Bacterial isolates from animals, foods and clinical samples with resistances to one or more antibiotics are being found frequently each year. Selective pressure exerted by antibiotic growth promoters in food animals has been considered a main cause for the development of antibiotic resistance and antibiotic use has been strongly criticized as a serious public health threat. The gastrointestinal tract of animals not only serves as a reservoir of zoonotic agents but also as a spot for exchange of genetic information between pathogenic and commensal bacteria. Humans get infections from resistant bacteria either through the food chain, contaminated water or by direct contact with animals. In this situation much of the concern has been directed against the use of antibiotic growth promoters in animals. The removal of synthetic antibiotics from animal diets created other problems such as a decline in animal welfare and an increase in the use of therapeutic antibiotics. So there is a need for new alternatives to antimicrobial drugs to overcome resistance development and related problems. Plants and plant-derived compounds have long been considered to posess antimicrobial activity since they were frequently used in ancient medicine as natural remedies to treat human infections. Identifying new sources of natural antimicrobials and inhibitors of resistance development will yield novel therapeutic drugs and extend the useful life of existing antibiotics. In the present work, individual and combined effects of five essential oils (eugenol, thymol, carvacrol, cinnamaldehyde, allyl isothiocyanate (AIT)) and a formaldehyde-based feed additive, Termin 8, with antibiotics against 4 antibiotic resistant bacteria with known determinants for resistance were tested using broth microdilution and the checker board assay. The bacteria showed considerable susceptibility towards these antimicrobials and a significant reduction in the minimum inhibitory concentrations (MICs) of antibiotics was noted when paired combinations of antibiotic and antimicrobial were used. The synergistic interaction was further confirmed by the extent of decrease in logarithmic count or viable population (Log DP). Although most of the combinations were synergistic by fractional inhibitory concentration (FIC) values, fewer combinations showed synergistic interaction when Log DP was considered. Gram-positive bacteria were more sensitive to the antimicrobials than Gram-negative bacteria. In combination studies, carvacrol was more effective and showed synergistic interaction with at least three antibiotics. When used alone, AIT was more effective and the concentration needed to exhibit antimicrobial action was much lower when compared to other compounds. An in vitro study was conducted to assess the antibacterial effects of Termin 8 and thymol in chicken caecal digesta and poultry feed samples by using a thin agar layer (TAL) method. Concentrations greater than the MIC of both the compounds was required to exert antimicrobial activity in the feed and digesta samples. The natural antimicrobials and Termin 8 had significant inhibitory effects on the drug resistant bacteria and synergistically enhanced the efficacy of antibiotics when used in combination. Further studies are needed to test their effectiveness in animal models.

Biological control is an intriguing alternative to the use of chemical pesticides as it represents a safer, more environmentally friendly approach to managing plant pathogens. Pseudomonas chlororaphis strain PA23 was isolated from soybean root tips and it was found to be an excellent antagonist of sclerotinia stem rot. Our studies have shown that pyrrolnitrin (PRN) is the key metabolite required for S. sclerotiorum inhibition, while phenazine (PHZ) is important for biofilm establishment. For this reason, research efforts were directed towards elucidating the mechanisms governing PA23-mediated antibiotic production. To determine how these compounds were regulated, QS-deficient strains and an rpoS mutant were generated. The QS-deficient strains no longer inhibited the fungal pathogen S. sclerotiorum in vitro and exhibited reduced PRN, PHZ and protease production. Analysis of transcriptional fusions revealed that RpoS has a positive and negative effect on phzI and phzR, respectively. In a reciprocal manner, RpoS is positively regulated by QS. Characterization of a phzRrpoS double mutant showed reduced antifungal activity as well as PRN and PHZ production, similar to the QS-deficient strains. Furthermore, phzR but not rpoS was able to complement the phzRrpoS double mutant for the aforementioned traits, indicating that the Phz QS system is a central regulator of PA23-mediated antagonism. GacS/GacA, PsrA, RpoS and the PhzI/PhzR QS are members of a complex regulatory hierarchy that influence secondary metabolite production in PA23. An additional system, termed Rsm, was identified, adding yet another layer of complexity to the regulatory network. The Rsm system in PA23 appears to be comprised of a single small non-coding regulatory RNA termed RsmZ, and two RNA binding proteins RsmA and RsmE. We discovered that the expression of rsmZ, rsmA and rsmE all require GacA. In addition, both PsrA and QS were shown to positively regulate rsmZ transcription. For rsmE, GacA may indirectly regulate expression through PsrA, RpoS and QS, as all three regulators control rsmE transcription. Furthermore, we believe that the positive effects of PsrA and QS on rsmE transcription are likely mediated through RpoS as only RpoS show direct activation of rsmE in an E. coli background.

