Background: Staphylococcus aureus is an important trigger of flares in atopic dermatitis. The exact mechanisms by which S. aureus induces inflammatory responses and cell death in the skin epithelium is unclear. The aim of this thesis was to elucidate the cellular and molecular mechanisms by which S. aureus induces it's pathogenic effects on keratinocyte and fibroblast cell lines. Methods: Human keratinocytes (HEKa), and mouse embryonic fibroblasts (MEF) from the NC/Nga dermatitis prone mouse strain were used to investigate the induction of Th2-promoting cytokines (IL-33 and TSLP) and cell death by S. aureus. Cytokine levels were measured by ELISA and cytotoxicity by flow cytometry. Results: Live, but not killed S. aureus or other staphylococcal species, induced release of Th2-promoting cytokines (IL-33 and TSLP) and necrosis in both human and mouse cell lines. Cytokines were not induced by TLR2 ligands, and anti-TLR2 antibodies did not inhibit release, suggesting that the TLR2 pathway was not involved. By contrast, the release of cytokines was induced by a secreted, heat-labile factor/s and could be blocked by protease and PAR2 inhibitors, suggesting that the protease-PAR2 pathway was critical. NC/Nga mouse fibroblasts that lacked soluble IL-33 (sST2) receptor were more sensitive to the effects of S. aureus than control MEF. Conclusions: S. aureus is unique amongst staphylococcal species in it's ability to induce an inflammatory response and cytotoxicity in human keratinocytes and mouse fibroblasts. The protease-PAR2 pathway is critical to this bioactivity. Development of specific inhibitors of this pathway may provide novel therapies for treating S. aureus -induced eczema flares.

Staphylococcus aureus is a major opportunistic pathogen causing a wide range of infections in humans and animals. Methicillin-resistant S. aureus (MRSA) has traditionally been regarded as a strictly human problem, initially confined to the healthcare settings and later a matter of concern in the general community too. All this changed in 2005 with the isolation of a specific MRSA clone, assigned to Clonal Complex (CC)398, from pigs and pig farmers in the Netherlands. These findings triggered worldwide investigation, showing the presence of this livestock-associated (LA)-MRSA clone in a variety of farm animals and in persons in contact with affected animals. Furthermore, the capacity of LA-MRSA CC398 to cause infections in humans and animals has been well documented. Recently, MRSA with a divergent mecA-homologue gene variant has been discovered in bovines and humans. Together, these emerging MRSA strains from animal sources have raised new questions as to their origin and inter-host transmission, as well as raised global concern in both veterinary and human medicine about health risks posed by MRSA prevailing in livestock.<p>In the present work, we aimed to investigate the extent and molecular epidemiology of the LA-MRSA reservoir in animal and human populations, including on livestock farms and in acute-care hospitals in Belgium. As a secondary objective, the presence of methicillin susceptible S. aureus (MSSA) CC398, from which MRSA CC398 could locally emerge by acquisition of the Staphylococal Cassette Chromosome mec (SCCmec) element, was assessed. To this end, we undertook an extensive and systematic cross-sectional survey of S.aureus and MRSA carriage among humans and animals on pig, veal calf, dairy cattle, beef cattle, broiler and horticulture farms. A questionnaire, completed by all farm residents, was used to assess occupational risk factors for human MRSA CC398 carriage. Bacterial genetic characterisation was done by spa typing, SCCmec typing and multi-locus sequence typing (MLST). Antimicrobial susceptibility profiles were determined; the presence of resistance genes and toxin genes were determined by PCR. A second set of S. aureus clinical isolates from two national surveys organised in 2005 and 2008 were characterised using the same methods.<p>Carriage of MRSA CC398 was highly prevalent in animals and humans on pig and veal calf farms and to a significantly lesser extent on beef, dairy, broiler and horticulture farms (Chapter 5.1). Persons who work with pigs or veal calves on a daily basis are at significantly higher risk for MRSA CC398 carriage compared to farm-exposed persons who work with them less regularly or never. In accordance with the results from the present work as well as those from others, it appears important to assess the impact of interventions at farm-level that aim to reduce the MRSA carriage rate in animals, as this would also reduce the risk for MRSA carriage in farmers and relatives.