In der vorliegenden Studie wurde die Sanierung einer durch S. aureus verursachten Eutergesundheitsstörung in einem hessischen Milchviehbestand mittels klassischer Sanierungsmaßnahmen über einen Zeitraum von 18 Monaten begleitet. Durch konsequente Einhaltung der Sanierungsmaßnahmen nach dem Fünf-Punkte-Plan und die räumliche Trennung der Herde in eine S. aureus-positive und -negative Gruppe sank die S. aureus-Prävalenz im Betrieb innerhalb von 15 Monaten von 30% auf <1%. Nach 18 Monaten waren erstmalig keine Neuinfektionen mehr mit S. aureus zu verzeichnen. Zusätzlich zu den im Abstand von drei Monaten durchgeführten Viertelanfangsgemelk (VAG)- Gesamtbestandsuntersuchungen wurden Umweltproben bakteriologisch auf das Vorhandensein von S. aureus untersucht. Diese Untersuchungen verliefen ausschließlich mit negativem Ergebnis. Die im Untersuchungszeitraum asservierten 218 S. aureus-Isolate aus diesem Betrieb wurden genotypisch mittels PCR untersucht. Thermonuklease-Gen, Protein A-Gen (IgG-bindende Region) und Polymorphismen bei Protein A-Gen (Xr-Region) sowie Koagulase-Gen ermöglichten die Unterscheidung in sechs Typen. Zusätzlich wurden 80 dieser Isolate mittels Pulsfeldgelelektrophorese (Pfge, Gold Standard) typisiert. Es konnten zwei Pfge-Typen gefunden werden: Pfge-Typ I mit insgesamt 10 Subtypen (bei 78 Isolaten) und Pfge-Typ II (bei zwei Isolaten). Der Pfge-Typ II wurde ausschließlich bei einem Zukaufstier nachgewiesen, das vor der Einstallung in diesen Betrieb nicht zytobakteriologisch untersucht worden war. Die 12 unterschiedlichen Pfge-Typen bzw. -Subtypen wurden zusätzlich mittels Staphylokokken-Protein A- (spa)-Typisierung untersucht. Dabei zeigte sich, dass alle Subtypen des Pfge-Typs I dem spa-Typ t2067 zugeordnet werden konnten und der Pfge-Typ II dem spa-Typ t2112 entsprach. 92 weitere bovine S. aureus-Mastitisisolate wurden möglichst randomisiert über das Landesgebiet Hessens entnommen und mittels dieser Typisierungsmethode untersucht. Die Isolate stammten ebenfalls aus S. aureus-Problembetrieben und wurden aus VAG von (sub-) klinischen Mastitiden in Reinkultur isoliert. Insgesamt konnten 28 spa-Typen unterschieden werden. Durch den Algorithmus BURP wurden 57 dieser Isolate dem Clonal Complex CC543 zugeordnet und waren demnach genetisch eng miteinander verwandt. Der in dem Sanierungsbetrieb vorherrschende spa-Typ t2067 war dem CC543 nicht zuzuordnen. Die Anwendung der spa-Typisierung in der Routinediagnostik und damit die Möglichkeit der laborübergreifenden, möglichst zentralisierten Datendokumentation der Ergebnisse könnten zukünftig die Identifizierung besonders häufig vorkommender und damit hochkontagiöser S. aureus-Stämme ermöglichen.

