Thesis (M. Tech. Environmental health) -- Central University of technology, Free State, 2011 / Microbial pathogens play an important role in the food industry where they could cause disease and subsequently significant economic losses. Limited information is available on the situation with regard to Listeria in food products in South Africa. However, much research is being done in the rest of the world on Listeria indicating serious problems as a result of resistance development against various antimicrobial agents, including the organic acids. It is hypothesised that the situation with regard to resistance development may be more serious than generally admitted. Isolation of 200 different food samples was done by using a slightly modified EN ISO 11290-1/A1:2004 standard method. Identification of presumptive positive colonies was confirmed as Listeria by API (Analytical profile index) Listeria. API positive cultures were subjected to 16S rDNA sequencing to compare and confirm identification. Isolates and standard strains were screened for resistance to food preservatives such as organic acids and antibiotics used in the current treatment regime for Listeria infections. The organisms evaluated included isolated strains namely Listeria monocytogenes, Listeria welshimeri, Listeria innocua and their corresponding ATCC (American type culture colletion) strains. An agar dilution method as described by the Clinical and Laboratory Standard Institute (CLSI) was used to determine the minimum inhibitory concentrations (MICs) of 11 antibiotics and 13 organic acids and salts for all the isolates. Overall antibiotic susceptibility patterns of all the isolates indicated high level susceptibility to all the antibiotics tested. Susceptibility to all the organic acids was notably reduced at pH 7 in all the isolates and control strains. Eight highly susceptible strains were selected for induction and represented each of the species isolated. These isolates were exposed to increasing concentrations of three antibiotics and three organic acids. MICs were again determined for all the induced strains for five antibiotics and three organic acids. Proteins extracted from the induced strains were separated on discontinuous SDS-PAGE slab gels to generate total protein profiles. Notable variations were observed in MICs, although induction with antibiotics as well as organic acids did not result in general resistance development. However, evidence was provided that continuous exposure to antimicrobial agents may cause Listeria spp. to develop resistance to different antimicrobial agents. Further research and in depth studies on mechanisms involved in the development of resistance to food preservatives would, therefore, be required. Finally, it is concluded that Listeria monocytogenes may be a possible threat in the Central South African food industry, which deserves more attention. The situation may actually pose a problem that is overseen, because only a small percentage of people that get sick from food, would seek medical advice.

Bacterial infections are a major cause of morbidity and mortality in both the community and nosocomial settings, particularly among the elderly and chronically ill. Sepsis is the body’s response to antigens and toxins released by the invasive pathogenic organisms that cause infection. When infection is not effectively controlled, sepsis may develop and progress to severe sepsis and septic shock. Early diagnosis and treatment is pivotal for survival in severe sepsis and particularly, septic shock. Our research focuses on developing a novel treatment strategy for septic shock by using single, ultra-high doses of aminoglycosides. In this project, the effect of a single, ultra-high dose of gentamicin in clearing bacteria from the blood and reducing the bacterial burden in vital organs was evaluated in a rat model of E. coli (Bort strain) induced peritonitis with severe sepsis/septic shock. Serum cytokine levels and serum lactate levels were serially measured. Further, the potential adverse effects of ultra-high dosing of aminoglycoside antibiotics in a short-term (9 h) invasive study and long term (180 days) non-invasive study were assessed. Neuromuscular paralyses due to ultra-high doses of aminoglycosides were assessed. In addition, renal injury markers such as serum creatinine and urinary Neutrophil Gelatinase Associated Lipocalin (NGAL) were assayed. The auditory and vestibular function was also assessed after ultra-high dosing of aminoglycoside in the long-term study. We conclude that animals can tolerate ultra-high doses of aminoglycosides with appropriate support. Animals were under neuromuscular paralysis for 28 – 50 minutes and were on ventilator support after single ultra-high doses (80 and 160 mg/kg) of aminoglycoside antibiotics (gentamicin and tobramycin). There was no significant acute or delayed renal or ototoxicity associated with the single, ultra-high dose aminoglycoside therapy. Histology studies of the kidneys and the cochlea of single, ultra-high aminoglycoside dosed animals and untreated control animals were performed after 180 days (6 months). Results indicated that there were no morphological differences between the treated