The Streptococcus Anginosus/Milleri group (SMG) colonize mucosal surfaces in humans but are also associated with numerous respiratory and invasive infections. These infections are often polymicrobial in nature, with obligate anaerobes often being isolated. The group consists of three species, S. anginosus, S. constellatus and S. intermedius. SMG are considered to be lactic acid bacteria, producing acids such as lactate, formate and acetate as byproducts of their metabolism. Their genomes have been recently sequenced but little is known about their metabolism. Understanding the basis of their metabolism is beneficial in determining optimal growth conditions and mechanisms associated with their pathogenicity. The isolation of obligate anaerobes from SMG polymicrobial infections suggests that they have anoxic microenvironments. There is also some evidence for synergy between SMG species and anaerobes. While cooperation might be occurring with certain anaerobes, streptococci also produce inhibitors such as hydrogen peroxide and short peptides called bacteriocins. These give streptococci a competitive advantage in polymicrobial commensal communities such as the oral cavity. The Streptococcus invasion locus controls bacteriocin production in Group A streptococci and has been identified in SMG species as well. It is unknown if SMG have mechanisms to compete with closely related streptococci. The goal of my thesis is to characterize the cooperative and competitive interactions of S. intermedius with other species. In chapter 2, we characterized the in vitro metabolism of S. intermedius under aerobic (5% CO2) and anaerobic conditions. Using a transcriptomic and metabolomic approach, we mapped the pathways involved in S. intermedius B196 metabolism. We found that there was a minimal upregulation of core pathways including carbohydrate metabolism under anaerobic conditions. Under aerobic conditions, oxidative stress genes were induced. An increased growth rate was also observed anaerobically. In chapter 3, I demonstrated that Streptococcus strains, including S. intermedius, can deplete oxygen and create an anaerobic environment. Certain strains could support the viability of the obligate anaerobe Prevotella melaninogenica in broth cultures under hypoxic conditions, while others inhibited Prevotella by producing hydrogen peroxide. S. intermedius B196 has an alkylhydroperoxidase system (ahpCF), which is thought to endogenously detoxify peroxides. An S. intermedius ahpCF mutant produced hydrogen peroxide and inhibited P. melaninogenica in coculture. Complementation in S. intermedius restored P. melaninogenica viability in coculture. I demonstrated that the ahpCF peroxide detoxification system directly protects S. intermedius from peroxides and indirectly affects a polymicrobial community. In chapter 4, we used a subcutaneous abscess model in BALB/c mice to demonstrate that S. intermedius promotes P. melaninogenica survival during co-infection in comparison to a P. melaninogenica mono-infection. S. intermedius induced abscesses appeared to induce apoptosis, necrosis and NETosis in neutrophils that infiltrated the site of infection. Our results demonstrate the complexity of SMG infections. In chapter 5, I demonstrated that S. intermedius B196 produces inhibitors of other SMG in response to stimulation with the pheromone peptide SilCR. This is the first case of S. intermedius inhibiting a closely related SMG strain. A bioinformatic analysis was done on the sil system in SMG. The system is associated with a genetically heterogeneous bacteriocin cluster which can carry any combination of sixteen putative open reading frames, six of which are putative bacteriocins. Together, my thesis outlines that S. intermedius has specific mechanisms of cooperation and competition. These allow it to cooperate with obligate anaerobes such as P. melaninogenica and inhibit other SMG species. Oxygen depletion, hydrogen peroxide production and bacteriocin production are only three factors addressed in this thesis. However, there are many factors involved in shaping a polymicrobial environment with SMG species. More research in SMG polymicrobial interactions is required to fully understand SMG pathogenicity. / Thesis / Doctor of Philosophy (PhD)

Enterotoxin production by Staphylococcus species other than Staphylococcus aureus has been reported. Staphylococcus strains (104 in toto) representing twelve species and subspecies were examined for enterotoxins using a commercial staphylococcal enterotoxin ELISA immunoassay (TECRA, International Bioproducts). Staphylococcus intermedius (24 strains) and S. aureus (7 strains) were positive with this test. Western blots of S. aureus exoproteins demonstrated proteins of ∼30 kD, consistent with known staphylococcal enterotoxins. The major antigen in all S. intermedius strains, a 75 kD protein, was not analogous to previously described staphylococcal enterotoxins. This protein was unique to S. intermedius. Gel filtration data indicate that the protein is a subunit of a larger protein in vivo. The 75 kD protein cross-reacts with several enterotoxin antibodies. It is unclear whether the protein is a toxin, but its homology with S. aureus enterotoxins may indicate a shared toxic region, or this protein may create false positive results in screening for enterotoxin.

