Thesis (MScMedSc (Pathology. Medical Microbiology))--University of Stellenbosch, 2009. / Water is used for various purposes and contamination can have severe implications if untreated. One of the most common and cost effective water disinfectants, especially used in developing countries, is chlorine. However, microorganisms have developed different mechanisms in response to environmental stress conditions, such as the viable but nonculturable (VBNC) effects possibly displayed in this study, enabling them to survive. Chlorine may also exert several effects on microorganisms, such as the expression of multi-substrate efflux pumps, decreased membrane permeability and transport inhibition that may lead to chlorine tolerance and antimicrobial resistance. In a descriptive and comparative study, the molecular characteristics of E. coli strains isolated from environmental waters in the Western Cape and the possible relationship between chlorination and antimicrobial resistance were investigated. Water and biofilm samples were exposed to chlorine, as well as efflux pump inhibitor (EPI) concentrations, and surviving E. coli strains were tested for their phenotypic characteristics including antimicrobial susceptibility profiles and morphological types. Candidate genes possibly involved in resistance to antimicrobials, disinfection and efflux pumps were detected with polymerase chain reaction (PCR) and sequenced. Sequencing analysis and homology searches were done and E. coli strains were typed as either Enteropathogenic E. coli strains (EPEC) or Enterotoxigenic E. coli strains (ETEC) on the presence of virulence genes. All water and biofilm sources examined were heavily polluted with E. coli, and a high enumeration level of this indicator organism of faecal contamination was recorded. Chlorine tolerance was found to be associated with antimicrobial resistance. Addition of EPI with exposure to chlorine decreased enumeration levels of these organisms, suggesting that efflux pumps may play a role in tolerance to chlorine. Several morphological patterns were described amongst the E. coli strains and a change in this was recorded after exposure to chlorine. Highly resistant antibiograms displayed by the isolated strains included ampC β-lactamase producing E. coli strains and extended spectrum β-lactamases (ESBLs). Amplification of the candidate genes selected for heatshock, oxidative stress genes and efflux pump were most frequently detected while the structural genes involved in fluoroquinolones (FQs) resistance were detected less frequently in the selected strains. Sequencing of these amplified candidate genes demonstrated various changes in amino acid sequences, including one common mutational pathway taken by E. coli when exposed to stress conditions. Further homology searches of the sequenced candidate genes illustrated similarities in 19 pathogenic and 14 non-pathogenic E. coli as well as 3 Shigella strains. Detection of virulence genes found three EPEC strains (bfpA, eaeA), two EPEC (eaeA), ten EPEC (bfpA) and one ETEC strain (st) amongst the isolates. This study underlines the need for monitoring our water sources, which poses a public health risk due to incomplete chlorination, antimicrobial resistance and the spread of clinically relevant pathogenic strains.

Bibliography / Thesis (MMed (Pathology. Medical Microbiology))--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Acinetobacter baumannii has emerged as one of the most troublesome nosocomial pathogens globally. This organism causes infections that are often extremely difficult to treat because of the widespread resistance to the major antibiotic groups. Colonization or infection with multidrugresistant A. baumannii is associated with the following risk factors: prolonged hospital stay, admission to an intensive care unit (ICU), mechanical ventilation, and exposure to broad spectrum antibiotics, recent surgery, invasive procedures, and severe underlying disease. A. baumannii has been isolated as part of the skin flora, mostly in moist regions such as axillae, groin and toe webs. It has also been isolated from the oral cavity and respiratory tract of healthy adults. Debilitated hospitalized patients have a high rate of colonization, especially during nosocomial Acinetobacter outbreaks. This organism is an opportunistic pathogen as it contains few virulence factors. Clinical manifestations of A. baumannii include nosocomial pneumonia, nosocomial bloodstream infections, traumatic battlefield and other wound infections, urinary tract infections, and post-neurological surgery meningitis. Fulminant community-acquired pneumonia has recently been reported, indicating that this organism can be highly pathogenic. The number of multidrug-resistant A. baumannii strains has been increasing worldwide in the past few years. Therefore the selection of empirical antibiotic treatment is very challenging. Antibiotic combinations are used mostly as empirical therapy in critically ill patients. One rationale for the use of combination therapy is to achieve synergy between agents. The checkerboard and time-kill methods are two traditional methods that have been used for synergy testing. These methods are labor intensive, cumbersome, costly, and time consuming. The E-test overlay method is a modification of the E-test method to determine synergy between the different antibiotics. This method is easy to perform, flexible and time efficient. The aim of this study was to assess the in vitro activity of different combinations of colistin, rifampicin, imipenem, and tobramycin against selected clinical strains of A. baumannii using the checkerboard and the E-test synergy methods. The MICs obtained with the E-test and broth microdilution method were compared. The results of the disk diffusion for imipenem and tobramycin as tested in the routine microbiology laboratory were presented for comparison. Overall good reproducibility was obtained with all three methods of sensitivity testing. The agreement of MICs between the broth dilution and E-test methods was good with not more than two dilution differences in MIC values for all isolates, except one in which the rifampicin E-test MIC differed with three