Dissertations / Theses on the topic 'Staphylococcus aureus – Dissertations universitaires'

Les infections ostéo-articulaires à Staphylococcus aureus sont des pathologies fréquentes dont les conséquences peuvent aller de la simple altération cellulaire à un retard de la réparation osseuse ou une réponse inflammatoire excessive. Afin d’étudier ce phénomène, nous avons, dans un premier temps, développé deux modèles d’infections in vitro faisant interagir Staphylococcus aureus et des cellules osseuses primaires issues d’explants chirurgicaux humains. Ces cellules ont été préalablement cultivées dans un milieu standard ou un milieu ostéogénique afin d’obtenir 2 populations à des stades de maturation différents. L’étude de l’internalisation de Staphylococcus aureus, de la mortalité cellulaire et de la production de médiateurs inflammatoires pour ces 2 populations a permis d’établir si l’impact de Staphylococcus aureus variait en fonction de la maturation cellulaire. Dans un second temps, nous avons étudié l’impact de Staphylococcus aureus sur des cellules souches mésenchymateuses dérivées du cordon ombilical. En effet, dans le cadre d’une régénération osseuse en site infecté, les cellules souches mésenchymateuses pourraient être amenées à interagir avec Staphylococcus aureus. Nous avons donc caractérisé la capacité de ces cellules à internaliser Staphylococcus aureus, à survivre face à l’infection et à produire des médiateurs inflammatoires dans notre modèle in vitro d’infection aiguë. Ce projet nous a permis de valider nos modèles d’infection in vitro et de caractériser l’impact de Staphylococcus aureus sur différentes cellules du microenvironnement osseux, donnant ainsi de nouvelles pistes pour le développement de stratégies pour la lutte antibactérienne et l’ingénierie tissulaire osseuse
Staphylococcus aureus-related bone and joint infections are common diseases whose consequences can range from simple cell damage to delayed bone repair or excessive inflammatory response. To study this phenomenon, we have developed two models of in vitro infection with Staphylococcus aureus and primary bone-forming cells derived from human surgical explants. These cells have been previously cultured in a standard medium or osteogenic medium to obtain two populations at different stages of maturation. The study of Staphylococcus aureus internalization, cell death and production of inflammatory mediators in these 2 populations allowed us to establish whether the impact of Staphylococcus aureus varied depending on cell maturation. We also studied the impact of Staphylococcus aureus on mesenchymal stem cells derived from umbilical cord. In case of bone regeneration in infected site, mesenchymal stem cells may have to interact with Staphylococcus aureus. Therefore, we characterized the ability of these cells to internalize Staphylococcus aureus, to survive against the infection and to produce inflammatory mediators in our in vitro model of acute infection. This project allowed us to validate our in vitro infection models and to characterize the impact of Staphylococcus aureus on different cells in the bone microenvironment, providing new approaches for the development of antibacterial strategies and bone tissue engineering

Staphylococcus aureus est une bactérie à Gram positif responsable d'un nombre croissant de pathologies humaines. Notre étude a comme objectif d'étudier la production de cytokines par les cellules mononucléées phagocytaires (monocytes, macrophages péritonéaux et alvéolaires) en réponse à S. Aureus. Nous avons confirmé que la présence de TLR2 n'était pas indispensable pour la reconnaissance de S. Aureus par les macrophages péritonéaux. En revanche, l'activation du TLR2 et la phagocytose de la bactérie étaient requises simultanément pour les monocytes et les macrophages alvéolaires, afin d'obtenir une production optimale de cytokines. De plus, à l'aide d'inhibiteurs spécifiques, nous avons observé que p38 et la Pi3K avaient un rôle central dans ces deux voies d'activauon. Au contraire, Racl et ERK étaient important spécifiquement pour l'activation de la voie intracellulaire chez les macrophages péritonéaux ou alvéolaires. Nous avons cherché à identifier le récepteur intracellulaire permettant la détection de S. Aureus après phagocytose. La transfection d'un dominant-négatif de NOD2 dans la lignée macrophagique RAW 264. 7 inhibait fortement l'activation de NF-KB en réponse à S. Aureus. Mais en utilisant des cellules primaires ne possédant pas NOD2, nous avons observé que l'absence de ce récepteur n'avait pas d'incidence sur la production de cytokines. Enfin, l'injection en intra-nasal d'une souche de S. Aureus, n'a pas montré de différence entre les souris NOD2 déficientes et leurs congénères sauvages. Toutefois, les souris NOD2 déficientes semblaient se remettre plus rapidement de l'infection, que ce soit au niveau de la perte du poids que des lésions pulmonaires. En conclusion, nos travaux mettent en évidence une voie d'activauon des macrophages autre que le TLR2 et dépendante de la phagocytose. NOD2 ne semble pas jouer un rôle critique, lors de la réponse à S. Aureus, que ce soit in vitro ou in vivo. Enfin, nous avons pu observer qu'au sein même de la population mononucléée phagocytaire, il existe de grandes différences quant au rôle de la phagocytose dans la réponse à S. Aureus. Ceci souligne l'hétérogénéité de la réponse de ces phagocytes professionnels selon le compartiment dont ils proviennent
Staphylococcus aureus is a Gram positive bacteria leading to an increasing number of human pathologies. The goal of our work was to study the S. Aureus-induced cytokine production by phagocytic mononuclear cells (i. E monocytes, peritoneal and alveolar macrophages). We confirmed that TLR2 was dispensable for peritoneal macrophage response to S. Aureus. In contrast, TLR2 activation and phagocytosis were both required to obtain a full cytokine production in monocytes and alveolar macrophages. Furthermore, with specific inhibitors, we observed that p38 and Pi3K had a key role in both activation pathways. On the contrary, Racl and ERK were important for macrophages intracellular pathway. We then investigated which receptor was detecting S. Aureus after phagocytosis. Transfection of a dominant-negative form of NOD2 in RAW 264. 7 macrophagic cell line inhibited strongly NF-KB activation in response to S. Aureus. However, using NOD2-deficient primary cells, we observed that the absence of NOD2 did not alter cytokine production. Finally, NOD2-/- animals responses were comparable to wild-type after intra-nasal injection of S. Aureus. Nevertheless, NOD2-/- mice recovered faster than wild-type (weigh gain and pulmonary lesions). In conclusion, our work shows the existence of a TLR2-independant activation pathway that relies on phagocytosis. NOD2 does not play a critical role in S. Aureus response, both in vivo and in vitro. Finally, we show that, in phagocytic mononuclear cells, there is a great discrepancy in the role of phagocytosis in the response to S. Aureus. This work underlines the heterogeneity in the phagocytic cells response depending on the compartment they are derived from