Avermectins and milbemycins are commonly used in agro-ecosystems for the control of parasites in domestic livestock. As integral members of agro-ecosystems with importance in maintaining pasture health through dung burial behaviour, dung beetles are an excellent, non-target, bio-indicator taxon for examining potential detrimental effects of pesticide application. The current study uses the dung beetle species, Euoniticellus intermedius (Reiche), as a bio-indicator to test the relative toxicity of four different anthelmintics in dung residues. There have been numerous laboratory and field trials on these four anthelmintics but there has never been a laboratory trial comparing ivermectin, eprinomectin, doramectin and moxidectin under precisely the same conditions. The effects of avermectin and milbemycin toxicity are not confined to parasitic nematodes and arthropods, but also extend to a large variety of beneficial invertebrate species, which use the dung as a microhabitat and breeding resource. Over time, pesticide usage indirectly affects the rate of dung degradation through adverse effects on dung beetles. This potential problem constitutes the main focus of this study. The present and previous studies have indicated no significant effect on the survival of adults but a significant reduction in reproductive rate and reproductive success. Over time, reduced reproductive rate would result in decreased population sizes in the dung beetle community and, ultimately, a decrease in the rate of dung degradation and dung burial. It is, thus, vitally important to create awareness about the importance of dung beetles and sound farming practices for healthy agro-ecosystems. / Dissertation (MSc)--University of Pretoria, 2014. / lk2014 / Zoology and Entomology / MSc / Unrestricted

Objectives: Report a novel case of new-onset type 1 diabetes in a pediatric patient presenting with DKA and concurrent Streptococcus intermedius brain abscess. Case presentation: The following case report is that of a previously healthy 12 year-old girl presenting with new-onset type 1 diabetes with mild diabetic ketoacidosis and subsequently found to have a brain abscess. Over the course of her hospital stay, she developed seizures and was found to have a 1.3 × 1.0 × 1.2 cm right frontal parasagittal mass culture-positive for S. intermedius. Neurologic symptoms were unmasked once insulin treatment was initiated and ketosis improved, supporting the relationship between therapeutic ketosis and the management of medication-refractory epilepsy. Conclusions: This case both supports the relationship between therapeutic ketosis and the management of medication-refractory epilepsy and highlights the need to carefully consider comorbid conditions in patients with DKA and new onset neurological symptoms.

Recent years have seen a dramatic increase in first generation bio-fuel production, mainly driven by concerns of climate change and rising prices of transportation fossil fuels. Due to significant pressure on the few available food sources, second generation bio-fuels have entered the fray, as a sustainable alternative. This research‟s aim was to search for cellulolytic micro-organisms and enzymes from the gut of the dung beetle, Euoniticellus intermedius, (Coleoptera: Scarabaeida) that can be used in the production of second generation bio-fuels. Dung beetle larvae were dissected and the gut micro-flora cultured in cellulose medium. Bacterial growth and cellulase activity was monitored on a daily basis. DNA isolation was then done on the cellulose medium-cultured microbes and the isolated DNA cloned in E. coli. The clones were screened for cellulase activity using plate assays. A total of 7 colonies out of 160 screened colonies showed positive CMC (endo-β-1,4-glucanase) and MUC (cellobiohydrolase) activities. Sequencing of these positive colonies yielded mostly bacteria belonging to the Enterobacteriaceae family, most of which have not been previously reported to have cellulase activity. This study‟s findings prove that in addition to this dung beetle‟s gut being a fruitful source of microbial biodiversity, it is also a potential source of cellulolytic micro-organisms and enzyme activities that will aid the function and design of future bioreactors for the bio-fuel industry.