dilutions from the MIC obtained with the microdilution method. However, the categorical agreement between the methods for rifampicin was poor. Although MICs did not differ with more than two dilutions in most cases, many major errors occurred because the MICs clustered around the breakpoints. The combinations of colistin + rifampicin, colistin + imipenem, colistin + tobramycin, rifampicin + tobramycin, and imipenem + tobramycin all showed indifferent or additive results by the E-test method. No results indicating synergy were obtained for all the above-mentioned combinations. There was one result indicating antagonistic effect for the combination of colistin + tobramycin. The results of the checkerboard method showed results indicating synergy in four of the six isolates for which the combination of colistin and rifampicin was tested. The other two isolates showed indifferent/additive results. All the other combinations showed indifferent/additive results for all isolates except isolate 30 (col + tob) and isolate 25 (rif + tob) which showed synergism. No antagonistic results were observed by the checkerboard method. When the results obtained with the E-test and checkerboard methods were compared, it was noted that for most antibiotic combinations an indifferent/additive result was obtained. However, for the colistin + rifampicin combination, the checkerboard method showed synergism for 4 of 6 isolates, whereas the E-test method showed indifference and an additive result in one. For the rifampicin + tobramycin, and colistin + tobramycin combinations, synergism was also shown with the checkerboard method in one isolate for each combination. The E-test method however showed an indifferent and additive result respectively. . The E-test method was found to be a rapid, reproducible, easy-to-perform, and flexible method to determine synergistic antibiotic activity. This study was however limited by low numbers of isolates. This might explain why no synergistic results were obtained with the E-test method and few synergistic results with the checkerboard method. Genotypic analysis using pulse-field gel electrophoresis (PFGE) may be considered in future studies to determine relatedness of the isolates which will facilitate the selection of different strains for synergy testing. Furthermore, clinical studies are needed to establish whether in vitro synergy testing is useful in the clinical setting and whether the results of synergy testing will have any bearing on the clinical outcome of patients infected with multidrug resistant A. baumannii. / AFRIKAANSE OPSOMMING: Acinetobacter baumannii het wêreldwyd as een van die mees problematiese nosokomiale patogene verskyn. Hierdie organisme veroorsaak infeksies wat dikwels baie moeilik is om te behandel weens wydverspreide weerstandigheid teen major antibiotikagroepe. Kolonisasie of infeksie met multi-weerstandige A. baumannii word geassosieer met die volgende riskofaktore: verlengde hospitaalverblyf, toelating tot ‘n intensiewe sorgeenheid (ICU), meganiese ventilasie, blootstelling aan breëspektrum antibiotika, onlangse chirurgie, indringende prosedures en ernstige onderliggende siekte. A. baumannii kan deel vorm van die normale velflora, veral in die axillae, inguinale area en tussen die tone. Dit is ook al vanuit die mondholte en die respiratoriese traktus van gesonde volwassenes geïsoleer. Verswakte gehospitaliseerde pasiënte word veral gekoloniseer gedurende nosokomiale Acinetobacter uitbrake. Hierdie organisme is ‘n opportunistiese patogeen en bevat min virulensie faktore. Kliniese manifestasies van A. baumannii sluit nosokomiale pneumonie, nosokomiale bloedstroom infeksies, troumatiese slagveld- en ander wondinfeksies, urienweginfeksies en meningitis wat volg op neurologiese chirurgie in. Fulminerende gemeenskapsverworwe pneumonie is onlangs beskryf en dui aan dat hierdie organisme hoogs patogenies kan wees. Die aantal multi-weerstandige A. baumannii stamme het wêreldwyd toegeneem oor die laaste paar jare. Daarom is die seleksie van empiriese antibiotiese behandeling ‘n uitdaging. Antibiotika kombinasies word meestal as empiriese behandeling in ernstige siek pasiënte gebruik. Die beginsel hiervan is om sinergistiese werking tussen agente te verkry. Die “checkerboard” en “time-kill” metodes is twee tradisionele metodes van sinergisme toetsing. Hierdie metodes is werksintensief, duur en tydrowend. Die E-toets sinergisme metode is gebaseer op die E-toets metode. Hierdie metode is maklik, buigbaar en tydseffektief. Die doel van hierdie studie was om die in vitro aktiwiteit tussen verskillende antibiotika kombinasies van colistin, rifampisien, imipenem, en tobramisien teen geselekteerde kliniese A. baumannii isolate te toets met die “checkerboard” en E-toets sinergisme toetsing metodes. Die minimum inhibitoriese konsentrasies (MIKs) verkry met die E-toets en “broth microdilution” metode is ook vergelyk. Die resultate van die skyfie diffusie metode (die metode wat in die roetiene mikrobiologie laboratorium gebruik word) vir imipenem en tobramisien word ook verskaf vir vergelyking van die resultate van verskillende sensitiwiteitsmetodes. In oorsig is goeie herhaalbaarheid van resultate verkry met al drie metodes van sensitiwiteitstoetsing. Die ooreenstemming van MIKs tussen die “broth dilution” en E-toets metodes was goed en resultate het met nie meer as twee verdunnings in MIK waardes verskil nie. Daar is een uitsondering waar die rifampisien E-toets MIK waarde met drie verdunnings van die MIK waarde verkry met die “microdilution” metode verskil. Die ooreenstemming tussen die sensitiwiteitskategorie resultate tussen die twee metodes was egter swak vir rifampisien. Alhoewel die MIKs in die meeste gevalle met nie meer as twee verdunnings in waarde verskil het nie, was daar baie major foute aangetoon omdat die MIKs rondom die breekpunte geval het. Die kombinasies van colistin + rifampisien, colistin + imipenem, colistin + tobramisien, rifampisien + tobramisien, en imipenem + tobramisien het oorwegend slegs matige interaksie met die E-toets metode getoon. Geen sinergisme is verkry met enige van die antibiotika kombinasies met hierdie metode nie. Daar was egter een resultaat wat antagonisme