<p> MRSA CC398 isolates, especially those from veal calf farms, were frequently multi-resistant and thereby represent a reservoir of antimicrobial resistance determinants that could be transferred to other, more human-adapted staphylococci or other micro-organisms (Chapter 5.1). Additionally, this multi-resistance phenotype should be considered when applying empiric treatment of human staphylococcal infections in livestock-exposed persons. Only very few major “human-associated” virulence factors were detected, indicating a limited virulence capacity of LA-MRSA CC398 isolates. MRSA strains with the mecA homologue mecC, which is difficult to detect using conventional diagnostic methods, were found in beef and dairy cattle, but not in humans. <p>MSSA CC398 strains from which MRSA CC398 might locally emerge were frequently detected in humans and animals on pig, veal and broiler farms, all of which are commonly known to be affected by MRSA CC398 (Chapter 5.2). Three porcine MSSA CC398-t011 isolates harbored remnant DNA of a composite SCCmec V(5C2&5)c element, from which the mec gene complex was lacking. These findings indicate that the strains were previously involved in SCCmec recombination events. Processes similar to the one described here likely contribute to the enormous diversity of SCCmec elements observed in staphylococci. <p>Although LA-MRSA CC398 strains were frequently detected in livestock and livestock-exposed persons, they only represented a minority (~1%) of the MRSA strains from hospitalised patients. This suggests that this specific MRSA clone has not yet spread among Belgian patients without livestock contact (Chapter 5.3). However, similar to what has been seen in other countries, we observed a recent emergence of severe infections, caused by a human-adapted subclone of MSSA CC398, in hospitalised patients without livestock contact (Chapter 5.4). <p>Once more has S. aureus proven its versatility: it has optimally adapted to the selective pressure exerted by intensive animal farming by acquisition of mobile genetic elements, such as resistance determinants. Clearly MRSA is no longer a strictly human problem. Working across the human and veterinary health sectors will be essential to tackle the dissemination and pathogenic evolution of MRSA in livestock. <p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Made available in DSpace on 2014-06-11T19:30:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-06-07Bitstream added on 2014-06-13T19:40:32Z : No. of bitstreams: 1 silva_ncc_dr_botib.pdf: 675325 bytes, checksum: 770804101b6bf91187e2941b56d767bb (MD5) / Staphylococcus sp é agente comum da mastite bovina, causando grandes perdas econômicas na pecuária brasileira. Esse micro-organismo possui fatores de virulência bastante conhecidos como a produção de hemolisinas, leucotoxinas e superantígenos como a toxina do síndrome do choque tóxico e enterotoxinas. Além disso, várias espécies de Staphylococcus podem adquirir genes de resistência aos antibióticos β lactâmicos. O objetivo do estudo foi caracterizar molecularmente isolados de Staphylococcus sp provenientes de leite de vacas com mastite clinica e subclínica de várias regiões do estado de São Paulo. Foram realizadas coletas de leite de vacas com mastite clinica e subclínica de diferentes fazendas no estado de São Paulo e realizados testes fenótipicos de resistência a antimicrobianos e de virulência (Toxina de Panton Valentine, sindrome do choque tóxico e toxinas esfoliativas), além de testes moleculares como Pulse Field Gel Eletrophoresis (PFGE), Multiloccus Sequence Typing (MLST) e Spa typing para comparação entre as cepas epidemiologicamente importantes e da tipagem do cromossomo cassette estafilocócico em cepas meticilina reistentes. As cepas resistentes a meticilina apresentaram amplo perfil de resistência e genes de resistência importantes e pouco relatados, sendo observados genes como fexA, lsaE, lnuB. Foram detectados dois novos spatyping (t10852 e t10856), bem como um novo alelo yqiL e um novo ST (2493). Os ECNs apresentaram resistências à maioria dos antibióticos estudados e foi observado a uma deleção no gene ermC, que codifica a resistência à eritromicina. / Staphylococcus sp. is the common agent of bovine mastitis, causing big economic losses in Brazilian cattle. This micro-organism have virulence factors have been well known as the production of hemolysin, and leucotoxinas toxin superantigens such as toxic shock syndrome and enterotoxins. In addition, several Staphylococcus species can acquire antibiotic resistance genes β lactamics. The objective of the study is to characterize molecularly Staphylococcus sp. from milk of cows with subclinical mastitis in various regions of the state of São Paulo. Samples were coleted from milk of cows with subclinical and clinical mastitis from different farms in the state of São Paulo. Tests were performed phenotypic antimicrobial resistance and virulence (toxin Panton Valentine, toxic shock syndrome and exfoliative toxins), molecular tests as Eletrophoresis Pulse Field Gel Electrophoresis (PFGE), Multiloccus Sequence Typing (MLST) and spa typing for comparison between epidemiologically important strains, as well as the typing of strains in staphylococcal cassette chromosome methicillin reistentes. Methicillin-resistant strains showed broad resistance profile and resistance genes important and underreported were detected as fexA, lsaE, lnuB. Two new spatyping (t10852 and t10856) were detected as well as a new allele yqiL and a new ST (2493). The ECN showed resistance to most antibiotics and was observed the deletion in ermC gene encoding resistance to erythromycin.

