involved in the alternative pathway for DGA catabolism. Interestingly, DGA also induced expression of SPI-2 T3SS, suggesting that DGA may also influence nutritional virulence of Salmonella. In summary, this is the first report describing the global transcriptional profiling of TYR and DGA catabolic pathways of Salmonella. This study will contribute to the better understanding of the role of TYR and DGA in metabolic adaptation and virulence of Salmonella.

The occurrence of Salmonella, extended-spectrum ß-lactamase producing Escherichia coli and carbapenem resistant non-fermenting Gram-negative bacteria in a backyard poultry flock environment.

Increase in the number of small-scale backyard poultry flocks in the USA has substantially increased human-to-live poultry contact, leading to increased public health risks of the transmission of multi-drug resistant (MDR) zoonotic and food-borne bacteria. The objective of this study was to detect the occurrence of Salmonella and MDR Gram-negative bacteria (GNB) in the backyard poultry flock environment. A total of 34 backyard poultry flocks in Washington State (WA) were sampled. From each flock, one composite coop sample and three drag swabs from nest floor, waterer-feeder, and a random site with visible faecal smearing, respectively, were collected. The samples were processed for isolation of Salmonella and other fermenting and non-fermenting GNB under ceftiofur selection. Each isolate was identified to species level using MALDI-TOFF and tested for resistance against 16 antibiotics belonging to eight antibiotic classes. Salmonella serovar 1,4,[5],12:i:- was isolated from one (3%) out of 34 flocks. Additionally, a total of 133 ceftiofur resistant (CefR ) GNB including Escherichia coli (53), Acinetobacter spp. (45), Pseudomonas spp. (22), Achromobacter spp. (8), Bordetella trematum (1), Hafnia alvei (1), Ochrobactrum intermedium (1), Raoultella ornithinolytica (1), and Stenotrophomonas maltophilia (1) were isolated. Of these, 110 (82%) isolates displayed MDR. Each flock was found positive for the presence of one or more CefR GNB. Several MDR E. coli (n = 15) were identified as extended-spectrum ß-lactamase (ESBL) positive. Carbapenem resistance was detected in non-fermenting GNB including Acinetobacter spp. (n = 20), Pseudomonas spp. (n = 11) and Stenotrophomonas maltophila (n = 1). ESBL positive E. coli and carbapenem resistant non-fermenting GNB are widespread in the backyard poultry flock environment in WA State. These GNB are known to cause opportunistic infections, especially in immunocompromised hosts. Better understanding of the ecology and epidemiology of these GNB in the backyard poultry flock settings is needed to identify potential risks of transmission to people in proximity.

Identification of common highly expressed genes of Salmonella Enteritidis by in silico prediction of gene expression and in vitro transcriptomic analysis.

Chickens are the reservoir host of Salmonella Enteritidis. Salmonella Enteritidis colonizes the gastro-intestinal tract of chickens and replicates within macrophages without causing clinically discernable illness. Persistence of S. Enteritidis in the hostile environments of intestinal tract and macrophages allows it to disseminate extra-intestinally to liver, spleen, and reproductive tract. Extra-intestinal dissemination into reproductive tract leads to contamination of internal contents of eggs, which is a major risk factor for human infection. Understanding the genes that contribute to S. Enteritidis persistence in the chicken host is central to elucidate the genetic basis of the unique pathobiology of this public health pathogen. The aim of this study was to identify a succinct set of genes associated with infection-relevant in vitro environments to provide a rational foundation for subsequent biologically-relevant research. We used in silico prediction of gene expression and RNA-seq technology to identify a core set of 73 S. Enteritidis genes that are consistently highly expressed in multiple S. Enteritidis strains cultured at avian physiologic temperature under conditions that represent intestinal and intracellular environments. These common highly expressed (CHX) genes encode proteins involved in bacterial metabolism, protein synthesis, cell-envelope biogenesis, stress response, and a few proteins with uncharacterized functions. Further studies are needed to dissect the contribution of these CHX genes to the pathobiology of S. Enteritidis in the avian host. Several of the CHX genes could serve as promising targets for studies towards the development of immunoprophylactic and novel therapeutic strategies to prevent colonization of chickens and their environment with S. Enteritidis.