getoon het vir die kombinasie van colistin + tobramycin. Die resultate van die “checkerboard” metode het sinergisme getoon in vier van die ses isolate wat vir die kombinasie van colistin en rifampisien getoets was. Die ander twee isolate het slegs matige interaksie getoon. Al die ander kombinasies het ook slegs matige interaksie getoon, behalwe in isolaat 30 (col + tob) en isolaat 25 (rif + tob) waar die spesifieke kombinasies sinergisme getoon het. Geen antagonisme is waargeneem met die “checkerboard” metode nie. Met vergelyking van die E-toets en “checkerboard” metodes, is dit opmerklik dat vir die meeste van die antibiotika kombinasies slegs matige interaksie verkry is. Vir die colistin + rifampisien kombinasie toon die “checkerboard” metode egter sinergisme vir 4 uit 6 isolate, terwyl die E-toets metode slegs matige interaksie toon. Vir rifampisien + tobramisien, en colistin + tobramisien kombinasies is sinergisme getoon met die “checkerboard” metode in een isolaat vir elke kombinasie. Die E-toets metode het slegs matige interaksie getoon. Die E-toets sinergisme metode was vinnig, herhaalbaar en maklik om uit te voer. Hierdie studie word egter beperk deur lae getalle van isolate. Dit mag verklaar waarom geen sinergistiese resultate met die E-toets metode verkry is nie en die min sinergistiese resultate met die “checkerboard” metode. Genotipiese analiese met “pulse-field gel electrophoresis” mag in aanmerking geneem word in toekomstige studies om die verwantskap tussen isolate te bepaal wat die seleksie van verskillende stamme vir sinergisme toetsing sal vergemaklik. Verder, kliniese studies is nodig om te bepaal of in vitro sinergisme toetsing van waarde is en of die resultate van sinergisme toetsing ‘n rol speel in die kliniese uitkoms van pasënte geïnfekteer met multiweerstandige A. baumannii. / The National Health Laboratory Serivice

Bibliography / Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Overview: Mycoplasmas and ureaplasmas are not routinely diagnosed and are under researched in South Africa. Prevalence, population shifts especially concerning genital flora and implications in infection or other conditions are unknown. Information pertaining to Mycoplasma pneumoniae in respiratory disease is similarly lacking. There is little information on antimicrobial susceptibilities and resistance development against Sexually Transmitted Infections (STI) syndromic management approaches. Aims: a) Elucidate mycoplasmal and ureaplasmal prevalence and contributing factors concerning cervical colonisation or preterm delivery in conjunction with HIV and Chlamydia trachomatis b) Investigate prevalence of M. pneumoniae in respiratory infections in conjunction with HIV, Mycobacterium tuberculosis and Pneumocystis jiroveci. c) Determine antimicrobial susceptibilities of mycoplasmas and ureaplasmas and analyse resistance genes. d) Assess the inter-generic transfer potential of resistance gene (tetM) between Ureaplasma spp. and Neisseria gonorrhea. Genital setting: The prevalence of genital mycoplasmas, ureaplasmas and Chlamydia on women attending their first prenatal visit, in conjunction with preterm labour or HIV status was investigated. For preterm labour (2003), 199 women were monitored for preterm delivery (<37 weeks); for colonisation and HIV (2005), 219 women were screened. Microbial detection was performed on DNA extracted from endocervical swabs employing PCR techniques. Colonisation was seen to be highest in the 14-20 year group from 2003. In women aged ±21 years, co-colonisation was 13% although there was a shift from co-colonisation with Mycoplasma hominis and Ureaplasma spp. in 2003 to other dual/triple combinations in 2005. Overall major trends from both collection periods were that the prevalence of Ureaplasma spp. tended to be higher in women ±26 years, whilst prevalence of C. trachomatis and M. hominis were lower. No association was evident between colonisation with M. hominis, U. urealyticum, Ureaplasma parvum and labour outcome. HIV status had no effect on the prevalence/co-colonisation of M. hominis, Ureaplasma spp. or C. trachomatis. Respiratory setting: Studies were conducted to determine the prevalence of community acquired atypical pneumonias in adults (M. pneumoniae and P. jiroveci) and neonates (mycoplasmas, ureaplasmas and Chlamydia trachomatis) in order to improve treatment management programmes in the Port Elizabeth region. Sputum specimens from 102 adult patients presenting with pneumonia/symptoms of pneumonia admitted to hospitals were assessed by PCR. Details of patient’s gender, age, HIV and Mycobacterium tuberculosis status were provided by the hospitals. Women were seen to be at high risk for community-acquired P. jiroveci colonisation. Overall, prevalence of P. jiroveci was 52.9% (54/102 patients). P. jiroveci was mainly associated with HIV (25/74) (P. jiroveci and HIV positive patients in patient sample for which clinical data and HIV status was available) and co-infection with M. tuberculosis was observed in 12 HIV cases and one HIV negative patient. No DHPS (20) or DHFR (17) resistance associated mutations were found in P. jiroveci. M. pneumoniae was detected in one patient. For prevalence studies (2007-2008) on atypical pneumonia in neonates, 69 endotracheal aspirates were obtained. PCR detection of M. hominis, U. urealyticum and C. trachomatis was performed and U. parvum detected in two specimens. Antibiotic susceptibilities and resistance genes: The following investigations on clinical isolates of U. parvum and U. urealyticum were conducted (i) antibiotic susceptibility profiles, (ii) detection of drug target gene mutations, or gene acquisitions and (iii) inter-generic resistance gene transfer potential to Neisseria gonorrhoeae. Culture techniques applied to 132 endocervical specimens provided 66 Ureaplasma cultures (35 U. parvum, 9 U. urealyticum, 22 U. parvum + U. urealyticum). MIC determinations to ofloxacin, erythromycin, tetracycline, doxycycline, azithromycin and josamycin were performed. Thirty-seven ureaplasma cultures were fully susceptible to all antibiotics tested; 21 showed intermediate resistance to erythromycin, azithromycin and ofloxacin; while seven were resistant to