Please read the abstract in the section 00front of this document / Dissertation (MSc(Microbiology))--University of Pretoria, 2005. / Microbiology and Plant Pathology / unrestricted

Animal hides are the basic raw material of the leather industry and they undergo rapid putrefaction unless "cured". This study investigated the role and interactive effects of three selected bacteria, Pseudomonas aeruginosa. Clostridium histoly ticum and Clostridium sporogenes in in-situ cattle hide degradation using a model system set up for the purpose. The system consisted of 3cm diameter hide pieces contained in sealed jars and sterilised by ethylene oxide to remove resident microbes and inactivate autolytic tissue enzymes. The inocula were prepared either as individual cultures or as combinations of two inocula or all three inocula. Degradative changes during storage at 30°C were measured for up to 8 days using ten different parameters. Initial trials confirmed that the selected inocula were readily isolated from raw hides and could outcompete resident populations to produce putrefactive decomposition. Growth rates and enzyme profiles of the organisms and the effects of nutrients and reductants on their relative denaturative effects were used to standardise the system. Trials on the effects of ethylene oxide indicated the suitability of the method for hide and collagen sterilisation. The findings of in-situ trials with the selected inocula confirmed previous studies of protein putrefaction in that a bacterial succession was evident involving aerobic proteolytic bacteria, micro-aerophilic proteolytic bacteria and strictly anaerobic amino acid degrading bacteria. However, this study showed that the micro-aerophilic collagenase producing C. histolyticum degraded hides at a far greater rate when inoculated on its own than when in the presence of either or both of the other two inocula. It also demonstrated a bacterial antagonism between the two clostridia in which C. sporogenes prevented degradative changes occurring for up to 4-6 days possibly due to cysteine production by C. sporogenes. These findings have implications for hide preservation since maintenance of aerobic conditions and suppression of spore outgrowth could be used to delay growth of collagenase producing clostridia. The use of C. sporogenes as a biocontrol agent is also postulated. The model system was also used to examine salted hides during storage and these studies indicated that Halobacteriaceae do not produce collagenase but that inadequately salted hides could possibly be subject to degradation by delsulfovibrios.

Pseudomonas aeruginosa is one of the most studied biofilm-forming organisms and has emerged as a model organism in the study of surface- and biofilm-induced gene expression. The transition from a planktonic to a biofilm mode of growth results in diverse changes in gene expression, which causes the attaching cells to become phenotypically and metabolically distinct from their planktonic counterparts. In this study, a proteomic approach was used to study differences in protein profiles obtained from 18-h old P. aeruginosa PAO1 (DSM 1707) planktonic, surface influenced planktonic (SIP) and biofilm populations grown in batch in the absence or presence of a glass wool substratum. Glass wool as an attachment substratum not only supported growth of biofilms, but it also allowed for the separation of the biofilm biomass from the surrounding surface influenced planktonic (SIP) cells for further characterisation. Comparative analysis of the respective proteomes indicated striking differences in the protein patterns of planktonic, biofilm and SIP cells and several uniquely expressed proteins were seen on the 2-DE protein maps of the respective populations. Whereas a general down-regulation of protein expression was seen in the biofilm cells, in SIP cells, expression of the proteins was generally up-regulated. The results confirmed that the biofilm population differs from the planktonic population and indicated that the SIP population is not merely a mixture of planktonic and biofilm cells but rather a unique phenotype. Several differentially expressed protein spots were selected and identified using a combination of N-terminal protein sequencing and peptide mass fingerprinting. The proteins comprised mostly of outer membrane or membrane-associated proteins. Based on these analyses, a mutant P. aeruginosa strain, deficient in outer membrane protein OprG, was generated and its ability to form biofilms on a glass wool substratum was compared with that of the wild-type P. aeruginosa strain. The mutant strain was attachment-proficient but biofilm-deficient, suggesting that OprG plays a role in P. aeruginosa biofilm development under the culturing conditions used in this study. / Thesis (PhD (Microbiology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

