Clinical S. aureus strains were gathered from four hospitals, two in Turkey (Hacettepe hospital 200 strains and Ankara Hospital 106 strains) and the other two from Libya (Aljalla Hospital 88 strains and Jamahyria Hospital 62 strains). The clinical specimens were collected form different sources including blood, urine, wound, pus, burn, sputum, semen, catheter and aspiration. Patients were aged between 0 to 84 years and from both sexes. Resistance to Methicillin was determined by measuring the Oxacillin MIC / this was done by using the oxacillin E-test, with resistance defined as an MIC of &gt / 2 &micro / g ml. In this study all isolates displayed an Oxacillin MIC of &amp / #8805 / 256&micro / g/ml. The MRSA strains were (56%) in Turkish hospitals, and (59%) in Libyan hospitals. The percentage of the VRSA and VISA in Libyan hospitals was (7%) and (26%) respectively, although the percentage of VRSA in Turkish hospitals was only 2% and there were no intermediately susceptible Staphylococcus aureus (VISA). Besides the MRSA isolates, Coagulase Negative Staphylococcus showing Methicillin resistance was collected from clinical isolates in thirteen patients in Turkish hospitals. In both countries, the majority MRSA isolates were multiresistant to more than five classes of antibiotics including / Ampicillin, Amoxicillin, Tetracycline, Erythromycin and Ciprofloxacin. Most of the MRSA isolates were from blood (68%), wounds (57%) and pus (50%).The results of genetic investigations indicated that the mecA gene was present in the majority of isolates in both countries / the community acquired MRSA type (ccr-BIV) was present in three samples out of thirty in Turkish hospitals and in one case out of twenty in Libyan hospitals / There was no case out of fifty specimens that carry the hospitals acquired MRSA type (ccr-BI, II, III) in both countries. Besides the Methicillin resistance gene, the incidence of Tetracycline resistance gene was quite high (tetM and tetK 50%) in Turkish hospitals isolates, and the prevalence of Panton-Valentine Leukocidin gene was high (PVL 70%) in Libyan hospitals specimens.

Staphylococcus aureus is a remarkably successful pathogen, accounting for an estimated 95,000 invasive infections annually in the U.S. alone. The burden of MRSA infections on public healthcare continues to rise, particularly with the continued spread of antibiotic resistant strains and the hyper-virulent CA-MRSA strains. The pathogenic nature of S. aureus can be attributed to the cache of virulence factors encoded within the genome of this organism. Typically, these are secreted toxins which directly interact with the host during infection, and facilitate pathogenesis. A previous screen in our laboratory investigating proteases in S. aureus identified a mutant in aminopeptidase Z as being attenuated in disease causation. Classically aminopeptidases function in the bioactivation/inactivation of proteins; and/or the utilization of imported peptides for cellular nutrition. We therefore hypothesize that cells deficient in one of these two processes would have decreased fitness levels, resulting in reduced virulence. We therefore sought to explore the role of PepZ in S. aureus; either in the processing of exogenous oligopeptides for nutrition, or in protein bioactivation/inactivation, and protein stability. We determined that S. aureus strains deficient in PepZ are less viable when cultured under conditions of starvation or while in competition for nutrients with the parent strain, and does not appear to be peptide driven. Using protein analysis approaches we have identified PepZ externalization, suggesting a potential for the aminopeptidase beyond the confines of the cell membrane. Additionally, we have also identified a potential role for PepZ in protein stability in this organism. Lastly, we present the essential role for PepZ in S. aureus virulence.

N-sec -butylthioloated β-lactam (NsβL) is a novel beta-lactam antimicrobial with a mechanism of action proposed to inhibit 3-oxoacyl-acyl carrier protein synthase (ACP) III (FabH), resulting in the inhibition of fatty acid synthesis. It has been suggested that NsβL inhibits FabH indirectly by inactivating coenzyme-A (CoA). CoA is an essential cofactor for numerous proteins involved in glycolysis, the citric acid cycle (TCA), and pyruvate metabolism, in addition to fatty acid biosynthesis. This study aimed to determine the effects of NsβL on a diverse array of laboratory and clinical Staphylococcus aureus isolates by analyzing the mode of resistance in spontaneous and adaptive mutant NsβL-resistant mutants. Phenotypic analysis of the mutants was performed, as well as sequence analysis of fabH; along with comparative proteomic analysis of intracellular proteomes. Our results indicate that NsβL resistance is mediated by drastic changes in the cell wall, oxidative stress response, virulence regulation, and those pathways associated with CoA. It is our conclusion that Nsβ L has activity towards CoA, resulting in wide-spread effects on metabolism, virulence factor production, stress response, and antimicrobial resistance.