Orientador: Maria de Lourdes Ribeiro de Souza da Cunha / Resumo: Infecções causadas por Staphylococcus aureus representam um dos principais problemas de saúde pública, com elevadas taxas de morbidade e mortalidade principalmente entre indivíduos com deficiência de sistema imunológico como os diabéticos, em especial aqueles que fazem uso diário de insulina. Além da alta patogenicidade e facilidade de aquisição de resistência aos antimicrobianos, o patógeno tem grande habilidade de colonizar indivíduos de forma assintomática, favorecendo sua disseminação e tornando esses indivíduos fonte de risco para infecções. O estudo teve como objetivo analisar a prevalência e fatores de risco para o carreamento nasal e orofaríngeo, bem como caracterizar o perfil de resistência, virulência e tipagem molecular de S. aureus sensível à meticilina (MSSA) e S. aureus resistente à meticilina (MRSA) isolados de indivíduos diabéticos insulino-dependentes do município de Botucatu, SP. S. aureus foram obtidos da nasofaringe e orofaringe de 312 indivíduos diabéticos insulino-dependentes da comunidade e analisados quanto à presença do gene mecA, genes das enterotoxinas (sea, seb, e sec-1), esfoliatinas A e B (eta e etb), toxina 1 da síndrome do choque tóxico (tst), leucocidina Panton–Valentine (pvl), hemolisinas alfa (hla) e delta (hld) e biofilme (operon icaADBC) através da técnica da reação em cadeia da polimerase (PCR) e a tipagem do SCCmec através de PCRmultiplex. O perfil de sensibilidade à oxacilina, cefoxitina, linezolida, quinupristina/dalfopristina e sulfam... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Infections caused by Staphylococcus aureus is a major public health problem and infections with this microorganism are associated with high morbidity and mortality rates, especially among individuals with immune deficiency disorders such as diabetes and particularly those who take insulin daily. In addition to its high pathogenicity and ability to acquire antimicrobial resistance, asymptomatic infection with this pathogen is common, favoring its dissemination and rendering these individuals a source of infection. The objective of this study was to analyze the prevalence and risk factors for nasal and oropharyngeal carriage, as well as to characterize the resistance profile, virulence, clonal profile and sequence type of methicillin-susceptible (MSSA) and methicillin-resistant S. aureus (MRSA) isolated from insulin-dependent diabetic individuals in the city of Botucatu, SP, Brazil. Staphylococcus aureus was collected from the nasopharynx and oropharynx of 312 community insulin-dependent diabetic individuals. The isolates were analyzed for the presence of the mecA, enterotoxin (sea, seb and sec-1), exfoliatin A and B (eta and etb), toxic shock syndrome toxin 1 (tst), Panton-Valentine leukocidin (pvl), alpha and delta hemolysin (hla and hld), and biofilm (icaADBC operon) genes by the polymerase chain reaction (PCR). SCCmec typing was performed by multiplex PCR. The susceptibility profile against oxacillin, cefoxitin, linezolid, quinupristin/dalfopristin and sulfamethoxazole/trim... (Complete abstract click electronic access below) / Doutor

Submitted by Erika Demachki (erikademachki@gmail.com) on 2017-01-06T18:29:27Z No. of bitstreams: 2 Dissertação - Jéssica de Oliveira Veloso - 2016.pdf: 2794813 bytes, checksum: 8bd9adfbbc795e1b905411f5c2a04d01 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-01-09T09:47:16Z (GMT) No. of bitstreams: 2 Dissertação - Jéssica de Oliveira Veloso - 2016.pdf: 2794813 bytes, checksum: 8bd9adfbbc795e1b905411f5c2a04d01 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-01-09T09:47:16Z (GMT). No. of bitstreams: 2 Dissertação - Jéssica de Oliveira Veloso - 2016.pdf: 2794813 bytes, checksum: 8bd9adfbbc795e1b905411f5c2a04d01 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-09-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Staphylococcus aureus is an important pathogen related to nosocomial infections, with high prevalence, morbidity and mortality rates. In this context, the Intensive Care Units (ICU) has been high-risk areas for the selection of multiresistant strains. The environment (objects, equipment and surfaces) of an ICU can also get contaminated, and microorganisms may remain viable for a long period of time, and can colonize patients, employees, visitors and other environments. The objectives of the study were to determine the prevalence of S. aureus contamination in patients and ICU environment of a university hospital in the city of Goiânia-GO, as well as to determine the antimicrobial susceptibility and virulence profile of the isolates and perform molecular typing of methicillin-resistant S. aureus (MRSA) isolates. The isolation and presumptive identification of S. aureus by phenotypic techniques and the confirmation of the species by detection of femA gene by PCR were performed. The isolates were subjected to diskdiffusion test for determining antimicrobial susceptibility profile, and those showing resistance to cefoxitin were subjected to E-Test® to determine the minimum inhibitory concentration (MIC) to oxacillin and vancomycin, as well as the mecA gene detection for identification of MRSA strains. In these isolates the SCCmec typing was performed. In all S. aureus isolates were detected virulence factors-coding genes and held the genetic comparison for determining the similarity profile by pulsed field gel electrophoresis. Fifty hundred and thirty six swabs were collected being 134 of patients and 402 of ICU environment. The