Orientador: Carla Raquel Fontana / Banca: Clóvis Wesley Oliveira de Souza / Banca: Ana Marisa Fusco Almeida / Resumo: A ocorrência de uma variedade de agentes patogênicos resistentes aos antibióticos atuais continua a ser um problema, especialmente quando as infecções bacterianas estão crescendo em biofilme. Neste estudo, propomos o uso da terapia fotodinâmica (TFD) como monoterapia e também associada à antibioticoterapia como um tratamento alternativo. O objetivo deste estudo foi analisar os efeitos da TFD mediada pelo azul de metileno (AM) em Staphylococcus aureus (ATCC 25923) e Escherichia coli (ATCC 25922), como biofilme e em fase planctônica. Diferentes concentrações de fotossensibilizador (400; 200; 100; 50; 25; 12,5; 6,25 μg/mL) e fluência (2,8; 5,6; 11.2 22,4J/cm²) foram testadas. Também realizamos experimentos que avaliaram o efeito sinérgico da terapia fotodinâmica e o antibiótico ciprofloxacina. Os efeitos bactericidas da TFD como monoterapia não foram estatisticamente aumentados com a concentração de fotossensibilizador, e bactérias em biofilmes foram menos afetadas que as da fase planctônicas. Entretanto, o efeito sinérgico da terapia fotodinâmica seguida de ciprofloxacina no biofilme aumentou a redução bacteriana. Em biofilme para S. aureus, utilizando 50 ug/mL de AM, irradiação 22,4 J/cm² e a concentração de 15.625 ug/mL de antibiótico, tivemos uma redução de 61,80%. Para E. coli, houve uma erradicação bacteriana em todas as concentrações testadas na TFD combinada com antibiótico, tanto a irradiação de 11,2 J/cm², como a irradiação 22,4 J/cm². O crescimento bacteriano foi observado apenas na concentração mais baixa do antibiótico testada, de 0,125 ug/mL, em concentrações de 50 e 25 ug/mL de AM. / Abstract: The occurrence of a variety of pathogens resistant to current antibiotics remains the major problem especially when bacterial infections are growing in biofilm. In this study, we propose the use of photodynamic therapy (PDT) as monotherapy and also associated with antibiotic therapy as an alternative treatment. The aim of this study was to analyze the effects of PDT mediated by methylene blue (MB) on Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) as biofilm and planktonic phase. Different concentrations of photosensitizer (400; 200; 100; 50; 25; 12,5; 6,25g/mL), and fluency of 2.8; 5.6; 11.2 22,4J/cm2 were tested. We also carried out experiments that evaluated the synergistic effect of photodynamic therapy and antibiotic ciprofloxacin. The bactericidal effects with PDT as monotherapy were not statistically increased with the concentration of photosensitizer and bacteria in biofilms were less than affect in the planktonic phase. Although, the synergistic effect of photodynamic therapy on biofilms followed by ciprofloxacin amplified the bacterial reduction. In biofilm for S. aureus, using 50 μg/ml MB, irradiation 22,4 J/cm² and the concentration of 15,625 μg/mL of antibiotic, got a reduction of 61,80%. For E. coli, there was a bacterial eradication at all concentrations tested in PDT combined with antibiotic, both the irradiation 11,2 J/cm² as the irradiation 22,4 J/cm². Bacterial growth was observed only in the lowest tested concentration of the antibiotic, 0,125 μg/mL, at concentrations of 50 and 25 μg/mL MB. / Mestre