Environmental pathogens, such as Escherichia coli and Streptococcus uberis, are currently the major cause of mastitis within dairy herds. This leads to severe financial losses, lower production rates and deterioration of the general health of the herd. E. coli mastitis is becoming a major threat to high milk-producing dairy herds. This is because of its increasing resistance to antibiotics, rendering antibiotic treatment regimes against E. coli infections mostly ineffective. The aim of this study was to develop a method to select mastitis causing E. coli isolates for the formulation of effective herd specific vaccines. Two methods, namely a genotyping method (Random Amplification of Polymorphic DNA; RAPD) and an antibiogram based method, were used. A dairy farm milking approximately 1000 Holstein cows in the Darling area, Western Cape Province, was selected for this study. The study was conducted over a period of 48 months and mastitis samples were analysed for mastitis pathogens. Antibiogram testing (disk diffusion method) and an in-house developed RAPD analysis method were used to analyse the E. coli isolates. A total of 921 milk samples were analysed from which 181 E. coli isolates were recovered. The number of all other common mastitis pathogens combined was 99 isolates (Streptococcus uberis 18, Streptococcus dysgalactiae 46, Streptococcus agalactiae 1, Staphylococcus epidermidis 21, Arcanobacterium pyogenes 13). All E. coli isolates, except for one, were resistant to at least three antibiotics. Antibiotic variance profiles were also highly erratic. The RAPD analysis revealed high levels of polymorphisms and clear epidemiological trends were observed over time. No similarities in the variance profiles between the antibiotic variance data and phylogenetic data were observed. Formalin inactivated autogenous vaccines were produced containing E. coli isolated from the herd. The vaccines were formulated using the RAPD or antibiogram data of the E. coli isolates. A total of 5 vaccines were formulated using RAPD data (Rvaccines) and one vaccine was formulated using antibiotic variance data (A-vaccine). The RAPD formulated vaccines were more effective than the antibiotic variance formulated vaccine. After each R-vaccination, the number of E. coli mastitis cases declined within the herd. The A-vaccinations seemed to have had no effect, which lead to a rise in E. coli mastitis cases. RAPD analysis on new emerging isolates was able to detect genetic variation from vaccine strains, which in turn facilitated the formulation of new updated vaccines with higher effectiveness than the previous vaccine. Mastitis data prior to and after the vaccination period revealed significant higher incidences of mastitis in the herd than during the vaccination period. This study demonstrated that sufficient sampling practices coupled with a reliable genotyping method, resulted in the formulation of updatable vaccines which were highly effective in controlling E. coli mastitis within the herd. / Thesis (M Environmental Sciences)--North-West University, Potchefstroom Campus, 2012

Biological control is an intriguing alternative to the use of chemical pesticides as it represents a safer, more environmentally friendly approach to managing plant pathogens. Pseudomonas chlororaphis strain PA23 was isolated from soybean root tips and it was found to be an excellent antagonist of sclerotinia stem rot. Our studies have shown that pyrrolnitrin (PRN) is the key metabolite required for S. sclerotiorum inhibition, while phenazine (PHZ) is important for biofilm establishment. For this reason, research efforts were directed towards elucidating the mechanisms governing PA23-mediated antibiotic production. To determine how these compounds were regulated, QS-deficient strains and an rpoS mutant were generated. The QS-deficient strains no longer inhibited the fungal pathogen S. sclerotiorum in vitro and exhibited reduced PRN, PHZ and protease production. Analysis of transcriptional fusions revealed that RpoS has a positive and negative effect on phzI and phzR, respectively. In a reciprocal manner, RpoS is positively regulated by QS. Characterization of a phzRrpoS double mutant showed reduced antifungal activity as well as PRN and PHZ production, similar to the QS-deficient strains. Furthermore, phzR but not rpoS was able to complement the phzRrpoS double mutant for the aforementioned traits, indicating that the Phz QS system is a central regulator of PA23-mediated antagonism. GacS/GacA, PsrA, RpoS and the PhzI/PhzR QS are members of a complex regulatory hierarchy that influence secondary metabolite production in PA23. An additional system, termed Rsm, was identified, adding yet another layer of complexity to the regulatory network. The Rsm system in PA23 appears to be comprised of a single small non-coding regulatory RNA termed RsmZ, and two RNA binding proteins RsmA and RsmE. We discovered that the expression of rsmZ, rsmA and rsmE all require GacA. In addition, both PsrA and QS were shown to positively regulate rsmZ transcription. For rsmE, GacA may indirectly regulate expression through PsrA, RpoS and QS, as all three regulators control rsmE transcription. Furthermore, we believe that the positive effects of PsrA and QS on rsmE transcription are likely mediated through RpoS as only RpoS show direct activation of rsmE in an E. coli background.

