Molecular detection of enterotoxins E, G, H and I in Staphylococcus aureus and coagulase-negative staphylococci isolated from clinical samples of newborns in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ocorrência de mastite bovina causada por Staphylococcus sp., Streptococcus sp. E Candida sp. em uma propriedade rural no município de Indianópolis – Minas Gerais

The objective of this study was to evaluate the occurrence of bovine mastitis by Staphylococcus sp., Streptococcus sp. and Candida sp. in a rural area of Indianopolis, Minas Gerais. It was realized the California Mastitis Test (CMT) in six collect, a total 671 of milk sample positive. Then the microbiological examination was performed, where the results revealed the presence of 137 milk samples with microbial multiplication. These, showed the presence of Staphylococcus aureus (45.2% of strains), other coagulase negative Staphylococcus (10.2%), Staphylococcus epidermidis (9.4%), Staphylococcus simulans (5.8%), other coagulase negative (15.3%), Streptococcus agalactiae (7.2%), other Streptococcus sp. (5.1%) and yeasts (1.4%). It was found that 100% of Staphylococcus were susceptible to rifampicin, gentamicin and ciprofloxacin; but, resistant to penicillin, tetracycline, and oxacillin. Regarding antimicrobial susceptibility Streptococcus, were employed, except to clindamycin, erythromycin and tetracycline. We conclude that there is a great necessity of proper hygiene practices and taking prophylactic measures taken in order to reduce the infection of animals caused by infectious microorganisms and resistance.

Detection of enterotoxin and toxic shock syndrome toxin 1 genes in Staphylococcus, with emphasis on coagulase-negative staphylococci

The detection of staphylococcal enterotoxins is decisive for the confirmation of an outbreak and for the determination of the enterotoxigenicity of strains. Since the recognition of their antigenicity, a large number of serological methods for the detection of enterotoxins in food and culture media have been proposed. Since immunological methods require detectable amounts of toxin, molecular biology techniques represent important tools in the microbiology laboratory. In the present study, polymerase chain reaction (PCR) was used to identify genes responsible for the production of enterotoxins and toxic shock syndrome toxin 1 (TSST-1) in S. aureus and coagulase-negative staphylococci (CNS) isolated from patients and the results were compared with those obtained by the reverse passive latex agglutination (RPLA) assay. PCR detection of toxin genes revealed a higher percentage of toxigenic S. aureus strains (46.7%) than the RPLA method (38.3%). Analysis of the toxigenic profile of CNS strains showed that 26.7% of the isolates produced some type of toxin, and one or more toxin-specific genes were detected in 40% of the isolates. These results suggests the need for further studies in order to better characterize the pathogenic potential of CNS and indicate that attention should be paid to the toxigenic capacity of this group of microorganisms.

Detecção do gene mecA em estafilococos coagulase negativa resistentes à oxacilina isolados da saliva de profissionais da enfermagem

Estafilococos coagulase negativa estão frequentemente associados às infecções nosocomiais e os profissionais da saúde podem ser reservatório e dissemina-los no hospital e comunidade. O objetivo desse estudo foi identificar espécies de estafilococos coagulase negativa isolados da saliva de profissionais da enfermagem, determinar o perfil de resistência e detectar o gene mecA. Foram selecionados 100 estafilococos coagulase negativa, sendo 41 identificados como Staphylococcus epidermidis, 25 Staphylococcus saprophyticus, 18 Staphylococcus haemolyticus, 8 Staphylococcus cohnii, 4 Staphylococcus lugdunenses, 3 Staphylococcus capitis, e 1 Staphylococcus Simulans. Desses, 32% apresentaram resistência à oxacilina, 84,4% à mupirocina, 32% à cefoxitina, e todos sensíveis a vancomicina. Dos estafilococos coagulase negativa resistentes à oxacilina, 93,7% desenvolveram-se no agar oxacilina (6µg/ml) e o gene mecA foi detectado em 75%. Os resultados sinalizam que maiores investimentos devem ser direcionados a identificação das espécies de estafilococos coagulase negativa nas instituições de saúde e na comunidade.

Avaliação do potencial de formigas (Hymenoptera: formicidae) como vetores mecânicos de bactérias do gêneroSstaphylococcus no ambiente hospitalar.