and untreated control animals. Terminal deoxy-nucleotidyl transferase dUTP nick end labeling (TUNEL) assay of kidney tissue indicated that there was no apoptosis of endothelial cells in the tubular and glomerular regions with single, ultra- high dose of aminoglycosides consistent with an absence of ultrahigh dose induced nephrotoxicity. In the septic shock model, the E. coli Bort was below the limit of detection from the blood of the animals within minutes after single, ultra-high dose aminoglycoside administration. After necropsy, bacterial load was determined from all the vital organs and peritoneal fluid (site of infection). The bacterial levels were below the detection limit from the kidneys and there was a significant reduction in bacterial counts from all the remaining organs compared to the infected control animals. A decrease in serum cytokine and serum lactate levels compared to baseline was observed after ultra-high doses of aminoglycosides in the septic shock animals. Our studies have indicated that the ultra-high dose gentamicin is well tolerated by rats. It is highly effective in clearing E. coli Bort from the blood and reducing the bacterial burden in the organs in an experimental model of bacterial peritonitis/septic shock. Further studies in larger animals such as rabbits, sheep, pigs or dogs are required to confirm these results. If these findings are replicated in larger animals, this therapy may be developed further from ‘lab to bedside’ to treat septic shock patients in intensive care units (ICUs). / October 2015

Este estudo identificou a presença e a viabilidade de Streptococcus spp., Propionibacterium acnes e Enterococcus faecalis antes e após os procedimentos endodônticos, utilizando o método de reação de cadeia de polimerase (PCR) baseado em RNA ribossômico (rRNA) e seus respectivos genes (rDNA). Foram coletadas amostras de 20 dentes com infecção primária antes (S1) e após o preparo químico-cirúrgico (S2), e depois do emprego do Ca(OH)2 como medicação intracanal (S3). DNA e RNA foram extraídos da mesma amostra de canal radicular e utilizados como moldes para reação de PCR com iniciadores específicos para a região 16S rRNA das espécies analisadas. Streptococcus espécies foram detectados em 20% e 25% das amostras S1 utilizando os métodos baseados em rRNA e rDNA, respectivamente; enquanto P. acnes foi detectado apenas pela análise de rRNA, estando presente em 10% das amostras S1. Após o preparo químico-cirúrgico, Streptococcus spp. foram detectado em 10% das amostras S2 quando se utilizou rDNA, porém não foi detectado pelo método baseado em rRNA, indicando ausência de células viáveis. Por outro lado, P. acnes foi detectado por ambos os métodos nas amostras S2, com prevalência de 10% e 5% quando se utilizou rRNA e rDNA como molde para PCR, respectivamente. Nas amostras S3, P. acnes foi a única espécie detectada nos ensaios baseados em rRNA, presente em 10% dos casos, enquanto o método baseado em rDNA falhou em detectar essa espécie. Por sua vez, E. faecalis não foi detectado em nenhuma amostra pelos métodos utilizados. Portanto, concluise que a suscetibilidade bacteriana aos procedimentos endodônticos varia entre as espécies Gram-positivas. Enquanto Streptococcus spp. foram suscetíveis, P. acnes persistiu ativo em canais radiculares após o preparo químico-cirúrgico e medicação intracanal. Esses dados sugerem que há necessidade de novas estratégias para a eliminação de espécies resisitentes ao tratamento endodôntico. / This study identified the presence and viability of Streptococcus spp., Enterococcus faecalis and Propionibacterium acnes before and after endodontic procedures, using the polymerase chain reaction (PCR) based on ribosomal RNA (rRNA) and their genes (rDNA ). Samples of 20 teeth with primary infection were collected before (S1) and after chemical-surgical preparation (S2), and after Ca(OH)2 as temporary dressing (S3). DNA and RNA were extracted from the same root canal sample and used as templates for PCR with specific primers for 16S rRNA region of the analyzed species. Streptococcus species were detected in 20% and 25% of the S1 samples using methods based on rRNA and rDNA, respectively; while P. acnes was only detected by analysis of rRNA, present in 10% of the samples S1. After chemicalsurgical preparation, Streptococcus spp. were detected in 10% of S2 samples when using rDNA as template, but they were not detected by the method based on rRNA, indicating the absence of viable cells. Furthermore, P. acnes was detected by both methods in samples S2, with a prevalence of 10% and 5% when using as template rRNA and rDNA for PCR, respectively. In S3 samples, P. acnes was the only species detected in assays based on rRNA, present in 10% of cases, while the rDNA-based method failed to detect this species. E. faecalis was not detected in any sample by the methods used. Therefore, it is concluded that bacterial susceptibility to endodontic procedures may vary among Gram-positive species. While Streptococcus spp. were susceptible, P. acnes persisted active in root canals after chemicalsurgical preparation and dressing. These data suggest the need for new strategies to eliminate resisitant species to endodontic treatment.