Introdução: a coleta de culturas de vigilância é uma das estratégias utilizadas no controle de infecções causadas por Staphylococcus aureus, especialmente S. aureus resistente a meticilina (MRSA). Estas culturas são utilizadas para determinar portadores assintomáticos e prevenir a disseminação do patógeno para outros pacientes através da tomada de medidas de isolamento do portador. Neste contexto, tem-se demonstrado que a descolonização de portadores pode reduzir o risco de infecções estafilocócicas em certas ocasiões. O sítio anatômico mais comumente analisado são as narinas anteriores, mas continuamos a nos questionar se seria necessária a cultura de outros sítios anatômicos para este fim. Objetivos: este estudo objetivou avaliar o desempenho de diferentes sítios de cultura de vigilância em determinar a colonização de gestantes e recém-nascidos (RN) e determinar os fatores associados a colonização nasal por S. aureus. Metodologia: este é um estudo descritivo, desenvolvido no Hospital das Clínicas de São Paulo, Brasil, um hospital terciário universitário. Os pacientes envolvidos no estudo são gestantes durante trabalho de parto e seus recém-nascidos. A coleta de material de seu em quatro sítios anatômicos para os recém-nascidos: narinas anteriores, orofaringe, períneo e umbigo, no momento do parto, no terceiro dia e semanalmente. Para as gestantes, foram coletados quatro sítios anatômicos: narinas anteriores, anus, períneo e orofaringe. Apenas a primeira cultura positiva foi considerada, os pacientes colonizados nas narinas foram comparados àqueles colonizados apenas em sítios extranasais e os fatores de risco para colonização por S. aureus foram determinados. Resultados: foram incluídas 392 gestantes e 382 recém-nascidos. A colonização materna por S. aureus foi 53% (MSSA 49% e MRSA 9%). A colonização de RN foi 47% (MSSA 39% e MRSA 9%). Entre os RN, o melhor sítio de coleta foi o umbigo (64% para MSSA e 68% para MRSA) e a melhor associação foi narinas anteriores mais umbigo (86% para MSSA e 91% para MRSA). Entre as gestantes o melhor sítio foi narinas anteriores (MSSA 59% e MRSA 67%) e a melhor associação de sítios foi narinas anteriores mais orofaringe (83% para MSSA e 80% para MRSA). Dentre os fatores de risco, apenas o número de moradores na mesma residência foi associado à colonização materna por S. aureus (2,0+0,6 vs 3,6+1,8; p: 0,04). Conclusão: nosso estudo confirma a necessidade da coleta de vários sítios para assegurar a sensibilidade das culturas de vigilância. Não há fatores associados a colonização nasal que distinguem portadores nasais dos colonizados em sítios extranasais. Os programas de controle de infecção baseados em culturas de vigilância nasal podem ser comprometidos / Introduction: Surveillance cultures are one of the strategies used to control Staphylococcus aureus infections, especially methicillin-resistant S. aureus (MRSA). These cultures are used to determine asymptomatic carriers and prevent spread of the organism to other patients by putting carriers under isolation precautions. Also some authors demonstrated that decolonization of carriers can reduce the risk of staphylococcal infections under certain conditions. The most commonly cultured body sites are the anterior nares but the challenge remains to determine whether routine culturing of other body sites is necessary. Objectives: the study objective to evaluate the performance of surveillance cultures at various body sites in determining S.aureus colonization in pregnant women and their newborns (NB) and determine factors associated with nasal colonization. Methods: This is a descriptive study, developed on Hospital das Clinicas, São Paulo, Brazil, a tertiary-care university hospital. Patients enrolled: pregnant women during labor and their newborns. Material collection: For NB four sites were evaluated: nares, oropharynx, perineum and umbilical stump at birth, 3rd day and weekly. For pregnant women four sites during labor: anterior nares, anus, perineum and oropharynx. Only the first positive culture was considered. Nasally