Development and Evaluation of a Commercial Direct-Fed Microbial (Zymospore®) on the Fecal Microbiome and Growth Performance of Broiler Chickens under Experimental Challenge Conditions.

Direct-fed microbials (DFM) are added to broiler chicken diets in order to promote the proliferation of beneficial intestinal bacterial populations, which may lead to gains in performance efficiency and, potentially, reduce the level of enteric pathogens in the broiler chickens. The selection and laboratory evaluation of Bacillus subtilis strains as well as the experimental trial results of a novel Bacillus-based commercial DFM product are described. Fifteen wild-type Bacillus subtilis strains were characterized and assayed for their enzyme production capability, spore resistance to pH, salinity, and temperature, and ability to inhibit the growth of E. coli and Salmonella spp. The final DFM formulation was evaluated and compared to an antibiotic growth promoter (AGPs) in two experimental trials. In Experiment 1, broilers were given a defined challenge of Eimeria spp. and Clostridium perfringens to induce intestinal dysbiosis. The optimal dose of the DFM was determined to be 0.3 kg/ton of feed. At this dose, the broilers fed the DFM performed as well as the Flavomycin®-fed broilers. Further, intestinal microbiome analysis indicates that the use of the DFM enhances bacterial diversity of the gut flora by day 5 of age, increasing levels of lactic acid bacteria (LAB) and Clostridiales by 25 days of age, which may enhance the digestion of feed and promote growth of the birds. In Experiment 2, the broilers were raised on recycled litter and given an undefined challenge orally to mimic commercial growth conditions. In this trial, the DFM performed as well as the bacitracin methylene disalicylate (BMD)-11%-fed birds. The results of the present studies suggest that this novel DFM, Zymospore®, improves the performance of broiler chickens under experimental challenge conditions as effective as an AGP, providing a safe and effective substitute to the poultry industry.

Streptococcus gordonii Poised for Glycan Feeding through a MUC5B-Discriminating, Lipoteichoic Acid-Mediated Outside-In Signaling Circuit.

Many oral bacteria employ cell wall-anchored adhesins to bind to the salivary films coating the teeth and mucosal surfaces. Surface binding prevents clearance and facilitates catabolism of salivary film glycoproteins. We asked whether Streptococcus gordonii adhesin expression changes in response to surface salivary cues using a eukaryote-like, outside-in recognition and signaling circuit. To determine whether the cues were discriminated, S. gordonii was tested during cell adhesion and biofilm formation on a MUC5B-rich or lower-molecular-mass salivary fraction or an uncoated abiotic surface. Cells were recovered and analyzed for differences in gene expression and proteins in cell wall fractions. In salivary-free conditions, planktonic S. gordonii presented three prominent cell wall LPXTG-motif proteins, SGO_1487, SGO_0890, and MbpA (mucin-binding protein A; SGO_0707). During biofilm formation on MUC5B-coated surfaces, MbpA, a MUC5B-binding protein, and key genes in the tagatose and quorum-sensing pathways were strongly promoted. The response to MUC5B required the two-component system (TCS), streptococcal regulator of adhesins sensor and regulator (SraSR, SGO_1180/81), lipoteichoic acid (LTA), and the homologous paired adhesins, SspA and SspB (SspAB). LTA appears to link the outside signal (MUC5B) to intramembrane SraSR. Tagatose pathway gene expression may poise cells to metabolize MUC5B glycans and, with a quorum-sensing gene (luxS), may direct formation of a consortium to facilitate glycan cross-feeding by S. gordonii. We now show that a Gram-positive bacterium discriminates specific surface environmental cues using an outside-in signaling mechanism to apparently optimize colonization of saliva-coated surfaces. IMPORTANCE All organisms throughout the tree of life sense and respond to their surface environments. To discriminate among mucosal surface environmental cues, we report that Streptococcus gordonii recognizes a high-molecular-weight mucin glycoprotein, MUC5B, using the paired adhesins SspAB and lipoteichoic acid; the latter bridges the outside signal to an intramembrane two-component system to transcriptionally regulate a MUC5B-specific adhesin and genes that may facilitate glycan catabolism.