tetracycline, three of which were also resistant to doxycycline and one also resistant to azithromycin. Concerning ofloxacin resistance directed at quinolone resistance determining regions, a substitution of Ser83Leu in ParC was demonstrated in one intermediately-resistant Ureaplasma (MIC 4 µg/ml) while a triple substitution of Asp112Glu in GyrA along with Ala125Thr and Ala136Thr in ParC was found in six further intermediately-resistant strains. No mutations were found in strains with MICs 1 µg/ml. No mutations were detected in 23S rRNA operons, L4 or L22 proteins. TetM and int-Tn genes were found in seven tetracycline-resistant strains. On screening 59 tetracycline-susceptible and -intermediate strains, eleven whilst possessing an int-Tn gene lacked a large region of tetM and 48 only contained small regions of tetM. The tetM genes of the seven tetracycline-resistant strains were sequenced and comparisons performed against GenBank sequences of Neisseria gonorrhoeae, Streptococcus pneumoniae and U. urealyticum. For five strains tetM was seen to be highly mosaic in structure containing regions that were similar to those of the GenBank strains and others that were unique. In the tetM leader region, four hot spot recombination sites were identified that could certainly influence the formation of the mosaic structures, upstream insertion sequences/open reading frames and transposon regions that regulate expression. On characterising the int-Tn genes of the seven tetracycline-resistant strains, three types were present indicating transposons from different origins had integrated into ureaplasma genomes. Reciprocal tetracycline resistance gene transfer between ureaplasmas and N. gonorrhoeae were unsuccessful. However, low-level tetracycline resistance (MICs 4-8 µg/ml) was transferred to a U. parvum recipient from one U. urealyticum and three U. parvum donors that carried tetM with MICs 16-64 µg/ml. On tetM PCR analysis, tetM was not detected in the transformants. Conclusions: The importance of genital mycoplasmas, ureaplasmas and C. trachomatis in long term aetiologies requires further investigations, certainly in relation with syndromic management regimens that fail to reduce colonisation rates. The high prevalence of P. jiroveci, the presence of M. pneumoniae in cases of pneumonia and detection of U. parvum in two cases of neonatal pneumonia investigated emphasises that in the absence of definitive diagnoses, it is crucial to monitor treatment responses carefully, especially when first line antibiotic preferences are ß-lactams, in order to ensure adequate and informed delivery of medical care. The finding of transposon and/or tetM regions in all ureaplasmas investigated with or without full expression of tetracycline resistance, in conjunction with tetM gene diversity, certainly places ureaplasmas strongly in the picture for intra- and inter-generic exchange of antibiotic resistance genes. / AFRIKAANSE OPSOMMING: Oorsig: Mikoplasma en ureaplasma word nie roetineweg gediagnoseer nie en in Suid Afrika is nog min navorsing daaroor gedoen. Prevalensie, populasie verskuiwings, veral in genital flora, en die impliksies van infeksie en ander toestande is onbekend. Inligting rakende Mycoplasma pneumoniae in respiratoriese siekte is ook gebrekkig. Daar is min inligting beskikbaar rakende die antimikrobiale vatbaarheid en die ontwikkeling van weerstandigheid gesien teen die benadering tot sindromiese hantering van seksueel oordraagbare siektes. Doelwitte: a) Om inligting te verskaf oor die prevalensie van mikoplasma en ureaplasma en bydraende faktore betreffende voortydige kraam tesame met MIV en Chlamydia trachomatis. b) Ondersoek van die prevalensie van M. pneumoniae in respiratoriese infeksies tesame met MIV, Mycobacterium tuberculosis en Pneumocystis jiroveci. c) Bepaling van die antimikrobiale vatbaarheid van mikoplasma en ureaplasma en analisevan weerstandigheids gene. d) Bereken die inter-genetiese oordrag potensiaal van weerstandigheids gene (tetM) tussen Ureaplasma spp. en Naisseria gonorrhoeae. Genitale omgewing: Die prevalensie van genitale mikoplasma, ureaplasma en Chlamydia in vroue tydens hul eerste prenatale besoek, tesame met vroegtydige kraam en MIV status is ondersoek. In voortydige kraam (2003), is 199 vroue gemonitor vir voortydige kraam (<37 weke); vir kolonisasie en MIV (2005), is 219 vroue getoets. Mikrobiale toetsing is gedoen deur DNS te win vanaf endoservikale deppers met PKR tegnieke. Kolonisasie was die hoogste in die ouderdomsgroep 14.20 jaar, in 2003. In vroue van ±21 jaar was medekolonisasie 13% alhoewel daar en verskuiwing was van mede-kolonisasie met Mycoplasma hominis en Ureaplasma spp. in 2003 tot ander dubbel/trippel kombinasies in 2005. Die oorkoepelende tendens in altwee die tydperke van waarneming was dat die prevalensie van Ureoplasma spp. geneig was om hoër te wees in vroue ±26 jaar, terwyl prevalensie van C. trachomatis en M. hominis laer was. Geen assosiasie kon getoon word tussen koloniesasie met M. hominis, U. urealyticum, Ureaplasma parvum en uitkoms van kraam nie. MIV status het geen effek gehad op die prevalensie/mede-kolonisasie van M. hominis, Ureaplasma spp. of C. Trachomatis nie. Respiratories: Studies is gedoen om die prevalensie van gemeenskaps verworwe atipiese pneumonie in volwassenes (M. pneumoniae en P. jiroveci) en neonate (mikoplasma, ureaplasma en Chlamydia trachomatis) te bepaal om behandeling en hantering programme in die Port Elizabeth area te verbeter. Sputum monsters van 102 volwasse pasiënte wat presenteer het met pneumonie of simptome van pneumonie en wat tot hospitale toegelaat was, is ontleed. Besonderhede van die pasiënte se geslag, ouderdom, MIV en Mycobacterium tuberculosis status is deur die hospitale verskaf. PKR is gedoen met inleiers gerig teen die volgende gene: P. jiroveci vir die aantoning van mitokondriale groot subeenheid RNS en vir die analise van mutasies vir ko-trimoksasool weerstandigheid dihydropteroaat sintetase (DHPS) en dihydrofolaat reduktase (DHFR); M. pneumoniae vir die aantoning van P1 adhesien en 16S rRNS. Vroue het ‘n hoë risiko vir gemeenskapsverworwe P. jiroveci kolonisasie gehad. In die algemeen was die prevalensie van P. jiroveci 52.9% (54/102 