Flagelinas de bactérias gram-negativas ativam o sistema imune inato resultando em efeitos adjuvantes para antígenos co-administrados. No entanto, não existem relatos sobre as propriedades adjuvantes de flagelinas de bactérias gram-positivas. O objetivo do projeto foi estudar os efeitos imunomoduladores da flagelina Hag de B. subtílis. Inicialmente foi purificada a Hag e subseqüentemente avaliada sua propriedade imunológica quando administrada junto com a ovalmbumina (OVA) pelas vias intra-nasal (i.n.) ou subcutânea (s.c.) em camundongos Balb/C e C57BL/6. Os resultados demonstraram as propriedades imunomoduladoras da Hag nas duas linhagens em relação à indução de anticorpos antígeno-específicos. Não foi possível demonstrar a ativação de linfócitos TCD4+ e CD8+ antígenos-específicos em animais Balb/C imunizados com OVA e Hag tanto pela via s.c. como pela i.n.. Resultados mais promissores do efeito adjuvante frente a células T foram encontrados em animais C57BL/6. Os resultados abrem perspectivas para o estudo do potencial da flagelina Hag de B. subtilis como adjuvante vacinal. / Flagellin of gram negatives bacteria actives the innate immune system resulting in adjuvants effects against coadministrated antigens. However, there is not information about immunological properties of flagellins produced by gram positive bacteria. This study aimed the evaluation of immunomodulator effects of Hag flagellin from B. subtilis. Vaccine formulations based in purified Hag and Ovalbumine protein (OVA) were administrated intranasal (i.n.) and subcutaneously (s.c.) in Balb/C and C57BL/6 mice and then the population of B and T lymphocytes were monitored for production of antibodies and citocines and/or surface markers expression. Results showed the immunomodulatory properties of Hag in both mice lineages regarding the induction of antigen specific antibodies when immunized with OVA and Hag by i.n. and s.c. via. Most promising results concerning the adjuvant effects observed on T cells activation were found in C57BL/6 mice. The results obtained open future perspectives for the study of the potential use of B. subtilis Hag flagellin as adjuvant in vaccines.

Os bastonetes Gram positivos irregulares (BGPIs) compõem um grupo de espécies bacterianas com ampla diversidade fenotípica e que podem estar presente no meio ambiente, na microbiota humana e de animais. A identificação acurada de BGPIs em nível de gênero e espécie empregando métodos bioquímicos convencionais é bastante limitada, sendo recomendado, portanto, o uso de técnicas moleculares. No presente estudo, foram identificadas amostras de BGPIs oriundas de espécimes clínicos de humanos, de produtos farmacêuticos e de áreas limpas através da análise de sequencias do gene 16S rRNA e de outros genes conservados (housekeeping genes). Os resultados obtidos pelo sequenciamento dos genes 16S rRNA e rpoB demonstraram C. striatum multi-resistente (MDR) como responsável por surto epidêmico em ambiente hospitalar da cidade do Rio de Janeiro. Quinze cepas de C. striatum foram isoladas em cultura pura a partir de secreção traqueal de pacientes adultos submetidos a procedimentos de entubação endotraqueal. A análise por eletroforese em gel de campo pulsado (PFGE) indicou a presença de quatro perfis moleculares, incluindo dois clones relacionados com cepas MDR (PFGE I e II). Os dados demonstram a predominância de PFGE I entre cepas MDR isoladas de unidades de terapia intensiva e enfermarias cirúrgicas. Uma potencial ligação causal entre a morte e a infecção por C. striatum MDR (PFGE tipos I e II) foi observada em cinco casos. Adicionalmente, acreditamos que este seja o primeiro estudo de identificação de espécies de Nocardia relacionadas com infecções humanas pela análise da sequencia multilocus (MLSA) no Brasil. Diferente dos dados observados na literatura (1970 a 2013) e obtidos pelos testes fenotípicos convencionais, a caracterização molecular de quatro lócus (gyrB-16S-secA1-hsp65) permitiu a identificação das espécies N. nova, N. cyriacigeorgica, N. asiatica e N. exalbida/gamkensis relacionadas com quadros de nocardiose em humanos. Cepas de N. nova isoladas de diferentes materiais clínicos de um único paciente apresentaram padrões de susceptibilidade antimicrobianos idênticos e dois perfis PFGE, indicando a possibilidade de quadros de co-infecção por N. nova em humanos. Em outra etapa da investigação, amostras