Members of the Staphylococcus genus are a major health issue in the clinical environment and can cause a wide range of disease in humans. However, this genus is also found as a part of the normal flora in humans, usually on the skin, nasal cavities, or on the linings of the throat. Normal flora members of the Staphylococcus genus become an opportunistic infection when there is breach in the physical barriers or the immune status of the human host. Another challenge with the Staphylococcus genus is the increase in drug resistant strains, such as Methicillin resistant Staphylococcus aureus (MRSA), making simple infections difficult to treat and leading to severe toxic shock or even death. Recently, there have been numerous studies demonstrating normal flora bacterial interactions inhibiting bacteria that are potentially harmful to humans. Our research lab has previously demonstrated that the benign bacterium Alcaligenes faecalis has inhibitory effects against Staphylococcus aureus. In the present study, we wanted to explore: 1) if the inhibitory effect of A. faecalis would translate to other Staphylococcus species (S. capitis, S. saprophyticus, and S. epidermidis); 2) if this inhibitory effect was found in other Alcaligenes species (A. viscolactis). To determine this possible interactions, two parallel experimental projects were undertaken. A. faecalis and A. viscolactis were tested for their interactions with Staphylococcus species on both agar and liquid medium. For agar medium analysis, Staphylococcus lawns were grown on agar plates and either Alcaligenes cells, heat killed Alcaligenes, or Alcaligenes cell free supernatant were spotted onto the lawns and observed and scored for zones of inhibition (ZOI). It was demonstrated live cells of both A. faecalis and A. viscolactis were needed to produce ZOI on Staphylococcus lawns and that all Staphylococcus species were inhibited. For liquid medium analysis, Staphylococcus species were either inoculated alone (control) or in a co-culture with Alcaligenes, serially diluted, and colony forming units (CFU) were enumerated. Both A. faecalis and A. viscolactis inhibited all Staphylococcus species in liquid culture. Based on the results from these experiments, it is our conclusion that: 1) Alcaligenes faecalis and Alcaligenes viscolactis both possess the ability to inhibit Staphylococcus growth; 2) all Staphylococcus species are inhibited by Alcaligenes, but at varying levels. The exact mechanism of how Alcaligenes can inhibit Staphylococcus species is unknown, which will require further studies to analyze and understand the exact mechanism in order to create effective therapeutic targets to combat these increasingly resistant strains of Staphylococcus.

<p>Aim Because of the major problems that certain Vibrio specie, especially Vibrio harveyi, can cause the aquaculture industries a rapid method to identify Vibrio isolates is required. Early diagnosis of a V. harveyi infection could facilitate disease surveillance, treatment and prevention in cultured marine animals. Therefore, the use of PCR to aid in the identification of Vibrio is increasing and a way of extracting DNA in a cheap, fast and easy way is also of an important requirement to facilitate rapid diagnosis.</p><p>Methods This report comprises biochemical profiling and PCR methods in the characterisation of four isolates of V. harveyi and single isolates of V. tubiashii, V. alginolyticus, V. anguillarum, V. splendidus, V. tapetis and V. parahaemolyticus.</p><p>Strains were examined for adherence to a Hep-2 cell line. Four different DNA extraction methods were evaluated and compared. The detection limits and the analytical limits of two PCR methods for Vibrio were determined.</p><p>Results The overall findings were that the use of a greater range of biochemical substrates than are in the API 20E is necessary to identify Vibrio strains, and that none of the strains tested adhered to Hep-2 cells. All extraction methods successfully produced DNA with the kit method giving the purest samples. RNA was a contaminant of the other techniques but this could be overcome by treating extracts with RNase. The rapid microwave extraction method gave appropriate PCR amplicons when tested.</p><p>Conclusion PCR determination of the VH-sequence in combination with VHA and a distinguishable colonial morphology may be a good choice for the identifying of Vibrio harveyi.</p>