Biosolids and Tillage Practices Influence Soil Bacterial Communities in Dryland Wheat.

Class B biosolids are used in dryland wheat (Triticum aestivum L.) production in eastern Washington as a source of nutrients and to increase soil organic matter, but little is known about their effects on bacterial communities and potential for harboring human pathogens. Moreover, conservation tillage is promoted to reduce erosion and soil degradation. We explored the impacts of biosolids or synthetic fertilizer in combination with traditional (conventional) or conservation tillage on soil bacterial communities. Bacterial communities were characterized from fresh biosolids, biosolid aggregates embedded in soil, and soil after a second application of biosolids using high-throughput amplicon sequencing. Biosolid application significantly affected bacterial communities, even 4 years after their application. Bacteria in the families Clostridiaceae, Norcardiaceae, Anaerolinaceae, Dietziaceae, and Planococcaceae were more abundant in fresh biosolids, biosolid aggregates, and soils treated with biosolids than in synthetically fertilized soils. Taxa identified as Turcibacter, Dietzia, Clostridiaceae, and Anaerolineaceae were highly abundant in biosolid aggregates in the soil and likely originated from the biosolids. In contrast, Oxalobacteriaceae, Streptomyceteaceae, Janthinobacterium, Pseudomonas, Kribbella, and Bacillus were rare in the fresh biosolids, but relatively abundant in biosolid aggregates in the soil, and probably originated from the soil to colonize the substrate. However, tillage had relatively minor effects on bacterial communities, with only a small number of taxa differing in relative abundance between traditional and conventional tillage. Although biosolid-associated bacteria persisted in soil, potentially pathogenic taxa were extremely rare and no toxin genes for key groups (Salmonella, Clostridium) were detectable, suggesting that although fecal contamination was apparent via indicator taxa, pathogen populations had declined to low levels. Thus, biosolid amendments had profound effects on soil bacterial communities both by introducing gut- or digester-derived bacteria and by enriching potentially beneficial indigenous soil populations.

Genomic organization and role of SPI-13 in nutritional fitness of Salmonella.

Salmonella pathogenicity island 13 (SPI-13) contributes to the virulence of Salmonella. The majority of the SPI-13 genes encode proteins putatively involved in bacterial metabolism, however, their functions largely remain uncharacterized. It is currently unknown if SPI-13 contributes to metabolic fitness of Salmonella and, if so, what are the metabolic substrates for the protein encoded by genes within SPI-13. We employed Phenotype Microarray (Biolog, USA) to compare the metabolic properties of SPI-13 deficient mutant (ΔSPI-13) and the WT parent strain of non-typhoidal Salmonella enterica sub sp. enterica serovar Enteritidis (S. Enteritidis). The results of Phenotype Microarray revealed that SPI-13 is required for efficient utilization of two micronutrients, namely, d-glucuronic acid (DGA) and tyramine (TYR), as sole sources of carbon and/or nitrogen. By systematic deletion of the individual gene(s), we identified specific genes within SPI-13 that are required for efficient utilization of DGA (SEN2977-80) and TYR (SEN2967 and SEN2971-72) as sole nutrient sources. The results show that SPI-13 mediated DGA and TYR metabolic pathways afford nutritional fitness to S. Enteritidis. Comparative genomics analysis of the SPI-13 locus from 247 Salmonella strains belonging to 57 different serovars revealed that SPI-13 genes specifically involved in the metabolism of DGA and TYR are highly conserved in Salmonella enterica. Because DGA and TYR are naturally present as metabolic byproducts in the gastrointestinal tract and other host tissues, we propose a metabolic model that shows that the role of SPI-13 mediated DGA and TYR metabolism in the nutritional fitness of Salmonella is likely linked to nutritional virulence of this pathogen.

Dimethyl adenosine transferase (KsgA) contributes to cell-envelope fitness in Salmonella Enteritidis.