Evaluation of a subunit vaccine candidate (Biotech Vac Cox) against Eimeria spp. in broiler chickens.

This study evaluated growth performance and cross-protection against Eimeria spp. using a subunit coccidia vaccine in 2 independent challenge experiments. In both trials, chickens were challenged with E. acervulina, E. maxima, and E. tenella oocysts. In Exp 1, 1000-day-old chickens were allocated in one of 2 treatments 1) Control group; 2) Biotech Vac Cox group. The vaccine was orally gavaged on d 2 and 16 of life and coccidia challenge was on d 21. Performance parameters were evaluated on d 21, 35, and 42. On d 34, coccidia lesions were scored. Oocysts per gram of feces (OPG) were evaluated on d 28, 35, and 42. In Exp 2, 900-day-old chickens were assigned in one of 2 treatments 1) Control group; 2) Biotech Vac Cox group. The vaccine was orally gavaged on d 2 and 16 of life and coccidia challenge was on d 21. Performance parameters were evaluated on d 21, 27, 35, and 42, and lesion scores and OPG at d 27. In Exp 1, chickens vaccinated had significantly lower feed intake (FI) at d 21 and feed conversion ratio (FCR) at d 35 compared to control chickens (P < 0.05). Vaccinated chickens showed a significant reduction (P ≤ 0.05) in OPG for E. maxima to nondetectable levels and for all coccidian species at d 42 compared to control chickens. In Exp 2, the chickens vaccinated showed a significant increase in BW, BW gain (BWG) and reduction in FCR on d 27, 35, and 42 (P ≤ 0.05). Vaccinated chickens had significantly lower (P ≤ 0.05) lesion scores for all 3 Eimeria species. Moreover, vaccinated chickens had a reduction in total OPG of 35.50% (P = 0.0739). Studies to evaluate the serological and mucosal immune response are currently being evaluated. This inactivated, orally delivered subunit vaccine offers significant cross-protection to Eimeria spp. and eliminates the needs to treat broilers with live oocysts, enhanced ease of use, and greater biosecurity to producers.

Assessment of Novel Water Applied Prebiotic to Evaluate Gut Barrier Failure and Performance in Two Commercial Trials in Brazil. A Pilot Study With an Economic Perspective.

The present study evaluated the effect of administration of a water applied prebiotic on gut barrier failure (Experiment 1) and performance in broiler chickens under commercial conditions (Experiment 2). Experiment 1, one thousand four hundred and forty day-of-hatch Ross broiler chickens were assigned to one of two experimental groups (n = 30 replicate pens/treatment; n = 24 chicks/pen). Birds in the treated group received the prebiotic orally in the drinking water (0.2ml/bird) on days 3 and 17 of age. The second group served as the untreated control group. On d 18, intestinal samples were analyzed by qRT-PCR to determine the expression of MUC2, IL-8, TGF-ß4, and ZO-1. On d 17, d 28, and d 35 blood samples were collected to determine circulating endotoxin levels. On d 28, mucosal intestinal scrapping was collected to measure relative total sIgA levels. At d 42, liver samples were collected to evaluate liver bacterial translocation. In Experiment 2, the prebiotic was evaluated in two commercial trials. Chickens were raised under normal production conditions and fed a 3-phase commercial basal diet with enramycin (7 g/ton). In Trial 1, 8,974,237 broiler chickens were treated with the prebiotic. The prebiotic was administered in the drinking water (0.2 mL/bird) following the manufacture label instructions at day three and seventeen of life. Production parameters were compared to historical information from the company over the same broiler operation and production cycles. For trial 2, 921,411 broiler chickens were treated with the prebiotic as in Trial 1. In Experiment 1, treated chickens showed a significant (P < 0.05) increase in mRNA expression of MUC2, TGF-ß4, IL-8, ZO-1, and sIgA, but a significant reduction of serum endotoxin levels and incidence of liver lactose positive bacterial translocation when compared to non-treated chickens. In both trials of Experiment 2, a significant reduction in total mortality was observed in the treated chickens when compared with the historical farm data. Economic analysis utilizing the total percent of mortality revealed a $1: $2.50 USD and $1: $4.17 USD return for Trial 1 and Trial 2, respectively. The results suggest that the prebiotic positively influences gastrointestinal integrity and performance.