pasiënte). P. jiroveci was hoofsaaklik geassosieerd met MIV (25/74) (P. jiroveci en MIV positiewe pasiënte in die pasiënt monster waarvoor daar kliniese data en MIV status bekend was) en mede-infeksie met M. tuberculosis is gesien in 12 MIV gevalle en een MIV negatiewe pasiënt. Geen DHPS (20) of DHFR (17) weerstandigheids geassosieerde mutasies is gevind in P. Jiroveci nie. M. pneumoniae was aangetoon in een pasiënt. Vir prevalensie studies (2007-2008) op atipiese pneumonie in neonate is 69 endotrageale aspirate verkry. PKR toetsing vir M. hominis, U. urealyticum en C. trachomatis is gedoen met ‘primers’ soos voorheen gepubliseer. Ureaplasma parvum is aangetoon in twee neonate met PKR met negatiewe kultuur resultate. Antibiotika sensitiwiteite en weerstandigheids gene: Die volgende toetse is gedoen op kliniese isolate van U. parvum en U. urealyticum (i) antibiotika sensitiwiteits profiele, (ii) aantoning van teiken geen mutasies, of geen aanwinste en (iii) potensiaal vir inter-generiese weerstandigheids geen oordrag na Neisseria gonorrhoeae. Kultuur tegnieke toegepas op 132 endoservikale monsters het 66 Ureaplasma kulture gelewer (35 U. parvum, 9 U. urealyticum, 22 U. parvum + U. urealyticum). MIK bepaling vir ofloksasien, eritromisien, tetrasiklien, doksisiklien, azitromisien en josamisien is gedoen. Sewe-en-dertig kulture was ten volle sensitief vir alle antibiotika wat getoets is; een-en twintig het intermediere weerstandigheid teenoor eritromisien, azitromisien en ofloksasien getoon, terwyl sewe weerstandig was vir tetrasiklien, drie daarvan was ook weerstandig vir doksisiklien. Wat betref ofloksasien weerstandigheid gemik teen kwinoloon weerstandigheids bepalende gebiede, is vervanging van Ser83Leu in ParC gedemonstreer in een intermedier weerstandige Ureaplasma (MIK 4 µml) terwyl en trippel vervanging van Asp112Glu in GyrA saam met Ala125Thr en Ala136Thr in ParC gevind is in ses ander intermedier weerstandige stamme. Geen mutasies is gevind in stamme met MIKs van MICs 1 µg/ml nie. Geeneen van die ureaplasma was weerstandig vir eritromisien/azitromisien nie en geen mutasies is gevind in 23S rRNA operons , L4 of L22 proteine nie. TetM en int- Tn gene is gevind in sewe tetrasiklien weerstandige stamme. 58 Tetrasiklien sensitiewe en .intermediere stamme is getoets, waarvan elf en int-Tn geen gekort het sowel as en groot deel van tetM, terwyl 48 slegs klein dele van TetM bevat het. Die tetM gene van die sewe tetrasiklein-werstandige stamme se geenvolgorde is bepaal en vergelykings is getref teenoor die GenBank volgordes van Neisseria gonorrhoeae, Streptococcus pneumoniae en U. urealyticum. In vyf stamme is gevind dat die tetM geen hoogs mosaiek in struktuur was met areas wat ooreenstem met die in GenBank stamme, en ander areas wat uniek is. In die tetM leier area, is vier ehot spot f herkombinasie areas geidentifiseer wat sekerlik die vorming van die mosaiiek strukture kon beinvloed, asook transposon areas wat geenuitdrukking bepaal. Met karakterisering van die int-Tn gene van die sewe tetrasikleinweerstandlige stamme, was drie tipes teenwoordig waarin transposons vanaf verskillende oorsprong aangedui was, geintegreerd met die ureaplama genome. Resiprokale tetrasiklien weerstandigheids geen oordrag tussen ureaplasma en n. gonorrhoea was nie suksesvol nie. Lae-vlak tetrasiklien weerstandigheid (MIK fs van 4 . 8 µg/ml) is wel suksesvol oorgedra na en U. parvum ontvanger vanaf een U. urealyticum en drie U. parvum ontvangers wat tetM gedra het met MIKs van 16-64 µg/ml. Met die analise van tetM met PKR, kon tetM nie aangetoon word in die transformante nie. Gevolgtrekkings: Die belang van genitale mykoplasma, ureaplasma en C. trachomatis in langtermyn etologie benodig verdere ondersoek, veral in die lig van die sindromiese behandeling regimes wat nie kolonisasie verminder nie. Die hoe prevalensie van P. jiroveci, die teenwoordigheid van M. pneumoniae in gevalle van pneumonie en die aantoning van U. parvum in twee gevalle van neonatale pneumonie benadruk dat, in die afwesigheid van en definitiewe diagnose, dit noodsaaklik is om respons tot behandeling sorgvuldig te moniteer, veral indien die eerste lyn antibiotika keuse ß-laktam antimikrobiale middels of kefalosporiene is, sodat behoorlike en ingeligde gesondheidsorg gelewer kan word. Die bevinding van transposon en/of tetM gebiede in alle ureaplasma wat ondersoek is met of sonder volle uitdrukking van tetrasiklien weerstandigheid, in samehang met tetM diversiteit, plaas verseker ureaplasma sterk in die prentjie vir intra- en inter-generiese uitruiling van antibiotika weerstandigheids gene. / Nelson Mandela Metropolitan University / National Research Foundation (NRF Thuthuka) / Medical Research Council

Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH SUMMARY: Background: Primary immunodeficiency disorders (PID) disrupt the proper functioning of the immune system. The prototypic PID is X-linked Agammaglobulinemia (XLA). This disorder is caused by mutations in the Bruton tyrosine kinase (Btk) gene and results in an arrest in B cell development which leads to a profound reduction of all classes of serum immunoglobulins (i.e antibodies). Patients with a lack of antibodies experience recurring bacterial infections during early childhood that can be fatal if not treated. Intravenous gammaglobulin replacement therapy (IVIg) is the standard treatment for XLA. It provides passive immunity thereby reducing the number and severity of infections as well as limiting many of the infectious complications. Early detection and treatment of XLA allows affected individuals to live a relatively normal life. Objective: The purpose of this study was to determine the molecular basis of XLA in South Africa using a direct sequence-based method to detect abnormalities in the Btk gene to aid clinical diagnosis of the disease. Methods : Male patients with a clinical diagnosis of XLA were included in this study. Genetic analysis was used to explore the exonic region of the Btk gene of 5 unrelated male patients and compared to 10 healthy controls. Family members were followed up to determine carrier status, where possible. Results: Mutational analysis revealed Btk abnormalities in 4 of the 5 