@On appelle infections nosocomiales, les maladies infectieuses contractées au cours d'une hospitalisation qui n'étaient ni en incubation ni présentes à l'admission du patient. Elles peuvent être d'origine endogène ou exogène : le patients lui-même, le personnel, le matériel, les surfaces et l'environnement. Cette contamination peut résulter d'un manque d'efficacité des procédures de nettoyage-désinfection, déficience souvent attribuée à l'état dans lequel se trouve ces germes indésirables (biofilms). Pour réduire ces risques de contamination deux axes ont été abordés : d'une part les prédictions d'adhésion entre le support en acier inoxydable AISI 304 (constituant majeur du matériel en bloc opératoire) et quatre souches responsables d'infections nosocomiales (E. Coli, S. Aureus, P. Aeruginosa et E. Hirae) et d'autres parts, l'évaluation de l'efficacité ainsi que les conditions d'utilisation d'un nouveau désinfectant OXSILÒ 320N sur des cellules planctoniques et des biofilms. L'étude physicochimique a montré toute la complexité des interactions impliquées dans la phase initiale d'adhésion. Si en théorie, il est possible d'enrayer l'adhésion bactérienne à un support en modifiant ses caractéristiques de surface, notre étude montre qu'il est impossible de limiter l'adhésion simultanée des 4 souches bactériennes étudiées au support AISI 304. Face à une telle situation, il est donc impératif d'optimiser les procédures de nettoyage-désinfection. OXSILÒ320N possède une activité de spectre 4 selon la norme AFNOR NF T 72-150 sur des cellules planctoniques à la concentration de 3,13%. Par contre les biofilms sont plus résistants que leurs homologues planctoniques. Ceci serait dû à la présence de mécanismes de défense connus. Selon les recommandations d'utilisation de SODIFRA les conditions optimales pour obtenir un niveau "zéro" de contamination sont : 12,52% d'OXSILÒ320N et un temps de contact de 10 min évitant tout risque infectieux entre deux opérations de courtes ou de longues durées en blocs opératoires. Ces résultats ont mis en évidence la nécessité d'utiliser ce désinfectant à une concentration appropriée et de ne pas négliger les conséquences d'une utilisation en infradose même si celle-ci autorise selon les normes AFNOR une activité bactéricide
@Nosocomial infections are hospital acquired infections. They are not present or incubating when the patient is admitted to the hospital, and are either endogenous or exogenous. Endogenous ones are caused by organisms present in the patient own flora and exogenous infections are caused by organisms originating from medical devices, hospital staff, or environment. This contamination might be the result of a lack of efficiency in cleaning and disinfection procedures, those deficiencies being often attributed to the state in which these harmful microorganisms are found (biofilms). To reduce the risks of contamination, two parts have been developed in this thesis: at first, predictions of adhesion between stainless steel AISI 304 (major component of equipment in operating room) and four nosocomial strains (E. Coli, S. Aureus, P. Aeruginosa et E. Hirae), and in the second part, we determine, the efficient concentration of a new disinfectant OXSILÒ 320N in order to eliminate biofilms. Physico-schemical studies demonstrated the complexity of interactions involved in the initial phase of adhesion. We suggest that a change of the stainless steel surface properties could theoretically limit bacterial adhesion. However it was nearly impossible to limit adhesion between the four studied bacterial strains and the support AISI 304. It is then necessary to optimise preventive hygienic conditions. The bactericidal concentration of OXSILÒ 320N against planktonic cells, according to AFNOR Norm NF T 72-150, was 3,13%. Furthermore, biofilms were commonly more resistant than their planktonic counterparts due to the presence of known resistance mechanisms. According to the SODIFRA recommendations, the optimal conditions, required to avoid any contamination, were a concentration 12,52% and 10 min contact. These conditions can eliminate infection risks during short or long laps between two interventions in an operating room. These results revealed the necessity to use this disinfectant at appropriate concentration and but also the consequences of using it in infradose, although this concentration is considered as an efficient concentration by AFNOR Norm

Since 1999, Methicillin-resistant Staphylococcus aureus (MRSA) outbreaks have occurred in many correctional facilities. Even after the Federal Bureau of Prisons developed clinical practice guidelines on the management of MRSA within correctional facilities, the prevalence of MRSA decreased only insignificantly. Other researchers suggested infection control compliance was equally as important as developing clinical practice guidelines in reducing the incidence of MRSA. Several studies identified the healthcare professionals' nonadherence and inconsistencies to clinical practice guidelines as contributors to MRSA transmission. Accordingly, this project was designed to develop evidence-based recommendations for improving nurse professionals' adherence to MRSA practice guidelines in correctional settings. Using the health belief model as the theoretical framework, this project examined the nurse professionals' perceptions as well as their level of knowledge regarding MRSA by using an original instrument, Knowledge and Health Beliefs Regarding MRSA Questionnaire. The study employed a quantitative design with a purposeful sample of 36 participants using social media. Through descriptive statistical analysis, it was determined that MRSA training and education were the greatest barriers among the nurse professionals in taking MRSA preventive action (64%, n = 23). Based on the findings, assessing the educational needs of the nurse professionals must become the priority when designing infection control programs. This study contributes to social change by recognizing the potential health impact of MRSA and cautions that if public health officials do not control MRSA within correctional settings, such behavior can affect the transmission of MRSA both nationally and globally.

Chiropractic training involves many hours of skin contact, and chiropractors have manual contact with millions of patients annually, but chiropractic has only had professional clinical hygiene guidance since 2010. Methicillin-resistant Staphylococcus aureus (MRSA) is the most common cause of cultured skin and soft tissue infection (SSTI) in the United States. Using the epidemiologic triad of person, place, and time as a framework, this quantitative, cross-sectional study obtained the first assessment of MRSA SSTI incidence among chiropractic students and its association with infection control behaviors (hand and table hygiene, sharing gowns, and sharing lotion) and initiation of patient care. The study obtained surveys from 312 students attending half (9/18) of U.S. chiropractic campuses. Associations were assessed by Ï?2 and Fisher's exact test. Stratum specific effects were assessed. Two logistic regression models were produced. The results were that attendance at Campus 6 was associated with postmatriculation MRSA SSTI in univariate analysis, p = 0.010. There was an interaction between campus attended, sharing lotion, and postmatriculation MRSA SSTI, with the Mantel-Haenszel pooled estimate varying significantly from unity, Ï?2 (1) = 6.75, p = 0.009. No other association between any assessed factor and MRSA SSTI was detected. Logistic regression models were significant (p < 0.05), but the composing variables were not. For social change, chiropractic colleges should instruct students and chiropractic associations could encourage members not to share massage lotions and emollients during the practice of manual therapy to help prevent MRSA SSTI.