Evaluation of a Novel Mucosal Administered Subunit Vaccine on Colostrum IgA and Serum IgG in Sows and Control of Enterotoxigenic Escherichia coli in Neonatal and Weanling Piglets: Proof of Concept.

The purpose of the present study was to evaluate the ability of a novel experimental subunit vaccine (ESV), induce colostrum IgA and serum IgG in sows, and to control enterotoxigenic Escherichia coli (ETEC) disease in neonatal and weanling piglets. The vaccine was tested in three experiments. Experiment 1 consisted of two independent trials. In each trial, 20 pregnant sows/groups were vaccinated intramuscularly (IM) with a commercial E. coli vaccine or intranasally with ESV at weeks 11 and 13 of pregnancy. Blood and serum samples were obtained within 12 h post-partum. In Experiment 1, intranasal vaccination with ESV significantly increased the sample-to-positive (S/P) ratio of secretory IgA in the colostrum of sows (P < 0.01, trial 1; P < 0.05, trial 2) compared to the IM vaccine. In Experiment 2, twenty-five 3-day old piglets were randomly allocated into two groups, control (n = 13) or ESV (n = 12) and were oral gavaged with the respective treatments on days 3 and 14 of life. On days 17-19, all piglets were challenged using a mixed ETEC culture via oral gavage. Within 72 h, all control group animals developed disease consistent with colibacillosis. Conversely, the ESV treated group remained disease free over the 7-day observation period and had significant increases in body weight gain compared to the control group piglets. In Experiment 3, thirty 28-day old piglets were randomly allocated, control (n = 15) or ESV (n = 15), and on days 33 and 43 of life, piglets were either given by oral gavage 2.0 mL saline (control group) or 2.0 mL ESV. At days 46 and 47 of life, all pigs were challenged with a mixed culture of ETEC and observed for clinical signs of disease. Results of Experiment 3 were similar to those observed in Experiment 2. This study indicates the ESV can induce better levels of colostrum secretory IgA in pregnant sows than IM vaccination, which may be protective to neonatal piglets. Further, the vaccine can protect piglets as early as 3 days of age from an ETEC infection. Importantly, the data suggest a single vaccine could be used across the farrowing, suckling, and weaning program to protect against pathogenic E. coli.

The Mbeya Antimicrobial Stewardship Team: Implementing Antimicrobial Stewardship at a Zonal-Level Hospital in Southern Tanzania.

Background: In 2017, Mbeya Zonal Referral Hospital (MZRH) and the University of South Carolina (UofSC) agreed to collaboratively strengthen antimicrobial prescribing in the southern highlands of Tanzania and train a new generation of clinicians in responsible antimicrobial use. Methods: Key stakeholders and participants were identified and the Mbeya Antimicrobial Stewardship Team (MAST) was created. The team identified assets brought by the collaborators, and four investigations of baseline needs were developed. These investigations included (a) a baseline clinician survey regarding antimicrobial resistance and stewardship, (b) a serial chart review of inpatient antimicrobial prescribing practices, (c) an investigation of antimicrobial resistance rates using existing isolates at the MZRH laboratory, and (d) a survey of antimicrobial availability at community pharmacies in the city. Results: 91% of physicians believe antimicrobial resistance is problem in Tanzania, although only 29% of physicians were familiar with the term "antimicrobial stewardship". Escherichia coli isolates had resistance rates of over 60% to the commonly used agents ciprofloxacin, trimethoprim-sulfamethoxazole, and ceftriaxone. Thirteen out of 14 community pharmacies offered over-the-counter antibiotics for upper respiratory symptoms. Conclusions: International antimicrobial stewardship collaborations can successfully identify opportunities and needs. Evaluating the team's efforts to improve patient outcomes will be essential.

