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O leite é um alimento muito importante na alimentação humana, mas, quando contaminado com resíduos de antibióticos, pode ser considerado adulterado e constituir risco à saúde pública. Desta forma, é importante que este alimento apresente condições sanitárias adequadas e risco à saúde minimizado ou inexistente. O presente estudo teve como objetivos identificar os resíduos de antibióticos comumente encontrados em leite cru produzido em propriedades rurais no Rio Grande do Sul (RS), avaliar a presença de resíduos de antibióticos nas amostras analisadas quanto ao limite máximo de resíduos (LMR) descrito no Programa Nacional de Controle de Resíduos e Contaminantes (PNCRC), averiguar o conhecimento dos responsáveis pela ordenha do leite quanto aos medicamentos utilizados em seu rebanho, bem como, identificar os resíduos de antibióticos comumente avaliados, na rotina, e a motivação para a escolha destes, em plataformas de recebimento de leite cru em estabelecimentos industriais, inspecionados pelo serviço oficial do RS. Para tanto, foram realizados dois estudos exploratório-descritivos. No primeiro, em 2013, foi realizada coleta de dados a campo, mediante aplicação de questionário referente à administração de medicamentos veterinários, bem como, da análise, no Laboratório Nacional Agropecuário, de 388 amostras de leite cru, oriundas de propriedades rurais distribuídas no Estado. No segundo estudo, que ocorreu de 2014 a 2015, foi aplicado questionário referente aos testes de detecção de resíduos de antibióticos em 36 estabelecimentos industriais, sob inspeção estadual. Os resultados do primeiro estudo demonstraram que 96 (24,7%) amostras apresentaram algum resíduo de antibiótico dos 45 analitos pesquisados. Destas, 91 (23,5%) estavam conformes, segundo a legislação vigente e, apenas, 5 (1,3%) foram suspeitas de violação do LMR descrito no PNCRC. Dos resíduos de antibióticos identificados, os da classe dos macrolídeos (n=51; 13,1%), seguidos das quinolonas (n=49; 12,6%) e das tetraciclinas (n=18; 4,6%) foram os comumente encontrados em leite cru produzido no RS. Quanto ao conhecimento dos responsáveis pela ordenha, infere-se que 22,6% não registraram ou controlaram a administração de medicamentos ao rebanho leiteiro, embora, 87,4% (n=339) tenha assegurado descartar o leite durante o período residual. Ainda, apenas 3,4% (n=13) dos entrevistados consideraram a bula do medicamento ou a orientação de veterinário (n=12; 3,1%), para o descarte do leite, guiando-se, principalmente, pela indicação da cooperativa a qual estiverem associados (55,9%; n=217). No segundo estudo, dos 36 estabelecimentos participantes, 83% foram classificados como fábrica de laticínios, sendo os antibióticos dos grupos dos betalactâmicos (100%) e das tetraciclinas (69%) os comumente pesquisados. Quanto à escolha do teste para a pesquisa de resíduos de antibióticos, esta foi influenciada pela praticidade e rapidez na execução do mesmo (67%) e não no conhecimento, especificamente, dos antibióticos utilizados pelos fornecedores de leite à indústria (22%). Conclui-se que o leite consumido no RS, quanto à presença de resíduos de antibióticos, no período da coleta, apresentou resultados compatíveis com aqueles identificados no PNCRC, oferecendo baixo risco à saúde pública. Mesmo assim, esforços devem ser direcionados quanto à melhoria das boas práticas agropecuárias nas propriedades rurais e às empresas quanto aos critérios de escolha dos testes de detecção de resíduos de antibióticos. / Milk is an important food for human consumption, but when contaminated with antibiotic residues may be considered adulterated and risk to public health. Thus, it is important that this food presents sanitary conditions and minimized or zero risk to the public health. This study aimed to identify the commonly antibiotic residues found in raw milk produced in rural properties in Rio Grande do Sul (RS), to evaluate the conformity of samples regarding the maximum residue limit (MRL) established by the National Plan for Control of Residues and Contaminants in Animal in milk (PNCRC), to verify the knowledge level of people responsible for the milking process, in regard to the medications used in the herd and the withdrawal period, as well to identify the antibiotic residues commonly screened during the dairy processing routine, and the reasons for selecting them, at raw milk receiving points in the dairy plant, inspected by official services in the RS. Therefore, it held two exploratory and descriptive studies. In the first one, the milk samples collection occurred in 2013. At the moment of collection, a questionnaire was employed in order to evaluate the use of veterinary medicines by people responsible for milking. The 388 samples of raw milk, from rural properties distributed along the state were analyzed at the National Agricultural Laboratory. The second one, was from 2014 to 2015, which was administered questionnaire regarding the testing of antibiotic residues in 36 industrial establishments under state inspection. In the first study, 388 samples were analyzed in which 96 (24.7%) contained some antibiotic residue from the 45 analytes searched; 91 of these samples (23.5%), were in conformance with the current legislation. Only a total of five samples (1.3%) were suspected of violating the MRL described in the PNCRC. The antibiotic residues commonly found in the raw milk produced in RS were the macrolides (n=51; 13.1%), followed by quinolones (n=49; 12.6%) and the tetracyclines (n=18; 4.6%). The analysis of degree of knowledge of those responsible for milking demonstrates that 22.6% do not record or control the drug administration to the dairy herd. However, 87.4% (n=339) of the producers ensured to perform the discharge of the milk during the residual period, although, only 3.4% (n=13) of them consider the instructions present in the medicine bottle or the veterinary instructions (n=12; 3.1%) for this procedure, being guided by the cooperative in which they are associated (n=217; 55.9%). In the second study, the results showed that from the 36 participating facilities, 83% were classified as dairy plants, in which it was observed that the most commonly screened antibiotics were those belonging to the beta lactam group (100%) and tetracyclines (69%), whereas the selection of which antibiotic residues to screen, at the milk receiving point of the plant, was influenced by the practicality and quickness in performing the screening (67%) rather than by specific knowledge on which antibiotics were used by milk suppliers (22%). It was concluded that the milk consumed in RS was in accordance with the previously results obtained by the PNCRC in terms of the presence of antibiotic residues during the collection period, offering negligible health risk. However, efforts should be directed to farmers on good agricultural practices and to industries in regard to criteria for choice of tests for antibiotic residue detection.