prevalence of colonization by S. aureus was 12.7% (68/536), being 13.4% (18/134) for patients and 12.4% (50/402) for the environment. The highest resistance rate presented was to penicillin (85.3%) followed by erythromycin (69.1%) clindamycin (66.2%) and sulfamethoxazole-trimethoprim (54.4%). Fifty-six isolates (82.4%) were classified as multiresistant. The prevalence of MRSA was 20.6% (14/68), and seven isolates (10.3%) presented intermediate susceptibility to vancomycin (VISA). The inducible resistance phenotype (iMLSb) was found in 11 strains (16.2%) and the constitutive resistance (cMLSb) in 25 (36.8%). Eleven isolates showed genes encoding for at least one virulence factor and were detected six virulence profiles. Of the 14 MRSA strains, six (42.9%) were SCCmec type IV, five (35.7%) SCCmec type I, two (14.3%) SCCmec type II and one (7.1%) SCCmec type III. PFGE analysis showed genetic diversity among the isolates, although a cluster grouped 16 isolates showing the spread of the bacteria among patients and environment. One MRSA isolate showed genetic relationship to the USA300 strain and two isolates MRSA/VISA were similar and another identical to the clone USA400. The results suggest that the prevalence of S. aureus and MRSA remains high in health institutions, especially in the ICU, with high rates of antimicrobial resistance and pathogenic potential. The detection of these microorganisms in the environment shows risk of cross-transmission primarily via health professionals. Identification of isolates with genetic background of strains acquired in the community alert to a flow of intra and inter- hospital and community environment. In addition, it is believed that environmental surfaces can be acting as reservoirs of genes of resistance and virulence as well as potential sources of contamination to patients, professionals and environments. / Staphylococcus aureus é um importante patógeno relacionado a infecções nosocomiais, com elevadas taxas de prevalência, morbidade e mortalidade. Nesse contexto, as Unidades de Terapia Intensiva (UTI) tem sido áreas de alto risco para a seleção de cepas multirresistentes. O ambiente (objetos, equipamentos e superfícies) de uma UTI também pode se contaminar, e os microrganismos podem permanecer viáveis por um longo período de tempo, podendo colonizar pacientes, trabalhadores, visitantes e contaminar ainda outros ambientes. Os objetivos do estudo foram determinar a prevalência de colonização por S. aureus em pacientes e ambiente da UTI de um hospital universitário na cidade de Goiânia-GO, bem como o perfil de susceptibilidade antimicrobiana e perfil de virulência dos isolados e realizar a tipagem molecular dos S. aureus resistentes à meticilina (MRSA). Foi realizado o isolamento e identificação presuntiva de S. aureus por técnicas fenotípicas e a confirmação da espécie pela detecção do gene femA por PCR. Os isolados foram submetidos ao teste de disco-difusão para determinação do perfil de suscetibilidade antimicrobiana e aqueles que apresentaram resistência à cefoxitina foram submetidos ao E-test® para determinação da concentração inibitória mínima à oxacilina e vancomicina, assim como, à detecção do gene mecA para identificação das cepas MRSA. Nestes isolados foi realizada a tipagem do SCCmec. Em todos os S. aureus isolados foi realizada a detecção dos genes codificadores de fatores de virulência e a comparação genética para determinação do perfil de similaridade por eletroforese em gel em campo pulsado. Foram coletados 536 swabs sendo 134 de pacientes e 402 de ambiente de UTI. A prevalência de colonização por S. aureus foi de 12,7% (68/536), sendo 13,4% (18/134) para pacientes e 12,4% (50/402) para o ambiente. A maior taxa de resistência apresentada foi à penicilina (85,3%) seguida da eritromicina (69,1%), clindamicina (66,2%) e sulfametoxazol-trimetoprim (54,4%). Cinquenta e seis isolados (82,4%) foram considerados multirresistentes. A prevalência de MRSA foi de 20,6% (14/68), houve ainda a existência de sete (10,3%) isolados com suscetibilidade intermediária à vancomicina (VISA). O fenótipo de resistência induzível (iMLSb) foi encontrado em 11 isolados (16,2%) e o de resistência constitutiva (cMLSb) em 25 (36,8%). Onze isolados apresentaram genes codificadores para pelo menos um fator de virulência pesquisado, sendo detectados seis perfis de virulência. Das 14 cepas MRSA, seis (42,9%) foram SCCmec tipo IV, cinco (35,7%) SCCmec tipo I, duas (14,3%) SCCmec tipo II e uma (7,1%) SCCmec tipo III.A análise de PFGE revelou diversidade genética entre os isolados, apesar de que um cluster agrupou 16 isolados mostrando a disseminação da bactéria entre pacientes e fômites. Um isolado MRSA mostrou relacionamento genético à cepa USA300 e dois isolados MRSA/VISA foram semelhantes e outro idêntico ao clone USA400. Os resultados sugerem que a prevalência de S. aureus e MRSA permanece elevada em instituições de saúde, especialmente em UTI, com elevadas taxas de resistência antimicrobiana e potencial patogênico. A detecção desses microrganismos no ambiente evidencia risco de transmissão cruzada desses patógenos, principalmente via profissionais da saúde. A identificação de isolados com background genético de cepas adquiridas na comunidade alerta para um fluxo de disseminação intra e inter-ambiente hospitalar e comunitário. Além disso, acredita-se que as superfícies ambientais podem estar atuando como reservatórios de genes de resistência e virulência, bem como fontes potenciais de contaminação de pacientes, profissionais e ambientes.