de BGPIs obtidos de ambientes de salas limpas que não puderam ser identificadas por critérios convencionais foram submetidas a análise da sequência do gene 16S rRNA e caracterizadas 95,83% em nível de gênero e 35,42% em espécies. Para gêneros mais encontrados no estudo, foram analisados os genes rpoB e recA de dezessete cepas de Microbacterium e utilizado o MLSA para a identificação de sete cepas identificadas como Streptomyces. Os ensaios permitiram a identificação de três cepas de Microbacterium e de uma única amostra de Streptomyces ao nível de espécie. A análise da sequencia do gene rpoB também se mostrou eficaz na identificação de espécies de cepas de Corynebacterium. Finalmente, para as cepas ambientais pertencentes à classe Actinobacteria os dados morfológicos, bioquímicos e genotípicos permitiram documentar a cepa 3117BRRJ como representante de uma nova espécie do gênero Nocardioides, para o qual o nome Nocardioides brasiliensis sp. nov. e as cepas 3712BRRJ e 3371BRRJ como representante de um novo gênero e espécie para o qual o nome Guaraldella brasiliensis nov. foi proposto. / Irregular Gram positive rods (coryneform bacteria; IGPRs) comprise a group of species that has a wide phenotypic diversity and makes the conventional identification limited and that may be present in the environment, humans and animals hosts. In order to provide further accurate identification of these microorganisms in a genus and species terms it is recommended the use of molecular methods. In this study, we analyzed the 16S rRNA gene sequences and other conserved genes (housekeeping) in order to elucidate the identification of clinical isolates, pharmaceutical and clean room areas. We have documented a nosocomial outbreak caused by multidrug-resistant (MDR) C. striatum in Rio de Janeiro. C. striatum identification was confirmed by 16S rRNA and rpoB gene sequencing. C. striatum was mostly isolated in pure culture from tracheal aspirates of adults undergoing endotracheal intubation procedures. The analysis by pulsed-field gel electrophoresis (PFGE) indicated the presence of four PFGE profiles, including two related clones of MDR strains (PFGE I and II). The data demonstrated the predominance of PFGE I, mostly isolated from patients of intensive care units and surgical wards. A potential causal link between death and MDR C. striatum (PFGE I and II) infection was observed in five cases. We also performed the identification of Nocardia species of human infections by multilocus sequence analysis (MLSA) and characterized their antimicrobial and phenotypic profiles. An overview from 1970 to 2013 of the case reports on Nocardia species related to human infections, except mycetomas, in Brazil, was also accomplished. Molecular characterization by four-locus (gyrB-16S-secA1-hsp65) has provided the species identification N. nova, N. cyriacigeorgica, N. asiatica and N. exalbida/gamkensis. N. nova strains isolated from different clinical specimens of one patient showed identical antimicrobial susceptibility patterns. PFGE analysis performed to determine the genetic relatedness of these N. nova strains two distinct profiles, which were designated A and B. This is the first report on identification of Nocardia species by MLSA in Brazil. The IGPRs obtained in clean room environments that could not be identified by conventional criteria were studied by 16S rRNA gene sequence analysis that allowed the identification of 95.83% at genus level and of 35.42% at the species level. The most common genus found in the clean room environments were Microbacterium and Streptomyces. The analysis of rpoB and recA genes sequence of seventeen Microbacterium strains contributed to the identification of three strains at species level. MLSA of seven Streptomyces strains identified a single sample at the species level. Moreover, the rpoB gene sequence analysis was effective in identifying Corynebacterium strains at species level. A new genus and species were found among clean room environmental strains belonging to the Actinobacteria class. Based on the morphological, genotypic and biochemical data presented in this study the 3117BRRJ strain represent a novel specie of the genus Nocardioides, for which the name Nocardioides brasiliensis sp. nov. was proposed and the 3712BRRJ and 3371BRRJ represent a new genus and species for which the name Guaraldella brasiliensis nov. were proposed.