The planktonic shrimp Acetes intermedius is an ecologically and commercially important species. Since it was described by Omori(1975) as a new species, there have been relative few studies focused on the reproduction of this species. The aim of this study is to examine the spawning, egg hatching, larval development of A. intermedius and the related environmental factors. The newly released eggs of Acetes intermedius were round, 200 um in diameter in average, transparent, demersal and were usually green in color. The cleavage pattern of developing egg is holoblastic. Larval stages could be divided into four stages: nauplius, protozea, zoea and postlarva. Nauplius, protozoea and zoea can be further divided into 4, 3 and 2 substages, respectively. At water temperature 30 ¢J and salinity 25 psu, hatching of nauplius was around 10-11 h after spawning while protozoea, zoea and postlarva occurred around 29h, 5-6 and 7 days after spawning, respectively. Newly released eggs were introduced directly into different temperature-salinity combinations. The results revealed that eggs failed to hatch at 15¢J at all salinities tested and at salinities 0 to 10 psu at all temperatures examined. Hatching success started to decrease at salinity 15 psu at 30¢J and at 20 psu at both 25¢J and 20¢J. When salinity was not lower than 25 psu, the hatching success reaches nearly 90 % in average and is generally similar among temperatures tested. Hatching started at about 10, 14 and 28h after spawning at temperatures 30, 25, 20¢J, respectively. At salinity 25 psu, hatching time was the shortest at both 25 and 30¢J, whereas it was similar among salinities at 20¢J. Eggs and nauplii I hatched at 30 ¢J, 30 psu were incubated in the different temperature-salinity combinations. It took eggs incubated at salinities ranging from 20 to 35 psu about 28, 45 h and 5 days to develop into protozoea I at 30, 25 and 20 ¢J, respectively. Nauplii usually started to metamorphose earlier at 25 and 30 psu than at 35 and 20 psu at 25 and 30¢J while no significant trend in metamorphosis time was found among different salinities at 20¢J. Nearly 90% of nauplii metamorphosed into protozoea I at 25 and 30¢J while the metamorphosis rate of nauplii was greatly reduced at 20¢J. The metamorphosis rate was usually higher around 20-25 psu and lower at 15 and 35 psu in all temperatures examined. While spawning, the gravid females swam near the water surface and were rapidly beating pleopods. The eggs were directly released into water. Spawning usually took place at night but the timing is generally peaked around midnight. The gravid females spawned in the early spring before midnight while they spawned after midnight in summer. The spawning timing is delayed at low temperature (24¢J) while it is not affected by high temperature (30¢J). The salinity did not show any effects on spawning but the hatching success of eggs released at 15 and 35 psu was significantly lower than those at 20-30 psu. The gravid females were tended to be endogenous timed to spawn in the night since spawning was not affected by absence of presence of light.

Thesis (M. Sc. (Ichthyology and Fisheries Science))--Rhodes University, 2006.