Methicillin-resistant Staphylococcus aureus (MRSA) strains have spread in Saudi Arabia, increasing morbidity, mortality, and financial burdens. Recent studies have suggested the phenotyping methods typically used to classify MRSA as either health care MRSA (HA-MRSA) or community-associated MRSA (CA-MRSA) cases are unreliable, because they lack concordance with the results of genotyping. Yet the expense associated with genotyping precludes its use in the Saudi Aramco population in Saudi Arabia. The absence of a standardized and affordable method to classify MRSA into CA-MRSA and HA-MRSA has been a challenge for infection control programs in Saudi Arabia. The objective of this quantitative, secondary data analysis was to determine the most reliable phenotyping approach to strain identification using John Hopkins Aramco hospital data. The ecological and antibiotics selection pressure theories framed this research. The results of concordance, and sensitivity and specificity tests, suggested hospital admission profiles and susceptibility pattern were the most reliable phenotypic predictors of genotype-based classifications. Multiple logistic regression for susceptibility pattern (OR = 15.47, p < .001) and hospital admission profile (OR = 2.87, p = .008) confirmed those results, whereas all other variables were not found to be statistically significant. These results can be used to clarify the epidemiological and molecular factors that affect the transition of MRSA from health care facilities to the Saudi Aramco community. Implications for positive social change include faster and more reliable classification of MRSA to aid in disease surveillance and the selection of appropriate treatments to reduce MRSA-related morbidity and mortality.

Staphylococcus is a significant cause of human infection and mortality, worldwide. Currently, there are greater than 60 taxa within Staphylococcus, and nearly all are pathogenic. The collective potential for virulence among species of Staphylococcus heightens the overall clinical significance of this genus and argues for a thorough understanding of the evolutionary relationships among species. Within Staphylococcus, aureus is the most common cause of human infection, where nasal carriage of this bacterium is a known risk factor for autoinfection. The predisposition to infection by nasal carriers of S. aureus, and the ease with which strains are transferred between individuals, suggests that nasal carriage is a major vector for the transmission of virulent strains throughout the community. This hypothesis, however, has not been assessed in any great detail to identify the genetic relationships between clinical isolates of S. aureus and those strains being carried asymptomatically throughout the community. Also lacking within this field is a unified and robust estimate of phylogeny among species of Staphylococcus. Here, we report on a highly unified species phylogeny for Staphylococcus that has been derived using multilocus nucleotide data under multiple Bayesian and maximum likelihood approaches. Our findings are in general agreement with previous reports of the staphylococcal phylogeny, although we identify multiple previously unreported relationships. Regardless of methodology, strong nodal support and high topological agreement was observed with only minor variations in results between methods. Based on our phylogenetic estimates, we propose that Staphylococcus species can be evolutionarily clustered into 15 groups, and six species groups. In addition, our more defined phylogenetic analyses of S. aureus revealed strong genetic associations between both nasal carriage strains and clinical isolates. Genetic analyses of hypervariable regions from virulence genes revealed that not only do clinically relevant strains belong to identical genetic lineages as the nasal carriage isolates, but they also exhibited 100% sequence similarity within these regions. Our findings indicate that strains of S. aureus being carried asymptomatically throughout the community via nasal colonization are genetically related to those responsible for high levels of infection and mortality. Due to nasal carriage of S. aureus being a risk factor for autoinfection, standardized preoperative decolonization has become a major consideration for the prevention of nosocomial infection. Toward this end, we have identified the macrocyclic ?-defensin analogue RC-101 as a promising anti-S. aureus agent for nasal decolonization. RC-101 exhibited bactericidal effects against S. aureus in both epithelium-free systems, and ex vivo models containing human airway epithelia. Importantly, RC-101 exhibited potent anti-S. aureus activities against all strains tested, including USA300. Moreover, RC-101 significantly reduced the adherence, survival, and proliferation of S. aureus on human airway epithelia without any noted cellular toxicity or the induction of a proinflammatory response. Collectively, our findings identify RC-101 as a potential preventative of S. aureus nasal colonization.
ID: 030646199; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Thesis (Ph.D.)--University of Central Florida, 2011.; Includes bibliographical references (p. 140-159).
Ph.D.
Doctorate
Molecular Biology and Microbiology
Medicine
Biomedical Sciences

Methicillin-resistant staphylococcus aureus (MRSA) has become resistant to antibiotics. The purpose of this quantitative, retrospective cohort study was to examine the relationship between length of hospitalization and invasive MRSA infection rates among different racial and ethnic groups in the 8 service planning areas (SPAs) of Los Angeles County. Cane, O'Connor, and Michie's theoretical domain framework was used. Secondary data from the Healthcare-Associated (HA) Infections Program of the California Department of Public Health were analyzed. For the first research question, a Pearson correlation analysis was conducted to assess the relationships between length of hospitalization and invasive HA-MRSA infection rates and counts. Length of hospital stay was not correlatedwith HA-MRSA infection rates; however, it was strongly and positively correlated with HA-MRSA infection counts. For the second research question, a one-way ANOVA was conducted on the infection count rate data, with SPA as the between-subjects factor. The results were statistically significant, indicating that HA-MRSA infection counts varied among the 8 SPAs. The findings might help medical professionals better understand the risk factors associated with MRSA infections. In doing so, findings may relieve some of the burden on the U.S. health care system and improve the overall quality of life of the patients involved.

The major research interest of our laboratory is focused on the replication-specific DNA polymerase III (pol III) family in Gram+ bacteria, and has used Bacillus subtilis (BS) as the primary model enzyme for study. The long range objective of the work of the laboratory is to gain a deeper understanding of the structure and function of Gram+ bacterial DNA polymerase IIIs, a structurally unique class of DNA-dependent DNA polymerase which are uniquely susceptible to inhibition by a specific class of dGTP analogs. The project described in this thesis dissertation deals specifically with the pol III of the Gram+ organism Staphylococcus aureus, and involves the isolation and characterization of DNA pol III from this clinically relevant pathogenic bacterium. A homology-based strategy was devised to clone the structural gene specifying DNA polymerase III of Staphylococcus aureus, SA polC. SA polC was found to contain a 4305-bp open reading frame (ORF) encoding a 162.4 kDa polypeptide, and mapped between Ω1074[Tn551] and recA/ngr on the genome map of S. aureus NCTC 8325. The 1435 codon ORF was engineered into the E. coli expression plasmid pBS(KS) under the control of the lac promoter and its repressor. The translational signals of SA polC were reengineered using expression cassette PCR (ECPCR) to optimize the in vitro expression of SA polC in E. coli. Derepression of E. coli transformants carrying the recombinant vector generated high level expression of active recombinant pol III. The recombinant SA pol III was purified to greater than 98% homogeneity and was shown by N-terminal amino acid analysis to be the bona fide product of the 4305-bp SA polC ORF. The physical and catalytic properties of recombinant SA pol III and its responsiveness to inhibitors of the HPUra type were similar to those of Bacillus subtilis (BS) pol III. Comparative structural analysis of the primary structure of SA pol III and the pol IIIs of B. subtilis and the Gram+ relative Mycoplasma pulmonis indicated strong conservation of essential catalytic domains and a novel zinc-finger motif. Comparison of the primary structures of E. coli pol III and these three Gram+ enzymes suggested a specific evolutionary relationship between the pol IIIs of Gram+ and Gram- bacteria.