Staphylococci are opportunistic pathogens responsible for a range of infections. Many staphylococcal species are frequently found to be resistant to antibiotics. The environment is considered a potential reservoir of genes conferring antibiotic resistance, which known as the 'resistomes'. Monitoring the dissemination of antibiotic resistant staphylococci is instrumental to mitigating this global health risk. The overall aim of this study was to generate informative data regarding dissemination of antibiotic resistance in environmental and public settings. This included looking into the distribution, epidemiology characteristic and transfer of oxacillin resistant determinant mecA; gaining an insight into genomic features that contribute to multiple antibiotic resistance and pathogenicity of one S. epidermidis isolate; and understanding the stress responses in mediating oxacillin resistance in S. aureus. The use of MALDI-TOF MS allowed identification of staphylococci to species level. MALDI-TOF MS data were used for taxonomic analysis of staphylococci, and taxonomic data were then combined with isolation sites and antimicrobial susceptibility profiles to aid the understanding of dissemination of environmental resistant staphylococci. The widespread dissemination of antibiotic resistant staphylococci in the environment was demonstrated. 12% of staphylococci harboured mecA gene. Community associated SCCmec types IV and V were more prevalent than nosocomial associated SCCmec types I, II, and III in the environment. 52% of SCCmec were non-typable. In addition, 14 new environmental S. epidermidis MLST types were reported. 9 antibiotic resistant determinants that were responsible for the resistant to 7 antimicrobial classes have been identified in environmental S. epidermidis 118 (G6_2). Proteomic analysis revealed that stress responses, including SOS response, stringent response and heat shock response, mediate oxacillin resistance in S. aureus. These results demonstrate widespread multiple drug resistance in different staphylococcal species isolated from non-healthcare environments. This uncontrolled dissemination of multidrug resistant bacteria poses a potential public health threats.