Bacterial infections are becoming progressively more difficult to treat due to antibiotic resistance and the decreasing rate at which new antibiotics are brought to market. The Turos laboratory has attempted to tackle this problem for the last 15 years with the discovery of N-thiolated β-lactams leading to the design of polyacrylate nanoparticles to deliver these and other drugs. Chapter 1 discusses many reported instances of utilizing different types of polymeric nanoparticles to deliver antibiotics. Poly(lactic-co-glycolic acid) (PLGA), poly(alkyl cyanoacrylate) (PACA), poly(styrene-co-butylacrylate), and chitosan are the main polymers used to make nanoparticles. Chapter 2 describes the synthesis, antibacterial activity, and mechanism of action of N-acyl ciprofloxacins, which have demonstrated in vitro bioactivity against Staphyloccocus aureus, methicillin-resistant Staphylococcus aureus, Bacillus anthracis, Enterococcus faecalis, Bartonella, and E. coli. Antimicrobial activity was found to diminish with increasingly lipophilic acyl groups of the derivatives. The N-acyl ciprofloxacins were found to utilize the same mechanism of action as the parent drug, ciprofloxacin, however, several exhibited lower mutation frequencies. Chapter 3 discusses the use of the N-acyl ciprofloxacins as probes for experimentation on the poly(vinyl benzoate) nanoparticles. These compounds were incorporated into poly(vinyl benzoate) nanoparticles, also designed in the Turos laboratory, to determine the effects of the lipophilic acyl groups on drug loading and drug release. N-acyl ciprofloxacins with higher lipophilicities (predicted logP values) experienced higher drug loading than the less lipophilic counterparts. However, the nanoparticles were unable to release large amounts of entrapped drug. N-acyl ciprofloxacins with increased hydrophilicity were found to either not be incorporated at all, or in incredibly small amounts. Drug release studies of these drugs indicate that possible the hydrophobic compounds that were associated with the nanoparticles were done so via adsorption onto the surface resulting in a burst release of drug. The work is concluded in Chapter 4, followed by experimental procedures and spectral data in Chapters 5 and 6.

It has been reported by the American Burn Association that 4,000 people die every year due to burn injury. After survival of the initial trauma, the next major obstacle that must be overcome is combating bacterial infection, the primary cause of mortality for burn victims (Chapter 1). The polyacrylate nanoparticle drug delivery system was created to provide a water-based solution for delivery of highly lipophilic antimicrobials; such as N-thiolated β-lactams, however, with the success of this system for these antimicrobials, it was extended towards other, commercially-available water-soluble antimicrobials through acrylation of the drug monomers, including those with observed bacterial resistance (Chapter 2). Various antibiotics were incorporated into this polyacrylate nanoparticle delivery system by either encapsulation or covalent attachment, and the antibacterial activity was determined in vitro (Chapter 3). Since current treatment of burn wound infections calls for numerous antimicrobials in order to combat the vast array of microbes that may be present in the wound, a multi-drug conjugated nanoparticle system was constructed and analyzed for antibacterial activity against many pathogens commonly found in burn wounds (Chapter 4). In vitro antibacterial assays suggest that the nanoparticle delivery system rejuvenated the activity of penicillin-based antibiotics against formerly resistant microbes, such as methicillin-resistant Staphylococcus aureus. The multi-drug conjugated nanoparticle emulsion had the added benefit of forming a drug-conjugated polyacrylate polymer film through air-drying and polymer coalescence. Upon topical application to a skin abrasion in a mouse model, a protective barrier was created over the wound. This film exhibits mechanical properties similar to elastin, a pliant biological material, giving it the elasticity and flexibility required to move and interact with the wound in the same fashion as intact skin (Chapter 5). This film also permits diffusion of essential nutrients and small molecules (such as oxygen and water) required for wound healing. The emulsion was able to be combined with other biological materials, such as collage, to form a biocomposite material expressing the most optimal properties from each constituent (Chapter 6). In vitro cytotoxicity analysis (Chapter 7) and in vivo toxicity studies (Chapter 8) produced positive results indicating that the multi-drug conjugated nanoparticle emulsion is a promising new treatment for the burn wound and other topical skin and soft tissue infections.