The bacterium Staphylococcus aureus has been an important human ailment for centuries, and with the overuse of antibiotics, methicillin resistant Staphylococcus aureus (MRSA) has emerged as a deadly, costly pathogen worldwide. Healthy carriers can become sick or can spread MRSA without symptoms. The amount of asymptomatic colonization among healthy college students and risk factors for colonization by MRSA are not well understood. According to the epidemiologic triangle model, the host (students who take antibiotics or have a history of skin infections), the infectious agent (MRSA) and the environment (direct contact with people, animals, or objects that may harbor MRSA) all play an important role in this disease. This study explored MRSA colonization rates among healthy students at a community college and explored the possibility that students exposed to sources of MRSA might have a higher colonization rate. Using a cross-sectional quantitative design with stratified sampling, risk factors to include student's discipline, gender, race, work, and leisure exposure were surveyed. In tandem, Mannitol Salt Agar and MRSA Select Agar were inoculated from nasal swabs to identify students colonized by MRSA. The data were analyzed using contingency tables and Chi Squares. Significant risk factors identified included students who had a major that involved touching shared equipment and/or those who were in majors such as nursing, students who had close contact with animals, and students who had a skin infection. The implication for positive social change include improved awareness of MRSA colonization and risk factors which can lead to better prevention strategies and increased awareness among the student population.

Staphylococcus aureus (SA) is a major human pathogen that colonizes the anterior nares in 30% of the human population. Though nasal carriage of SA is a known risk factor for the subsequent spread of SA infections, the dynamics of SA nasal colonization is poorly understood. Our research focuses on understanding the host and bacterial factors that might contribute to the human nasal colonization by SA. In an attempt to elucidate the host response to SA, we performed an autologous human in vivo nasal colonization study, which showed decreased survival rates of SA in hosts who elicited a robust immune response. We also identified a significant correlation between SA nasal colonization and the expression of host proinflammatory, chemotactic and growth factors. Additionally, we functionally disrupted a major autolysin, atl a surface expressed bacterial protein that plays multiple roles in cell separation, adhesion and biofilm formation of SA. Microscopic analysis of the ?atl strains showed phenotypic differences, including cell clumping and cluster formation due to defective cell separation, which confirmed the functional loss of atl. Subsequent analysis of the ?atl and wild-type strains revealed that there was no significant difference in their ability to adhere to human nasal epithelial cells (hNEC) in an ex vivo hNEC model. Similarly, our competitive in vivo human nasal colonization study, in which equal colony-forming units of each wild-type and ?atl SA strain were inoculated in the anterior nares of donors, showed similar survival rates between wild-type and ?atl. These results suggest that Atl might not be directly involved in the adherence and colonization of SA to the anterior nares. Furthermore, our study suggests that host factors might play a predominant role in determining SA colonization to human anterior nares.
M.S.
Masters
Molecular Biology and Microbiology
Medicine
Biotechnology

Thesis (PhD)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Numerous studies conducted internationally have identified and described several endemic methicillin-susceptible Staphylococcus aureus (MSSA) clones. However, only some of these clones are associated with methicillin resistance (CC5, CC8, CC22, CC30 and CC45). To date, studies reporting on the population structure of S. aureus isolated in South Africa represent limited demographic areas, focus on methicillin-resistant S. aureus (MRSA) only and have displayed little emphasis on virulence. This study was undertaken to elucidate the population structure of S. aureus isolated from specific clinical sources at Tygerberg hospital, and to investigate specific host-pathogen interactions of representative isolates. Consecutive non-repetitive clinical S. aureus isolates were collected over one year (September 2009/2010) with patient demographics and limited clinical information. Strains were typed by PFGE and molecular markers (spa, multi-locus sequence typing (MLST), agr, Staphylococcal Chromosome Cassette mec and Panton-Valentine leukocidin (PVL)). Representative isolates were selected and investigated for the presence of virulence genes, adherence (to immobilised fibronectin [Fn], fibrinogen [Fg], collagens IV [CnIV] and VI [CnVI]), cellular invasion and cell death induction. Statistical association were determined between all in vitro results and methicillin-resistance, clonality, patient HIV status and bacterial PVL status. Fifteen percent of the isolates (n = 367) were MRSA. Forty four present of isolates were PVL+. agr I-IV and SCCmec I-V were identified. The MSSA population was diverse: ST22 (dominant), ST1865 and ST121 were PVL+. ST45, ST1863 and ST15 were PVL-. PVL- MRSA were diverse: ST612-MRSA-IV (dominant), ST5-MRSA-I, ST239-MRSA-III, ST36-MRSA-II and ST22-MRSA-IV. The genes fnbA/B (fibronectin-binding protein A/B), clfA/B (clumping factor A/B), eap (extracellular adherence protein), nuc (nuclease), coa (coagulase) and hld (delta toxin) were detected in all representative isolates. The CC8 and CC6 isolates adhered strongly to all ligands (100-700% of control, ligand dependent), while isolates of CC45, CC22 and CC88 adhered strongly only to Fg and Fn. The CC30, CC15, and CC12 isolates adhered extremely strongly to CnIV (>300%) and CC8, CC15, and C6 to CnVI (>200%). Isolates from CC30, CC8, CC15, CC6, CC12, CC97, CC88 and CC45 were highly invasive (>100%). ST121 was non-invasive (>50%). Isolates of CC5, CC30 and CC121 were non-cytotoxic (<50%), while isolates of CC22, CC8, CC15, CC45 and CC88 were very cytotoxic (>70%). No significant difference was observed in adherence or cell death induction of MRSA vs. MSSA clones or between isolates from HIV+ vs. HIV- persons. PVL- isolates displayed higher cellular invasiveness than PVL+ isolates. The presence of ST612-MRSA-IV, ST22-MRSA-V and ST8-MRSA-V points to local SCCmec acquisition, as we found MSSA isolates with the same spa types. Numerous MSSA clones were prevalent, but do not appear to have a major common genetic background with MRSA. PVL was highly prevalent among MSSA, indicating acquisition of PVL genes independently of SCCmec. The abilities to adhere to specific immobilised ligands in vitro were diverse and grouped with the genetic background, while the vast majority of isolates were invasive and induced significant cell death. We can conclude that the population of S. aureus at Tygerberg hospital is composed of a vast number of MSSA and MRSA clones, which display varying patters of adherence to selected ligands and of which, the majority clones are invasive and cytotoxic.
AFRIKAANSE OPSOMMING: Talle internasionale studies het verskeie endemiese metisillien vatbare Staphylococcus aureus (MSSA) klone geïdentifiseer en beskryf. Slegs 'n paar van hierdie klone word geassosieer met metisillien weerstandigheid (Klonale kompleks (KK) 5, KK8, KK22, KK30 en KK45). Studies oor die bevolking struktuur van S. aureus geïsoleer in Suid-Afrika is tot dusver beperk tot demografiese gebiede, fokus slegs op metisillien-weerstande S. aureus (MRSA) en het min klem op virulensie geplaas. Hierdie studie is onderneem om die bevolking struktuur van S. aureus, geïsoleer vanaf spesifieke kliniese bronne, in die pasiëntpopulasie van Tygerberg-hospitaal te ondersoek en om ondersoek in te stel na spesifieke gasheer-patogeen interaksies van verteenwoordigende isolate. Opeenvolgende, nie-herhalende en suiwer kliniese S. aureus isolate is versamel oor ´n periode van een jaar (September 2009/2010), tesame met pasiënt demografiese- en beperkte kliniese inligting. Stamme is deur PFGE en molekulêre merkers (spa, MLST, agr, SCCmec en PVL) beskryf. Verteenwoordigende isolate is gekies en ondersoek vir die teenwoordigheid van virulensie gene, aanhegting ( aan geïmmobiliseerde fibronektien [Fn], fibrinogeen [Fg], kollageen IV [CnIV] en kollageen VI [CnVI]), sellulêre indringing en die induksie van seldood. Statistiese assosiasies is bepaal tussen alle in vitro resultate en methicillin-weerstandigheid, klonaliteit, pasiënt MIV status en bakteriese PVL status. Fyftien persent van die isolate (n = 367) was MRSA. Vier-en-veertig van die isolate was PVL+. agrI-IV en SCCmec I-V is geïdentifiseer. Die MSSA bevolking was divers: ST22 (dominant), ST1865 en ST121 PVL +. ST45, ST1863 en ST15 was PVL+. PVL- MRSA was divers: ST612-MRSA-IV (dominant), ST5-MRSA-I, ST239-MRSA-III, ST36-MRSA-II en ST22-MRSA-IV. Die gene fnbA/B (fibronektien A/B), clfA/B (klontings faktor A/B), eap (ekstrasellulêre aanhegtings protein), nuc (nukease), coa (koagulase) en hld (delta toksien) was aangetref in alle verteenwoordigende isolate. Isolate van KK8 en KK6 het sterk aan alle ligande (100-700% van kontrole, ligand-afhanklike) aangeheg, terwyl isolate van KK45, KK22 en KK88 slegs sterk aand fibronektien en fibrinogeen aangeheg het. Isolate van KK30, KK15, en KK12 het baie sterk aan CnIV (> 300%) aangeheg en KK8, KK15, en KK6 and CnVI (> 200%). Isolate van KK30, KK8, KK15, KK6, KK12, KK97, KK88 en KK45 was hoogs indringend (> 100%). ST121 was nie-indringende (> 50%). Isolate van KK5, KK30 en KK121 was nie-sitotoksiese (<50%), terwyl isolate van KK22, KK8, KK15, KK45 en KK88 baie sitotoksies was (> 70%). Geen betekenisvolle verskil is waargeneem in die aanhegting of seldood induksie van MRSA teenoor MSSA klone of tussen isolate van MIV+ teenoor MIV- persone nie. PVL- isolate het hoër sellulêre indringing as PVL+ isolate vertoon. Die teenwoordigheid van ST612-MRSA-IV, ST22-MRSA-V en ST8-MRSA-V verwys na die plaaslike verwerwing van SCCmec, aangesien ons MSSA isolate beskryf het met dieselfde spa-tipes. Talle MSSA klone was algemeen, maar het nie 'n beduidende genetiese agtergrond met MRSA vertoon nie. PVL was baie algemeen onder MSSA isolate en die PVL gene is dalk onafhanklik van SCCmec verkry. Die vermoë om aan spesifieke geïmmobiliseer ligande in vitro aan te heg was divers en groepeer met die genetiese agtergrond, terwyl die meerderheid van die isolate indringend was en kon betekenisvolle sel dood veroorsaak. Ons kan aflei dat die bevolking van S. aureus by die Tygerberg hospitaal saamgestel is uit 'n groot aantal van MSSA en MRSA klone, wat verskillende patrone van aanhegting aan geselekteerde ligande vertoon en waarvan die meeste klone indringende en sitotoksies is.
DFG/NRF International Research and Training Group (IRTG) 1522 “HIV and associated infectious diseases in Southern Africa”
National Research Foundation
Medical Research Council, Medi-Clinic
Harry Crossley Fund (Stellenbosch University)
Stellenbosch University

Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: S. aureus causes serious infections in the hospital and community settings. The rate of MRSA infections are rapidly increasing worldwide. Currently, at Tygerberg hospital, approximately a third of S. aureus isolates are MRSA. This was the first epidemiological study of S. aureus conducted at Tygerberg Hospital that included prospective clinical data on patients with S. aureus bacteraemia together with spa typing of strains and the detection of the mecA and pvl genes in a multiplex PCR. Clonal cluster groups of S. aureus isolates were obtained by BURP analysis and compared to international important clones. The molecular epidemiology of hospital acquired (HA), health-care associated (HCA) and community acquired (CA) S. aureus bacteraemic strains at this hospital was examined. Lastly, repeat isolates of patients were collected to analyse any possible organism-related factors associated with persistent and recurrent bacteraemia. We investigated a total number of 113 S. aureus strains from 104 patients (70% MSSA, 30% MRSA). Repeat strains consisted of nine isolates (from 5 patients). All isolates were obtained from blood cultures collected during the period March 2008 to May 2009. Phenotypic and genotypic detection of methicillin resistance correlated well. According to the literature, most CA-MRSA strains are distinguishable from HA-MRSA strains based upon the presence of the PVL toxin. However, no CA-MRSA was detected in our study, therefore the association between HA-MRSA versus CA-MRSA strains could not be analysed. In this study, CA-MSSA was identified in 22% of all MSSA isolates versus 0% CA-MRSA. PVL positive strains were found in 22.7% of all MSSA isolates with no detection in MRSA isolates. It was noted that MRSA strains clustered in spa CC-701 and CC-012, whereas CC-002 only contained MSSA strains. Likewise HA-strains representing the majority of MRSA strains also clustered in spa CC-701 and CC-012. Forty nine spa types were identified in 89.3% of all isolates, whereas 9.7% of these strains were non-typeable. Five novel spa types were revealed. We detected a diverse number of spa-types that correlated to international clones. The most predominant spa type found in our setting was t037 (only in MRSA), followed by t891. According to the literature, t037 is associated to the Brazilian/Hungarian clone (SCCmec type III; ST 239). Our findings, as well as other South African studies, indicate that t037 has been identified in clinical strains from numerous provinces in South Africa. Interestingly, all isolates from spa type t891 were PVL positive MSSA. Bacteraemia cases were predominantly related to catheter sepsis, followed by skin and soft tissue infections (SSTI). Only one persistent bacteraemia case was identified related to a HA-SSTI. Recurrent bacteraemia cases were found in patients on dialysis for chronic renal failure and in burns patients related to intravascular catheter infections. The local epidemiology of S. aureus and the prevalence rate of different strains are important to investigate. The information provided contributes to the epidemiology of staphylococcal strains causing bacteraemia in our setting. These insights are useful for optimal diagnostic and therapeutic measures. The techniques developed can be used to identify outbreaks and recurrent infections.
AFRIKAANSE OPSOMMING: S. aureus veroorsaak ernstige infeksies in die hospitaalomgewing en in die gemeenskap. Wêreldwyd, neem metisillien-weerstandige S. aureus (MRSA) infeksies vinnig toe. Huidiglik by Tygerberg hospitaal is ongeveer ‘n derde van S. aureus isolate MRSA. Hierdie is die eerste epidemiologiese studie by Tygerberg hospitaal wat prospektiewe kliniese data van pasiënte met S. aureus bakteremie saam met spa tipering en aantoning van die mecA en pvl gene in ‘n multipleks PKR insluit. Klonale groepe (spa-CC) van MRSA en MSSA isolate is deur BURP analise verkry, en vergelyk met internasionaal belangrike klone. Die molekulêre epidemiologie van hospitaalverworwe (HA), gesondheidsorgverworwe (HCA) en gemeenskapsverworwe (CA) S. aureus bakteremie by hierdie hospitaal is ondersoek. Laastens, oorspronklike en daaropvolgende herhaal isolate is gekollekteer om moontlike organisme- faktore geassosieerd met persisterende en herhalende bakteremiese episodes te analiseer. Ons het in totaal 113 S. aureus isolate van 104 pasiënte ondersoek (70% MSSA, 30% MRSA). Nege isolate (van 5 pasiënte) was herhaal isolate. Alle isolate was afkomstig vanaf bloedkulture wat gedurende die periode Maart 2008 tot Mei 2009 gekollekteer is. Fenotipiese en genotipiese aantoning van metisillien weerstandigheid het goed gekorreleer. Volgens die literatuur kan die meeste CA-MRSA isolate van HA-MRSA isolate onderskei word op grond van die teenwoordigheid van die PVL toksien. Geen CA-MRSA is egter in ons studie gevind nie, dus kon die assosiasie tussen HA-MRSA en CA-MRSA isolate nie ondersoek word nie. CA-MSSA was in 22% van alle MSSA geidentifiseer teenoor 0% CA-MRSA. PVL is in MSSA isolate gevind (22.7% van alle MSSA) maar glad nie in MRSA nie. Dit is opgemerk dat MRSA isolate hoofsaaklik in spa CC 701 en CC-012 kloongroepe voorkom, teenoor kloongroep CC-002 wat slegs MSSA isolate bevat het. Soortgelyk het HA-isolate wat die meerderheid van MRSA isolate verteenwoordig het ook in kloongroepe 1 & 2 gegroepeer. Nege-en-veertig spa tipes is geïdentifiseer in 89.3% of alle isolate en 9.7% was nie-tipeerbaar. Vyf nuwe spa tipes is getoon. Ons het ‘n diverse aantal spa-tipes geïdentifiseer wat met internasionale klone gekorreleer het. Die mees dominante spa tipe in ons omgewing was t037 (slegs in MRSA), gevolg deur t891. Volgens die literatuur word t037 met die Brasiliaanse/Hongaarse kloon geassosieer (SCCmec tipe III; ST 239). Ons bevindings, asook ander Suid Afrikaanse studies, dui aan dat t037 in kliniese isolate vanaf talle provinsies in Suid-Afrika aangetoon is. Van belang is dat al die isolate van spa tipe t891 MSSA en PVL positief was. Bakteremiese gevalle was hoofsaaklik geassosieer met kateter-sepsis, gevolg deur vel en sagteweefsel infeksies (SSTI). Slegs een persisterende bakteremiese geval was geïdentifiseer geassosieer met HA-SSTI. Herhalende bakteremiese episodes is in pasiënte op dialise vir kroniese nierversaking en in brandwonde pasiënte met intra-vaskulêre kateter infeksies aangetoon. Die lokale epidemiologie van S. aureus en die prevalensie koers van verskillende stamme is van belang. Hierdie inligting dra by tot kennis van die epidemiologie van stafilokokkale stamme wat in ons omgewing bakteremie veroorsaak. Hierdie insigte is nuttig vir optimale diagnostiese en terapeutiese riglyne. Die tegnieke wat ontwikkel is, kan gebruik word om uitbrake en herhalende infeksies te identifiseer.