Este estudo identificou a presença e a viabilidade de Streptococcus spp., Propionibacterium acnes e Enterococcus faecalis antes e após os procedimentos endodônticos, utilizando o método de reação de cadeia de polimerase (PCR) baseado em RNA ribossômico (rRNA) e seus respectivos genes (rDNA). Foram coletadas amostras de 20 dentes com infecção primária antes (S1) e após o preparo químico-cirúrgico (S2), e depois do emprego do Ca(OH)2 como medicação intracanal (S3). DNA e RNA foram extraídos da mesma amostra de canal radicular e utilizados como moldes para reação de PCR com iniciadores específicos para a região 16S rRNA das espécies analisadas. Streptococcus espécies foram detectados em 20% e 25% das amostras S1 utilizando os métodos baseados em rRNA e rDNA, respectivamente; enquanto P. acnes foi detectado apenas pela análise de rRNA, estando presente em 10% das amostras S1. Após o preparo químico-cirúrgico, Streptococcus spp. foram detectado em 10% das amostras S2 quando se utilizou rDNA, porém não foi detectado pelo método baseado em rRNA, indicando ausência de células viáveis. Por outro lado, P. acnes foi detectado por ambos os métodos nas amostras S2, com prevalência de 10% e 5% quando se utilizou rRNA e rDNA como molde para PCR, respectivamente. Nas amostras S3, P. acnes foi a única espécie detectada nos ensaios baseados em rRNA, presente em 10% dos casos, enquanto o método baseado em rDNA falhou em detectar essa espécie. Por sua vez, E. faecalis não foi detectado em nenhuma amostra pelos métodos utilizados. Portanto, concluise que a suscetibilidade bacteriana aos procedimentos endodônticos varia entre as espécies Gram-positivas. Enquanto Streptococcus spp. foram suscetíveis, P. acnes persistiu ativo em canais radiculares após o preparo químico-cirúrgico e medicação intracanal. Esses dados sugerem que há necessidade de novas estratégias para a eliminação de espécies resisitentes ao tratamento endodôntico. / This study identified the presence and viability of Streptococcus spp., Enterococcus faecalis and Propionibacterium acnes before and after endodontic procedures, using the polymerase chain reaction (PCR) based on ribosomal RNA (rRNA) and their genes (rDNA ). Samples of 20 teeth with primary infection were collected before (S1) and after chemical-surgical preparation (S2), and after Ca(OH)2 as temporary dressing (S3). DNA and RNA were extracted from the same root canal sample and used as templates for PCR with specific primers for 16S rRNA region of the analyzed species. Streptococcus species were detected in 20% and 25% of the S1 samples using methods based on rRNA and rDNA, respectively; while P. acnes was only detected by analysis of rRNA, present in 10% of the samples S1. After chemicalsurgical preparation, Streptococcus spp. were detected in 10% of S2 samples when using rDNA as template, but they were not detected by the method based on rRNA, indicating the absence of viable cells. Furthermore, P. acnes was detected by both methods in samples S2, with a prevalence of 10% and 5% when using as template rRNA and rDNA for PCR, respectively. In S3 samples, P. acnes was the only species detected in assays based on rRNA, present in 10% of cases, while the rDNA-based method failed to detect this species. E. faecalis was not detected in any sample by the methods used. Therefore, it is concluded that bacterial susceptibility to endodontic procedures may vary among Gram-positive species. While Streptococcus spp. were susceptible, P. acnes persisted active in root canals after chemicalsurgical preparation and dressing. These data suggest the need for new strategies to eliminate resisitant species to endodontic treatment.

The engineering of bacterial surfaces has in recent yearsattracted a lot of attention with applications in manydifferent areas of bioscience. Here we describe the use of twodifferent surface display systems for the gram-positivebacteria Staphylococcus carnosus and Staphylococcus xylosus invarious biotechnological applications. Environmental microbiology currently attracts a lot ofattention since genetically engineered plants and bacteriamight be used as bioadsorbents for sequestration of toxicmetals. Bacterial surface display of metal-binding peptidesmight enable recycling of the biomass by desorption ofaccumulated heavymetals. In an attempt to recruitstaphylococcal display systems for bioremediation purposes,polyhistidyl peptides were successfullly displayed on thesurface of recombinant S. carnosus and S. xylosus cells.Whole-cell Ni2+-binding assays demonstrated that therecombinant cells had gained metal-binding capacity compared towild-type cells. Tailor-made, metal-binding staphylococci was created using apreviously constructed phage-display