This study was conducted to examine the rate of contamination and molecular characteristics of enteric bacteria isolated from a selection of food sources in Vietnam. One hundred and eighty raw food samples were tested and 60.8% of meat and 18.0% of shellfish samples were found to be contaminated with Salmonella spp. which belonged to variety of serogroups and serotypes. More than 90% of all food sources contained Escherichia coli and 32% of 50 shellfish samples were contaminated with Vibrio parahaemolyticus. PFGE was used to determine the degree of relatedness of Salmonella spp. There were 33 distinct PFGE patterns from 51 Salmonella spp. isolates tested, indicating that PFGE could be used as an alternative method for serotyping for use in epidemiology of Salmonella spp. Susceptibility of the isolates to 15 antimicrobial agents was investigated. Moderate to high frequencies of resistance to antibiotics were observed in Salmonella spp. and E. coli isolates and multi-resistance, defined as resistance to at least 4 antibiotics, was observed. All of the V. parahaemolyticus isolates were resistant to ampicillin/amoxicillin but not to other antibiotics. Betalactam TEM gene and tetracycline resistance tetA, tetB genes were widely distributed in both E. coli and Salmonella spp. isolates. Other resistance genes, including sulI, cmlA, aadA, aphA1, dhfrV, and aac(3)-IV were also present at high to moderate levels. Identification and characterisation of the mobile genetic elements, including identification of class 1 integrons and plasmids were carried out for multi-resistance isolates. The integrons harboured varying gene cassettes, including aadA1, aadA2, aadA5, aacA4, dhfrXII, drfA1 and dhfrA17, blaPSE1 and catB3. Thirty-five percent of Salmonella spp. isolates and 76% of E. coli isolates harboured plasmids of more than 95 kb. Transfer of resistance phenotypes between the isolates via conjugation and phage transduction was also demonstrated. Salmonella genomic island 1 (SGI1), a 43-kb genomic region contains a 13-kb antibiotic resistance gene cluster, has been identified in an isolate of S. Albany from chicken. The presence of Salmonella spp. virulence genes was investigated to examine the pathogenicity potential of the isolates. The invA gene was present in all Salmonella spp. isolates and the plasmid virulence gene spvC was detected in one S. Typhimurium isolate only, on a 95 kb virulence plasmid. Invasion assays performed in vitro demonstrated that all Salmonella isolates were capable of invading human intestine INT407 cells. In addition, the investigation for the presence of 58 selected virulence genes showed that all the tested isolates contained at least one virulence gene and there were 16 genes which are associated with different pathotypes detected. The data obtained in this study indicates that raw food in Vietnam is a potential reservoirs for many pathogenic organisms, and confirms the role of food animals as a reservoir of multidrug resistant E. coli and Salmonella spp.