Pseudomonas aeruginosa is a dreaded opportunistic pathogen that causes severe and often intractable infections in immunocompromised and critically ill patients. This bacterium is also the primary cause of fatal lung infections in patients with cystic fibrosis and a leading nosocomial pathogen responsible for nearly 10% of all hospital-acquired infections. P. aeruginosa is intrinsically recalcitrant to most classes of antibiotics and has the ability to acquire additional resistance during treatment. In particular, resistance to the widely used β-lactam antibiotics is frequently mediated by the expression of AmpC, a chromosomally encoded β-lactamase that is ubiquitously found in P. aeruginosa strains. This dissertation delved into the role of a recently reported chromosomal β-lactamase in P. aeruginosa called PoxB. To date, no detailed studies have addressed the regulation of poxB expression and its contribution to β-lactam resistance in P. aeruginosa. In an effort to better understand the role of this β-lactamase, poxB was deleted from the chromosome and expressed in trans from an IPTG-inducible promoter. The loss of poxB did not affect susceptibility. However, expression in trans in the absence of ampC rendered strains more resistant to the carbapenem β-lactams. The carbapenem-hydrolyzing phenotype was enhanced, reaching intermediate and resistant clinical breakpoints, in the absence of the carbapenem-specific outer membrane porin OprD. As observed for most class D β-lactamases, PoxB was only weakly inhibited by the currently available β-lactamase inhibitors. Moreover, poxB was shown to form an operon with the upstream located poxA, whose expression in trans decreased pox promoter (Ppox) activity suggesting autoregulation. The transcriptional regulator AmpR negatively controlled Ppox activity, however no direct interaction could be demonstrated. A mariner transposon library identified genes involved in the transport of polyamines as potential regulators of pox expression. Unexpectedly, polyamines themselves were able induce resistance to carbapenems. In summary, P. aeruginosa carries a chromosomal-encoded β-lactamase PoxB that can provide resistance against the clinically relevant carbapenems despite its narrow spectrum of hydrolysis and whose activity in vivo may be regulated by polyamines.

The pyrimidine biosynthetic pathway is an essential pathway for most organisms. Previous research on the pyrimidine pathway in Pseudomonas aeruginosa (PAO1) has shown that a block in the third step of the pathway resulted in both a requirement for exogenous pyrimidines and decreased ability to produce virulence factors. In this work an organism with a mutation in the second step of the pathway, aspartate transcarbamoylase (ATCase), was created. Assays for pyrimidine intermediates, and virulence factors were performed. Results showed that the production of pigments, haemolysin, and rhamnolipids were significantly decreased from PAO1. Elastase and casein protease production were also moderately decreased. In the Caenorhabditis elegans infection model the nematodes fed the ATCase mutant had increased mortality, as compared to nematodes fed wild type bacteria. These findings lend support to the hypothesis that changes in the pyrimidine biosynthetic pathway contribute to the organism's ability to effect pathogenicity.

It has been shown previously in our lab that mutations in the pyrimidine pathway reduced the ability of Pseudomonas aeruginosa to produce virulence factors. Knockout mutations in pyrB, pyrC and pyrD genes of the pyrimidine pathway showed that virulence factor production was decreased. Pyoverdin, pyocyanin, hemolysin, iron chelation, motility, and adherence are all considered virulence factors. Here I further investigate the effects of mutations in the pyrimidine pathway by studying a pyrE mutant. I studied the effect of the pyrE mutation on the production of the above virulence factors. Just like the effect of pyrB, pyrC and pyrD mutations,the pyrE mutation also showed that the bacteria were deficient in producing virulence factors when compared to the wild type. The broader impact of this research would be the possibility of finding drugs that could treat patients infected with P. aeruginosa and possibly extend the lives of chronically infected patients with cystic fibrosis.

The express purpose for doing this project was to develop methods for the continuous culture of E. coli and P. aeruginosa as a mixed population, and to apply these methods in studying the effects of pH and substrate upon the growth of these two organisms in mixed continuous culture.