This study investigated aspects of the epidemiology of malaria in the Natal province of the Republic of South Africa. In this study the Collins English dictionary definition of epidemiology is used where it is defined as the branch of medical science concerned with the occurrence, transmission and control of an epidemic disease. Malaria has been a notifiable disease in the Republic of South Africa since 1958. Retrospective malaria case data from the Natal province as a whole was analyzed and the data from the KwaZulu and Natal areas of the province compared. Malaria cases were reported from 35 of the 65 magisterial districts in Natal province during the study period. In the Natal areas 91.5% of the cases were reported from eight districts and in the KwaZulu areas 96.4% of the cases came from three districts or as imports from Mozambique. The overall attack rate for both the Natal and KwaZulu areas using the total population figures for each area were very similar for the period 1986-1988 at 0.71 and 0.70 per 1000 head of population for the respective areas. The disease showed a distinct seasonal pattern in the KwaZulu areas with 86.9% of the cases being classified as indigenous and only 13.1% as imported. In the Natal areas, however, the seasonal pattern was not as marked and only 12.1% of the cases were recorded as indigenous and in excess of 82% as imported. Three species of the Anopheles gambiae complex were found to occur sympatrically in Natal province, namely: An. arabiensis, An. quadriannulatus and An. merus. Of these species An. arabiensis was found to occur at five localities during or after the notification of indigenous malaria cases from these areas. Due to the sympatric distribution of these species particular emphasis was placed on species identification and in particular the biting behaviour and control of An. arabiensis was investigated. The study found both morphological and behavioural differences between populations of An. arabiensis from those areas of the province with an intra-domiciliary residual insecticide vector control programme and those from the unsprayed areas. In the unsprayed areas the majority of the indoor resting An. arabiensis had fed on man whereas in the sprayed areas the majority of the indoor resting An. arabiensis were bovine fed. In the sprayed areas, however, the majority of the An. arabiensis caught leaving huts had fed on man. The percentage survival of bloodfed An. arabiensis caught leaving huts in the DDT sprayed area was in excess of 72%. The data strongly suggest that optimal control of An. arabiensis will not be achieved using the current control strategy of the annual application of intra-domiciliary DDT. / Thesis (Ph.D.)-University of Natal, 1990.

Following an outbreak of multi-resistant Haemophilus influenzae type b (Hib)infections in a tuberculosis hospital, this study was undertaken to determine carriage of Hib in 2 paediatric wards; to characterise all isolates of Hib, determine their antimicrobial susceptibility profile and the antibody response of the children to a conjugate vaccine. Prior to and one month after immunisation, oro- and nasopharyngeal swab specimens as well as venous blood were collected from each child. Isolates were tested for /3-lactamase and chloramphenicol acetyltransferase (CAT)production, their MIC's determined by the agar dilution method and characterisation of Hib isolates was performed by biotyping and analysis of outer membrane protein (OMP) profiles. An ELISA was also developed to determine serum antibody levels to polyribosyl-ribitol-phosphate (PRP), the capsular polysaccharide of Hib. The study population comprised a total of 135 children who had been hospitalised for treatment for tuberculosis. The patients were aged 4 months to 14 years with a median of 37,5 months. During the study period, none of the children developed invasive Hib disease. The overall carriage rate of Hib increased from 38% (51/135) before immunisation to 62% (84/135) after immunisation (P 0,15 /ig/ml. After immunisation, 34%(45) of patients increased their antibody levels to > 1,0 /xg/ml. There was no statistical difference between the mean antibody concentrations of patients who were colonised by Hib and those who were not (p = 0,58). The vaccine did not reduce carriage of Hib in this study population of children being treated for tuberculosis and the immune response to the vaccine was not optimal. Production of /3-lactamase and the prevalence of rifampicin resistance has implications for treatment and chemoprophylaxis in this population. OMP analysis showed a diversity of types. Multi-resistant strains causing invasive disease had the same OMP type as some multiresistant strains which colonised the children. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1996.