Work in the Turos group over the past five years has focused on the development of N-alkylthio beta-lactams, which show antibacterial activity against Staphylococcus (including MRSA), Bacillus, and others. These compounds do not function in the manner of the traditional beta-lactam antibiotics, but were thought to undergo an intracellular thiol-transfer to coenzyme A. In expanding the SAR of these novel compounds, it was found that N-alkylthio-2-oxazolidinones also exhibit antibacterial activity. Although CoA acts as the thiol-redox buffer in the genera most susceptible to the N-alkylthio beta-lactams, studies on Coenzyme A disulfide reductase (CoADR) show that the redox buffer is not affected by these compounds. However, the recent finding that fatty acid synthesis is affected by the N-alkylthio beta-lactams led to the discovery that these compounds act as prodrugs, and that the asymmetric CoA disulfides produced by in vivo thiol transfer are potent inhibitors of beta-ketoacyl-acyl carrier protein synthase III (FabH) through a novel thiol-disulfide exchange with the active site cysteine. Lactams 2a and 2g were also found to be potent inhibitors of this enzyme. In an effort to produce a CoA mixed-disulfide mimic which could cross the cell membrane, a series of simple aryl-alkyl disulfides were synthesized and tested against E. coli, S. aureus, and B. subtilis. Several of these compounds were found to be very potent antibacterials both in vitro and in vivo, with MICs less than 0.125 micrograms/mL. Comparison of the activities of these disulfides with those of acyl-CoA analogs and CoA mixed disulfides support the assertion that FabH is indeed the cellular target of these potent new compounds.

Microbes survive for an extended period of time on fomites. With new strains of pathogens constantly emerging, it is important to understand their survival and spread and to evaluate the efficiency of new disinfection methods. The purpose of this study was to determine the occurrence of pathogens on fomites, and evaluate different disinfection methods (household bleach and steam vapor). Fomites were sampled in a variety of environments for the presence of pathogens including methicillin intermediate- and resistant- Staphylococcus aureus, Clostridium difficile, Penecillium brevi-compactum, Alternaria alternata and novel H1N1 influenza A. Samples were assayed using both cultural and genetic techniques to determine the microbial occurrence. In disinfection studies, samples were collected before and after disinfectant application. Steam vapor was effective at reducing microbial occurrence by>90% with a minimal contact time of 10-20 seconds on hospital fomites. The material and shape of the surface had an effect on the reduction rate, due to access of the disinfecting agent. In addition, low levels of initial contamination limited the reduction rate. Many surfaces had a starting contamination level of less than 3 log10 and after disinfection the contamination levels, in 69% of the samples, were at or near the limit of detection. This suggests a higher reduction is feasible. Household bleach reduced the presence of mold spores and mycelium by >99% on gypsum-wall board. Reduction rates were influenced by the growth stage of mold, with the mycelium requiring additional disinfectant application to achieve the 99% reduction rate. Novel H1N1 influenza A was not recovered on any surfaces in day care facilities and elementary schools. This is consistent with previous studies looking at H1N1 influenza occurrence in similar environments, though it is different from H3N2 influenza A studies. Survival differences among varying influenza strains are expected to have an impact on pathogen spread and human health risks. Differences can be quantitatively evaluated and used to develop more advanced risk assessment models. Steam vapor and household bleach are effective at reducing risks of pathogens in the environment and are critical interventions in an overall strategy to minimize exposure and prevent disease.

Background: Catheter-associated bloodstream infections have been extensively studied in Intensive Care Unit Patients. However, less is known about the effect these infections have on non-ICU patients. This study attempted to elucidate the nature of the problem effecting non-ICU patients through descriptive analysis. Methods: Data were collected for all non-ICU patients with Staphylococcus aureus (S. aureus) infections at Grady Memorial Hospital in 2006. Demographic and hospitalization information was collected. The hospital epidemiologist responsible for surveillance of infection control programs determined outcomes attributable to the S. aureus infections. Data were entered into an Excel spreadsheet and then imported into EpiInfo 2007. Results: There were 91 S. aureus bloodstream infections documented, 38 infections in non-ICU patients and 52 infections in ICU patients. The descriptive information we accumulated offered potentially important different points between patients with MSSA and MRSA. We found more MRSA infection than MSSA infection in both the non-ICU and ICU patients. Additionally, in both groups those with MRSA infection had a length of stay at least twice as long as patients with MSSA, longer time from admission to positive blood culture, and longer time from positive blood culture to discharge. The most common type of morbidity found was sepsis, which was also the most common morbidity found in those who died. Conclusions: Our hypothesis that non-ICU patients would have increased morbidity and mortality directly attributable to the CA-BSI with S. aureus was proven to be false. Although still an issue, the morbidity and mortality was not as different as we thought it would be. While our hypothesis was proven false, this research provides information that would be well served through further studies.