Point-of-Care Ultrasound in Family Medicine Residencies 5-Year Update: A CERA Study.

BACKGROUND AND OBJECTIVES: In 2014, family medicine residency programs began to integrate point-of-care ultrasound (POCUS) into training, although very few had an established POCUS curriculum. This study aimed to evaluate the resources, barriers, and scope of POCUS training in family medicine residencies 5 years after its inception. METHODS: Questions regarding current training and use of POCUS were included in the 2019 Council of Academic Family Medicine Educational Research Alliance (CERA) survey of family medicine residency program directors, and results compared to similar questions on the 2014 CERA survey. RESULTS: POCUS is becoming a core component of family medicine training programs, with 53% of program directors reporting establishing or an established core curriculum. Only 11% of program directors have no current plans to add POCUS training to their program, compared to 41% in 2014. Despite this increase in training, the reported clinical use of POCUS remains uncommon. Only 27% of programs use six of the eight surveyed POCUS modalities more than once per year. The top three barriers to including POCUS in residency training in 2019 have not changed since 2014, and are (1) a lack of trained faculty, (2) limited access to equipment, and (3) discomfort with interpreting images without radiologist review. CONCLUSIONS: Training in POCUS has increased in family medicine residencies over the last 5 years, although practical use of this technology in the clinical setting may be lagging behind. Further research should explore how POCUS can improve outcomes and reduce costs in the primary care setting to better inform training for this technology.

An intramembrane sensory circuit monitors sortase A-mediated processing of streptococcal adhesins.

Bacterial adhesins mediate adhesion to substrates and biofilm formation. Adhesins of the LPXTG family are posttranslationally processed by the cell membrane-localized peptidase sortase A, which cleaves the LPXTG motif. This generates a short C-terminal peptide (C-pep) that remains in the cell membrane, whereas the mature adhesin is incorporated into the cell wall. Genes encoding adhesins of the oral bacterium Streptococcus gordonii were differentially expressed depending on whether the bacteria were isolated from saliva or dental plaque and appeared to be coordinately regulated. Deletion of sspA and sspB (sspAB), both of which encode LPXTG-containing adhesins, unexpectedly enhanced adhesion and biofilm formation. C-peps produced from a model LPXTG-containing adhesin localized to the cell membrane and bound to and inhibited the intramembrane sensor histidine kinase SGO_1180, thus preventing activation of the cognate response regulator SGO_1181. The absence of SspAB C-peps induced the expression of the scaCBA operon encoding the lipoprotein adhesin ScaA, which was sufficient to preserve and even enhance biofilm formation. This C-pep-driven regulatory circuit also exists in pathogenic streptococci and is likely conserved among Gram-positive bacteria. This quality control mechanism ensures that the bacteria can form biofilms under diverse environmental conditions and may play a role in optimizing adhesion and biofilm formation.

Using Future Employers' Expectations to Inform Global Health Fellowship Curricula.

BACKGROUND: Fellowships in global health are increasingly popular and seek to equip physicians with the skills necessary to be effective global health practitioners. Little objective guidance exists on which skills make graduates competitive applicants from the perspective of potential global health employers. OBJECTIVE: We sought to provide objective evidence for the qualifications that make applicants competitive for global health positions by analyzing the listed qualifications for current job openings in large global health organizations. METHODS: The websites of 48 global health employers were systematically searched for current job postings between May and August 2017. Jobs were included for analysis if a medical degree was listed among the primary degrees accepted, and the job was based primarily in a low- or middle-income country. RESULTS: A total of 5849 employment opportunities were posted during the search period, and 81 (1.4%) of these met inclusion criteria. Twenty-two (27%) jobs required and 35 (43%) preferred a relevant master's degree. Few jobs requested a candidate with a PhD and none mentioned tropical medicine training as a requirement or preference. Twenty-three jobs (28%) required and 19 (23%) preferred candidates to speak another language. Most jobs (69%, 56 of 81) required more than 5 years of relevant experience. Only 11 (13%) jobs were primarily clinical positions. CONCLUSIONS: For physicians pursuing a career in global health, most publicly searchable jobs require substantial previous experience and involvement in global health activities beyond clinical practice. Master's degree and language skills are frequently requested candidate qualifications.