patients leading to a definitive diagnosis of XLA. Two of the three mutations found in this study have been previously described while one mutation appears to be novel. The novel mutation is a one base pair deletion in exon 16 which leads to the truncation of the Btk protein. Despite the clinical findings suggestive of XLA, no mutation was identified in the exonic region of the Btk gene of the remaining patient, indicating that this patient might have a different form of PID. Maternal follow-up confirmed the maternal inheritance pattern as all mothers screened were carriers of the Btk mutation present in the affected individual. Discussion : Using a direct sequence-based method abnormalities were identified in the Btk gene of three patients. Molecular diagnosis coupled to clinical history of the patient provides a definitive XLA diagnosis. This study supports the use of molecular techniques in the diagnosis of PID and underlines the synergy that could be possible in a clinical setting. / AFRIKAANSE OPSOMMING: Agtergrond: Primêre immuungebrek siektes (PIGS) word gekenmerk aan ‘n gebrek aan teenliggame in die immuunsisteem wat lei tot herhaalde infeksies in jong kinders wat fataal kan wees indien dit nie vroegtydig behandel word nie. Die prototype van die bekende PIGS is X-gekoppelde Agammaglobulinemia (XGA). Die siekte word veroorsaak deur mutasies in die Bruton Tirosien kinase (Btk) geen en lei tot ʼn stilstand in B sel ontwikkeling en gevolglik ʼn vermindering van alle klasse van serum immuunoglobulins (teenliggaam). Intraveneuse gammaglobulien vervangingsterapie(IVIg) is die standaard behandeling vir XGA. Dit voorsien passiewe immunitiet en gevolglik verminder dit die getal en erns van infeksies en beperk baie van die aansteeklike komplikasies. Vroeë diagnose en behandeling van XGA laat toe dat geaffekteerde individue ʼn relatiewe normale lewe ly. Doel: Die doel van hierdie studie is om die molekulêre basis van XGA in Suid Afrika te ondersoek, deur gebruik te maak van direkte volgorde bepaling van die Btk geen in die hoop om die kliniese diagnose van die siekte aan te help. Metode : Manlike pasiente met ‘n kliniese diagnose wan XGA was by die studie ingesluit. Genetiese analise was gebruik om die “exonic” omgewing van die Btk geen te ondersoek van 5 onverwante manlike pasiente en vergelyk teenoor 10 gesonde kontrole. Waar moontlik was familie lede ogevolg om draers te bepaal. Resultaat: Mutasies in die Btk geen is geidentifiseer in 3 van die 4 pasiente, klinies gediagnoseer meet XGA. Die mutasies sluit 2 reeds beskryfde variante in en een nuwe mutasie, ‘n een basis paar delesie in ekson 16 van die Btk geen, Ten spyte van die kliniese profiel suggestief van XGA in die 5de pasient, was geen mutasies geidentifiseer in die “exconic” omgewing van die Btk geen nie, dit kan moontlik toegeskryf word aan die teenwoordigheid van ‘n ander vorm van PIGS in hierdie pasient. Opvolg analise op die DNA van die moeders van die pasiente het die moederlike oorerwings patroon van die siekte bevestig aangesien al die moeders draers van die geidentifiseerde mutasie in die Btk geen van die gaffekteerde individu was. Gevolgtrekking: Genetiese analise van die Btk geen blyk ʼn sensitiewe en spesefieke metode te wees om individue met XGA te diagnoseer. Hierdie studie ondersteun die gebruik van molekulêre metodes in die diagnose van PIGS en beklemtoon die moontlike sinergie wat kan bestaan tussen hierdie tipe benadering in die kliniese omgewing. / National Research Foundation / National Health Laboratory Services : Pathology Research Development Grant of NHLS Research Trust Grants

My first project was to investigate the role of Hepatocyte Nuclear Factor 1 (HNF1) and Nuclear Factor I (NFI) on alpha-fetoprotein (AFP) promoter activity during liver development. AFP is highly expressed in the fetal liver, silenced at birth, and remains at very low levels in the adult liver. A GA substitution located at -119 of the human AFP promoter is associated with hereditary persistence of AFP (HPAFP) expression in the adult liver (Hum Molec Genet, 1993, 2:379). The -120 region harbors overlapping binding sites for HNF1 and NFI. While it has been shown that the GA substitution increases HNF1 binding, the role of NFI in AFP regulation has not been investigated. This overlapping HNF1/NFI site is conserved in other mammals, including mice. In this study, I used a combination of biochemical, tissue culture, and animal studies to explore further the role of this HNF1/NFI site in AFP regulation. Transient co-transfections in Hep3B hepatoma cells indicate that HNF1 activates while NFI represses the mouse AFP promoter. EMSAs indicate that HNF1 and NF1 compete for binding to this site. Transgenes regulated by the wild-type AFP promoter are expressed at low levels in the adult liver. Transgenes with a GGAA mutation (similar to the G-A human mutation) are more active in the adult liver. My data indicate that HNF1 and NFI compete for binding to the -120 region of the AFP promoter and this competition is involved in postnatal AFP repression. My second project was to study the control of Elongation of very long chain fatty acids like 3 (Elovl3) in the liver by Zinc fingers and homeoboxes 2 (Zhx2). The Zhx2 gene was originally characterized in our lab based on its ability to control the developmental repression of several hepatic genes, including AFP (PNAS, 102:401). Zhx2 is a member of a small family of proteins found only in vertebrates that also includes Zhx1 and Zhx3. These proteins all contain two zinc fingers and four homeodomains, suggesting that they function as regulators of gene expression. My study shows that Zhx2 regulates Elovl3 expression in female liver. Mouse strain-specific differences in adult liver Elovl3 mRNA levels and transgenic mouse data indicate that Zhx2 activates Elovl3 expression in the female adult liver. I also demonstrate that Elovl3 is repressed in the regenerating liver and that the level of Elovl3 repression is controlled by alpha-fetoprotein regulator 2 (Afr2). In addition, I show that Elovl3 expression is reduced in liver tumors, fibrotic livers and fatty livers, raising the possibility that Elovl3 can serve as a marker for HCC and liver damage.

