RESUMO
Reducing salt content in foods such as cheeses, while limiting the growth of spoilage microorganisms and foodborne pathogens, is a difficult challenge. One method that may prove useful is use of staphylococcins, which are bacteriocins produced by staphylococci. Therefore, staphylococcin antimicrobial activity against six strains of S. aureus isolated from cheese was tested aiming at their industrial application in biopreservation of Minas fresh (Frescal) cheese with reduced sodium content. Three staphylococcins were selected for these tests: Pep 5, aureocin A53 and lysostaphin. All three staphylococcins proved to be bacteriolytic against all six strains of S. aureus. The antimicrobial activity of the partially purified staphylococcins was subsequently investigated against strains S. aureus Q1 and QJ3 in cheese matrices (6.0 log CFU/g) with different NaCl contents (control, a 25% reduction, and a 50% reduction), kept under refrigeration at 4⯰C, for 21â¯days. Both strains were shown to be of concern for food industry as they carry the SEA, SEB and SEH enterotoxin genes, and are resistant to ß-lactam drugs and moderate biofilm formers when grown in TSB. When used singly, Pep5, aureocin A53 and lysostaphin reduced approximately 95%, 99% and 99.99% of the viable cell counts, respectively, irrespective of the sodium content of the cheese matrix. The combined action of aureocin A53 and Pep5 resulted in an additional and significant reduction (pâ¯<â¯0.05) of ~1.0 log CFU/g when compared with the reduction caused by the use of either one singly. The combined action of lysostaphin and aureocin A53 or lysostaphin and Pep5 resulted in a reduction similar to or slightly smaller (pâ¯>â¯0.05) than that observed when lysostaphin was employed singly. Lysostaphin also proved to reduce the number of the staphylococcal viable cells to a level (~ 2.0 log CFU/g) at which enterotoxin production should not reach a sufficient quantity to cause food poisoning. Therefore, lysostaphin may have a practical application in the food industry to control staphylococcal contamination of Minas fresh cheese with a sodium content reduced up to 50%, providing consumers with more safe options to reduce their intake of sodium.
Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Queijo/microbiologia , Doenças Transmitidas por Alimentos/prevenção & controle , Lisostafina/farmacologia , Peptídeos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos , Enterotoxinas/análise , Doenças Transmitidas por Alimentos/microbiologia , Cloreto de Sódio/análise , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/metabolismoRESUMO
AIMS: The biofilm produced by Staphylococcus aureus isolates involved in clinical or subclinical bovine mastitis and the activity of nisin and lysostaphin against the preformed biofilm produced by these strains were investigated. METHODS AND RESULTS: Eighteen strains were tested and all produced biofilm. Eight strains with distinct biofilm composition were selected for the antimicrobial activity assays. The minimal inhibitory concentration of each bacteriocin was determined against the planktonic cells and ranged from 15·6 to 500 µg ml(-1) for nisin, and from 3·9 to 50 µg ml(-1) , for lysostaphin. Lysostaphin treatment (0·4 µg ml(-1) ) for 4 h caused a strong Staph. aureus 4181 biofilm detachment and death of the majority of the sessile cells, while nisin treatment (100 µg ml(-1) ) for the same time caused only a great reduction in cell viability. Additionally, combination of both bacteriocins for 4 h resulted in significant death of the sessile cells but no biofilm detachment. CONCLUSIONS: The treatment with lysostaphin alone or in combination with nisin was effective in killing most biofilm sessile cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The action of lysostaphin, either alone or in combination with nisin, against established staphylococcal biofilm may represent an alternative to bovine mastitis control. However, the duration of the treatment should be considered for its application so that the best effectiveness can be achieved.
Assuntos
Biofilmes/efeitos dos fármacos , Lisostafina/farmacologia , Mastite Bovina/tratamento farmacológico , Nisina/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Lisostafina/uso terapêutico , Testes de Sensibilidade Microbiana/métodos , Nisina/uso terapêutico , Plâncton/efeitos dos fármacos , Staphylococcus aureus/fisiologiaRESUMO
The combination of antimicrobial agents has been proposed as a therapeutic strategy to control bacterial diseases and to reduce the emergence of antibiotic-resistant strains in clinical environments. In this study, the interaction between the lantibiotic bovicin HC5 with chloramphenicol, gentamicin, nisin, lysostaphin and hydrogen peroxide against Staphylococcus aureus O46 was evaluated by MIC assays. The central composite rotatable design (CCRD), a robust and economic statistical design, was used to combine concentration levels of different antimicrobials agents with distinct mechanisms of action and the presence of significant interactions among the antimicrobials was determined by regression analysis. According to the adjusted model, there were no significant interactions between bovicin HC5 and gentamicin, lysostaphin, nisin or hydrogen peroxide. However, bovicin HC5 showed a significant interaction (P < 0.02) with chloramphenicol. This is the first study applying the CCRD approach to evaluate the combined effect of antimicrobials against S. aureus. Based on our results, this approach is an effective strategy to determine synergistic interactions between antimicrobial agents applied in human and veterinary medicine against bacterial pathogens.
Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Cloranfenicol/farmacologia , Sinergismo Farmacológico , Quimioterapia Combinada , Gentamicinas/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Lisostafina/farmacologia , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Nisina/farmacologia , Análise de RegressãoRESUMO
The evaluation of phagocytic and microbicidal activities of the blood neutrophils has been recognized as one of the important tools for investigating phagocytic dysfunctions in patients with recurrent infections. In the present study, these activities were examined in neutrophils and monocytes from healthy adults and patients affected by primary phagocytic dysfunctions by using a modified fluorochromic microbicidal assay, discriminating simultaneously the extracellular adherence, ingestion and intracellular killing of Staphylococcus aureus Cowan I. The assay employs acridine orange staining, as described in Bellinati-Pires et al. (1989) (AO assay), but was modified by the addition of an alternative leukocyte treatment with 0.5 U/ml of lysostaphin (LS) for 5 min at 37 degrees C, after phagocytosis (AO-LS assay). The LS treatment was standardized to eliminate staphylococci adhered to the outer surface of the phagocytes without affecting the determination of intracellular live or dead bacteria, as demonstrated in normal neutrophils and monocytes. Our purpose in this study was to compare AO and AO-LS assays in order to evaluate the effect of LS on the determination of actually ingested staphylococci and to provide a means for improving the fluorochromic assay for detecting phagocytic defects, as well as bactericidal disturbances. By using the AO-LS assay, decreased ingestion of staphylococci by neutrophils in Chediak-Higashi Syndrome (CHS) was demonstrated. However, increased staphylococci adherence, as well as ingestion, was observed in neutrophils or monocytes from chronic granulomatous disease (CGD) patients, comparing AO and AO-LS assays. Bactericidal defect, which is a common feature in CHS and CGD, was detected in neutrophils or monocytes in both assays. We emphasize that such alterations were deduced by comparing the patients' results with those obtained from their respective normal controls and with the normal range of values previously established for 160 healthy adults. No alteration was observed in hyper IgE syndrome phagocytes. Despite the possible penetration of LS into the leukocytes, as stated in other studies, we concluded that a short period of phagocyte incubation with this enzyme increased the sensitivity of the fluorochromic assay to detect phagocytic defect without affecting the determination of the bactericidal activity. Moreover, comparations between AO and AO-LS assays may be important in the study of the initial pathways of staphylococci phagocyte interaction, including adherence by non-phagocytic receptors.
Assuntos
Atividade Bactericida do Sangue , Lisostafina , Monócitos/imunologia , Neutrófilos/imunologia , Disfunção de Fagócito Bactericida/sangue , Disfunção de Fagócito Bactericida/diagnóstico , Disfunção de Fagócito Bactericida/etiologia , Adulto , Criança , Pré-Escolar , Estudos de Avaliação como Assunto , Feminino , Corantes Fluorescentes , Humanos , Imunoglobulina E/sangue , Masculino , Monócitos/microbiologia , Neutrófilos/microbiologia , Staphylococcus aureus/imunologiaRESUMO
Eleven methods for capsule detection of Staphylococcus aureus were compared. The most suitable of them were transmission electron microscopy, determination of the presence of clumping factor, determination of colonial morphology in serum-soft agar, estimation of cell volume and staining with safranine. The determination of clumping factor is a fast and effective method for presumptive diagnosis of capsulated strains, but need to be confirmed by another method. The cell volume estimation is useful for determination of capsule production in liquid cultures, while staining with safranine is suitable for genetic studies of capsule production. The other methods analyzed in this work (Indian ink staining, use of anticapsular antisera, determination of virulence for mice, lisostaphin susceptibility, resistance to phages and resistance to phagocytosis) were laborious, too slow, or need components and/or equipment not available in all laboratories. In addition, two methods of induction of capsule production were assayed, one in vitro by several passages in broth with 10% bovine serum and the other in vitro by intraperitoneal inoculation in mice. Both methods induced capsule production.
Assuntos
Cápsulas Bacterianas/análise , Técnicas Bacteriológicas , Carbono , Coloração e Rotulagem/métodos , Staphylococcus aureus/química , Testes de Aglutinação , Animais , Anticorpos Antibacterianos/imunologia , Cápsulas Bacterianas/imunologia , Corantes , Imunodifusão , Lisostafina/farmacologia , Camundongos , Microscopia Eletrônica , Fagocitose , Fenazinas , Coelhos , Fagos de Staphylococcus , Staphylococcus aureus/ultraestruturaRESUMO
Se han aislado 60 cepas del género Staphylococcus en el hospital Guillermo Almenara Irigoyen de secreciones de cátetes y urocultivos, los cuales fueron clasificdas siguiendo los lineamientos del esquema de KLOOS y Schcleides y el manual de Bergey identificandose a las siguientes especies: S.aureus (68.3 por ciento). s. saprolyticus (13.3 por ciento). s. epidermis (83 por ciento). S. Haemolyticus (5.0 por ciento), S.simulans (5.0 por ciento). En base al esquema de KLOOS y COL y combinando algunos caracteres fenotipicos esenciales con caracteres con caracteres secundarios se desarrolló un esquema simplificado de 4 pruebas bioquímicos: Determinación de la enzima coagulasa, sensibilidad o resistencia a la Lisostofina, prueba de termonucleasa y sensibilidad o resistencia a 5 ug de novabiocina, habiéndose encontrado 2 biovaves en S. aureus y S. epidermis, con resultados variables en la prueba de termonucleasa y coagulasa. s. saprophyticus, s. simulans, s. haemolyticus dieron resultados coincidentes con el esquema de KLOOS.