Introdução. Pseudomonas aeruginosa é um patógeno clinicamente importante. Existem diversos mecanismos de resistência aos antimicrobianos em P. aeruginosa, dentre eles a produção de enzimas (β-lactamases) e sistemas de efluxo se destacam, uma vez que são capazes de conferir resistência aos carbapenêmicos. Objetivo. Avaliar os sistemas de efluxo MexAB-OprM e MexXY em isolados clínicos de P.aeruginosa de pacientes atendidos no Hospital de Clínicas de Porto Alegre-RS e relacionar a expressão destes sistemas com a CIM de meropenem em isolados produtores e não produtores de metalo-beta-lactamases (MBL). Metodologia. Um total de 86 isolados de P. aeruginosa com suscetibilidade reduzida aos carbapenêmicos foram avaliados. A hiperexpressão dos sistemas MexAB e MexXY foi determinada fenotipicamente utilizando inibidor seletivo da bomba (PAβN). MBLs foi determinada por PCR utilizando primers específicos. Resultados. O fenótipo de hiperexpressão dos sistemas de efluxo estudados foi observado em 34 (47,8%) dos 71 isolados negativos para a produção de MBL e em 14 (93.3%) dos 15 isolados MBL positivos. Na presença de PaβN, todos os isolados não produtores de MBL apresentaram uma redução da CIM para meropenem para valores na faixa de suscetibilidade. Entretanto, das 13 P. aeruginosa produtoras de MBL que diminuíram a CIM, essa redução não foi para valores dentro da faixa de sensibilidade. Conclusão. Os isolados de P. aeruginosa não produtores de MBL apresentam resistência ao meropenem devido a hiperexpressão de MexAB-OprM. Na presença de PaβN, independente de apresentarem produção de MBL, o CIM de meropenem reduziu para valores ≤ 8 mg/L. Contudo, quando isolados não apresentavam MBL, a CIM de meropenem reduziu para níveis de sensibilidade. / Introduction. Bacterial efflux pump systems are resistance mechanisms which may lead to therapeutic failure of antibiotic treatment since many antimicrobial agents are substrate for these mechanisms. The aim of the study was to evaluate the expression of the MexAB, MexXY pump efflux and to determine its influence on meropenem MIC in carbapenemase producing and non-producing P. aeruginosa. Methods. A total of 86 non-repetitive clinical isolates of P. aeruginosa with reduced susceptibility to carbapenems were evaluated. Overexperession of MexAB and MexXY efflux systems were evaluated phenotypically using the PAβN selective inhibitor. Metallo-β-lactamases (MBL) were detected by PCR using specific primers. Results. The efflux pump-overexpressed phenotype was observed in 34 (47.8%) MBL-negative and in 14 (93.3%) MBL-positive isolates. In the presence of the PaβN, all non-producers of MBL presented a reduction of meropenem MICs to the range of susceptibility. In contrast, the 13 P. aeruginosa MBL-producing isolates decreased meropenem MICs at least 16-fold but this reduction did not reach the range of susceptibility. Conclusion. P. aeruginosa non-MBL-producing showed resistance to meropenem due to overexpression of MexAB-OprM. In the presence of PaβN, the isolates harboring or not MBL genes, the meropenem MICs were reduced to values ≤8 mg/L. However, when the isolates harbor MBL genes, the meropenem MIC values were reduced to the susceptibility.