colonized patients were compared with colonized only extra-nasally and risk factors to S. aureus colonization were determined. Results: 392 pregnant women and 382 NB were included. S. aureus colonization was 53% among pregnant women (MSSA 49% and MRSA 4%). S. aureus colonization among NB was 47% (MSSA 39% and MRSA 9%). For NB patients, the best body site was the umbilical stump (64% for MSSA and 68% for MRSA). The best combination in NB was nares plus umbilical stump (86% for MSSA and 91% for MRSA). Among pregnant women, the best body site was the anterior nares (MSSA 59% and MRSA 67%). The best combination was nares plus oropharynx, (83% for MSSA and 80% for MRSA). Only the smaller number of household members was associated with MRSA carriage in pregnant women (2.2±0.6 vs 3.6±1.8; p: 0.04). Conclusion: Our study confirms the need for multiple culture sites to assure sensitivity. No features distinguish nasal carriers from only extra-nasal colonized people. Control programs relying mainly on nasal surveillance cultures may be compromised

Staphylococcus aureus is the leading cause of infective endocarditis in the United States. Infective endocarditis (IE) is defined as an infection of the endocardium, typically involving the heart valves. The hallmark features of IE are vegetations. Vegetations are cauliflower-like, stratified biofilms of bacteria and host factors that develop on the valve leaflets of the heart. The mechanisms of how vegetations form are not well understood, and as a consequence the bacterial factors that are important for development of IE are not well defined. My studies focus on the role of a family of S. aureus exoproteins known as superantigens and their role in IE. Superantigens (SAgs) are a class of secreted virulence factors that have been extensively studied for their role in systemic diseases such as toxic shock syndrome (TSS), pneumonia, and food poisoning. The SAg protein family is comprised of 23 distinct members designated as staphylococcal enterotoxin (SE) or enterotoxin-like (SEl) and toxic shock syndrome toxin-1 (TSST-1). The term superantigen is derived from the ability of SAgs to interact with the immune system, resulting in a nearly 3000-fold increase in activation when compared to standard antigens. SAgs have a defined structure that is composed of 2 domains, a carboxy-terminal beta-grasp domain and amino-terminal oligosaccharide/oligonucleotide binding (OB) fold. Defined groups of SAgs are associated with S. aureus strains isolated from specific diseases, but few studies have been done to determine the role of SAgs in diseases outside of TSS and food poisoning. The enterotoxin gene cluster (egc) is a group of 6 SAgs (selo, selm, sei, selu, seln, and seg) assembled into an operon-like cluster that is present in the majority of S. aureus strains isolated from IE patients. My studies have determined that the egc is able to induce vegetations when expressed in avirulent S. aureus strains. This is the first time the egc has been directly associated with IE. I further characterized the capacity of the individual egc proteins to induce vegetations. Four (selo, selm, sei, and selu) of the 6 egc SAgs were able to induce vegetation formation. This is the first time the individual egc proteins have been characterized and directly associated with IE. I also demonstrated that the egc proteins may not be exclusively expressed as a single polycistronic transcript but that selu and seg contain promoter elements that may drive their individual expression. Lastly, I provide evidence that the egc SAgs may be regulated by MgrA, a global regulator of S. aureus associated with virulence factor expression.