No description available.

Neisseria gonorrhoeae, the causative agent of the STI gonorrhea, is not preventable by vaccination and is rapidly developing resistance to antibiotics. One important strategy for gonococcal survival in the host is iron acquisition in the face of nutritional immunity. To overcome iron limitation, the gonococcus expresses TonB dependent transporters (TdTs), outer membrane proteins that facilitate nutrient acquisition. Of the TdTs, the transferrin (Tf), lactoferrin (Lf), and hemoglobin (Hb) receptors hijack iron directly from host proteins, and studies have already shown that the Tf receptor is essential for the initiation of human infection. Given that the TdTs are virulence factors, they are widely conserved across strains, and are not subject to antigenic variation, they are ideal targets for novel therapeutics and vaccine development. As such, studies exploring these proteins and their potential as vaccine candidates and antimicrobial targets are needed. In this study we report that loops of the Tf receptor protein TbpA are not strongly immunogenic, and the antibodies raised against them are incapable of inhibiting TbpA-Tf interactions on the gonococcal cell surface. We also report that the loop 3 helix motif of TbpA is a critical functional domain for Tf-binding and iron uptake; however, no single residue was identified that was essential for these functions. In addition, we report the development of a platform for the structure-function analysis of HpuA, a member of the poorly studied Hb receptor. We also present evidence that novel small molecules may be able to inhibit TbpA-Tf interaction, presenting the Tf receptor as a novel, species-specific antimicrobial target. Finally, we demonstrated that a novel drug, OSU-03012, has antimicrobial activity against the gonococcus through down-regulation of DnaK, a protein chaperone. These findings suggest that DnaK, a widely conserved protein, may be a universal target for antimicrobial development. These studies provide insight into the structure function relationship of TbpA, the drug potential of DnaK, and lay the framework for future investigations of the TdTs for use in a multi-antigen vaccine.

Introdução: Atualmente o uso de fluoroquinolonas na prática clínica tem sido associado a um aumento da incidência da infecção com bactérias resistentes as quinolonas, especialmente Escherichia coli. O Brasil tem uma das mais altas taxas de resistência as quinolonas entre os países da America Latina. Diferentes mecanismos de resistência estão envolvidos no desenvolvimento de resistência as quinolonas. Os principais mecanismos dividem se em três categorias: i) alteração na DNA girase e topoisomerase IV; ii) diminuição do acúmulo de antibióticos no interior da bactéria e iii) produção de proteínas protetoras da DNA girase e topoisomerase IV. No ano de 2010 foram observadas elevadas taxas de resistência às fluorquinolonas em Escherichia coli isoladas de hemoculturas de pacientes atendidos em hospitais privados da cidade de São Paulo, pelo Fleury Medicina e Saúde. As taxas de resistência à ciprofloxacina variaram de 26,1% a 43,9% em três hospitais diferentes. Considerando que há determinantes cromossômicos não transferíveis e determinantes plasmidiais transferíveis da resistência às fluorquinolonas, a avaliação da clonalidade dos isolados e dos determinantes genéticos da resistência às fluorquinolonas poderá contribuir para o entendimento de alguns dos fatores que possam contribuir para essa progressiva elevação nas taxas de resistência a essa classe de antimicrobianos. Objetivo: Avaliar o fenótipo, a diversidade genética, determinantes cromossômicos e plasmidiais da resistência a fluorquinolonas em Escherichia coli isoladas de corrente sanguínea. Materiais e métodos: Foram estudados 47 E. coli resistentes a ciprofloxacino isoladas de hemoculturas de pacientes atendidos em cinco centros hospitalares da cidade de São Paulo. A caracterização fenotípica foi realizada por determinação da concentração mínima inibitória para fluoroquinolonas. A confirmação genotípica da resistência foi confirmada por PCR para os genes qnrA, qnrB, qnrS. As regiões determinantes da resistência a fluorquinolonas dos genes gyrA, parC foram sequenciadas. A tipagem molecular foi realizada pela técnica de ERIC-PCR. Resultados e conclusões: Os genes qnrA e qnrS não foram detectados nos isolados avaliados neste estudo. Os genes qnrB foram detectados em 42,5% dos isolados. Em todos os isolados, exceto o F4991, foram detectadas as substituições S83L e D87N na GyrA Em todos os isolados, exceto o F4991, foram detectadas as substituições S80I em ParC. Foi detectada a substituição E84V em ParC 23,4% dos isolados. Foi observada a disseminação dos grupos clonais ERIC1 e ERIC2 entre hospitais e disseminação intrahospitalar dos grupos clonais ERICS, ERIC6 e ERIC7. / Introduction: The use of fluoroquinolones in clinicai practice has been associated with an increased incidence of infection with bacteria resistant to quinolones, especially Escherichia coli. Brazil has one of the highest rates of resistance to quinolones among the countries in Latin America. Different resistance mechanisms are involved in the development of resistance to quinolone. The main mechanisms fall into three categories: i) alteration in DNA gyrase and topoisomerase IV, ii) reduction in the accumulation of antibiotics within the bacterium and iii) production of proteins that protect DNA gyrase and topoisomerase IV. In the year of 2010 high rates of resistance to fluoroquinolones in E. coli were observed blood cultures isolates from of patients from private hospitais in São Paulo. Ciprofloxacin resistance rates ranged from 26.1 to 43% 9% at three different hospitais. There are chromosomal and transferable plasmid-determined resistance to fluoroquinolones. The evaluation of the clonality of the isolates and the genetic determinants of resistance to fluoroquinolones may contribute to the understanding of some of the factors that may contribute to the progressive increase in resistance rates to this class of antimicrobials. Objective: To evaluate the phenotype, genetic diversity, chromosomal and plasmid determinants of resistance to fluoroquinolones in E. coli isolated from bloodstream. Materiais and methods: We studied 47 ciprofloxacin resistant E. coli isolated from blood cultures of patients treated at five hospitais in the city of Sao Paulo. Phenotypic characterization was performed by determining the minimum inhibitory concentration for fluoroquinolones. Genotypic resistance was confirmed by PCR to genes qnrA, qnrB and qnrS. The quinolone resistance determining regions of genes gyrA and parC were sequenced. Molecular typing was performed using ERIC-PCR. Results and conclusions: The qnrA and qnrS genes were not detected in the isolates evaluated in this study. The qnrB genes were detected in 42.5% of isolates. In all isolates, except for F4991, substitutions were detected in gyrA S83L and D87N. In all isolates, except for F4991, substitution S80I were detected in ParC. E84V substitution in ParC was detecxted in 23.4% of isolates. We observed the spread of clonal groups ERIC1 and ERIC2 and between hospitais and alsdo intra-hospital spread of clonal groups ERIC5, and ERIC6 ERIC7.