La Broncho-Pneumopathie Chronique Obstructive (BPCO) est une maladie pulmonaire inflammatoire consécutive à l'exposition chronique à la pollution atmosphérique et surtout au tabagisme dans environ 90% des cas. Cette maladie se caractérise par une obstruction des bronches due à une hypersécrétion de mucus, une hypertrophie des muscles lisses, ainsi qu’une destruction de la paroi des alvéoles respiratoires amenant le patient à l’emphysème. Le stress induit par la fumée de cigarette provoque une activation de la barrière épithéliale pulmonaire associée à une altération de la réponse immunitaire responsable d’une susceptibilité accrue aux infections pulmonaires. De ce fait, les patients atteints de cette maladie développent des exacerbations principalement liées à ces infections bactériennes en particulier à Non-Typable Haemophilus influenza (NTHi) et Streptoccocus pneumoniae (Sp).La cytokine IL-22 est un acteur très important des défenses antibactériennes et du maintien de la barrière épithéliale. Cette cytokine appartient à la grande famille de l’IL-10, et à la sous-famille des cytokines IL-20 composée de l’IL-19, l’IL-20 et l’IL-24. L’IL-22 se lie au récepteur formé par les sous-unités IL-10Rb et IL-22Ra, tandis que les cytokines IL-19, IL-20 et IL-24 utilisent deux récepteurs associant l’IL-20Rb avec l’IL-20Ra ou l’IL-22Ra. Il a été démontré que les cytokines de la famille IL-20 (IL-19, IL-20, IL-24) agissent sur la clairance bactérienne au cours d’une infection cutanée par Staphylococcus aureus (Myles et al., 2013), en inhibant la production des cytokines IL-17 et IL-22. De plus, des précédents travaux au laboratoire, ont montré un défaut de l’expression des cytokines IL-17 et IL-22 qui participaient à la susceptibilité à l’infection chez les souris atteintes de BPCO (Pichavant et al., 2015). Enfin, nos données actuelles montrent que l'exposition à la fumée de cigarette augmente l'expression des cytokines de la famille IL-20 et que l'inhibition de cette voie permet de bloquer le développement d'épisodes d'exacerbation chez des souris BPCO.L'objectif de cette thèse est de préciser le rôle des cytokines IL-20 dans la réponse à l'infection bactérienne (Sp, NTHi) tant dans un contexte physiologique qu'au cours d’un contexte mimant la BPCO. Pour cela, nous nous focaliserons sur le rôle de l’épithélium pulmonaire tant dans la production que dans la fonction de ces cytokines en contexte infectieux.Pour répondre à ces questions, nous avons analysé l’expression des cytokines IL-20 par l’épithélium pulmonaire in vitro et ex vivo dans un modèle murin mimant l’exacerbation de la BPCO ainsi que dans des biopsies pulmonaires de patients fumeurs atteints ou non de BPCO. Dans un second temps nous avons évalué la modulation par un anticorps bloquant le récepteur des cytokines IL-20 (anti-IL-20Rb) au cours de la réponse anti-infectieuse de l'épithélium dans nos modèles in vivo (souris infectées par Sp) et in vitro (cellules épithéliales de trachées murines). Nous avons en parallèle évalué l'implication des cytokines IL-20 dans la réparation épithéliale.L’ensemble des résultats acquis au cours de la thèse nous a permis de démontrer l'implication des cytokines IL-20 et de préciser leur rôle sur l’épithélium pulmonaire au cours de l'infection bactérienne ainsi que dans la pathologie de la BPCO. De plus, les résultats obtenus avec l’anticorps neutralisant anti-IL-20Rb dans ces contextes d’infections et de BPCO, font de celui-ci une potentielle piste thérapeutique pour le traitement des lésions dues à l’infection
Chronic Obstructive Pulmonary Disease (COPD) is an inflammatory lung disease due to chronic exposure to air pollution and especially to cigarette smoke exposure in approximately 90% of the cases. This disease is characterized by obstruction of the bronchi due to hypersecretion of mucus, hypertrophy of the smooth muscles, and destruction of the alveolar wall leading the patient to emphysema. The stress induced by cigarette smoke exposure causes activation of resident cells including pulmonary epithelial cells and an alteration of the immune system responsible for an increased susceptibility to pulmonary infections. As a result, patients with this disease develop exacerbations especially du to Non-Typable Haemophilus influenza (NTHi) and Streptoccocus pneumoniae (Sp).The IL-22 cytokine plays a key role in antibacterial defenses and maintenance of the epithelial barrier. This cytokine belongs to the large IL-10 family, and to the IL-20 cytokine subfamily also including IL-19, IL-20 and IL-24. IL-22 binds to the receptor formed by the IL-10Rb and IL-22Ra subunits, while the IL-19, IL-20 and IL-24 cytokines binds to IL-20Rb associated with either IL-20Ra or IL-22Ra subunits. IL-20 cytokines (IL-19, IL-20, IL-24) have been shown to impair bacterial clearance during cutaneous infection with Staphylococcus aureus (Myles et al., 2013), by inhibiting the production of IL-17 and IL-22 cytokines. In addition, previous work in the laboratory showed a defect in the expression of IL-17 and IL-22 cytokines contributing to the susceptibility to infection in COPD mice (Pichavant et al., 2015). In fact, our current data show that exposure to cigarette smoke increases cytokine expression of the IL-20 family and that inhibition of this pathway blocks the development of exacerbation episodes in COPD mice.The aim of this thesis is to clarify the role of IL-20 cytokines in the response to bacterial infections (Sp, NTHi) both in a physiological context and in a context mimicking COPD. To do so, we will focus on the role of pulmonary epithelium both in the production and function of these cytokines in infectious context.To answer these questions, we analyzed the expression of IL-20 cytokines by pulmonary epithelium in vitro and ex vivo in a mouse model mimicking the COPD exacerbation as well as in pulmonary biopsies of smokers and non-smokers patients and of COPD patients. In a second step we evaluated the modulation by an IL-20 receptor blocking antibody (anti-IL-20Rb) of the anti-infectious response in our in vitro (murine tracheal epithelial cells) and in vivo models (Sp-infected mice). In parallel, we evaluated the involvement of IL-20 cytokines in the epithelial repair.All the results acquired during the thesis allowed us to demonstrate the expression of IL-20 cytokines and to demonstrate their role on the pulmonary epithelium during bacterial infection as well as in COPD. In addition, the results obtained with the anti-IL-20Rb neutralizing antibody in these contexts of infections and COPD, suggests a potential therapeutic application for respiratory infection