Point-of-Care Ultrasound as Part of a Short-Term Medical Mission to Rural Nicaragua.

OBJECTIVES: The lack of access to diagnostic imaging in resource-limited settings (RLSs) poses a worldwide problem. Advances in ultrasound (US) imaging technology bridge this gap, particularly when examinations are performed by physicians and integrated into the patient encounter, termed point-of-care ultrasound (POCUS). Because the number of physicians participating in short-term medical missions (STMMs) is increasing, the authors sought to characterize how the use of POCUS would affect care delivered as part of a 1-week outreach trip in rural Nicaragua. METHODS: In February 2017, as part of an ongoing collaboration among the University of South Carolina, the Medical University of South Carolina, and OneWorld Health, the authors conducted an observational prospective study of all of the patients who received a POCUS examination as part of standard clinical practice during an STMM to Sébaco, Nicaragua. The goal was to determine how often POCUS changed medical management. In addition, the number and types of scans performed were recorded to assess the most common reasons for POCUS use. RESULTS: More than 1100 patients were seen, and a total of 79 POCUS examinations were performed on 59 patients by 2 physicians with extensive POCUS training. Eighty percent of the patients were women, with an average age of 40.5 years (range 1.6-87 years). The use of US changed management for 35.6% of total patients examined (N = 21), divided among changes in diagnosis, pharmacotherapy, new referral, or referral not needed. The average time to perform a POCUS examination was 6.0 minutes. A wide range of POCUS examinations were performed, with lung, gallbladder, obstetric/gynecologic, and cardiac examinations performed most often. CONCLUSIONS: Incorporating POCUS by trained physicians in an RLS as part of an STMM was successful and often changed management. As interest in nonemergency and noncritical care POCUS increases and proliferation of low-cost, accurate, handheld US devices continues, it is probable that more physicians traveling to RLSs will use POCUS as part of STMMs, positively affecting patient care.

A D-enantiomer of the antimicrobial peptide GL13K evades antimicrobial resistance in the Gram positive bacteria Enterococcus faecalis and Streptococcus gordonii.

Antimicrobial peptides represent an alternative to traditional antibiotics that may be less susceptible to bacterial resistance mechanisms by directly attacking the bacterial cell membrane. However, bacteria have a variety of defense mechanisms that can prevent cationic antimicrobial peptides from reaching the cell membrane. The L- and D-enantiomers of the antimicrobial peptide GL13K were tested against the Gram-positive bacteria Enterococcus faecalis and Streptococcus gordonii to understand the role of bacterial proteases and cell wall modifications in bacterial resistance. GL13K was derived from the human salivary protein BPIFA2. Minimal inhibitory concentrations were determined by broth dilution and a serial assay used to determine bacterial resistance. Peptide degradation was determined in a bioassay utilizing a luminescent strain of Pseudomonas aeruginosa to detect peptide activity. Autolysis and D-alanylation-deficient strains of E. faecalis and S. gordonii were tested in autolysis assays and peptide activity assays. E. faecalis protease inactivated L-GL13K but not D-GL13K, whereas autolysis did not affect peptide activity. Indeed, the D-enantiomer appeared to kill the bacteria prior to initiation of autolysis. D-alanylation mutants were killed by L-GL13K whereas this modification did not affect killing by D-GL13K. The mutants regained resistance to L-GL13K whereas bacteria did not gain resistance to D-GL13K after repeated treatment with the peptides. D-alanylation affected the hydrophobicity of bacterial cells but hydrophobicity alone did not affect GL13K activity. D-GL13K evades two resistance mechanisms in Gram-positive bacteria without giving rise to substantial new resistance. D-GL13K exhibits attractive properties for further antibiotic development.