combinatorial proteinlibrary based on a fungal cellulose-binding domain (CBD)derived from the cellobiohydrolase Cel7A of Trichoderma reseii.Novel metal-binding CBDs were generated through a phagemediated selection procedure. Selected CBD variants, now devoidof cellulose binding, were randomly selected and sequenceanalysis of selected variants revealed a marked preference forhistidine residues at the randomized positions. Surface displayof these novel CBD variants resulted in recombinantstaphylococci with increased metal-binding capacity compared tocontrol strains, indicating that this could become a generalstrategy to engineer bacteria for improved binding to specificmetal ions. Directed immobilization of cells with surface displayedheterologous proteins have widespread use in modernbiotechnology. Among other things they could provide aconvenient way of generating biofilters, biocatalysts orwhole-cell diagnostic devices. It was therefore investigatedwhether directed immobilization of recombinant staphylococci oncotton fibers could be achieved by functional display of afungal cellulose-binding domain (CBD). Recombinant S. carnosuscells with surface anchored CBDs from Trichoderma reseii Cel6Awere found to efficiently bind to cotton fibers creating almosta monolayer on the fibrous support. The co-expression of thisCBD together with previously described metal-binding proteinson the surface of our staphylococci would create means fordeveloping effective bioadsorbents for remediationpurposes. The original plasmid vector, designed for heterologoussurface display on recombinant S. carnosus cells has exhibitedproblems related to structural instability, possibly due to thepresence of a phage f1 origin of replication in the vectorsequence. This would be a problem if using the vector systemfor library display applications. Therefore, novel surfacedisplay vectors, lacking the phage ori were constructed andevaluated by enzymatic and flow cytometric whole-cell assays.One such novel vector, pSCXm, exhibited dramatically increasedplasmid stability with the retained high surface density ofexpressed heterologous proteins characteristic for the originalS. carnosus display vector, thus making it potentially moresuitable for library display applications. The successful engineering of our staphylococcal displaysystem encouraged us to further evaluate the potential to usethe staphylococcal system for display of combinatorial proteinlibraries and subsequent affinity based selections using flowcytometric cell sorting. A model system of recombinant S.carnosus cells with surface displayed engineered protein Adomains was constructed. It was demonstrated that target cellscould be sorted essentially quantitatively from a moderateexcess of background cells in a single sorting-step.Furthermore, the possibility of using staphylococcal surfacedisplay and flow cytometric cell sorting also for specificenrichment of very rare target cells by multiple rounds ofcell-sorting and in between amplification was demonstrated. <b>Key words:</b>affibody, albumin binding protein, bacterialsurface display, cell immobilization, bioremediation,combinatorial protein engineering, flow cytometry,Gram-positive, metal binding, staphylococcal protein A,Staphylococcus carnosus, Staphylococcus xylosus, whole-celldevices

<p>The engineering of bacterial surfaces has in recent yearsattracted a lot of attention with applications in manydifferent areas of bioscience. Here we describe the use of twodifferent surface display systems for the gram-positivebacteria Staphylococcus carnosus and Staphylococcus xylosus invarious biotechnological applications.</p><p>Environmental microbiology currently attracts a lot ofattention since genetically engineered plants and bacteriamight be used as bioadsorbents for sequestration of toxicmetals. Bacterial surface display of metal-binding peptidesmight enable recycling of the biomass by desorption ofaccumulated heavymetals. In an attempt to recruitstaphylococcal display systems for bioremediation purposes,polyhistidyl peptides were successfullly displayed on thesurface of recombinant S. carnosus and S. xylosus cells.Whole-cell Ni2+-binding assays demonstrated that therecombinant cells had gained metal-binding capacity compared towild-type cells.</p><p>Tailor-made, metal-binding staphylococci was created using apreviously constructed phage-display combinatorial proteinlibrary based on a fungal cellulose-binding domain (CBD)derived from the cellobiohydrolase Cel7A of Trichoderma reseii.Novel metal-binding CBDs were generated through a phagemediated selection procedure. Selected CBD variants, now devoidof cellulose binding, were randomly selected and sequenceanalysis of selected variants revealed a marked preference forhistidine residues at the randomized positions. Surface displayof these novel CBD variants resulted in recombinantstaphylococci with increased metal-binding capacity compared tocontrol strains, indicating that this could become a generalstrategy to engineer bacteria for improved binding to specificmetal ions.