Successful treatment of canine pyoderma has become increasingly difficult due to the development of antimicrobial resistance and recurrence of infection. The development and spread of antimicrobial resistance has major implications because treatment failures have been associated with increased mortality, morbidity and costs related to disease. All canine skin samples submitted to Vetdiagnostix Veterinary Pathology Services for microbiological culture and sensitivity between January 2007 and June 2010, from which Staphylococcus intermedius was isolated, were selected for this investigation. A total of 319 samples from dogs of various ages, sexes and breeds from various locations throughout South Africa were obtained. In addition to the antimicrobial susceptibility data, data relating to dog signalment (age, gender, breed), case history and any other aspects of the history provided e.g. diagnosis, sample type and geographical location of the patient were collected. The number of skin samples yielding Staphylococcus intermedius was high in dogs up to the age of 6 years and, then decreased with only a few cases in dogs aged 11 years or older. The distribution of samples collected in 2010 was unusual in that there were two peaks, one in dogs two years old or less and one in 6 to 9-year-old dogs. With the exception of skin samples taken in 2008, a high percentage of dogs were under the age of one year old. 2008 had a lower percentage of affected dogs less than one year of age compared to the other years. Staphylococcal pyoderma is more common in dogs below the age of five years. Dogs above this age are less likely to contract this condition. The genders tended to be equally distributed throughout the years, with almost equal proportions of affected males and females present. The Bull Terrier types and Shepherd types were grouped separately as they were over-represented and are known to be prone to pyoderma. Large short haired dogs were consistently worse affected throughout the years sampled, followed by the Bull Terrier types. In 2010, small short haired breeds were worse affected compared to the numbers between 2007 and 2009. The monthly distribution, with the exception of 2009, tended to be consistent throughout the years. The unusual temporal distribution in 2009 could be associated with a general distribution in the number of samples compared to the other years sampled. Samples tended to be mainly from practices located in the KwaZulu-Natal province of South Africa. This is most likely due to the fact that the laboratory is located in this province, with a courier network that arranges collection from these practices. Practices in other regions send their samples to the laboratory via private courier companies and the South African Post Office. Antimicrobial resistance of S. intermedius was greatest to ampicillin followed by tetracycline and then potentiated sulphonamides. The results also showed that, in general, antimicrobial resistance was low. Very few methicillin resistant isolates were detected. Temporal trends were not noted, with the exception of ampicillin where isolates became more susceptible and potentiated sulphonamides (co-trimoxazole) where isolates were becoming more resistant. Staphylococcus intermedius is significantly less resistant to erythromycin, clindamycin, cephalexin, oxacillin, amoxicillin-clavulanic acid, enrofloxacin, marbofloxacin and gentamicin, with most strains being susceptible to these drugs. Resistance to penicillin and tetracycline is frequently found in Staphylococcus intermedius and is on the increase. Resistance to most other antimicrobials, particularly newer generation antimicrobial agents such as the fluoroquinolones, is still comparatively low. In general, both the Kirby-Bauer and broth dilution MIC tests yielded similar results for the antimicrobial agents tested. The main difference between the two tests was evident in the over-estimation of resistance by the Kirby-Bauer test in the cases of ampicillin, co-trimoxazole, penicillin and doxycycline. This could be related to the instability of these particular drugs in vitro. Inoculum densities may also have played a role, with denser inocula producing smaller zone sizes for the drugs tested. Using the MIC method, all of the isolates tested were found to be completely sensitive to ticarcillin, oxacillin, amoxicillinclavulanic acid, imipenem, ceftiofur, chloramphenicol, doxycycline, gentamicin, amikacin and co-trimoxazole. Of the isolates tested using the MIC method, between 2-40% showed some level of resistance to the following antimicrobials: erythromycin, penicillin, ampicillin, enrofloxacin, clindamycin and marbofloxacin. The highest level of resistance observed was shown to erythromycin. The increase in resistance to the lincosamides, lincomycin, clindamycin and erythromycin may be attributed to the increased use of these drugs in the last decade. Knowledge of trends in bacterial resistance is important for veterinarians when determining treatment for canine skin infections. The information obtained from the analysis of the antimicrobial susceptibility profiles of Staphylococcus intermedius isolated from canine pyoderma cases will provide veterinarians with valuable information on choosing the most appropriate drug to treat S. intermedius skin infections as well as re-enforcing the need for the prudent use of antimicrobial drugs in companion animals. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Veterinary Tropical Diseases / unrestricted

Numerous species of the genus Staphylococcus other than Staphylococcus aureus are important pathogens in human clinical practice and veterinary medicine. With improved methods of identification and more precise classification, we have speciated over 500 strains of staphylococci representing 17 species and subspecies of non-S. aureus Staphylococcus. We have examined these strains for possible virulence factors which may play a role in their pathogenesis. Using transmission electron microscopy (TEM), we have demonstrated that small to large amounts of glycocalyx are found on staphylococcal cells. Animal models have shown that staphylococci cause abscess formation in the presence or absence of a foreign body implant. Molecular characterization of cell extracts of Staphylococcus intermedius show that this species elaborates a protein which is serologically similar to the enterotoxins of Staphylococcus aureus in ELISA tests, but differs markedly in other characteristics.

The purpose of this study was to evaluate the antimicrobial potential of the propolis extract, natural substance known for its therapeutic properties, against to 67 microbial isolates of canine otitis. The Minimum Bactericidal Concentration (MBC) of the propolis extract against the isolates of coagulase-positive Staphylococcus and the Minimum Fungicide Concentration (CFM) for Malassezia sp. isolates were determined using the broth microdilution technique. The MBC was 15.1mg mL-1, while the MFC was 2.4mg mL-1. Thus was shown the antimicrobial potential of the propolis extract tested against these two agents and the lower bactericidal and fungicidal concentration needed for the inhibition of them. / O objetivo do presente estudo foi avaliar o potencial antimicrobiano do extrato de própolis, substância natural conhecida pelas suas propriedades terapêuticas, frente a 67 isolados microbianos da otite canina. A Concentração Bactericida Mínima (CBM), do extrato de própolis, frente a isolados de Staphylococcus coagulase positiva e a Concentração Fungicida Mínima (CFM) para isolados de Malassezia pachydermatis foram determinadas utilizando-se a técnica de microdiluição em caldo. A CBM foi de 15,1mg mL-1, enquanto a CFM foi de 2,4mg mL-1.Dessa forma foi demonstrado o potencial antimicrobiano do extrato de própolis testado contra esses dois agentes e a menor concentração bactericida e fungicida necessária para a inibição dos mesmos.