The SaeRS Two-Component System Controls Survival of Staphylococcus aureus in Human Blood through Regulation of Coagulase.

The SaeRS two-component system plays important roles in regulation of key virulence factors and pathogenicity. In this study, however, we found that the deletion mutation of saeRS enhanced bacterial survival in human blood, whereas complementation of the mutant with SaeRS returned survival to wild-type levels. Moreover, these phenomena were observed in different MRSA genetic background isolates, including HA-MRSA WCUH29, CA-MRSA 923, and MW2. To elucidate which gene(s) regulated by SaeRS contribute to the effect, we conducted a series of complementation studies with selected known SaeRS target genes in trans. We found coagulase complementation abolished the enhanced survival of the SaeRS mutant in human blood. The coa and saeRS deletion mutants exhibited a similar survival phenotype in blood. Intriguingly, heterologous expression of coagulase decreased survival of S. epidermidis in human blood. Further, the addition of recombinant coagulase to blood significantly decreased the survival of S. aureus. Further, analysis revealed staphylococcal resistance to killing by hydrogen peroxide was partially dependent on the presence or absence of coagulase. Furthermore, complementation with coagulase, but not SaeRS, returned saeRS/coa double mutant survival in blood to wild-type levels. These data indicate SaeRS modulates bacterial survival in blood in coagulase-dependent manner. Our results provide new insights into the role of staphylococcal SaeRS and coagulase on bacterial survival in human blood.

The Staphylococcus aureus AirSR Two-Component System Mediates Reactive Oxygen Species Resistance via Transcriptional Regulation of Staphyloxanthin Production.

Staphylococcus aureus is an important opportunistic pathogen and is the etiological agent of many hospital- and community-acquired infections. The golden pigment, staphyloxanthin, of S. aureus colonies distinguishes it from other staphylococci and related Gram-positive cocci. Staphyloxanthin is the product of a series of biosynthetic steps that produce a unique membrane-embedded C30 golden carotenoid and is an important antioxidant. We observed that a strain with an inducible airR overexpression cassette had noticeably increased staphyloxanthin production compared to the wild-type strain under aerobic culturing conditions. Further analysis revealed that depletion or overproduction of the AirR response regulator resulted in a corresponding decrease or increase in staphyloxanthin production and susceptibility to killing by hydrogen peroxide, respectively. Furthermore, the genetic elimination of staphyloxanthin during AirR overproduction abolished the protective phenotype of increased staphyloxanthin production in a whole-blood survival assay. Promoter reporter and gel shift assays determined that the AirR response regulator is a direct positive regulator of the staphyloxanthin-biosynthetic operon, crtOPQMN, but is epistatic to alternative sigma factor B. Taken together, these data indicate that AirSR positively regulates the staphyloxanthin-biosynthetic operon crtOPQMN, promoting survival of S. aureus in the presence of oxidants.

The evolution of an integrated ultrasound curriculum (iUSC) for medical students: 9-year experience.

Interest in ultrasound education in medical schools has increased dramatically in recent years as reflected in a marked increase in publications on the topic and growing attendance at international meetings on ultrasound education. In 2006, the University of South Carolina School of Medicine introduced an integrated ultrasound curriculum (iUSC) across all years of medical school. That curriculum has evolved significantly over the 9 years. A review of the curriculum is presented, including curricular content, methods of delivery of the content, student assessment, and program assessment. Lessons learned in implementing and expanding an integrated ultrasound curriculum are also presented as are thoughts on future directions of undergraduate ultrasound education. Ultrasound has proven to be a valuable active learning tool that can serve as a platform for integrating the medical student curriculum across many disciplines and clinical settings. It is also well-suited for a competency-based model of medical education. Students learn ultrasound well and have embraced it as an important component of their education and future practice of medicine. An international consensus conference on ultrasound education is recommended to help define the essential elements of ultrasound education globally to ensure ultrasound is taught and ultimately practiced to its full potential. Ultrasound has the potential to fundamentally change how we teach and practice medicine to the benefit of learners and patients across the globe.