</p><p>Directed immobilization of cells with surface displayedheterologous proteins have widespread use in modernbiotechnology. Among other things they could provide aconvenient way of generating biofilters, biocatalysts orwhole-cell diagnostic devices. It was therefore investigatedwhether directed immobilization of recombinant staphylococci oncotton fibers could be achieved by functional display of afungal cellulose-binding domain (CBD). Recombinant S. carnosuscells with surface anchored CBDs from Trichoderma reseii Cel6Awere found to efficiently bind to cotton fibers creating almosta monolayer on the fibrous support. The co-expression of thisCBD together with previously described metal-binding proteinson the surface of our staphylococci would create means fordeveloping effective bioadsorbents for remediationpurposes.</p><p>The original plasmid vector, designed for heterologoussurface display on recombinant S. carnosus cells has exhibitedproblems related to structural instability, possibly due to thepresence of a phage f1 origin of replication in the vectorsequence. This would be a problem if using the vector systemfor library display applications. Therefore, novel surfacedisplay vectors, lacking the phage ori were constructed andevaluated by enzymatic and flow cytometric whole-cell assays.One such novel vector, pSCXm, exhibited dramatically increasedplasmid stability with the retained high surface density ofexpressed heterologous proteins characteristic for the originalS. carnosus display vector, thus making it potentially moresuitable for library display applications.</p><p>The successful engineering of our staphylococcal displaysystem encouraged us to further evaluate the potential to usethe staphylococcal system for display of combinatorial proteinlibraries and subsequent affinity based selections using flowcytometric cell sorting. A model system of recombinant S.carnosus cells with surface displayed engineered protein Adomains was constructed. It was demonstrated that target cellscould be sorted essentially quantitatively from a moderateexcess of background cells in a single sorting-step.Furthermore, the possibility of using staphylococcal surfacedisplay and flow cytometric cell sorting also for specificenrichment of very rare target cells by multiple rounds ofcell-sorting and in between amplification was demonstrated.</p><p><b>Key words:</b>affibody, albumin binding protein, bacterialsurface display, cell immobilization, bioremediation,combinatorial protein engineering, flow cytometry,Gram-positive, metal binding, staphylococcal protein A,Staphylococcus carnosus, Staphylococcus xylosus, whole-celldevices</p>

Streptococcus equi is the causative agent of strangles, a prevalent and highly contagious disease of horses. Despite the animal suffering and economic burden associated with strangles, little is known about the molecular basis of S. equi virulence. Here we have investigated the contributions of a specific lipoprotein and the general lipoprotein processing pathway to the abilities of S. equi to colonize equine epithelial tissues in vitro and to cause disease in both a mouse model and the natural host in vivo. Colonization of air interface organ cultures after they were inoculated with a mutant strain deficient in the maturase lipoprotein (prtM138-213, with a deletion of nucleotides 138 to 213) was significantly less than that for cultures infected with wild-type S. equi strain 4047 or a mutant strain that was unable to lipidate preprolipoproteins (lgt190-685). Moreover, mucus production was significantly greater in both wild-type-infected and lgt190-685-infected organ cultures. Both mutants were significantly attenuated compared with the wild-type strain in a mouse model of strangles, although 2 of 30 mice infected with the lgt190-685 mutant did still exhibit signs of disease. In contrast, only the prtM138-213 mutant was significantly attenuated in a pony infection study, with 0 of 5 infected ponies exhibiting pathological signs of strangles compared with 4 of 4 infected with the wild-type and 3 of 5 infected with the lgt190-685 mutant. We believe that this is the first study to evaluate the contribution of lipoproteins to the virulence of a gram-positive pathogen in its natural host. These data suggest that the PrtM lipoprotein is a potential vaccine candidate, and further investigation of its activity and its substrate(s) are warranted.