Submitted by Haroudo Xavier Filho (haroudo.xavierfo@ufpe.br) on 2016-04-14T18:46:13Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Tacilene Luzia Silva.pdf: 1235794 bytes, checksum: f6e18d8a1d865ebf8536ae8b8a1666f3 (MD5) / Made available in DSpace on 2016-04-14T18:46:13Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação Tacilene Luzia Silva.pdf: 1235794 bytes, checksum: f6e18d8a1d865ebf8536ae8b8a1666f3 (MD5) Previous issue date: 2015-02-13 / CNPq / Pseudomonas aeruginosa é uma bactéria Gram negativa, oportunista e ubíqua, frequentemente associada a infecções graves em pacientes imunocomprometidos. Em razão do aumento de resistência dessa bactéria aos múltiplos antimicrobianos, surgem à preocupação e a procura por novas alternativas terapêuticas, com as substâncias bioativas de origem natural representando uma importante fonte para obtenção desses medicamentos. O objetivo do presente estudo foi determinar a atividade sinérgica do timol e agentes antimicrobianos frente a cepas de Pseudomonas aeruginosa multirresistentes e avaliar os efeitos dessa interação sobre a biossíntese de biofilme e de piocianina. Para isso, numa primeira etapa foi determinada a concentração inibitória e bactericida mínima do timol e de antimicrobianos (Polimixina B, ceftazidima, piperacilina/tazobactam, cefepima, ciprofloxacino e meropenem) frente a dez cepas de Pseudomonas aeruginosa. O estudo da interação entre o timol e os agentes antimicrobianos foi realizado pelo método do tabuleiro de xadrez. Os critérios utilizados para avaliar a atividade sinérgica foram definidos pelo Índice da Concentração Inibitória Fracionada (FIC índex). A partir dos melhores valores do FIC índex das associações timol/antimicrobiano foram avaliadas a atividade sobre a produção de biofilme e piocianina. Três cepas (LFBM 01, LFBM 02, LFBM 16) apresentaram um perfil de resistência ao meropenem e cefepima e um efeito sinérgico foi observado entre o timol e meropenem ou cefepima sobre essas cepas. A associação timol/cefepima inibiu a biossíntese do biofilme em até 99,76%, e a associação timol/meropenem mostrou ser mais eficaz na inibição da piocianina cujos valores foram de até 84,33%. O timol associado ao meropenem ou cefepima, age sinergicamente, inibindo cepas de Pseudomonas aeruginosa multirresistentes e interferindo na biossíntese de biofilme e piocianina. / Pseudomonas aeruginosa is a Gram negative bacteria, opportunistic and ubiquitous, often associated with severe infections in immunocompromised patients. Due to the increased resistance of the bacteria to multiple antibiotics, there are the concerns and the search for new therapeutic alternatives, with the bioactive substances of natural origin represents an important source for obtaining these drugs. The aim of this study was to determine the synergistic activity of thymol and antimicrobials agents multiresistant Pseudomonas aeruginosa strains and evaluate the effects of this interaction on the biofilm biosynthesis and pyocyanin. For this, a first step was determined and the minimum inhibitory concentration of thymol and bactericidal antibiotics (polymyxin B, ceftazidime, piperacillin / tazobactam, cefepime, ciprofloxacin and meropenem) compared to ten strains of Pseudomonas aeruginosa. The study of the interaction between the thymol and antimicrobial agents was carried out by the checkerboard method. The criteria used to evaluate the synergistic activity were defined by the Index of Fractional Inhibitory Concentration (FIC index). From the best FIC index values of associations thymol / antimicrobial activity were evaluated on the production of biofilm and pyocyanin. Three strains (LFBM 01, LFBM 02, LFBM 16) showed an meropenem resistance profile and cefepime and a synergistic effect was observed between the thymol and meropenem or cefepime on these strains. The thymol / cefepime combination inhibited biofilm biosynthesis up to 99.76%, and thymol association / meropenem was more effective in inhibiting pyocyanin with values of up to 84.33%. The thymol associated with meropenem or cefepime, acts synergistically by inhibiting multidrug-resistant Pseudomonas aeruginosa strains and interfering in the biosynthesis of biofilm and pyocyanin.

The opportunistic pathogen Pseudomonas aeruginosa utilizes a variety of virulence factors to infect a wide range of hosts. Virulence genes in this organism are many times controlled by two-component regulatory systems consisting of a sensor histidine kinase and a response regulator giving them high importance in research. Orphan response regulators consist of genes that have been proposed to be a response regulator but have not been studied to determine if they do work in a two-component regulatory system or not. Investigating these orphan response regulators could potentially lead to the finding of another regulator of virulence genes. Non-polar deletions were designed using splicing of genomic segments by overlapping extension. A variety of phenotypic assays, liquid-killing assays with the nematode C. elegans, and virulence assays with macrophages were utilized to determine if these orphans were different from the wild-type strain PAO1. If attenuated, these genes can be further studied to find new and novel regulators of virulence in Pseudomonas aeruginosa.