Point of Care Ultrasound in Family Medicine Residency Programs: A CERA Study.

BACKGROUND AND OBJECTIVES: Point-of-care (POC) ultrasound is increasingly used by clinicians across multiple medical specialties. Current perceptions and prevalence of POC ultrasound practice and training in family medicine residency programs has not been described. METHODS: Questions were included in the 2014 Council of Academic Family Medicine Educational Research Alliance (CERA) survey of family medicine residency directors. The survey included questions regarding current use and current curricula regarding POC ultrasound. It also asked rank order questions of perceived benefits and perceived barriers to expanding such training. RESULTS: Fifty percent (n=224) of residency program directors completed the 2014 CERA survey. Few programs (2.2%) reported an established ultrasound curriculum. However, 29% indicated they have started a program within the past year, and 11.2% reported starting the process of establishing such training. Ultrasound assistance for procedural guidance was the most commonly reported (44%) use out of seven POC examples. The three leading perceived benefits of POC ultrasound were: making a more rapid diagnosis, the potential to save health care costs, and the potential to improve patient outcomes. The three leading barriers to expanding training were a lack of appropriately trained faculty, limited access to ultrasound equipment, and a lack of comfort in interpreting images without radiologist review. CONCLUSIONS: A small, but rapidly growing, number of family medicine residencies currently use POC ultrasound. Further research is needed to explore how POC ultrasound can improve patient outcomes in the ambulatory setting and to develop appropriate training methods for this technology.

The AirSR two-component system contributes to Staphylococcus aureus survival in human blood and transcriptionally regulates sspABC operon.

To date, genes identified and transcriptionally regulated by the AirSR TCS have been involved in energy production and cellular homeostasis of the staphylococcal cell. It is well accepted that the state of cellular metabolism impacts the expression of virulence factors in Staphylococcus aureus. For this reason, we conducted experiments to determine if the AirSR TCS contributes to the pathogenesis of S. aureus using an antisense RNA interference technology, an inducible overexpression system, and gene deletions. Depletion of AirSR by antisense RNA expression or deletion of the genes, results in significant decrease in bacterial survival in human blood. Conversely, overexpression of AirR significantly promotes survival of S. aureus in blood. AirR promotes the secretion of virulence factors that inhibits opsonin-based phagocytosis. This enhanced survival is partially linked to the transcriptional regulation of the sspABC operon, encoding V8 protease (SspA), staphopain B (SspB) and staphostatin B (SspC). SspA and SspB are known virulence factors which proteolytically digest opsonins and inhibit killing of S. aureus by professional phagocytes. This is the first evidence linking the AirSR TCS to pathogenesis of S. aureus.

Sensing and Adapting to Anaerobic Conditions by Staphylococcus aureus.

A highly adaptive commensal organism, Staphylococcus aureus, possesses an array of genes that allow the bacterium to survive and grow in a wide variety of niches. Several of these niches are known to be or become anaerobic during the course of an infection; additionally, biofilms that develop, commonly on implanted medical devices, become anaerobic. The metabolic capability of S. aureus provides the organism with the essential nutrients needed to continue to grow, divide, and thwart the host immune system in the presence or absence of oxygen. In order to utilize the ATP-producing pathways and maintain cellular health S. aureus has evolved a series of regulatory systems that regulate these ATP-producing pathways. In this review, we discuss the protein signaling systems that sense, indirectly and directly, anaerobic conditions, their sensory mechanisms and signals, and outline the genes that are altered due to the absence of oxygen and the subsequent response by the bacterial cell. The switch from aerobic to anaerobic growth in S. aureus is complex and highly regulated, with some metabolic pathways regulated by multiple regulatory systems to ensure maximal utilization of each pathway and substrate.