Thesis (D. Tech. (Biomed. Tech.)) -- Central University of Technology free State, 2010
Consumer demands for fresh, microbiologically safe foods with high organoleptical and nutritional quality has led to the development of novel food preservation technologies as alternatives or enhancements to traditional preservation techniques. An example of these novel preservation technologies is high hydrostatic pressure (HHP) processing. It involves the applications of static pressure of 50 to 1 000 mega Pascals (MPa) to solid or packaged liquid foods, with varying holding times. The combination of factors to enhance preservation is increasingly being used in industry, e.g. the use of different temperatures and additives (hurdles) can enhance the preservative effect of HHP. In this study the influence of HHP on organism viability and growth response was assessed. The organisms evaluated included Escherichia coli O111, Listeria monocytogenes (UAFSBCC) and Staphylococcus aureus (ATCC 25923), in peptone water, which was subjected to HHP of 200 MPa for 15 minutes at 8 and 50 ºC respectively. Subsequent to the mentioned pressurisation, sub-culturing was performed and growth responses were evaluated at 0, 6, 18, 24, 30, 42 and 48 hours. Bacterial survival and growth response was measured by means of intact cell count, colony forming units and optical density. From the results it was eminent that bacterial cells were only sublethally injured and were able to repair within 48 hours of enriched sub-culturing. E. coli O111 proved to be most sensitive to HHP with Staphylococcus aureus (ATCC 25923) most resistant. This study also proved that bacterial concentration and inactivation rate are inversely proportionate to each other. Subsequent to growth and cell repair assessments, E. coli O111 was selected as a model to evaluate the effect of sublethal HHP on the liberation and toxicity of bacterial endotoxins (free and cell wall bound). It is also known that different extraction procedures extract different lipopolysaccharides (LPS) fractions and therefore LPS was extracted from the test broth by a combination method of Folch, Lees & Sloane-Stanley, and Venter and Ivanov. The extraction yielded a biphasic system, LPS with reduced lipid content in the upper phase (aqueous) and LPS with increased lipid content in the lower phase (organic). Following extraction the Limulus amebocyte lysate (LAL) test was performed to quantify the concentration (assumed) of LPS in the aqueous and organic phases. Free LPS was detected within six hours in the supernatant in the high and low bacterial loads, moreover the toxicity response of post HHP cell damage was more pronounced at 50 ºC (hurdle) than that observed for the treatments at 8 ºC (hurdle) and more so in the organic phases. The latter implied that HHP not only resulted in quantity LPS variation but also in structural change. However membrane repair was apparently complete after 48 hours, as differences in toxicity were no longer evident. Furthermore, the use of a porcine IL-6 ELISA assay was evaluated as an alternative for the customary LAL as a biomarker for pyrogenic substances in matrixes. Porcine whole blood was challenged for IL-6 production by LPS in the samples from the organic and aqueous systems. A porcine IL-6 enzyme-linked immunosorbent assay was used to assess IL-6 expression in whole blood after being challenged with LPS. From the results it emanated that HHP caused in a change in LPS structure which resulted in a decreased IL-6 expression in whole blood, indicating that structural adaptation of the cell membrane in response to HHP influenced the ability of LPS to stimulate macrophages and monocytes. Therefore, further research and development would be required to evaluate the influence of post HHP LPS on human IL-6 expression. When comparing the porcine IL-6 with the LAL no correlation in toxicity could be established in any of the treatment parameters. Finally it can be concluded that HHP had an influence in the structural morphology of LPS. These structural changes could result in LPS being more toxic, it could also have an effect on the accuracy of immunological assessments, the ability to form biofilm, and susceptibility to phages.