Bacterial isolates from animals, foods and clinical samples with resistances to one or more antibiotics are being found frequently each year. Selective pressure exerted by antibiotic growth promoters in food animals has been considered a main cause for the development of antibiotic resistance and antibiotic use has been strongly criticized as a serious public health threat. The gastrointestinal tract of animals not only serves as a reservoir of zoonotic agents but also as a spot for exchange of genetic information between pathogenic and commensal bacteria. Humans get infections from resistant bacteria either through the food chain, contaminated water or by direct contact with animals. In this situation much of the concern has been directed against the use of antibiotic growth promoters in animals. The removal of synthetic antibiotics from animal diets created other problems such as a decline in animal welfare and an increase in the use of therapeutic antibiotics. So there is a need for new alternatives to antimicrobial drugs to overcome resistance development and related problems. Plants and plant-derived compounds have long been considered to posess antimicrobial activity since they were frequently used in ancient medicine as natural remedies to treat human infections. Identifying new sources of natural antimicrobials and inhibitors of resistance development will yield novel therapeutic drugs and extend the useful life of existing antibiotics. In the present work, individual and combined effects of five essential oils (eugenol, thymol, carvacrol, cinnamaldehyde, allyl isothiocyanate (AIT)) and a formaldehyde-based feed additive, Termin 8, with antibiotics against 4 antibiotic resistant bacteria with known determinants for resistance were tested using broth microdilution and the checker board assay. The bacteria showed considerable susceptibility towards these antimicrobials and a significant reduction in the minimum inhibitory concentrations (MICs) of antibiotics was noted when paired combinations of antibiotic and antimicrobial were used. The synergistic interaction was further confirmed by the extent of decrease in logarithmic count or viable population (Log DP). Although most of the combinations were synergistic by fractional inhibitory concentration (FIC) values, fewer combinations showed synergistic interaction when Log DP was considered. Gram-positive bacteria were more sensitive to the antimicrobials than Gram-negative bacteria. In combination studies, carvacrol was more effective and showed synergistic interaction with at least three antibiotics. When used alone, AIT was more effective and the concentration needed to exhibit antimicrobial action was much lower when compared to other compounds. An in vitro study was conducted to assess the antibacterial effects of Termin 8 and thymol in chicken caecal digesta and poultry feed samples by using a thin agar layer (TAL) method. Concentrations greater than the MIC of both the compounds was required to exert antimicrobial activity in the feed and digesta samples. The natural antimicrobials and Termin 8 had significant inhibitory effects on the drug resistant bacteria and synergistically enhanced the efficacy of antibiotics when used in combination. Further studies are needed to test their effectiveness in animal models.