Não consta resumo na publicação. / Abstract not available.

O Staphylococcus aureus é um patógeno extremamente versátil tanto em termos de resistência a antimicrobianos quanto em virulência. O S. aureus resistente a oxacilina (ORSA) adquire a resistência a toda a classe de beta-Iactâmicos através de um cassete cromossômico (SCCmec) que carrega o gene mecA, mas pode carregar outros genes de resistência. A soma desses genes de resistência e de virulência torna o S. aureus um grave problema para hospitais do mundo inteiro, que nos últimos vem se estendendo também à comunidade. Foram estudados 50 isolados de ORSA, dentre os quais 15 pertencentes ao clone endêmico brasileiro (CEB) e 3 cepas SCCmec tipo IV isoladas entre 1995 e 1999. Adicionalmente, 32 amostras ORSA SCCmec tipo IV isoladas no Hospital de Clínicas de São Paulo. As amostras foram analisadas quanto ao perfil de sensibilidade a antimicrobianos, classificação do tipo de SCCmec, perfil de virulência quanto a toxinas e adesinas, classificação do grupo agr (locus regulatório dos genes de virulência) e sua funcionalidade, avaliação da expressão dos genes de toxinas e genotipagens por PFGE e MLST. Observou-se que as cepas CEB são multiresistentes. Já as cepas SCCmec IV apresentam um perfil de sensibilidade maior, uma vez que possuem um tipo de SCCmec que não carrega outros genes de resistência além do gene mecA. As amostras CEB SCCmec IV não apresentaram grandes diferenças no conteúdo de toxinas e adesinas. Apenas as cepas SCCmec IV isoladas entre 1995 e 1999 apresentaram um maior conteúdo de genes de virulência que as isoladas no HC. As cepas SCCmec IV isoladas no Brasil não são altamente virulentas como descrito em outros países. Não possuem fatores de virulência como a Leucocidina Panton-Valentine, toxinas exfoliativas e enterotoxinas. Por outro lado, possuem a alfa-hemolisina e a leucocidina LukD-LukE, toxina ainda pouco estudada, que vem sendo apresentada em pesquisas como causa de lesões oculares graves e diarréias pós-antibioticoterapia. Não foi possível estabelecer uma relação entre o tipo de agr e o perfil de virulência das cepas, uma vez que os perfis foram muito semelhantes mesmo entre cepas de grupos agr diferentes. / Staphylococcus aureus is an extremely successful pathogen for it is both highly resistant to antibiotics in addition to being virulent. Methicillin-resistant Staphylococcus aureus (MRSA) acquires resistance to the beta-Iactam antibiotics through the acquisition of a chromosomal cassette (SCCmec) which carries the mecA gene, and can carry other resistance genes. The presence of these genes in S. aureus makes it a serious problem in hospitaIs worldwide. In spite of usually being restricted to the nosocomial environment, over the last few years MRSA has been spreading throughout the community. Fifty nosocomial MRSA strains were studied, including 15 belonging to the Brazilian endemic clone (BEC), 3 type IV SCCmec strains isolated between 1995-1999, and 32 type N SCCmec isolates from the \"Hospital de Clínicas (HC) de São Paulo\". The isolates were analyzed as to their susceptibility profile, SCCmec type, virulence and expression profile (toxins and adhesins), agr group classification and functionality, PFGE and MLST profiles. BEC isolates proved to be multiresistant to antibiotics. Type IV SCCmec strains presented a susceptibility profile to a number of drugs of different antimicrobial classes. BEC and type N SCCmec strains did not present significant differences in their virulence profiles. Only the type IV SCCmec strains isolated in 1995-1999 presented a greater virulence profile than those isolated in the HC. Type IV SCCmec strains isolated in Brazil were not highly virulent as described in other countries. Brazilian isolates usually do not possess virulence factors such as the Panton-Valentine leukocidin, exfoliative toxins and enterotoxins. On the other hand, they usually possess alpha-hemolysin and the LukED leukocidin, which is still very poorly studied that have been presented in papers like cause of serious ocular lesions and post-antimicrobial therapy diarrhea. A relation between the agr type and the virulence profile was not established, for virulence profiles were very similar even between isolates belonging to different agr groups.