Thesis (PhD (Microbiology))--University of Stellenbosch, 2009.
Multidrug resistant strains of Staphylococcus aureus is presenting an increasing threat, especially immune compromised individuals. Many of these strains have developed resistance to newly approved drugs such as quinupristin-dalfopristin, linezolid and daptomycin. The search for alternative treatment, including bacteriocins (ribosomally synthesized antimicrobial peptides) of lactic acid bacteria is increasing . Lactococcus lactis subsp. lactis F10, isolated from freshwater catfish, produced a new nisin variant active against clinical strains of S. aureus. The operon encoding nisin F is located on a plasmid and the structural gene has been sequenced. The lantibiotic is closely related to nisin Z, except at position 30 where valine replaced isoleucine. The antimicrobial activity of nisin F against S. aureus was tested in the respiratory tract of Wistar rats. Non-immunosuppressed and immunosuppressed rats were intranasally infected with S. aureus K and then treated with either nisin F or sterile physiological saline. Nisin F protected immunosuppressed rats against S. aureus, as symptoms of an infection were only detected in the trachea and lungs of immunosuppressed rats treated with saline. The safety of intranasally administered nisin F was also evaluated and proved to have no adverse side effects. The potential of nisin F as an antimicrobial agent to treat subcutaneous skin infections was evaluated by infecting C57BL/6 mice with a bioluminescent strain of S. aureus (Xen 36). Immunosuppressed mice were treated with either nisin F or sterile physiological saline 24 h and 48 h after infection with subcutaneously injected S. aureus Xen 36. Histology and bioluminescence flux measurements revealed that nisin F was ineffective in the treatment of deep dermal staphylococcal infections. Non-infected and infected mice treated with nisin F had an influx of polymorphonuclear cells in the deep stroma of the skin tissue. This suggested that nisin F, when injected subcutaneously, may have modulated the immune system. Nisin F proved an effective antimicrobial agent against S. aureus-related infections in the respiratory tract, but not against subcutaneous infections. The outcome of nisin F treatment thus depends on the route of administration and site of infection.

M. Tech. (Department of Chemistry, Faculty of Applied and Computer Sciences), Vaal University of Technology
The present study includes the use of a green synthetic method to prepare copper and silver nanoparticles using chitosan, aqueous extracts of Camellia sinensis, Combretum molle and Melia azedarach linn leaves. This study aims to investigate the influence of capping and precursor concentration on the properties of silver nanoparticles with emphasis on the medicinal plants chosen. The effect of capping agent on the properties of copper nanoparticles is also investigated. The phytochemical properties of plant extracts and the antimicrobial activity of the synthesized particles were also studied; this was achieved by using microdilution bioassay. Decoction method was used to extract secondary metabolites from plant leaves. Preliminary phytochemical screening carried out on the aqueous extracts of the plant leaves showed the presence of tannins, proteins, flavonoids, phenols, and carbohydrates. The total phenolic and flavonoids content of the aqueous extract was determined using spectroscopic methods. The highest phenolic content was found in the aqueous extract of Combretum molle (135 mg/g), and the highest flavonoid content was found in the aqueous extract of Camellia sinensis (0.4 mg/g). Characterization was done by a combination of spectroscopic, microscopy and XRD techniques. Both the size and shape of the synthesized silver nanoparticles were dependent on the identity of the capping molecule, precursor and capping agent concentration as depicted from their TEM and XRD results. Silver nanoparticles were found to be predominantly spherical. The capping agent concentration was also found to influence the degree of agglomeration, with an increase in capping agent concentration giving lesser agglomeration. FTIR spectral analysis showed that silver nanoparticles interact with bioactive compounds found in the plants through the hydroxyl functional group. Other shapes including diamond were observed for the effect of precursor concentration. The XRD micrographs revealed a face-centered cubic geometry and the phase remained the same with an increase in precursor concentration. The synthesized silver nanoparticles were all blue shifted compared to the bulk material. The TEM results revealed that copper nanoparticles with different sizes and shapes were successfully synthesized. All the prepared copper and silver nanoparticles showed satisfactory antifungal and antibacterial activity against Candida albicans, Cryptococcus neoformans, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumonia and Pseudomonas aeruginosa. The capping molecules used in this study also showed some antibacterial and antifungal activity against the selected strains. However nanoparticles performed better than these capping molecules. Both silver and copper nanoparticles were found to be more active against gram-negative bacteria compared to gram-positive bacteria. Amongst all the prepared silver nanoparticles Combretum molle capped nanoparticles were found to be the most active nanoparticles. Also with copper nanoparticles, it was found that Combretum molle capped nanoparticles were the most active nanoparticles. Between the two metal nanoparticles, silver nanoparticles showed high antibacterial and antifungal activity compared to copper nanoparticles. The antioxidant activity of silver nanoparticles was assessed using 2.2-diphenyl-1-picrylhydrazyl. Silver nanoparticles were found to have some antioxidant activity. However, the capping molecules were found to be more active than the synthesized nanoparticles. This observation is attributed to the presence of some bioactive compounds in the plant extracts.

The effect of water on growth and survival of Staphylococcus aureus was investigated using liquid (17O) and solid-state (1H) Nuclear Magnetic Resonance (NMR) spectroscopy. Different growth media, solute types, and methods of water activity (aw) adjustment (i.e. moisture versus solute variations) were studied. For the growth studies (>0.75 aw), mostly all water present was detected by the NMR and was highly mobile. Dependence of S. aureus growth was only partly dependent on the NMR signal intensity (amount of mobile or detected water) and partly dependent on the solute types. When aw was adjusted by changing moisture content, the use of brain heart infusion (BHI) or chicken meat media (CMM) did not affect the general conclusion. However, CMM resulted in an increased viscosity particularly at lower moisture content and partly influenced the NMR water mobility results. In general, it is postulated from this work that there is a critical amount of mobile water (based on 17O NMR intensity) of ∼40 g water detected/100 g sample below which S. aureus is significantly inhibited. For the survival study (<0.75 aw), the mobile proton signal was primarily due to the amount of water protons. Upon hydration, the onset NMR mobility increase also correlated with the monolayer value of water. A substantial increase in proton mobility (T2) was observed upon further increase in moisture content. Survival of S. aureus in a freeze-dried gum mixture was dependent on proton mobility, amount of mobile protons, and aw. Added mannitol and raffinose both protected the cells from osmotic-related death. The critical aw's at which cell death dramatically increased were in a similar aw range when proton mobility also increased. This suggested that molecular mobility facilitated the cell damage brought about by osmotic stress and in this case may serve as an indicator of water availability. Thus, molecular mobility plays a critical role in controlling cell survivability at a low moisture condition.