Gentamicin C complex is a mixture of five structurally similar aminoglycoside antibiotics, gentamicins C1, C1a, C2, C2a, and C2b, produced by the actinomycete bacterium Micromonospora echinospora. It is established in clinical use and despite significant toxicity remains valuable to treat severe Gram-negative bacterial infections. There is a pressing need to develop novel versions of such antibiotics to combat the rise of resistance among pathogens. Engineering of the pathway requires a detailed knowledge of the genes, enzymes, and intermediates involved. The final steps of gentamicin biosynthesis begin at gentamicin X2, the last common intermediate of the C complex. 6'-C-Methylation generates two branches, with analogous reactions happening in both. Candidate genes and enzymes for the steps from the first 6'-C-methylated intermediate, G418, to an aminated metabolite JI-20B have already been described, but none for the subsequent loss of two hydroxyl groups from Ring II, or the N-methylation that then occurs. A novel separation method using dynamic countercurrent chromatography was successfully applied to the difficult purification of gentamicin metabolites. The results described here allowed a detailed mechanism to be proposed for almost the entire pathway from G418 to the C complex, and by analogy for the unbranched pathway, too. The last step of the pathway is 6'-N-methylation of gentamicins C1a and C2. Genome mining and cell-free assays were used by the group of Professor Yuhui Sun (Wuhan University) to identify genL, a methyltransferase gene encoded elsewhere on the M. echinospora genome and capable of this catalysis. Here, in vitro reactions with recombinant GenL confirmed its function, and its kinetic parameters were measured with its substrates. The full mechanistic pathway for the late stages of gentamicin C complex biosynthesis has therefore now been elucidated.

The aim of this project was to study the in vitro release of cephalothin, vancomycin and strontium from hydroxyapatite particles and its PMMA composite. The hydroxyapatite spheres containing strontium were prepared in the laboratory. The in vitro release study for the hydroxyapatite was carried out in phosphate buffer saline solution (PBS) with differing pH value at 37 °C for five days and the PMMA composites for 21 days. All of the releases showed a burst release within the first 24 hours followed by a slow release. The pH value of the release medium had influence on the release rate to some extent for the antibiotic release and the acidic solution had a more significant impact on the strontium release. All of the composite groups had a much lower strontium release rate than the strontium release from the hydroxyapatite spheres.

Page 1 of 11 (page number not for citation purposes) BMC Microbiology Research article Open Access Assessing the suitability of antibiotic resistance markers and the indirect ELISA technique for studying the competitive ability of selected Cyclopia Vent. rhizobia under glasshouse and field conditions in South Africa Amy C Spriggs1 and Felix D Dakora*2 Address: 1Botany Department, University of Cape Town, Private Bag, Rondebosch 7701, Cape Town, South Africa and 2Chemistry Department, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africa Email: Amy C Spriggs - amy.spriggs@sanbi.org; Felix D Dakora* - dakorafd@tut.ac.za * Corresponding author Abstract Background: Symbiotic N2 fixation in legumes is constrained by many factors, including the paucity of suitable soil rhizobia To maximise growth of legume species therefore often requires the application of effective rhizobia as inoculants. But where native strains out-compete introduced rhizobia for nodule formation, it is important that the competitiveness of selected strains is tested in the field and glasshouse prior to their recommendation as commercial inoculants. However the methodology for strain identification inside nodules has often proved difficult and thus limited this field of research. In this study, the suitability of the antibiotic resistance technique (both intrinsic low-resistance fingerprinting and high-resistance marking) and the serological indirect ELISA method were assessed for their ability to detect selected Cyclopia rhizobia under glasshouse and field conditions. The four rhizobial strains that were used, namely PPRICI3, UCT40a, UCT44b and UCT61a, were isolated from wild Cyclopia species growing in the Western Cape fynbos of South Africa. Results: The test strains formed two distinct groups with regard to their intrinsic resistance to the antibiotics streptomycin sulphate and spectinomycin dihydrochloride pentahydrate, making it impossible to use intrinsic antibiotic resistance to distinguish strains from within the same intrinsic resistance group. The use of strains marked with double antibiotic resistance was also investigated. A number of these strains lost their antibiotic marker tags after one plant passage; and some also lost their competitive ability. The indirect ELISA technique provided a more satisfactory method of identifying selected Cyclopia strains under both field and glasshouse conditions. The primary antibodies raised against strains UCT40a, UCT61a and UCT44b gave absorbance readings that were unambiguously negative (0.30 OD405), while those of strain PPRICI3 were ambiguous (0.50 OD405) with many false positive readings (1.0 A405). The indirect ELISA method showed a high level of analytical sensitivity in glasshouse experiments and there were no cross-reactions between the four test strains. The method was also suitable for detecting three of the four test strains in competition studies under field conditions, and can also be used to identify some strains under field conditions. Conclusion: The antibiotic marker method was found unsuitable for identifying Cyclopia rhizobia in competition experiments in both glasshouse and field conditions. However, the indirect ELISA technique was found suitable for identifying these strains in glasshouse studies. The method was also appropriate for identifying strains UCT40a, UCT44b and UCT61a, but not strain PPRICI3, in field competition studies.