Gram-positive bacteria are part of the normal flora of fish from different aquatic environments. They are mesophilic bacteria and demonstrate optimum growth at ambient temperature. In the sub-tropics, marine fish are caught from seas at temperatures of 16 to 34C, they are usually not iced and are handled at ambient temperature. It was hypothesized that under these conditions Gram-positive bacteria will be abundant in sub-tropical marine fish and will have roles in the spoilage of fish. A review of literature showed that there is a gap in understanding the Gram-positive bacterial populations in sub-tropical marine fish. This is partly due to the fact that the selective media used for isolating Gram-positive bacteria have limitations. Ecological and speciation studies have revealed that the ecology and speciation of many Gram-positive bacteria have not been clearly elucidated. The effect of ambient storage on the individual genera and species of Gram-positive bacteria in fish has been rarely studied. The spoilage potential of Gram-positive bacteria of marine fish origin has not been clearly determined. Therefore, the main aims of this study were to isolate Gram-positive bacteria from fresh and ambient-temperature-stored sub-tropical marine fish, speciate the isolates and study the spoilage potential of the isolates. The practical components of this study were conducted in four parts. The first part dealt with validation of tryptone soya agar with 0.25% phenylethyl alcohol (PEA-TSA) to enumerate Gram-positive bacteria. The second part enumerated Gram-positive bacteria from the muscles, gills and gut of Pseudocaranx dentex (Silver Trevally), Pagrus auratus (Snapper) and Mugil cephalus (Sea Mullet) stored at 25C for 15 hours using PEA-TSA. The third part dealt with the speciation of the isolates using appropriate methods such as polymerase chain reaction, 16S rRNA gene sequence, the VITEK JR system and conventional biochemical methods. In the fourth part, the isolates were assayed qualitatively for their ability to produce volatile sulphur compounds (VSC), reduce trimethylamine oxide and decarboxylate histidine, lysine and ornithine at mesophilic temperature, 32C. Initial studies indicated that PEA-TSA significantly (P< 0.05) reduced the total aerobic bacterial count of fish whereas control Gram-positive bacteria were not affected (P> 0.05). Gram-positive aerobic bacterial counts (GABC) significantly (P< 0.05) increased in the muscles and gills during ambient storage for 15 hours. Within each species, no significant (P> 0.05) differences were found in GABC between muscles and gills. Moreover, there were no significant differences (P> 0.05) in GABC between fish species during storage. In total, 390 bacteria were isolated from the fresh and stored fish; 339 isolates (87%) were found to be Gram-positive. Two hundred and sixty-six isolates (78%) of Gram-positive bacteria were identified to fall into 13 genera, namely Staphylococcus, Micrococcus, Bacillus, Virgibacillus, Brevibacillus, Corynebacterium, Streptococcus, Enterococcus, Aerococcus, Exiguobacterium, Carnobacterium, Vagococcus and Sporosarcina and 30 species. In fresh fish, Staphylococcus epidermidis and Micrococcus luteus were the most frequent isolates. The effect of storage at 25C for 15 hours resulted in a change of Gram-positive bacterial populations; while S. epidermidis, S. xylosus and Bacillus megaterium were no longer present, S. warneri, B. sphaericus, Brevibacillus borstelensis, Enterococcus faecium and Streptococcus uberis increased. Three species, E. faecium, Str. uberis and B. sphaericus, were the most prevalent at the end of storage. Micrococcus luteus and S. warneri were the most prevalent isolates from Pseudocaranx dentex, but E. faecium and Str. uberis were the most frequently isolated from Pagrus auratus and Mugil cephalus. With respect to different parts of the fish body, E. faecium, Str. uberis and B. sphaericus were the most frequent isolates from the muscles, E. faecium, Str. uberis from the gills and M. luteus from the gut. Among the 228 isolates examined, Br. borstelensis 73, Br. borstelensis 291, Str. uberis 339, Vagococcus fluvialis 31 and Vag. fluvialis 132 produced VSC from sodium thiosulphate, cysteine and methionine. However, strains varied in sulphur source utilization. Exiguobacterium acetylicum 5, Exiguobacterium spp. 191, Carnobacterium spp. 338, Br. borstelensis 73, Br. borstelensis 291, Str. uberis 30, Str. uberis 339, Vag. fluvialis 31 and Vag. fluvialis 132 reduced TMAO. No histidine decarboxylase activity was found in the Gram-positive bacterial species tested. Lysine and ornithine were decarboxylated mainly by different strains of S. warneri, S. epidermidis and M. luteus. During ambient storage of fish, the frequency of lysine-decarboxylating bacteria increased and became more diverse after 5 hours of storage. Among fish species examined, the frequencies of lysine- and ornithine-decarboxylating bacteria were higher and more diverse in Pseudocaranx dentex than in Pagrus auratus and Mugil cephalus. This study found that Gram-positive bacteria were abundant and diverse in sub-tropical marine fish; however, their frequencies were affected by fish habitat and fish body part. Ambient temperature storage determined which Gram-positive bacterial species were dominant. With the exception of one isolate of S. aureus, Gram-positive bacteria isolated from sub-tropical marine fish caught from unpolluted water were not potential pathogens. The study also showed that Gram-positive bacteria had greater ability to decarboxylate lysine and ornithine than to produce VSC or reduce TMAO, and the spoilage potential of a bacterial species was a strain-dependent behaviour. This is a significant study as it is the first study on this aspect sub-tropical marine fish. It validated a selective medium that can be used to enumerate most Gram-positive bacteria from a marine environment. Most of the Gram-positive bacterial species from sub-tropical marine fish identified in this study were documented for the first time. The effects of ambient storage and the spoilage potential of Gram-positive bacteria from sub-tropical marine were clearly elucidated.