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Includes bibliographical references., A previous study of 100 methicillin-resistant Staphylococcus aureus (MRSA) isolates, collected between January 2007 and December 2008 from five Cape Town hospitals, identified ST612-MRSA-IV as the predominant MRSA. This raised the question of whether methicillin-susceptible S. aureus (MSSA) isolates with the same sequence type (ST) were present in hospitals in this city. Nineteen MSSA isolates, collected during the same period, and from four of the hospitals in Cape Town, as the previously characterized MRSA isolates, were screened for the presence of ST612-MSSA. spa typing and multi-locus sequence typing (MLST) identified one isolate, MS14, as ST612-MSSA, spa type t064.

Acinetobacter baumannii is a major pathogen causing nosocomial infections, particularly in critically ill patients. This organism has acquired the propensity to rapidly develop resistance to most antibiotics. At several hospitals within Cape Town, tobramycin and colistin remain frequently the only therapeutic options. The Vitek2 automated susceptibility testing (AST) is used in the clinical laboratory to determine selected susceptibility profiles. The suspicion of a possible AST-related technical error when testing for susceptibility to tobramycin in A. baumannii precipitated this study.

Comprehensive molecular epidemiological data are prerequisite for establishing control over methicillin-resistant S. aureus (MRSA) in the hospital setting; however, there is currently a paucity of molecular epidemiological data available on MRSA from South Africa. A molecular characterisation of one hundred MRSA isolates collected between January 2007 and December 2008 from patients in five hospitals in Cape Town was carried out in this study.

This study describes the molecular characteristics of STEC isolated from the pastoralist community of Nyabushozi in Southwestern Uganda. Faecal samples or rectal swabs of children with diarrhoea obtained in phases 1 and 2 were investigated for the presence of STEC by PCR detection of stx genes. During phase 1, cattle reared on range which were associated with households of sick children were investigated in parallel to the children for STEC excretion. STEC was isolated from E. coli in 7 of 80 (8.8%) children and in 15 of 216 (6.9%) bovines in phase 1. Similarly, STEC was isolated from 11 of 142 (7.7%) E. coli carrying children and 3 of 45 (6.7%) water samples in phase 2. Molecular characterization further ascertained the genetic relatedness of STEC. PFGE pro les of up to 10 colonies obtained from an individual source (child, bovine or water) and in total 185 STEC colonies were analysed. Nine pro les from 43 colonies (phase 1) and 15 pro les from 38 colonies (phase 2) obtained from children were not or were distally related, indicating the genetic diversity of clinical STEC. The intra-host analysis of STEC pro les revealed that strains from 11 of the 13 children exhibited multiple clonal subgroups. The 101 colonies from 15 bovines clustered in 18 di erent pro les. Clonal subgroups were observed in multiple STEC colonies from 11 of 12 bovines. Closely related pro les indicated that STEC isolated from two children (Hh2 and Hh4) was acquired from bovines or their environment. While none of the clinical or bovine STEC were related to 5 genetically diverse water strains. A single isolate of STEC representing each PFGE pro le in association with stx gene content was serotyped for the O antigen. Twenty four bovine STEC were typed into 10 O serogroups including O8, O76, O111 and O113, which were also identi ed among the clinical STEC. The 25 clinical STEC belonged to 15 serogroups of which O29, O149 and O176 are being reported for the rst time as clinical STEC. STEC xxi Abstract O166 was isolated from a child and water during the same sampling, indicating the potential health hazard of drinking STEC-contaminated water. The production of Shiga toxin (Stx) investigated using Duopath Verotoxin detection kits showed that a majority of STEC from di erent sources produced Stx1 or Stx2 or both Stx. Using PCR or PCR-RFLP assays, stx2 and eae gene types were analysed. Variant stx2 vhc was most prevalent and closely associated with stx2d2 in clinical and bovine STEC. The frequency of eae-positive STEC among clinical and bovine STEC was 15 of 25 (60%) and 14 of 24, (58.3%), respectively. eae- 2/ was predominant among the bovine STEC, eae- / in clinical STEC, while eae- 1 was associated with STEC from di erent sources including water. Previously undescribed eae-positive serogroups O28ac, O113, O142 and O158 were identi ed. Studies of the genetic background showed that both clinical and bovine STEC obtained in phase 1 predominantly belonged to phylogenetic group A and B1, while phase 2 clinical and water STEC belonged to group D and A, respectively. Seropathotype classi cation of clinical STEC, separated most strains (20 of 24 strains) into seropathotype D. These STEC belonged to phylogenetic groups A, B1 and D. Thus, the characterised genetic attributes of STEC from Nyabushozi suggests that the pathogens have the potential to cause a wide spectrum of childhood illnesses ranging from mild to bloody diarrhoea and haemolytic uraemic syndrome. xxii

Includes bibliographical references (leaves 60-78).

Bibliography leaves 122-136.

