Staphylococcal slime layers and biofilm from different origins / Slime layer e biofilme de Staphylococcus de diferentes origens

The genus Staphylococcus comprises some of the most important pathogenic bacteria for both humans and animals. It is responsible for bovine mastitis and canine otitis, besides being present in the microbiota of animals and as a contaminant in food. Its pathogenesis is related to the formation of capsule and biofilm, which contribute to its infectivity. The objective of this study was to observe the production of slime layer and formation of biofilm, which are related to the resistance to antimicrobial agents and presence of icaA and icaD genes, in 41 isolates of Staphylococcus spp. from different origins, provided by the Universidade Federal de Pelotas (UFPEL), Laboratório Regional de Diagnóstico (LRD). Strains of Staphylococcus spp. were cultivated in Congo red agar for capsule detection. Biofilm formation was detected using the 96-well microplate testing. Antimicrobial susceptibility testing was performed using the plate diffusion method. Part of the analyzed samples produced slime layer (36.6%) and formed biofilm (17.1%). However, six of those that formed biofilms were susceptible to the eight antibiotics tested in the antibiogram. In tests to determine the minimum bactericidal and inhibitory concentrations, gentamicin resistance of biofilm-forming strains was greater than that of non-forming strains. Ampicillin was the least effective antimicrobial drug (51%), followed by tetracycline (71%), neomycin (73%), and erythromycin (73%). Some isolates presented the icaA (6) and icaD (11) genes. Therefore, we suggested that the origin of an isolate can determine its expression of virulence factor and resistance to certain antibiotics.(AU)

O gênero Staphylococcus abrange algumas das bactérias patogênicas mais importantes tanto para humanos como para animais. Ele é responsável pela mastite bovina e otite canina, além de estar presente na microbiota de animais e como contaminante em alimentos. Sua patogênese está relacionada à formação de cápsula e biofilme, que contribuem para sua infectividade. O objetivo deste estudo foi observar a produção de slime layer e a formação de biofilme, que estão relacionados à resistência a antibicrobianos e à presença dos genes icaA e icaD, em 41 isolados de Staphylococcus spp. de diferentes origens fornecidos pelo Laboratório Regional de Diagnóstico (LRD) da Universidade Federal de Pelotas (UFPEL). Os isolados de Staphylococcus spp. foram cultivados em ágar vermelho do Congo para detecção de cápsulas. A formação de biofilme foi detectada usando o teste de microplaca com 96 poços. O teste de susceptibilidade antimicrobiana foi realizado usando o método de difusão em placa. Parte das amostras analisadas produziram slime layer (36,6%) e formaram biofilme (17,1%). Entretanto, seis daquelas que formaram biofilmes foram sensíveis aos oito antibióticos testados no antibiograma. Em testes para determinar as concentrações bactericidas e inibitórias mínimas, a resistência à gentamicina de cepas formadoras de biofilme foi maior que aquela das cepas não formadoras. O antimicrobiano menos eficaz foi a ampicilina (51%), seguida por tetraciclina (71%), neomicina (73%) e eritromicina (73%). Alguns isolados apresentaram os genes icaA (6) e icaD (11). Portanto, sugerimos que a origem de um isolado pode determinar sua expressão de fator de virulência e resistência a certos antibióticos.(AU)

Detection of methicillin resistance and slime factor production of Staphylococcus aureus in bovine mastitis

This study aimed to detect methicillin resistant and slime producing Staphylococcus aureus in cases of bovine mastitis. A triplex PCR was optimized targetting 16S rRNA, nuc and mecA genes for detection of Staphylococcus species, S. aureus and methicillin resistance, respectively. Furthermore, for detection of slime producing strains, a PCR assay targetting icaA and icaD genes was performed. In this study, 59 strains were detected as S. aureus by both conventional tests and PCR, and 13 of them were found to be methicillin resistant and 4 (30.7%) were positive for mecA gene. Although 22 of 59 (37.2%) S. aureus isolates were slime-producing in Congo Red Agar, in PCR analysis only 15 were positive for both icaA and icaD genes. Sixteen and 38 out of 59 strains were positive for icaA and icaD gene, respectively. Only 2 of 59 strains were positive for both methicillin resistance and slime producing, phenotypically, suggesting lack of correlation between methicillin resistance and slime production in these isolates. In conclusion, the optimized triplex PCR in this study was useful for rapid and reliable detection of methicillin resistant S. aureus. Furthermore, only PCR targetting icaA and icaD may not sufficient to detect slime production and further studies targetting other ica genes should be conducted for accurate evaluation of slime production characters of S. aureus strains.