In a hospital environment, these bacteria can be spread by insects such as ants, which are characterized by high adaptability to the urban environment. Staphylococcus is a leading cause of hospital infection. In Europe, Latin America, USA and Canada, the group of coagulase negative staphylococci (CoNS) is the second leading cause of these infections, according to SENTRY (antimicrobial surveillance program- EUA). In this study, we investigated the potential of ants (Hymenoptera: Formicidae) as vehicle mechanics of Staphylococcus bacteria in a public hospital, in Natal-RN. The ants were collected, day and night, from June 2007 to may 2008, in the following sectors: hospitals, laundry, kitchen, blood bank. The ants were identified according to the identification key of Bolton, 1997. For the analysis of staphylococci, the ants were incubated in broth Tryptic Soy Broth (TSB) for 24 hours at 35 º C and then incubated on Mannitol Salt Agar. The typical colonies of staphylococci incubated for 24 hours at 35 ° C in Tryptic Soy Agar for the characterization tests (Gram stain, catalase, susceptibility to bacitracin and free coagulase). The identification of CoNS was performed through biochemical tests: susceptibility to novobiocin, growth under anaerobic conditions, presence of urease, the ornithine decarboxylation and acid production from the sugars mannose, maltose, trehalose, mannitol and xylose. The antimicrobial susceptibility examined by disk-diffusion technique. The technique of Polymerase Chain Reaction was used to confirm the presence of mecA gene and the ability to produce biofilm was verified by testing in vitro using polystyrene inert surface, in samples of resistant staphylococci. Among 440 ants, 85 (19.1%) were carrying coagulase-negative staphylococci (CoNS) of the species Staphylococcus saprophyticus (17), Staphylococcus epidermidis (15), Staphylococcus xylosus (13), Staphylococcus hominis hominis (10), Staphylococcus lugdunensis (10), Staphylococcus warneri (6), Staphylococcus cohnii urealyticum (5), Staphylococcus haemolyticus (3), Staphylococcus simulans (3), Staphylococcus cohnii cohnii (2), and Staphylococcus capitis (1). No Staphylococcus aureus was found. Among the isolates, 30.58% showed resistance to erythromycin. Two samples of CoNS (2.35%), obtained from the ant Tapinoma melanocephalum collected in the post-surgical female ward, S. Hominis hominis and S. lugdunensis harbored the mecA gene and were resistant to multiple antibiotics, and the specie S. hominis hominis even showed to be a biofilm producer. This study proves that ants act as carriers of multidrug-resistant coagulase-negative Staphylococci and biofilm producers and points to the risk of the spreading of pathogenic microorganisms by this insect in the hospital environment

Avaliação do potencial de formigas (Hymenoptera: formicidae) como vetores mecânicos de bactérias do gêneroSstaphylococcus no ambiente hospitalar.

In a hospital environment, these bacteria can be spread by insects such as ants, which are characterized by high adaptability to the urban environment. Staphylococcus is a leading cause of hospital infection. In Europe, Latin America, USA and Canada, the group of coagulase negative staphylococci (CoNS) is the second leading cause of these infections, according to SENTRY (antimicrobial surveillance program- EUA). In this study, we investigated the potential of ants (Hymenoptera: Formicidae) as vehicle mechanics of Staphylococcus bacteria in a public hospital, in Natal-RN. The ants were collected, day and night, from June 2007 to may 2008, in the following sectors: hospitals, laundry, kitchen, blood bank. The ants were identified according to the identification key of Bolton, 1997. For the analysis of staphylococci, the ants were incubated in broth Tryptic Soy Broth (TSB) for 24 hours at 35 º C and then incubated on Mannitol Salt Agar. The typical colonies of staphylococci incubated for 24 hours at 35 ° C in Tryptic Soy Agar for the characterization tests (Gram stain, catalase, susceptibility to bacitracin and free coagulase). The identification of CoNS was performed through biochemical tests: susceptibility to novobiocin, growth under anaerobic conditions, presence of urease, the ornithine decarboxylation and acid production from the sugars mannose, maltose, trehalose, mannitol and xylose. The antimicrobial susceptibility examined by disk-diffusion technique. The technique of Polymerase Chain Reaction was used to confirm the presence of mecA gene and the ability to produce biofilm was verified by testing in vitro using polystyrene inert surface, in samples of resistant staphylococci. Among 440 ants, 85 (19.1%) were carrying coagulase-negative staphylococci (CoNS) of the species Staphylococcus saprophyticus (17), Staphylococcus epidermidis (15), Staphylococcus xylosus (13), Staphylococcus hominis hominis (10), Staphylococcus lugdunensis (10), Staphylococcus warneri (6), Staphylococcus cohnii urealyticum (5), Staphylococcus haemolyticus (3), Staphylococcus simulans (3), Staphylococcus cohnii cohnii (2), and Staphylococcus capitis (1). No Staphylococcus aureus was found. Among the isolates, 30.58% showed resistance to erythromycin. Two samples of CoNS (2.35%), obtained from the ant Tapinoma melanocephalum collected in the post-surgical female ward, S. Hominis hominis and S. lugdunensis harbored the mecA gene and were resistant to multiple antibiotics, and the specie S. hominis hominis even showed to be a biofilm producer. This study proves that ants act as carriers of multidrug-resistant coagulase-negative Staphylococci and biofilm producers and points to the risk of the spreading of pathogenic microorganisms by this insect in the hospital environment