<p>Bacteria of the genus <i>Bartonella</i> inhabit the red blood cells of many mammals, including humans, and are transmitted by blood-sucking arthropod vectors. Different species of <i>Bartonella</i> are associated with different mammalian host species, to which they have adapted and normally do not cause any symptoms. Incidental infection of other hosts is however often followed by various disease symptoms, and several <i>Bartonella</i> species are considered as emerging human pathogens.</p><p>In this work, I have studied the genomic diversity within and between different <i>Bartonella</i> species, with focus on the feline-associated human pathogen <i>B. henselae</i> and its close relatives, the similarly feline-associated <i>B. koehlerae</i> and the trench-fever agent <i>B. quintana</i> which is restricted to humans.</p><p>In <i>B. henselae</i>, the overall variability in sequence and genome content was modest and well correlated, suggesting low levels of intra-species recombination in the core genome. The variably present genes were located in the prophage and the genomic islands, which are also absent from <i>B. quintana</i> and <i>B. koehlerae</i>, indicating multiple independent excision events. In contrast, diversity of genome structures was immense and probably associated with rearrangements between the repeated genomic islands located around the terminus of replication, possibly to avoid the host’s immune system. In both <i>B. henselae</i> and the mouse-associated species <i>B. grahamii</i> a large portion of the chromosome was manifold amplified in long-time cultures and packaged into phage particles, allowing for different recombination rates for different chromosomal regions.</p><p>In B<i>. quintana</i>, diversity was studied by sequencing non-coding spacers. The low variability might be due to the recent emergence of this species. Surprisingly, also this species displayed high variability in genome structures, despite its lack of repeated sequences.</p><p>The results indicate that genome rearrangements and gain or loss of mobile elements are major mechanisms of evolution in <i>Bartonella</i>.</p>

Bacteria of the genus Bartonella inhabit the red blood cells of many mammals, including humans, and are transmitted by blood-sucking arthropod vectors. Different species of Bartonella are associated with different mammalian host species, to which they have adapted and normally do not cause any symptoms. Incidental infection of other hosts is however often followed by various disease symptoms, and several Bartonella species are considered as emerging human pathogens. In this work, I have studied the genomic diversity within and between different Bartonella species, with focus on the feline-associated human pathogen B. henselae and its close relatives, the similarly feline-associated B. koehlerae and the trench-fever agent B. quintana which is restricted to humans. In B. henselae, the overall variability in sequence and genome content was modest and well correlated, suggesting low levels of intra-species recombination in the core genome. The variably present genes were located in the prophage and the genomic islands, which are also absent from B. quintana and B. koehlerae, indicating multiple independent excision events. In contrast, diversity of genome structures was immense and probably associated with rearrangements between the repeated genomic islands located around the terminus of replication, possibly to avoid the host’s immune system. In both B. henselae and the mouse-associated species B. grahamii a large portion of the chromosome was manifold amplified in long-time cultures and packaged into phage particles, allowing for different recombination rates for different chromosomal regions. In B. quintana, diversity was studied by sequencing non-coding spacers. The low variability might be due to the recent emergence of this species. Surprisingly, also this species displayed high variability in genome structures, despite its lack of repeated sequences. The results indicate that genome rearrangements and gain or loss of mobile elements are major mechanisms of evolution in Bartonella.

Staphylococcus aureus has long been recognized as a leading cause of nosocomial infection. However, several recent publications have demonstrated this pathogen as the cause of community-acquired severe wound infections and necrotizing pneumonia in otherwise healthy individuals. These highly virulent endemic clones have been reported in several locations in the United States and Canada. The rapid spread of the organism, the ability of certain clones to cause serious infection, and the antibiotic resistance of the endemic clones, illustrates the importance of infection control measures. In this study we examined three S. aureus typing techniques; pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and Staphylococcal protein A (spa) sequencing for subspeciation of community-acquired methicillin-resistant S. aureus (CA-MRSA). It is hypothesized that PFGE will result in a higher level of discrimination among the strains, while MLST and spa typing will result in highly portable data that lacks the discriminatory power of PFGE. Thirty CA-MRSA isolates that were obtained from Florida and Washington State were characterized by molecular typing methods. Whole genome restriction analysis was performed by PFGE using the SmaI enzyme. Sequence-based typing analyses, MLST and spa typing, were performed by polymerase chain reaction (PCR) followed by sequencing. PFGE data was analyzed using the BioNumerics® software package and sequence-based data was analyzed using DNAstar®. MLST Alleles were assigned using the online MLST database (www.mlst.net) and spa types were assigned using the Ridom SpaServer (www.ridom.de/spaserver). Molecular characterization of the 30 isolates resulted in 21 pulsotypes, four MLST sequence types (STs), and six spa types. Combining data from both MLST and spa typing resulted in only seven strain categories, many of which grouped isolates that are not epidemiologically linked. These data demonstrate that techniques such as MLST and spa typing are not well suited for tracking isolates with limited evolutionary diversity such as the CA-MRSA epidemic clones.