Fluoroquinolone susceptible and resistant Campylobacter coli were isolated from pigs on two separate pig farms. C. coli are enteric pathogens of humans and animals and although diarrhoea resulting from C. coli and C. jejuni is generally a self-limiting disease, in severe cases, fluoroquinolones are the choice antibiotic for treatment. The presence of fluoroquinolone resistant C. coli strains in the food chain is cause for concern as this may be a source of resistant strains in humans. Sixty-one isolates were included in the study: 26 were susceptible to nalidixic acid and ciprofloxacin and 35 were resistant to these antibiotics. Fifty-five strains were obtained from pigs on farm A, while 6 strains were obtained from pigs on farm B, the source farm of pigs to farm A. Serotyping and flaA typing were carried out to study the epidemiology of the isolates. Serotyping identified 0:24 (11/61) as the most frequent serotype isolated, followed by 0:5 (7/61). Common serotypes 0:48, 0:54 and 0:59 were identified in strains from both farms. A high number of the strains were non-typeable (23/61) but were distinguished by flaA typing. RFLP analysis of the flaA gene revealed 13 distinct profiles in strains from farm A, and 4 profiles in strains from farm B, of which only 1 was unique to farm B. Profile 1 was the commonest profile observed with 31 % (17 /55) of flaA typed strains in this profile. There was an association between 0:24, profile 6, and resistance. Resistant and sensitive pairs were isolated from 15 pigs; flaA profiles of each of 4 pairs were identical, suggesting selection of resistant mutants from previously sensitive populations. An investigation of the molecular basis of the fluoroquinolone resistance identified a Thr-86 to Ile mutation in GyrA, the primary target of these antibiotics.

Bacterial resistance to antibiotics is a common and important clinical problem. Beta lactam resistance in Gram negative bacilli is mediated predominantly by beta lactamases, enzymes able to hydrolyse the beta lactam ring. The commonest plasmid mediated beta lactamases in the Enterobacteriaceae are those related to either TEM-1 or SHY-1. Although TEM-1, TEM-2 and SHY-1 do not have activity against extended spectrum beta lactams, their derivatives (TEM-3 and SHY-2 onwards) are able to confer resistance to one or more of these antibiotics. A problem encountered in clinical microbiology laboratories is the lack of a reliable method for the detection of ESBLs, along with the lack of a quick, reliable method of differentiating TEM-related genes from SHY -related genes. The primary aim of this study was to evaluate two molecular techniques for the detection of SHY and TEM-related genes in clinical isolates. The study sample consisted of 209 clinical isolates of enteric Gram negative bacilli, isolated at Groote Schuur Hospital microbiology laboratory. The isolates had all been selected on the basis of resistance to one or more of the extended spectrum beta lactams. These isolates were all identified, and the susceptibility of each to a variety of beta lactam antibiotics determined. Using this information, 45 isolates, belonging to different genera and with differing antimicrobial sensitivity patterns, were selected for this pilot study. These 45 isolates consisted of 24 Klebsiella spp., 14 Enterobacter spp., 3 Citrobacter spp., 2 Salmonella spp., 1 Pantoea agglomerans and 1 Serratia marcescens.

Acinetobacter baumannii is responsible for an increasing number of nosocomial infections in patients receiving intensive care and comprehensive antibiotic resistance of these organisms hampers treatment of infections due to A.baumannii. The molecular basis of antibiotic resistance in A.baumannii has not been extensively investigated. A few studies have demonstrated the role of plasmids and transposons in resistance in this organism, but there is little data on the role of integrons and integron-associated antibiotic resistance. This study was undertaken to determine the incidence of integrons in clinical isolates of Acinetobacter from Groote Schuur Hospital (GSH), Cape Town and Universitas Hospital (UH), Bloemfontein, and to characterise the resistance genes carried in the variable regions of these integrons.

The genetic basis of aminoglycoside resistance in clinical isolates of Acinetobacter was investigated. The aadB genes cloned from two clinical isolates, strain CHA and strain SUN, were sequenced. Analysis of the sequencing data indicated that both genes were contained on gene cassettes, which had integrated at secondary sites on a plasmid isolated in each strain. Gene cassettes are usually associated with integrons, and cassettes recombined at secondary sites are thought to be stably integrated. However, conduction assays indicate that the aadB gene cassette described in this study is potentially mobile. Outside of an integron, transcription of the structural gene on a cassette is dependent on insertion of the cassette downstream of correctly aligned promoter sequences. A number of putative promoters were identified upstream of the aadB structural gene. Primer extension studies were carried out to study the regulation of aadB. These experiments showed that in Acinetobacter the aadB gene is regulated by a promoter consisting of a -10 hexamer only. Similar experiments showed that, in Escherichia coli, the same gene is transcribed from a different promoter, which is typical of those recognized by the major RNA polymerase in this organism. Thus, the transcription signals recognized in Escherichia coli were different from those recognized in Acinetobacter. Naturally occurring plasmids in clinical isolates of Acinetobacter have not been fully characterized: pRAY was characterized by sequencing. An analysis of the sequencing data identified features consistent with an origin of replication. Moreover, this analysis suggests that pRAY may be mobilizable. This work, in part, has been published in FEMS Microbiology Letters (Segal and Elisha, 1997).