Maternal obesity and gestational weight gain in rural versus urban dwelling women in South Carolina.

PURPOSE: An unhealthy prepregnancy weight and/or gaining an inappropriate amount of weight during pregnancy increase the risk for poor pregnancy and birth outcomes. To our knowledge, no studies to date have examined differences in prepregnancy body mass index (BMI) and gestational weight gain (GWG) patterns by rurality. METHODS: The 2004-2006 South Carolina birth certificate data (n = 132,795) were used. Rurality of residence was determined using Rural-Urban Commuting Area (RUCA) codes. Mothers were categorized as underweight (<18.5 kg/m(2)), normal weight (18.5-24.9), overweight (25.0-29.9), and obese (≥30.0) using their prepregnancy BMI and as having inadequate, adequate, or excessive GWG according to the Institute of Medicine's 2009 GWG guidelines. Chi-square tests and adjusted multinomial logistic regression were used in analysis. FINDINGS: Rural women had higher odds of being overweight and obese compared to urban women. This relationship was found to be partially explained by the higher proportion of minorities living in rural areas. The relationship between GWG and residence type varied by BMI category. Specifically, among normal weight women, rural women had increased odds of inadequate GWG. Among overweight women, rural women had decreased odds of excessive GWG. In obese women, rural women had decreased odds of both inadequate and excessive GWG. CONCLUSIONS: Rural women were more likely to have an unhealthy prepregnancy weight than urban women. However, rural residence was found to be protective against unhealthy GWG in overweight and obese women. Future research exploring reasons for these findings and confirmation of these results in other populations is necessary.

The essential yhcSR two-component signal transduction system directly regulates the lac and opuCABCD operons of Staphylococcus aureus.

Our previous studies suggested that the essential two-component signal transduction system, YhcSR, regulates the opuCABCD operon at the transcriptional level, and the Pspac-driven opuCABCD partially complements the lethal effects of yhcS antisense RNA expression in Staphylococcus aureus. However, the reason why yhcSR regulon is required for growth is still unclear. In this report, we present that the lac and opuC operons are directly transcriptionally regulated by YhcSR. Using real-time RT-PCR we showed that the down-regulation of yhcSR expression affected the transcription of lacA encoding galactose-6-phosphotase isomerase subunit LacA, and opuCA encoding a subunit of a glycine betaine/carnitine/choline ABC transporter. Promoter-lux reporter fusion studies further confirmed the transcriptional regulation of lac by YhcSR. Gel shift assays revealed that YhcR binds to the promoter regions of the lac and opuC operons. Moreover, the Pspac-driven lacABC expression in trans was able to partially complement the lethal effect of induced yhcS antisense RNA. Likewise, the Pspac-driven opuCABCD expression in trans complemented the growth defect of S. aureus in a high osmotic strength medium during the depletion of YhcSR. Taken together, the above data indicate that the yhcSR system directly regulates the expression of lac and opuC operons, which, in turn, may be partially associated with the essentiality of yhcSR in S. aureus. These results provide a new insight into the biological functions of the yhcSR, a global regulator.

Identification of predominant SNPs as a novel method for genotyping bovine Staphylococcus aureus isolates.

Staphylococcus aureus is a formidable pathogen of both human and animal. Infection often gives rise to an economic loss resulting from the extended cost of treatment and hospitalization for humans, and loss of usable agriculture animal products from infected animals and treatment regiments. We describe here a protocol for the amplification and sequencing of predominant single nucleotide polymorphisms within the promoter region of hla (encoding α-toxin) that confers a hyper-producing α-toxin phenotype to S. aureus isolates associated with chronic and severe bovine mastitis infections. We validated our findings with a second round of analysis, confirming the SNPs as a valid genotypic marker for α-toxin hyper-producing bovine isolates. The identification of highly virulent isolates will allow for aggressive treatment of the infection and limit the disease and economic impact. With readily available reagents and facilities, this protocol can be completed in as little as 72 h once samples are isolated.