Salmonella is one of the most important enteropathogenic bacteria. It is responsible for about 5000 reported cases of human gastroenteritis each year in Sweden. Salmonellosis is a zoonotic disease, and the bacterium has the ability to infect a variety of both domestic and wild animal species. In studies of Swedish wild bird populations, we found that Black-headed gull may be the main reservoir for Salmonella in birds, and that Salmonella infection is expressed as carriage with no obvious disease manifestations. Black-headed gull is a migratory bird and can transport strains of Salmonella with virulence traits like antibiotic resistance, from sources outside Sweden. Genetic molecular methods, PFGE and IS200, also demonstrate that Black-headed gull play a role in the transmission chain of Salmonella in Sweden. In a study of the Swedish Peregrine Falcon population, Salmonella amager and Campylobacter jejuni were found. There were indications, based on serotyping of Salmonella and genetical typing by PFGE of Campylobacter that these isolates were transmitsted to the falcons from a human or domestic animal source. This bird of prey has sparse contact with humans but may be infected by Salmonella of human origin by feeding on other birds, like gull. Salmonella was found in penguins, albatrosses and mainly in seals in a study in Antarctica. Several features of the Salmonella serotypes found indicate a human source for Salmonella infection in these animals, and also a spread of Salmonella within and between animal species in Antarctica. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 2002</p> / digitalisering@umu

Erysipelothrix rhusiopathiae, a Gram-positive bacillus, has long been an important pathogen in veterinary medicine as well as a cause of serious disease in humans. Infections caused by this organism have economic impact on animal industries, causing erysipelas in swine and morbidities in other farmed animals. Human infections are commonly erysipeloid (skin cellulitis) and occasionally septicaemia or endocarditis. Little is known of the diagnosis, epidemiology and pathogenesis of such infections in Western Australia. The aims of this thesis were to establish new diagnostic techniques for the detection and recovery of E. rhusiopathiae, to describe the epidemiology of Erysipelothrix infection in Western Australia in humans and animals, and to characterize virulence-associated characteristics, especially focusing on the neuraminidase produced by the organism. A protocol using 48 h Brain Heart Infusion enrichment followed by subculture to selective agar containing antibiotics achieved the highest recovery rate of 37% in a seafood survey. Twentyone isolates of Erysipelothrix spp., of which 19 were identified as E. rhusiopathiae, were obtained. Two published PCR assays for differentiating E. rhusiopathiae and other Erysipelothrix species were evaluated and the best PCR detection rate achieved was 67% following selective enrichment. The PCR method was 50% more sensitive than the culture method. Epidemiological surveys using the above methods showed that E. rhusiopathiae infection is present in farmed animals in Western Australia. The PCR positive frequencies (3.3-3.7%) and isolate recovery rate (2.8-3.3%) in samples from pig and sheep abattoirs and carcass washings indicate a potential threat to the economy of the farmed animal industry as well as a public health concern with the occurrence of E. rhusiopathiae in meat for consumption. Positive PCR results (1.1%) from human skin swabs of patients with cellulitis and wounds may suggest the existence of Erysipelothrix colonization in the general population. Genetic relatedness of 92 isolates of Erysipelothrix species from various sources was analyzed and a total of 64 distinct PFGE patterns identified. Isolates were further classified into 20 clonal groups based on pattern similarities, and most E. rhusiopathiae were clustered into six groups. A few patterns of other Erysipelothrix species were clustered into separate groups from E. rhusiopathiae but shared greater than 70% similarity with E. rhusiopathiae. The genetic relatedness of colonial variants was well demonstrated using this method. PFGE typing promises to be a useful tool for epidemiological and taxonomic studies of Erysipelothrix. Several virulence-associated factors were characterized in 86 isolates of Erysipelothrix spp. A rapid and sensitive peanut lectin hemagglutination assay for neuraminidase was developed and the influence of media, incubation