Antral mucosal biopsies were examined microbiologically and histologically for the presence of Campylobacter pylori in 224 patients with upper gastrointestinal symptoms. One hundred and eighty three (83%) patients were found to harbour Campylobacter pylori in their gastric mucosa. Campylobacter pylori was strongly associated with the presence of histological gastritis (93%) and was detected in only 10% of 30 patients whose gastric biopsies showed normal histology. Endoscopically diagnosed duodenal lesions were more strongly associated with the presence of Campylobacter pylori than were gastric lesions (p<0.001). A variety of laboratory methods were evaluated to determine the sensitivity and specificity to detect the presence of Campylobacter pylori. Histology was the most sensitive and specific method to detect the presence of Campylobacter pylori. Although culture was highly specific, it was less sensitive than histology in detecting Campylobacter pylori in gastric antral mucosal specimens. The "conventional" gastric urease assay, although specific, needs be performed under controlled conditions (37°C) for optimal results. The "one-minute" urease assay was more sensitive than the "conventional" gastric urease assays and was highly specific. ELISA to detect specific-IgG antibodies to Campylobacter pylori was a moderately sensitive non-invasive method to detect Campylobacter pylori infection, but was non-specific. / Thesis (M.Med.)-University of Natal, 1988.

Microbiological and molecular epidemiological studies were conducted on 36 multi-resistant Haemophilus influenzae strains, isolated from paediatric patients, over a 26 month period (April 1986 to May 1988). The majority of strains (80,5%) had been isolated from blood and cerebrospinal fluid. More than 80% of isolates tested belonged to biotype II and 90% were of serotype B. Minimal inhibitory concentrations against 6 antibiotics (ampicillin, chloramphenicol, tetracycline, rifampicin, streptomycin and cefotaxime) confirmed the presence of multi-resistant strains. Resistance to rifampicin was confirmed in 6 (16,7%) strains. All strains were susceptible to cefotaxime. Ten transconjugants analysed with respect to their plasmid content were shown to harbour an identical 41 MDa plasmid. Restriction endonuclease digests of these plasmids with Eco R1 and Sst1 revealed almost identical restriction patterns. Outer membrane protein profiles of 19 strains revealed the predominance of one particular subtype. By combining the microbiological and molecular epidemiological findings, it is concluded that one strain of H. influenzae type b is responsible for the nosocomial acquisition of infections amongst paediatric patients. The implifications of these findings are discussed. / Thesis (M.Med.)-University of Natal, Durban, 1988.

Tuberculosis (TB) is one of the most prevalent infectious diseases worldwide and causes high morbidity and mortality, despite the widespread availability of effective antibiotics against most strains of Mycobacterium tuberculosis, which is the causative agent of TB. One of the primary reasons that hinder TB control is that many cases of active disease go undetected or are discovered late. This is, in large part, due to the relative insensitivity and limited specificity, amongst other limitations, of the current TB diagnostics tests. Moreover, M. tuberculosis infection can be asymptomatic and latent, or cause active disease. Therefore, an ideal or effective TB diagnostic needs to distinguish between these two states. The aim of this study was to develop novel diagnostic reagents for M. tuberculosis using phage displayed peptides and nucleic acid aptamers with a view to discerning latent from active TB. Using a linear (X12) and constrained (CX7C) phage display libraries, five rounds of selection (biopanning) were performed. Ten phage displayed peptides that bind to the mycobacteria surface were selected. These phage clones were identified using both random clone picking and high throughput (HTP) sequencing. A phage clone displaying the CPLHARLPC peptide was identified by HTP sequencing as the most enriched, representing 82.49% of the selected CX7C phage population. Further characterization showed that it bound better to different mycobacteria species, including M. tuberculosis, than the unselected phage library. Moreover, using surface plasmon resonance (SPR) technology, the chemically synthesised CPLHARLPC peptide was shown to bind M. tuberculosis H37Rv whole cell lysate and not non-mycobacteria lysates. In addition, using the systematic evolution of ligands by exponential enrichment (SELEX) protocol and SPR technology, 2'-Fluoro-pyrimidine-RNA aptamers were selected against the mycobacteria ESX-3 secreted protein, ESX-G. At least five aptamers were identified after five rounds of selection. Two of these aptamers, GH43 and GH78, not only bound EsxG with high affinities, KD 8.04 ± 1.90 nM and KD 78.85 ±9.40 nM respectively, but also preferentially bound EsxG better than the EsxA homologue. Taken together, these findings suggest that a combination of phage display, SELEX and HTP sequencing can be a useful tool for the identification of specific detection reagents that can bind to mycobacteria and its associated targets. These reagents could be exploited to develop alternative molecular probes for TB diagnostics. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.