Staphylococcus aureus causes significant illnesses throughout the world, including toxic shock syndrome (TSS), pneumonia, and infective endocarditis. Major contributors to S. aureus illnesses are secreted virulence factors it produces, including superantigens and cytolysins. Rabbit cardiac physiology is considered similar to humans, and rabbits exhibit susceptibility to S. aureus superantigens and cytolysins. As such, rabbits are an excellent model for studying pneumonia, infective endocarditis, and sepsis, We examined the ability of USA200, USA300 and USA400 strains to cause vegetations and lethal sepsis in rabbits. USA200, TSST-1+ strains that produce only low amounts of Α-toxin, exhibited modest LD50 in sepsis (1x108-5x108) colony-forming units (CFUs), and 3/4 caused significant IE. USA200 strain MNPE, which produces high levels of Α-toxin, was both highly lethal (LD50 5x106 CFUs) and effective in causing IE. In contrast, USA300 strains were highly effective in causing lethal sepsis (LD50s 1 x 106 and 5 x 107 CFUs) but were minimally capable of causing IE. USA400 strains were both highly lethal (LD50s of 1 x 107 and 5 x 107 CFUs) and highly effective causes of IE. Additional studies investigated the role of phenol soluble modulins in infection. We showed that PSMs are important for the ability of S. aureus to cause sepsis but not infective endocarditis. Additionally, immunization against PSMs did not protect rabbits from lethal infection. Our studies show that clonal groups of S. aureus differ in abilities to cause infective endocarditis and lethal sepsis and suggest that secreted virulence factors, including superantigens and cytolysins, account for some of these differences. This thesis also investigates the use of superantigens and cytolysins as staphylococcal vaccine candidates. We generated three TSST-1 mutants; G31S/S32P, H135A, and Q136A. All rabbits administered these TSST-1 toxoids generated strong antibody responses (titers>10,000) that neutralized native TSST-1 in TSS models, both in vitro and in vivo. These TSST-1 mutants lacked detectable residual toxicity. Additionally, the TSST-1 mutants exhibited intrinsic adjuvant activity, increasing antibody responses to a second staphylococcal antigen (Β-toxin). This effect may be due to TSST-1 mutants binding to the immune co-stimulatory molecule CD40. The superantigens TSST-1 and SEC and the cytolysin Α-toxin are known to contribute to staphylococcal pneumonia. Immunization of rabbits against these secreted toxins provided complete protection from highly lethal challenge with a USA200 S. aureus strain producing all three exotoxins; USA200 strains are common causes of staphylococcal infections. The same three exotoxins plus the cytolysins Β-toxin and Γ-toxin contribute to infective endocarditis and sepsis caused by USA200 strains. Immunization against these five exotoxins protected rabbits from infective endocarditis and lethal sepsis. Additionally, a heptavalent vaccine composed of the pentavalent units plus SEB and SE-l X protected rabbits from lethal pneumonia caused by USA100 strain 209. Passive immunization using pooled sera protects previously non-immunized rabbits from lethal pneumonia due to MNPE. These data suggest that immunization against toxoid proteins of S. aureus exotoxins protects from serious illnesses, and concurrently superantigen toxoid mutants provide endogenous adjuvant activity.

Variovorax paradoxus EPS is a gram-negative rod isolated from the sunflower rhizosphere at CSUSB. Preliminary research has shown that Variovorax paradoxus EPS has anti-staphylococcal activity in liquid and solid co-culture. Anti-staphylococcal activity of Wild type and V. paradoxus EPS 𝚫4519 on 0.5% YE agar with embedded S. aureus AH1710 supports the idea that a soluble molecule is responsible for this activity, as the agar acted as a physical barrier between V. paradoxus EPS and S. aureus colonies. Preliminary genetic analysis of V. paradoxus EPS identified three loci that suitable candidates for the synthesis of a potential anti-staphylococcal small molecule. Preliminary data failed to detect expression at two of the three identified loci and a strain with a mutation at the third locus continues to produce anti-staphylococcal activity. We hypothesize that the microbial agent is expressed at a different locus or loci that have not yet been identified. These gene products are responsible for the synthesis of the microbial agent and are controlled by exposure to Staphylococcus aureus. Optimal growth conditions were identified for V. paradoxus EPS and S. aureus to demonstrate the formation of a zone of inhibition on Tryptic Soy Agar. The use of a V. paradoxus EPS Δ 4519 transposon library at optimal growth conditions allowed us candidate mutants with altered antimicrobial activity phenotypes. We identified 28 insertion sites that resulted in altered antimicrobial activities, which will allow us to identify the genes involved in this biosynthetic pathway.