conditions and pH on the production of the enzyme was investigated. All 61 isolates of E. rhusiopathiae produced neuraminidase in cooked meat broth with titres between 1:10 and 1:320, with no significant difference in titre among isolates from different sources. The enzyme activity was not detected in non-pathogenic Erysipelothrix spp. Capsule was produced by 78.7% of isolates of E. rhusiopathiae but not by other species, while both hyaluronidase and haemolysin were produced by non-pathogenic Erysipelothrix spp. It was concluded that neuraminidase and capsule are most likely to be virulence factors of E. rhusiopathiae. The gene encoding neuraminidase was cloned from the type strain E. rhusiopathiae ATCC 19414. The cloned fragment was a functional partial nanH gene with a mol% G+C of 39.7. The predicted amino acid sequence displayed homology with many microbial neuraminidases and contained conserved sequences found in most bacterial neuraminidases. Southern hybridization experiments demonstrated that the gene was present as a single copy on the bacterial genomic DNA. A neuraminidasenegative mutant vector was constructed by insertional inactivation using a tetM cassette. This has provided starting material for developing a neuraminidase-deficient E. rhusiopathiae mutant, which will permit the study of the role of neuraminidase in pathogenesis. Based on the cloned sequence, a sensitive neuraminidase-specific nested PCR technique was designed and optimized. The specificity was tested in 61 isolates of E. rhusiopathiae, 25 Erysipelothrix species, and 62 other species of neuraminidaseproducing and non-producing bacteria. All isolates of E. rhusiopathiae were PCR positive and all other bacteria were negative; thus this PCR is a highly specific method suitable for application in clinical investigations of Erysipelothrix infection. In conclusion, the present study has contributed new knowledge of the biology of Erysipelothrix spp. and current occurrence of Erysipelothrix infections in Western Australia, as well as to the understanding of pathogenesis of E. rhusiopathiae. Development of several new cultural and molecular approaches in combination with other established techniques will facilitate future studies of the epidemiology, taxonomy and pathogenesis of this bacterial species.

Thesis (MSc Food Sc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Listeria monocytogenes is a foodborne pathogen that has been isolated from a variety of food sources. It is the cause of the food-borne disease, listeriosis that shows symptoms such as meningitis, encephalitis and abortion. Different strains of L. monocytogenes exist and not all are thought to be pathogenic to humans. The aim of this study was to evaluate and compare conventional methods, culturing on selective (Oxford agar) and chromogenic (RAPID’L.mono agar) media, as well as speciesspecific and multiplex polymerase chain reaction (PCR) methods for the detection and identification of 94 L. monocytogenes isolates from various areas in an avocado processing facility, as well as the final product. To achieve a better understanding of the genetic diversity of the confirmed L. monocytogenes strains isolated from the avocado facility, two subtyping techniques, PCR-restriction fragment length polymorphism (PCR-RFLP) and pulsed-field gel electrophoresis (PFGE), were employed. All of the isolates were identified as Listeria species on both Oxford and RAPID’L.mono agar. On the RAPID’L.mono agar, 76 of the 94 isolates produced colonies typical of L. monocytogenes, with the remaining 18 showing colonies typical of L. innocua (n=13) and L. ivanovii (n=5). The species-specific PCR successfully amplified a 730 base pairs region of the hly gene of 80 of the 94 isolates. For the same 80 isolates the multiplex PCR successfully amplified 800, 517 and 238 base pair (bp) fragments of the inlA, inlC and inlJ genes, respectively. The remaining 14 isolates included the 13 isolates identified as L. innocua, as well as an isolate identified as L. monocytogenes on RAPID’L.mono. The results obtained on the Oxford agar showed a 100 % positive correlation when compared to the PCR results in identifying Listeria species, while the RAPID’L.mono had a 4 % false negative result in identifying L. monocytogenes compared to the PCR results. Sixty-four of the confirmed L. monocytogenes isolates were subtyped using PCRRFLP and PFGE. For the PCR-RFLP analysis, a 733 bp fragment of the inlA gene was successfully amplified for all of the