INTRODUCTION: National obesity rates are leading to higher rates of Type 2 Diabetes, increasing the number of people at risk of invasive infections with Methicillin Resistant Staphylococcus aureus (iMRSA) upon onset of ESRD and hemodialysis. However, an association between adiposity and risk of iMRSA has not been researched. AIM: The purpose of this study is to describe the epidemiological characteristics of an iMRSA cohort in the Atlanta metro area between 2005-2008; to examine BMI-related health outcomes within the cohort; and to compare proportions of BMI categories in this cohort to BRFSS data. METHODS: Surveillance data collected by the CDC EIP program on iMRSA in Atlanta, Georgia was used. BMI was calculated for each eligible case. Statistical analysis was carried out in SPSS. Univariate and multivariate binary logistic regression analysis was performed on select variables. A p-value of < 0.05 and a 95% confidence interval (CI) were used to determine significance. BRFSS BMI data for Georgia was compared to the study population. RESULTS: Overweight and obese cases were more likely to be diagnosed with diabetes by regression analysis. Cases with diabetes were at greater odds of having undergone dialysis within the previous year (univariate OR=2.3, p=0.000; multivariate OR=2.5, p=0.000). The proportion of iMRSA patients with diabetes is much greater (42.8%) than in the general population of the United States (7.8-10.7%). DISCUSSION: The results indicate that there may be a higher risk for iMRSA in overweight and obese individuals, particularly if other adiposity-related health problems are present.