Este estudo objetivou a detecção de Staphylococcus aureus resistente a meticilina e produtor do fator slime em casos de mastite bovina. Um PCR triplex foi otimizado, com alvo no genes 16SrRNA, nuc e mecA para detecção de Staphylococcus spp, S. aureus e resistencia a meticilina, respectivamente. Para detecção das cepas produtoras do fator slime, empregou-se um PCR com alvo nos genes icaA e icaD. No estudo, 59 cepas foram identificadas como S. aureus por testes convencionais e PCR, sendo 13 resistentes a meticilina e quatro positivas para o gene mecA. Embora 22 das 59 cepas tenham sido produtoras do fator slime em Agar Vermelho Congo, no teste PCR somente 15 foram positivas para os genes icaA e icaD. Dezesseis e 38 das 59 cepas foram positivas para os genes icaA e icaD, respectivamente. Somente duas das 59 cepas foram positivas simultaneamente para resistência a meticilina e produção do fator slime, sugerindo falta de correlação entre estas características. Em conclusão, o PCR triplex otimizado neste trabalho mostrou-se ser um método rápido e confiável para detecção de S.aureus meticilina resistente. Por outro lado, somente PCR para os genes icaA e icaD pode não ser suficiente para detectar produção de fator slime e outros estudos com alvo em outros genes ica são necessários para um avaliação correta da produção do fator slime por S. aureus.

Adherence assays and Slime production of Vibrio alginolyticus and Vibrio parahaemolyticus

In this study we investigated the phenotypic slime production of Vibrio alginolyticus and Vibrio parahaemolyticus strains, food-borne pathogens, using a Congo red agar plate assay. Furthermore, we studied their ability to adhere to abiotic surfaces and Vero cells line. Our results showed that only V. alginolyticus ATCC 17749 was a slime-producer developing almost black colonies on Congo red agar plate. Adherence to glace tube showed that all V. alginolyticus strains were more adherent than V. parahaemolyticus. Only V. alginolyticus ATCC 17749 was found to be able to form biofilm on polystyrene microplate wells (OD570 = 0.532). Adherence to Vero cells showed that all tested strains were non adherent after 30 min, however after 60 min all the studied strains become adherent. The percentage of adherence ranged from1.23% to 4.66%.

Neste estudo, investigou-se a produção de muco por cepas de Vibrio alginolyticus e Vibrio parahaemolyticus através do teste em placa de ágar com vermelho congo. Estudou-se também a capacidade de adesão à superfícies abióticas e células Vero. Os resultados indicaram que somente V. alginolyticus ATCC 17749produziu muco, formando colônias quase negras nas placas de ágar com vermelho congo. O teste de adesão a tubos de vidro indicou que as cepas de V. alginolyticus foram mais aderentes do que as de V. parahaemolyticus. Somente V. alginolyticus ATCC 17749 foi capaz de formar biofilme nos poços das microplacas de poliestireno (OD570=0,532). Testes de adesão a células Vero mostraram que nenhuma das cepas apresentou adesão em 30 min, mas todas aderiram após 60 min. A porcentagem de adesão variou de 1,23% a 4,66%.

Bacterial biofilms with emphasis on coagulase-negative staphylococci

In addition to their capacity to attach to surfaces, various groups of microorganisms also produce an extracellular polymeric substance known as "slime". This slime forms a thin layer around cells known as biofilm. Thus, biofilm structure comprises bacterial cells and an extracellular polymeric substance. It also presents a defined architecture, providing the microorganisms with an excellent protective environment and favoring the exchange of genetic material between cells as well as intercellular communication. The ability to produce biofilm is observed in a large group of bacteria, including coagulase-negative staphylococci (CNS) which are the predominant microorganisms of normal skin flora and have been implicated as the causative agents of hospital infections. Bacteremia caused by these agents is common in immunodepressed persons, in patients with cancer, in adult and neonatal intensive care units (ICU) and in patients using catheters or other prosthetic devices. The pathogenicity of CNS infections is probably related to the production of slime, which adheres preferentially to plastic and smooth surfaces, forming a biofilm that protects against attacks from the immune system and against antibiotic treatment, a fact hindering the eradication of these infections. The main objective of the present review was to describe basic and genetic aspects of biofilm formation and methods for its detection, with emphasis on biofilm creation by CNS and its relationship with diseases caused by these microorganisms which are becoming increasingly more frequent in the hospital environment.