Coincident with human activity in recent decades, human-associated microorganisms have arrived to the Antarctic region, possibly linked to increasing presence of scientific bases and ship-borne tourists. In the Arctic, humans have been present for a very long time, and the few parts of the Arctic without human activities is decreasing with time. The studies in this thesis investigate the occurrence of different pathogens in Antarctic and Arctic wildlife, especially in birds. The first study shows the existence of Enteropatogenic Escherichia coli (EPEC) in Antarctic fur seals. The EPEC isolates were so called atypical EPECs, carrying the eae gene but lacking the bfp gene. This is the first record of a diarrheogenic E. coli in wild animals in the Antarctic. The second study displays that spreading of antibiotic resistance mechanisms appears to be much more efficient than previously was known. Enterococcus faecium isolated from Alaskan birds showed high resistance to vancomycin and teicoplanin, but also to ampicillin and ciprofloxacin. These isolates also carried vanA genes and the virulent esp gene, which places the isolates in the clinical clone CC17 and indicates the isolates had a human origin. Bacteria from birds that reside in the Bering Strait region in the third study, demonstrates that only six of 145 E. coli from 532 birds had reduced antibiotic susceptibility. Despite this, selective screen on E. coli showed only four ESBL-producing isolates. The four E. coli isolates carried CTX-M genes. One isolate belonged to the E. coli O25b - ST131 genotype, which is a successful clone with a global spread. In the fourth study, 123 seawater samples and 400 fresh penguin feces were analyzed. From these, 71 E. coli strains were isolated and only one E. coli from penguins was resistant to one antibiotic (cloramfenicol), whereas in E. coli from seawater, resistance against ampicillin, tetracycline, streptomycin and trim-sulfa were detected. E. coli carrying ESBL type CTX -M genes were also detected and Multilocus Sequencing Typing (MLST) showed six different sequence types (ST) previously reported in humans: ST131, ST227, ST401, ST410, ST685 and ST937. In the short time interval between the second study (2005) and the third study (2010) in relation to the fifth study (2012) we found a dramatic increase in antibiotic-resistant genes in the Arctic region. Enterococci, E. coli, and Kl. pneumoniae carried antibiotic resistance genes to an extent and variety not previously reported. E. coli from Arctic birds showed resistant to 1, 2, 3, 4, and 5 different antibiotics. Resistant gene type vanA was confirmed in enterococci and ESBL genes type TEM, SHV and CTX-M in E. coli and Kl. pneumoniae was detected. Multilocus Sequencing typing (MLST), indicating that both E. coli and Kl. pneumoniae carrying ESBL markers that connects them to the humans. In summary, the combined studies strengthen that bacteria that cause infections in humans could spread to relatively pristine environments. We concluded that human and associated antibiotic-resistant bacteria has reached a global level, then we showed that ESBL- carrying bacteria circulating nowadays also in the last ESBL-free continent, Antarctica.

The treatment of Staphylococcus aureus bloodstream infections (SABSI) remains a major challenge. With an emphasis on complicated methicillin–sensitive S. aureus (MSSA), a comprehensive analysis of initial antibiotic treatment was conducted. The influence of treatment gaps on clinical outcomes were examined. Strategies were developed to improve the use of available antibiotics. Patient- and infection-related variables predictive for end-of-treatment failure included higher Charlson Comorbidity Index and healthcare-associated infection. Treatment variables of shorter duration of optimal targeted, shorter duration of optimal or adequate and lower TSE score were also predictive for end-of-treatment failure when tested separately in their own models. Strategies to optimize the treatment of complicated MSSA BSI at minimum should include: 1) Initiating at least an adequate therapy within 24 hours following the index blood culture draw and 2) Maintaining uninterrupted treatment, especially during the initial 7 days including at least 4 days of cloxacillin or cefazolin.

This research aimed to measure the effect on health outcomes of implementing selected recommendations of the Grampian Joint Drug Formulary in primary care. Antibiotics used in the treatment of uncomplicated lower urinary tract infections (UTIs), ulcer healing agents and peripheral vasodilators were selected for study, thereby reflecting both acute and chronic prescribing. For the UTI study, 12 randomly selected high and low prescribers of trimethoprim, the recommended agent, each agreed to distribute 20 patient questionnaires. Following a period of 18 months and despite repeated contact with the GPs, only 89/480 (19%) questionnaires had been distributed. Patient response was, however, very high with 80 (90%) questionnaires returned. Health outcome measures identified that trimethoprim resulted in no or mild symptoms in 40/45 (91 %) of patients. These findings must be interpreted with caution due to the low level of questionnaire distribution and thus cannot be extrapolated to the total population of patients. In addition, the poor questionnaire distribution did not permit comparison between trimethoprim and non-recommended therapy. One hundred and eighty four patients receiving repeat prescriptions for ulcer healing agents were identified from one general practice. Therapy in 95 patients did not adhere to formulary recommendations. Changes to therapy were considered inappropriate in 11 patients due to factors such as severe depression and a further 8 were deemed unsuitable for participation for non-clinical reasons. The remaining 76 patients were contacted with 19 (25%) refusing to participate. Fifty seven patients were interviewed using the Glasgow Dyspepsia Severity Score and Short Form 36 (SF-36). Changes in health outcomes were measured for 21 patients where a change in therapy had taken place. These results were difficult to interpret due to the diversity of changes recommended and the lack of data relating to those patients not participating. Work involving peripheral vasodilators aimed to determine the effect on health outcomes of cessation of therapy. Forty five patients receiving these agents in 2 practices were identified, although 8 had not requested a prescription in the previous year. Two further patients were excluded from the study due to cancer and old age. The remaining 35 agreed to be interviewed using the Walking Impairment Questionnaire and SF-36. All patients were subsequently instructed to stop therapy for 2 months, although 6 (17%) refused to follow this instruction, one patient was seriously ill thus was excluded and 3 refused to be reinterviewed. Of the remaining 25 patients, no significant differences were observed in the domains studied. Seventeen patients (68%) expressed no desire to restart therapy, generating considerable savings. These results must be interpreted with caution since those not stopping therapy or refusing re-interview are likely to have responded differently to those completing the study. The measurement of health outcomes following formulary implementation deserves further work.