Thesis (M.S.)--California Polytechnic State University, 2009. / Title from PDF title page; viewed on September 23, 2009. Major professor: Lanny Griffin, Ph.D. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Biomedical Engineering." "June 2009." Includes bibliographical references (p. 43). Also available on microfiche.

Microorganisms, particularly bacteria, can associate together to form complex communities called biofilms. These communities are embedded in extracellular polymeric substances and can form on numerous surfaces such as implanted devices (catheters, central lines, joint replacement) in patients. These biofilms cause bloodstream and systemic infections that are difficult to treat and increase the chances of sepsis. Previously, our laboratory has identified a protein secreted by Klebsiella that has inhibitory effects on other members of the Enterobacteriacea bacterial family, namely Citrobacter and Enterobacter. Our current interest lies in the ability of the protein to potentially inhibit this bacterial family from establishing biofilms. In the present study, we wanted to explore: (1) if it is possible to form Citrobacter and Enterobacter biofilms in 6-well plates and on microscope coverslips; (2) if treating these biofilms with the secreted protein shows inhibition similar to previous planktonic cultures; (3) if these biofilms and inhibition could be visualized by a variety of staining techniques. To determine if it is possible to create Citrobacter and Enterobacter biofilms, bacteria were inoculated into 6-well plates, grown under static conditions in a 37°C incubator for 24 hours, and stained with crystal violet. Images showed that robust biofilms grew in the 6-well control plates while wells treated with the Klebsiella protein displayed reduced biofilms. To determine if it was possible to see Citrobacter and Enterobacter biofilms at a microscopic level, microscope slides were placed into 6-well plates, treated as above, and the slides were Gram stained. Images show thick biofilms consisting of Gram negative rods on control slides, while slides treated with the Klebsiella molecule become sparse and poorly grown. To determine if Citrobacter and Enterobacter biofilms could be fluorescently labeled for visualization, the same process was employed as above, but stained with a LIVE/DEAD cell viability kit where live cells fluoresce green and dead cells fluoresce red. Control slides showed bright thick green fluorescing biofilms while slides treated with the Klebsiella molecule had fewer green fluorescing cells and some red cells. From these observations, it is our conclusion that: (1) it is possible to grow Citrobacter and Enterobacter biofilms on both 6-well plates and microscope slides; (2) Citrobacter and Enterobacter biofilms can be visualized both by simple staining and fluorescent staining; (3) Citrobacter and Enterobacter biofilms are inhibited by Klebsiella secreted proteins. Currently, the identity of this protein is unknown. However, it is possible that this unknown protein could be of future use in the treatment of bacterial biofilms one identified.

The aim of this project was to identify growth phase-specific heat shock proteins of Mycobacterium smegmatis LR222. A growth curve was constructed using the ATP assay. This method was shown by B. A. Ntolosi to be the most accurate indicator of when the organism entered the various phases of growth. It was possible to determine that M. smegmatis LR222 entered the exponential phase of growth after a short lag phase of 4 to 8 hours and persisted in this phase for 20 to 22 hours. It then reached the stationary phase, which lasted for 40 to 46 hours. Protein heat shock assays were performed on growth phase-specific samples. This allowed the identification of a 43-46 kDa in molecular weight stationary phase protein on one-dimensional SOS-PAGE. The protein was induced in cells entering the stationary phase of growth and not by heat shock as it was induced under both the control and the heat shock temperatures. The protein was further characterised by two-dimensional gel electrophoresis, which demonstrated resolution into two, strongly age-associated, proteins. Subtractive RNA hybridisation was attempted in order to obtain a subtraction cDNA probe from a stationary phase RNA sample depleted of sequences common in both the exponential and the stationary phase samples. Ribosomal RNA was removed from the total RNA by the process of photobiotinilation. The mRNA was then used as a template to synthesise with reverse transcriptase single stranded cDNA. cDNA/mRNA denatured hybrids were hybridised to the exponential phase RNA sample. The new hybrids were "subtracted" by chemical cross-linking with DZQ and the unique cDNA used to produce by random primers a radioactively labelled probe. A M. smegmatis library was probed but unfortunately no signal was observed. Further adjustments and improvements to this technique are required before it can be used effectively.