Bacterial biofilms with emphasis on coagulase-negative staphylococci

In addition to their capacity to attach to surfaces, various groups of microorganisms also produce an extracellular polymeric substance known as "slime". This slime forms a thin layer around cells known as biofilm. Thus, biofilm structure comprises bacterial cells and an extracellular polymeric substance. It also presents a defined architecture, providing the microorganisms with an excellent protective environment and favoring the exchange of genetic material between cells as well as intercellular communication. The ability to produce biofilm is observed in a large group of bacteria, including coagulase-negative staphylococci (CNS) which are the predominant microorganisms of normal skin flora and have been implicated as the causative agents of hospital infections. Bacteremia caused by these agents is common in immunodepressed persons, in patients with cancer, in adult and neonatal intensive care units (ICU) and in patients using catheters or other prosthetic devices. The pathogenicity of CNS infections is probably related to the production of slime, which adheres preferentially to plastic and smooth surfaces, forming a biofilm that protects against attacks from the immune system and against antibiotic treatment, a fact hindering the eradication of these infections. The main objective of the present review was to describe basic and genetic aspects of biofilm formation and methods for its detection, with emphasis on biofilm creation by CNS and its relationship with diseases caused by these microorganisms which are becoming increasingly more frequent in the hospital environment.

Species identification, slime production and oxacillin susceptibility in coagulase-negative staphylococci isolated from nosocomial specimens

Ninety-two coagulase negative staphylococci (CNS) (forty-five of clinical origin and forty-seven of environmental origin), collected in a hospital in San Luis, Argentina, from March to June, 1999, were identified to species level by the ID 32 Staph and API Staph System (bioMérieux). Slime production was investigated by the quantitative and qualitative methods. Oxacillin susceptibility was determined by the disk diffusion test (1 µg), the agar dilution method (0.125 to 4 mg/ml) and agar screen (6 µg/ml). The presence of mecA gene was investigated by PCR. The clinical CNS species most commonly isolated were S. epidermidis, S. haemolyticus, S. hominis and S. saprophyticus. The frequency of slime production by clinical and environmental isolates was similar (25/45 and 27/47, respectively) and the results obtained by the quantitative and the qualitative methods correlated well. The mecA gene was detected in all S. epidermidis, S. haemolyticus and S. hominis isolates, which were resistant to oxacillin by the phenotypic methods. However, this gene was not present in S. klossii, S. equorum, S. xylosus and S. capitis strains. The gene was neither found in two out of the six S. saprophyticus isolates, in two out of three S. cohnii subsp. urealyticum isolates and in two out of five S. cohnii subsp. cohnii isolates, all of which resulted oxacillin resistant according to MIC. The gene was not found in oxacillin-susceptible strains either. Most of the CNS isolates (enviromental and clinical) that were slime producers were found to be oxacillin resistant, which makes the early detection of these microorganisms necessary to prevent their dissemination in hospitals, particularly among immunocompromised patients.

Noventa e duas amostras de Staphylococcus coagulase negativo (SCN), (45 amostras clínicas e 47 ambientais), coletadas em um hospital de San Luis, Argentina, durante o período de março a junho de 1999, foram identificadas até espécies, empregando-se os sistemas ID 32 Staph e API Staph (bioMérieux). A produção de "slime" foi investigada através de métodos quantitativo e qualitativo. A susceptibilidade à oxacilina foi determinada por métodos de difusão em discos (1 µg), diluição em ágar (0,125 a 4 mg/ml) e ágar screen (6 µg/ml). A presença do gene mecA foi pesquisada por PCR. As espécies de SCN de origem clínica mais comumente isoladas foram S. epidermidis, S.saprophyticus, S.hominis e S.haemolyticus. Observou-se uma freqüência similar na produção de "slime" nas amostras de origem clínica e ambiental (25/45 e 27/47, respectivamente), tendo havido concordância nos resultados obtidos pelos métodos quantitativo e qualitativo. O gene mecA foi observado em todas as cepas de S. epidermidis, S. haemolyticus e S. hominis que foram resistentes a oxacilina pelos métodos fenotípicos, mas não foi detectado em S. klossii, S. equorum, S. xylosus e S. capitis. O gene também não foi detectado em duas das seis amostras de S.saprophyticus, duas das três S. conhii subsp. urealyticum e duas das cinco S. conhii subsp. conhii, as quais resultaram resistentes à oxacilina segundo a CIM. Esse gene não foi encontrado nas cepas sensíveis à oxacilina. A maioria das amostras de SCN (ambientais e clínicas) que foram produtoras de slime foram resistentes à oxacilina, o que obriga a uma detecção precisa desses microorganismos e ao controle de sua disseminação em hospitais, particularmente entre pacientes imunocomprometidos.