Antibiotics are an important method for protecting human health. Unfortunately, the development of antibiotic resistance has decreased the effectiveness of antibiotics in treating disease and preventing deaths associated with bacterial infection. The objective of this dissertation research was to gain a better understanding of anthropogenic influences on occurrence of tetracycline resistance and use of traditional disinfection methods for the reduction of tetracycline resistant bacteria and genes. Culture based and molecular methods were used to evaluate the occurrence of tetracycline resistance in a rapidly urbanizing watershed, identify the dominant resistant organisms and resistance genes in the watershed, and evaluate the use of UV and chlorine to reduce the concentration of resistant bacteria and resistance genes. Results from this research showed that tetracycline resistance was prevalent and is maintained in this study area. Several bacterial species (Aeromonas, Acinetobacter, Chryseobacterium, E. coli, Pseudomonas, and Serratia) made up the resistant population. The results also indicated that tet(W) was the major resistance gene in this watershed and that a majority of the resistant bacteria were capable of transferring their resistance. Landuse did not cause a difference in occurrence of resistant bacteria or resistance genes which suggests that a rapidly urbanizing watershed could experience resistance. It was also identified that environmental media (sediment and water) influence the occurrence and prevalence of resistant bacteria and resistance genes. The results indicate that streambed sediment may act as a reservoir for resistance and resistance might be transported in the water. Finally, the results showed that neither UV nor chlorine disinfection were effective in reducing tet(W) concentrations though the results varied greatly among species. Results from this research indicate that preventing the occurrence and distribution of resistance gene in the environment is difficult, and resistance will most likely be maintained. Therefore, in order to prevent the spread of antibiotic resistance, it will be important to prevent antibiotic resistance from becoming established in the environment. This can be done by educating the public about the importance of misusing and mismanaging antibiotics. Additionally, classifying antibiotics for either human or veterinary use may help slow the development of resistance. This should prevent clinically important antibiotics from being used in sub-therapeutic doses, which could decrease the selective pressure in the environment. Also clinically relevant bacteria can be prevented from interacting with resistant bacteria in the environment by disinfecting human waste.

Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium that is also a major opportunistic human pathogen in nosocomial infections and cystic fibrosis chronic lung infections. These P. aeruginosa infections can be extremely difficult to treat due to the high intrinsic antibiotic resistance and broad repertoire of virulence factors, both of which are highly regulated. It was demonstrated here that the psrA gene, encoding a transcriptional regulator, was up-regulated in response to sub-inhibitory concentrations of antimicrobial peptides. Compared to wild-type and the complemented mutant, a P. aeruginosa PAO1 psrA::Tn5 mutant displayed intrinsic super-susceptibility to polymyxin B, a last resort antimicrobial used against multi-drug resistant infections, and indolicidin, a bovine neutrophil antimicrobial peptide; this super-susceptibility phenotype correlated with increased outer membrane permeability. The psrA mutant was also defective in simple biofilm formation, rapid attachment, and normal swarming motility, phenotypes that could be complemented by the cloned psrA gene. The role of PsrA in global gene regulation was studied by comparing the psrA mutant to wild-type by microarray analysis, demonstrating that 178 genes were up or down-regulated by greater than 2-fold (P ≤0.05). Dysregulated genes included those encoding known PsrA targets, the type III secretion apparatus and effectors, adhesion and motility genes and a variety of metabolic, energy metabolism and outer membrane permeability genes. This indicates that PsrA is a central regulator of antimicrobial peptide resistance and virulence. P. aeruginosa containing a mutation in the PhoQ sensor kinase-encoding gene was highly attenuated for persistence in a rat chronic lung infection model. In addition, the polymyxin B hyper-resistant phoQ mutant displayed reduced type IV pili-dependent twitching motility and was less cytotoxic towards human bronchial epithelial cells, indicating that the virulence defect observed could be due at least in part to these phenotypes. Using microarrays it was further demonstrated that PhoQ regulates a large number of genes that are PhoP-independent and that the phoQ mutation leads to up-regulation of PhoP- and PmrA regulated genes as well as other genes consistent with its virulence phenotypes.