isolates, followed by digestion with the restriction enzymes, AluI and Tsp509I. AluI produced three different banding patterns and Tsp509I produced two different banding patterns. Subtyping of the isolates using PFGE was carried out by macrorestriction of the genomic DNA with ApaI and AscI. The restriction fragments were resolved by PFGE and the fingerprints were classified into four clusters. In the combined analyses, cluster I contained forty-eight isolates (n=48), cluster II 1 isolate (n=1), cluster III fifteen isolates (n=15) and cluster IV 1 isolate (n=1). The PCR-RFLP results had a 98 % correlation with the PFGE results. The results of this study indicated inconsistencies between the results obtained by conventional and molecular detection methods for the identification of L. monocytogenes. Species-specific and multiplex PCR, however, proved useful to accurately detect and identify L. monocytogenes in a shorter period of time and could replace the use of conventional agar during identification. Both PCR-RFLP and PFGE proved useful in the subtyping of L. monocytogenes isolates with the PCR-RFLP being less expensive and results obtainable in a shorter period of time. / AFRIKAANSE OPSOMMING: Listeria monocytogenes is ‘n patogeen afkomstig van voedsel wat uit ‘n verskeidenheid voedselbronne geisoleer kan word. Dit is die oorsaak van die voedsel afkomstigde siekte, listeriosis met simptome soos harsingvliesontsteking, ensefalitis en aborsie. ‘n Verskeidenheid L. monocytogenes stamme bestaan, maar nie almal word as patogenies beskou nie. Die doel van hierdie studie was om konvensionele metodes, naamlik mikrobiologiese kweking op selektiewe (Oxford agar) en chromatografiese (RAPID’L.mono agar) media, sowel as spesies-spesifieke en multipleks polimerase ketting reaksie (PKR) metodes te evalueer en vergelyk vir die deteksie en identifikasie van 94 L. monocytogenes isolate geisoleer vanuit verskeie areas in ‘n avokado prosesseringsfasiliteit sowel as die finale produk. Om ‘n beter begrip van die genetiese diversiteit van die isolate wat as L. monocytogenes bevestig is te verkry, is twee subtiperingstegnieke, PKR-restriksiefragmentlengte polimorfisme (PKR-RFLP) en pulsveld jel-elektroforese (PVJE) toegepas. Beide Oxford en RAPID’L.mono agar het al die isolate as Listeria spesies geidentifiseer. Op die RAPID’L.mono agar het 76 van die 94 isolate kolonies tipies van L. monocytogenes gevorm, 13 kolonies was tipies van L. innocua (n=13) en vyf kolonies tipies van L. ivanovii (n=5). Die spesies-spesifieke PKR het ‘n 730 basis paar (bp) streek van die hly geen suksesvol geamplifiseer vir 80 van die 94 isolate. Die multipleks PKR het 800, 517 en 238 bp fragmente van die inlA, inlC and inlJ gene onderskeidelik, vir dieselfde 80 isolate suksesvol geamplifiseer. Die oorblywende 14 isolate het die 13 isolate wat as L. innocua geïdentifiseer is en die een isolaat wat as L. monocytogenes op RAPID’L.mono geïdentifiseer is ingesluit. Resultate verkry met die Oxford agar het 100 % ooreengestem met die PKR resultate vir die identifikasie van Listeria spesies. Die RAPID’L.mono het ‘n 4 % vals negatiewe resultaat gelewer in vergelyking met die PKR resultate. Vier-en-sestig van die bevestigde L. monocytogenes isolate is gesubtipeer deur PKR-RFLP en PVJE. Tydens die PKR-RFLP analise is ‘n 733 bp fragment van die inlA geen suksesvol geamplifiseer, gevolg deur vertering met die restriksie-ensieme, AluI and Tsp509I. AluI het drie verskillende bandpatrone opgelewer en Tsp509I twee verskillende bandpatrone. Subtipering deur PVJE is uitgevoer deur makro-restriksie van die genomiese DNA met ApaI en AscI. Die restriksie fragmente is geskei deur PVJE en die vingerafdrukke is in vier groepe geklassifiseer. Groep I het 48 isolate (n=48), groep II 1 isolaat (n=1), groep III 15 isolate (n=15) en groep IV 1 isolaat (n=1) gehad tydens die gekombineerde analise. Die PKR-RFLP resultate het 98 % ooreengestem met die van die PVJE. Die resultate van hierdie studie het teenstrydighede tussen die resultate van konvensionele en molekulêre deteksie metodes opgelewer vir die identifikasie van L. monocytogenes. Die spesies-spesifieke en multipleks PKR het egter beide goed te pas gekom vir die akkurate deteksie en identifikasie van L. monocytogenes en kan heel moontlik die gebruik van konvensionele agar tydens identifikasie vervang. Beide PKRRFLP en PVJE was nuttig vir die subtipering van L. monocytogenes isolate. PKR-RFLP is egter ‘n goedkoper tegniek en die resultate is in ‘n korter tydsperiode beskikbaar.