We previously reported that inactivation of a universally conserved dimethyl adenosine transferase (KsgA) attenuates virulence and increases sensitivity to oxidative and osmotic stress in Salmonella Enteritidis. Here, we show a role of KsgA in cell-envelope fitness as a potential mechanism underlying these phenotypes in Salmonella. We assessed structural integrity of the cell-envelope by transmission electron microscopy, permeability barrier function by determining intracellular accumulation of ethidium bromide and electrophysical properties by dielectrophoresis, an electrokinetic tool, in wild-type and ksgA knock-out mutants of S. Enteritidis. Deletion of ksgA resulted in disruption of the structural integrity, permeability barrier and distorted electrophysical properties of the cell-envelope. The cell-envelope fitness defects were alleviated by expression of wild-type KsgA (WT-ksgA) but not by its catalytically inactive form (ksgAE66A), suggesting that the dimethyl transferase activity of KsgA is important for cell-envelope fitness in S. Enteritidis. Upon expression of WT-ksgA and ksgAE66A in inherently permeable E. coli cells, the former strengthened and the latter weakened the permeability barrier, suggesting that KsgA also contributes to the cell-envelope fitness in E. coli. Lastly, expression of ksgAE66A exacerbated the cell-envelope fitness defects, resulting in impaired S. Enteritidis interactions with human intestinal epithelial cells, and human and avian phagocytes. This study shows that KsgA contributes to cell-envelope fitness and opens new avenues to modulate cell-envelopes via use of KsgA-antagonists.

Draft Genome Sequences of 12 Clinical and Environmental Methicillin-Resistant Staphylococcus pseudintermedius Strains Isolated from a Veterinary Teaching Hospital in Washington State.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is a globally emergent multidrug-resistant pathogen of dogs associated with nosocomial transmission in dogs and with potential zoonotic impacts. Here, we report the draft whole-genome sequences of 12 hospital-associated MRSP strains and their resistance genotypes and phenotypes.

Complete Genome Sequence of a Ciprofloxacin-Resistant Salmonella enterica subsp. enterica Serovar Kentucky Sequence Type 198 Strain, PU131, Isolated from a Human Patient in Washington State.

Strains of the ciprofloxacin-resistant (Cipr) Salmonella enterica subsp. enterica serovar Kentucky sequence type 198 (ST198) have rapidly and extensively disseminated globally to become a major food safety and public health concern. Here, we report the complete genome sequence of a CiprS. Kentucky ST198 strain, PU131, isolated from a human patient in Washington State (USA).

Genetically distinct lineages of Salmonella Typhimurium ST313 and ST19 are present in Brazil.

In sub-Saharan Africa, two genetically distinct lineages of multi-drug resistant non-typhoidal Salmonella (NTS) serovar Typhimurium sequence type 313 (ST313) are known to cause invasive disease among people. S. Typhimurium ST313 has evolved to become more human-adapted and is commonly isolated from systemic sites (eg., blood) from febrile patients in sub-Saharan Africa. Epidemiological studies indicate that S. Typhimurium is frequently isolated from systemic sites from human patients in Brazil, however, it is currently unknown if this pathogen has also evolved to become more invasive and human-adapted in this country. Here we determined genotypic and phenotypic divergence among clinical S. Typhimurium strains isolated from systemic and non-systemic sites from human patients in Brazil. We report that a subset (8/38, 20%) of epidemiologically diverse human clinical strains of S. Typhimurium recovered from systemic sites in Brazil show significantly higher intra-macrophage survival, indicating that this subset is likely more invasive. Using the whole genome sequencing and phylogenetic approaches, we identified S. Typhimurium ST313-lineage in Brazil that is genetically and phenotypically distinct from the known African ST313-lineages. We also report the identification of S. Typhimurium ST19-lineage in Brazil that is evolving similar to ST313 lineages from Africa but is genetically and phenotypically distinct from ST19-lineage commonly associated with the gastrointestinal disease worldwide. The identification of new S. Typhimurium ST313 and ST19 lineages responsible for human illnesses in Brazil warrants further epidemiological investigations to determine the incidence and spread of a genetically divergent population of this important human pathogen.

Draft Genome Sequences of 11 Salmonella enterica Serovar Typhimurium Strains Isolated from Human Systemic and Nonsystemic Sites in Brazil.

Salmonella enterica serovar Typhimurium strains isolated from systemic sites outside sub-Saharan Africa have been rarely sequenced. Here, we report the draft genome sequences of S Typhimurium sequence type 19 (ST19) (n = 9), ST1649 (n = 1), and ST313 (n = 1) strains isolated from human systemic (e.g., blood) and nonsystemic (e.g., stool and wounds) sites in Brazil.