Submitted by ELIANE PATRICIA LINO PEREIRA FRANCHI null (fliane24@yahoo.com.br) on 2016-03-16T12:22:17Z No. of bitstreams: 1 TeseEliane2016_final.pdf: 1593298 bytes, checksum: 660e47b876115bc8cd5e60c3800f6ecd (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-03-18T13:01:25Z (GMT) No. of bitstreams: 1 franchi_eplp_dr_bot.pdf: 1593298 bytes, checksum: 660e47b876115bc8cd5e60c3800f6ecd (MD5) / Made available in DSpace on 2016-03-18T13:01:25Z (GMT). No. of bitstreams: 1 franchi_eplp_dr_bot.pdf: 1593298 bytes, checksum: 660e47b876115bc8cd5e60c3800f6ecd (MD5) Previous issue date: 2016-02-26 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Diante da importância de S. aureus resistente à meticilina (Methicillin-resistant Staphylococcus aureus - MRSA) em feridas, este estudo objetivou estudar a prevalência, fatores de risco e epidemiologia molecular de S. aureus coletados de feridas e narinas de pacientes atendidos nas 17 Unidades Básicas de Saúde do município de Botucatu-SP, Brasil. Após a identificação dos isolados de S. aureus, foram realizados: teste de susceptibilidade à 13 drogas antimicrobianas, identificação do gene de resistência (mecA) e dos genes codificadores da Leucocidina PantonValentine- PVL (pvl), enterotoxinas A-E (sea, seb, sec, sed, e see), hemolisinas α, β e δ (hla, hlb e hld), esfoliatinas A, B e D (eta, etb e etd), biofilme (icaAD) e Toxina-1 da Sindrome do choque tóxico – TSST-1 (tst); tipagem molecular por Pulsed-Field Gel Eletroforese (PFGE), Multilocus sequence typing (MLST) e spa typing. Foram incluídos 171 pacientes, dos quais foram isolados 119 S. aureus. Amostras nasais foram coletadas apenas em 74 pacientes do total estudado. A prevalência de S. aureus e MRSA foi de 51,5% e 8,7%, respectivamente. No geral foram isolados 101 MSSA de 73 pacientes, destes 98 foram isolados de feridas e 21 de narinas; e 18 MRSA de 15 pacientes, sendo 4 isolados de narinas e 14 de feridas, com 6 MRSA com SCCmec tipo II e 12 com SCCmec tipo IV. Os isolados mostraram alto nível de resistencia a penicilina (85%), seguido pela eritromicina (27%), gentamicina (12%), clindamicina (11%), e levofloxacina (6%). Não houve resistência ao sulfametoxazol/trimetoprim, ácido fusídico, tigeciclina, quinupristina/dalfopristina e linezolida. A pesquisa por genes de virulência nos 119 isolados de S. aureus sensíveis e resistentes demonstrou que 42% possuem genes para enterotoxina A, 11% para enterotoxina B, 26% para enterotoxina C e 0,8% para enterotoxina D, 100% para o gene icaA, 95,8% para o gene icaD, 97,5% para o gene da hemolisina alfa, 65% para hemolisina beta, 95% para hemolisina delta, 4,2% para TSST-1 e 2,5% para o gene da PVL. Houve associação entre a presença de S. aureus nas narinas a nas feridas (p<0,01), o mesmo ocorreu para MRSA (p<0,01). A análise multivariada para S. aureus, demonstrou associação negativa com idade (OR: 0,94, IC95%: 0,90-0,98, p<0,01), uso de amoxicilina (OR: 0,16, IC95%: 0,04-0,60, p<0,01) e de ciprofloxacina (OR: 0,28, IC95%: 0,08-0,98, p=0,04). Por outro lado, observou-se associação positiva com uso de benzilpenicilina (OR:3,81, IC95%:1,23-11,82, p=0,02). Houve a formação de oito clusters, com predominância de MSSA que apresentaram as STs: 5, 30, 188, 1635 e spa t002. Os 18 MRSA foram caracterizados pelos STs: 5, 8 e 1176 e pelos spas t002, t008 e t062. Foram isoladas linhagens semelhantes aos clones internacionais USA300, USA500 e USA800. Nossos resultados demonstram a presença de clones importantes de MRSA resistentes e virulentos em pacientes atendidos nas diferentes UBSs estudadas. / Given the importance of methicillin resistant S. aureus (Methicillin-resistant Staphylococcus aureus - MRSA) and wounds, this study aimed to study the prevalence, risk factors and molecular epidemiology related to the presence of S. aureus sensitive and resistant to methicillin in wounds of patients who attended BHUs in a city in Sao Paulo state, Brazil. After the identification of S. aureus isolates was performed: susceptibility testing to 13 antimicrobial drugs, identification of the resistance gene (mecA) and the genes encoding Panton-Valentine Leukocidin - PVL (pvl), enterotoxins AE (sea , seb, sec, sed and see) hemolysins α, β and δ (hla, hlb and hld), esfoliatinas A, B and D (eta, etb and etd), biofilm (icaAD) and Toxin-1 syndrome of toxic shock - TSST-1 (tst); Molecular typing by Pulsed-Field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and spa typing. 171 patients were included and 119 S. aureus isolates. Nasal samples were collected only in 74 patients of the total sample. The prevalence of S. aureus and MRSA was 51.5% and 8.7%, respectively. Overall 101 MSSA were isolated from 73 patients, 98 of these were isolated from wounds and 21 nostrils; MRSA and 18 of 15 patients, 4 isolates from nostrils and 14 wounds, with six MRSA with SCCmec type II and 12 SCCmec type IV. The strains showed high-level resistance to penicillin (85%) followed by erythromycin (27%), gentamicin (12%), clindamycin (11%) and levofloxacin (6%). There was no resistance to sulfamethoxazole / trimethoprim, fusidic acid, tigecycline, quinupristin / dalfopristin and linezolid. The search for virulence genes in the 119 isolates of S. aureus sensitive and resistant, 42% demonstrated presence of genes sea, 11% to seb, 26% to sec , 0.8% to sed, 100% to icaA, 95.8% icaD, 97.5% to hla, 65% to hlb, 95% to hld, 4.2% to tst and 2.5% to pvl. There was an association between the presence of S. aureus in the nostrils to the wounds (p<0.01), the same was true for MRSA (p<0.01). Multivariate analysis for S. aureus, showed a negative association with age (OR: 0.94, 95% CI: 0.90-0.98, p<0.01), use of amoxicillin (OR: 0.16, 95% CI: 0.04-0.60, p<0.01) and ciprofloxacin (OR: 0.28, 95% CI: 0.08-0.98, p=0.04). On the other hand, there was a positive association with use of penicillin G (OR: 3.81, 95% CI: 1.23-11.82, p=0.02). There was the formation of eight clusters, with a predominance of MSSA presenting the STs: 5, 30, 188, 1635 and spa t002. The 18 MRSA were characterized by STs 5, 8 and spas 1176 and the t002, t008 and t062. Strains were isolated similar to international clones USA300, USA500 and USA800. Our results demonstrate the presence of important clones resistant and virulent MRSA patients studied in different UBS. / FAPESP: 2011/10146-7 / FAPESP: 2012/00257-9 / FAPESP: 2013/10975-9