Neutrophils (PMN) and group IIA phospholipase A2 (gIIA PLA2) are components of the innate immune system mobilized to sites of invasion by microorganisms such as Staphylococcus aureus. Although accumulating coincidentally in vivo, the in vitro anti-staphylococcal activities of PMN and gIIA PLA2 have thus far been separately studied. The goal of this thesis was to study the collaborative activity of PMN and gIIA PLA2 against S. aureus. We have identified and characterized the collaboration of PMN and gIIA PLA2 against S. aureus ingested by PMN. PMN induced conversion of bacterial phosphatidylglycerol into cardiolipin, but were unable to degrade S. aureus phospholipids without gIIA PLA2. PMN reduced by 10-fold the concentration of gIIA PLA2 needed to digest bacterial phospholipids alone. In addition to increased phospholipid degradation, collaboration of PMN and gIIA PLA2 caused greater bacterial killing and greater loss of bacterial green fluorescent protein fluorescence. The collaboration of PMN and gIIA PLA2 against S. aureus is dependent on catalytic activity and is specific to gIIA PLA2 as related secretory PLA2, groups IB, V, and X, show little or no phospholipid degradation of S. aureus either alone or in the presence of PMN. Synergy of PMN and gIIA PLA2 requires a functional NADPH oxidase and phagocytosis. Although addition of gIIA PLA2 after phagocytosis causes some bacterial phospholipid degradation, the greatest effect is observed when gIIA PLA2 is added before phagocytosis. An extracellular source of H2O2 can partially restore antibacterial activities to NADPH oxidase deficient PMN including the ability to collaborate with gIIA PLA2, supporting a role for reactive oxygen species in NADPH oxidase dependent antimicrobial functions of PMN. In contrast, iberiotoxin, an inhibitor of BK potassium channels had no effect of PMN antibacterial activities. Although H2O2 partially restored antibacterial activity to NADPH oxidase deficient PMN, extracellular H2O2 was not sufficient to increase S. aureus to gIIA PLA2 activity. In summary, PMN and gIIA PLA2 collaborate against S. aureus. These findings revealed collaboration between cellular oxygen-dependent and extracellular oxygen-independent host defense systems that may be important in the ultimate resolution of S. aureus infections.

Background. Surgical site infections (SSIs) cause many adverse outcomes for patients including increased length of hospital stay, hospital costs, morbidity, and psychological distresses. Staphylococcus aureus is one of the most common causes of SSIs in the United States. Objective. Identify risk factors for Staphylococcus aureus SSIs following breast operations. Design. Retrospective nested case-control study of SSIs among women undergoing breast operations. Setting. An academic health center. Patients. We studied patients undergoing breast operations at the University of Iowa Hospitals and Clinics from 7/1/2004 through 9/30/2015. Cases were patients who acquired SSIs meeting the National Healthcare Safety Network definition and whose SSIs were caused by S. aureus. We randomly selected two controls for each case from patients who had breast operation during the study period and did not meet the SSI definition. Controls were selected randomly from uninfected patients whose operations occurred during the same month and year as a case. Results. Forty two (1.2%) patients acquired S. aureus SSIs after 3494 breast operations. SSIs were identified a mean of 27.8 days after the breast operations; 54.76% were deep incisional infections. Poisson regression analysis revealed that S. aureus SSIs following breast operations at UIHC have been increasing at a statistically significant rate. Bivariable analysis identified several patient and procedure related risk factors that increased the risk for S. aureus SSIs. Patient-related factors included a diabetes mellitus, active skin disease, prior chemotherapy, breast cancer, hypertension, and preoperative hemoglobin. Procedure-related factors included ASA score > 2, a mastectomy followed by immediate reconstruction, sentinel lymph node biopsy (SLN), drain placement, procedure time, and estimated blood loss. A multivariable analysis of patient factors found only breast cancer maintained significance. A similar analysis of procedure factors found that drain placement remained significant. The combined model contained breast cancer, drain placement, and mastectomy followed by immediate reconstruction as significant variables. Conclusions. S. aureus SSIs following breast operations have been increasing at UIHC. Possible remediable risk factors include blood glucose levels, blood pressure, timing of chemotherapy, and drain placement and care. These results will help doctors at UIHC design interventions to prevent S. aureus SSIs following these procedures.