The MexCD-OprJ multidrug efflux pump of Pseudomonas aeruginosa confers resistance to a range of antimicrobials. Although not expressed under normal laboratory conditions, exposure to the membrane-active biocides, chlorhexidine or benzalkonium chloride, results in mexCD-oprJ expression. This suggests that membrane disruption provides the inducing signal. Consistent with this, increased mexCD-oprJ expression was demonstrated in the presence of additional membrane-damaging agents including polymyxin B, ethanol, SDS, EDTA, the organic solvents n-hexane and p-xylene, and the antimicrobial peptides melittin, V8 and V681. MexCD-OprJ expression was initially verified through increased resistance to known MexCD-OprJ antimicrobial substrates and subsequently using a mexC-lacZ transcriptional fusion and RT-PCR. Since the P. aeruginosa sigma factor AlgU is responsive to envelope stress, it was of interest to ascertain whether AlgU is capable of mediating this increased mexCD-oprJ expression. Thus, the impact of AlgU loss on mexCD-oprJ expression in response to membrane-damaging agents was assessed in a algU strain. In contrast with above, little or no mexCD-oprJ expression (assessed using resistance to MexCD-OprJ antimicrobial substrates, the mexC-lacZ transcriptional fusion and RT-PCR) occurred in response to membrane-damaging agents in the algU strain, consistent with AlgU playing a role in the envelope stress inducibility of mexCD-oprJ. Overall, envelope stress, and the ability to react to this stress through AlgU, appears to play an important role in mexCD-oprJ induction. This suggests an important role for MexCD-OprJ in alleviating envelope stress, independent of its ability to export and provide resistance to antimicrobials. A gene, PA4596, whose product shows substantial homology to the NfxB repressor of mexCD-oprJ expression, occurs downstream of mexCD-oprJ and shows AlgU-dependence and chlorhexidine inducibility, suggesting a role in the chlorhexidine-induced, AlgU-mediated expression of mexCD-oprJ. Thus, the impact of PA4596 loss on mexCD-oprJ expression was assessed. Paradoxically, the loss of PA4596 increases mexCD-oprJ expression in wild-type cells in response to chlorhexidine treatment (as assessed through RT-PCR), while its loss compromises mexCD-oprJ expression in an nfxB mutant. Nonetheless, this suggests that PA4596 is involved in the induction of mexCD-oprJ and that its ability to induce mexCD-oprJ differs depending on the state of nfxB. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-07-31 12:03:52.535

No description available.

Thesis (Ph.D. (Pharmacy Practice))--North-West University, Potchefstroom Campus, 2010.