Avaliação da colonização nasal por Staphylococcus spp. resistenteà oxacilina em alunos de enfermagem

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Determinação do perfil toxigênico em Staphylococcus aureos e estafilococos coagulase-negativa isolados de recém-nascidos pela técnica de RT-PCR

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Perfil clonal e fatores de patogenicidade de Staphylococcus spp. no prognóstico das peritonites em diálise peritoneal

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Detecção do operon ica da produção de biofilme, gene mecA de resistência à oxacilina e identificação de espécies de Staphylococcus spp. diretamente dos frascos de hemoculturas pela técnica de PCR multiplex

Pós-graduação em Doenças Tropicais - FMB

CARACTERIZAÇÃO DO STAPHYLOCOCCUS COAGULASE NEGATIVO ISOLADOS DE QUARTOS MAMÁRIOS DE VACAS COM MASTITE SUBCLÍNICA

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Características genotípicas de Staphylococcus coagulase-negativos e taxas de cura da mastite ovina

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Características genotípicas de Staphylococcus coagulase-negativos e taxas de cura da mastite ovina.

A mastite em ovelhas de dupla aptidão é reconhecida por afetar a qualidade do leite. Staphylococcus coagulase-negativos (SCN) são os principais micro-organismos responsáveis pela doença, e o tratamento ao final da lactação, pode contribuir para a cura e prevenção de casos subclínicos na lactação consecutiva. Entretanto, fatores de virulência e mecanismos de resistência apresentados por SCN podem reduzir as taxas de cura. Os objetivos deste trabalho foram identificar no leite de ovelhas tratadas e não tratadas à secagem com antimicrobianos, as espécies de SCN antes e após o tratamento e identificar nesses micro-organismos a presença dos genes mecA, icaA, icaC, icaD, bap, bhp, sea, seb, sec, sed e tsst-1, determinar o perfil clonal das principais espécies identificadas e relacionar os casos de cura após o tratamento com a presença/ausência dos respectivos genes. Sessenta ovelhas foram divididas em três grupos experimentais: G1, controle, metades mamárias que não receberam antimicrobiano; G2, metades mamárias em que foram administrados 10 mL de cloxacilina-benzatina 100 mg via intramamária / estrutura convencional; G3, metades mamárias em que foram administrados 86 mL de cloxacilina-benzatina 50 mg via intramamária / estrutura nanoencapsulada. As amostras de leite foram coletadas à secagem e aos 15 e 30 dias pós-parto da lactação seguinte. As análises para identificação das espécies de SCN foram realizadas por meio de testes bioquímicos e Internal Transcribe Spacer (ITS-PCR), e a pesquisa dos genes responsáveis pelos fatores de virulência e pela resistência à oxacilina foram realizados por meio da técnica de reação em cadeia de polimerase (PCR). Dentre as espécies identificadas S. warneri prevaleceram nos três grupos experimentais. Nenhuma amostra foi positiva para o gene mecA. O único gene relacionado com a produção de enterotoxinas encontrado foi o sec. Dentre os genes relacionados com a produção de biofilme, icaD foi o único identificado nos três grupos experimentais. Staphylococcus warneri, S. simulans e S. epidermidis apresentaram clones na mesma metade mamária no pré e pós-parto das ovelhas. A cloxacilina benzatina nanoparticulada 50mg / 86 mL foi eficiente para reduzir a mastite subclínica no pós-parto de ovelhas (P= 0,0192). Staphylococcus warneri, S. simulans, S. epidermidis e S. xylosus foram as espécies de maior ocorrência. Os genes icaA, icaC, icaD e bap foram encontrados no momento da desmama e no pós-parto, os genes sec e icaD estão associados à ausência de cura da mastite subclínica no pós-parto. Ovelhas em que foram isolados SCN portadores de genes responsáveis pela formação de biofilme não apresentaram resultados satisfatórios quando submetidas a esquemas de controle e ao tratamento da mastite subclínica. Os genes sec e icaD, estão associadas à ausência de cura microbiológica da mastite subclínica no pós-parto. Staphylococcus epidermidis e S. xylosus portador do gene bap estão associados à reinfecção.

Staphylococcus Aureus Enterotoxins A And B Inhibit Human And Mice Eosinophil Chemotaxis And Adhesion In Vitro.

Staphylococcus aureus aggravates the allergic eosinophilic inflammation. We hypothesized that Staphylococcus aureus-derived enterotoxins directly affect eosinophil functions. Therefore, this study investigated the effects of Staphylococcal enterotoxins A and B (SEA and SEB) on human and mice eosinophil chemotaxis and adhesion in vitro, focusing on p38 MAPK phosphorylation and intracellular Ca(2+) mobilization. Eosinophil chemotaxis was evaluated using a microchemotaxis chamber, whereas adhesion was performed in VCAM-1 and ICAM-1-coated plates. Measurement of p38 MAPK phosphorylation and intracellular Ca(2+) levels were monitored by flow cytometry and fluorogenic calcium-binding dye, respectively. Prior incubation (30 to 240 min) of human blood eosinophils with SEA (0.5 to 3 ng/ml) significantly reduced eotaxin-, PAF- and RANTES-induced chemotaxis (P<0.05). Likewise, SEB (1 ng/ml, 30 min) significantly reduced eotaxin-induced human eosinophil chemotaxis (P<0.05). The reduction of eotaxin-induced human eosinophil chemotaxis by SEA and SEB was prevented by anti-MHC monoclonal antibody (1 μg/ml). In addition, SEA and SEB nearly suppressed the eotaxin-induced human eosinophil adhesion in ICAM-1- and VCAM-1-coated plates. SEA and SEB prevented the increases of p38 MAPK phosphorylation and Ca(2+) levels in eotaxin-activated human eosinophils. In separate protocols, we evaluated the effects of SEA on chemotaxis and adhesion of eosinophils obtained from mice bone marrow. SEA (10 ng/ml) significantly reduced the eotaxin-induced chemotaxis along with cell adhesion to both ICAM-1 and VCAM-1-coated plates (P<0.05). In conclusion, the inhibition by SEA and SEB of eosinophil functions (chemotaxis and adhesion) are associated with reductions of p38 MAPK phosphorylation and intracellular Ca(2+) mobilization.

Maximum inhibitory dilution of mouthwashes containing chlorhexidine and polyhexamethylene biguanide against salivary staphylococcus aureus

OBJECTIVE: The aim of the present study was to determine the in vitro maximum inhibitory dilution (MID) of two chlorhexidinebased oral mouthwashes (CHX): Noplak®, Periogard®, and one polyhexamethylene biguanide-based mouthwash (PHMB): Sanifill Premium® against 28 field Staphylococcus aureus strains using the agar dilution method. MATERIALS AND METHODS: For each product, decimal dilutions ranging from 1/10 to 1/655,360 were prepared in distilled water and added to Mueller Hinton Agar culture medium. After homogenization, the culture medium was poured onto Petri dishes. Strains were inoculated using a Steers multipoint inoculator and dishes were incubated at 37ºC for 24hours. For reading, MID was considered as the maximum dilution of the mouthwash still capable of inhibiting microbial growth. RESULTS: Sanifill Premium® inhibited the growth of all strains at 1/40 dilution and of 1 strain at 1/80 dilution. Noplak® inhibited the growth of 23 strains at 1/640 dilution and of all 28 strains at 1/320 dilution. Periogard® showed inhibited growth of 7 strains at 1/640 dilution and of all 28 strains at 1/320 dilution. Data were submitted to Kruskal-Wallis statistical test, showing significant differences between the mouthwashes evaluated (p<0.05). No significant difference was found between Noplak® and Periogard® (p>0.05). Sanifill Premium® was the least effective (p<0.05). CONCLUSION: It was concluded that CHX-based mouthwashes present better antimicrobial activity against S. Aureus than the PHMB-based mouthwash.