Abstract Ingesting food contaminated by pathogens and/or their toxins can cause foodborne illness. In this sense, this study investigated the occurrence of enterotoxigenic S. aureus in frozen cheese bread dough and assessed the production and thermosensitivity of SEA in artificially contaminated cheese dough. E. coli counts were determined by MPN. Confirmation of the presence of S. aureus was carried out by biochemical and molecular identification. The detection of the genes sea, seb, sec, and sed was performed using the PCR. The detection of SEA in artificially contaminated cheese dough, before and after baking at 180 ºC for 35 minutes was performed using the RPLA. All samples contaminated with E. coli had NMP < 5.0×102 CFU / g. None of the isolated S. aureus strains expressed the classical enterotoxin genes sea, seb, sec, and sed. However, as there are 23 serologically distinct staphylococcal enterotoxins, we cannot rule out the possibility that strains expressed other enterotoxin-encoding genes. Laboratory tests showed the presence of SEA in cheese bread dough artificially contaminated with SEA-producing S. aureus after 8 h of incubation at 10 or 20 °C. Heat treatment at 180 °C for 35 minutes was not sufficient to inactivate SEA in artificially contaminated cheese bread previously incubated for 24 h at 10 or 20 °C. These results indicate a potential health risk to consumers of cheese bread and underscore the need for ingredient quality control and measures to prevent the multiplication of S. aureus during product manufacture and storage.

ABSTRACT Many infants are nurtured with milk supplied by human banks, whose bacteriological and physical-chemical profiles are a major issue. We investigated the bacteriological and physical-chemical characteristics, as well as genotypic and phenotypic and profiles of Staphylococcus species isolated from 240 samples of breast milk from a bank in a teaching hospital. Dornic acidity of milk revealed that 95.4% (229/240) had acceptable limits (< 8.0 oD). Caloric intake showed a wide variation in cream content (4%), fat (4%) and energy values (559.81 Kcal/L). Staphylococcus (105/186 or 56.5%) and Enterobacter (25/186 or 13.4%) were the most prevalent genera, although other microorganisms were identified, including Klebsiella pneumoniae and Pseudomonas aeruginosa. Amoxicillin/clavulanic acid (125/157 or 79.6%), vancomycin (115/157 or 73.2%), and cephalexin (112/157 or 71.3%) were the most effective antimicrobials. High resistance rates of isolates were found to penicillin G (141/157 or 89.8%), ampicillin (135/157 or 86%), and oxacillin (118/157 or 75.2%). Multidrug resistance to ≥ 3 antimicrobials occurred in 66.2% (123/186) of the isolates. Residues of microbial multiplication inhibitory substances were found in 85% (204/240) of samples. Among the coagulase-positive-CPS and negative-CoNS staphylococci, the mecA gene was detected in 53.3% (8/15) and 75% (30/40), respectively. Genes sea, seb and sec were detected in 20% (3/15) of CPS, while tsst-1 was detected in 13.34% (2/15). In addition, 13.3% (2/15) of S. aureus were toxin-producers. Genes sea, seb and sec were detected in 90% (36/40), 5% (2/40) and 15% (6/40) CoNS, respectively. Enterotoxin production was identified in 5% (2/40) of CoNS. The identification of multidrug-resistant bacteria, staphylococci species toxin-producers harboring methicillin-resistance genes, and residues of microbial multiplication inhibitory substances reinforce the need for a continuous vigilance of milk quality offered to infant consumption by human banks.

ABSTRACT The increasing rates of nosocomial infection associated with coagulase-negative staphylococci (CoNS) were the rationale for this study, aiming to categorize oxacillin-resistant CoNS species recovered from blood culture specimens of inpatients at the UNESP Hospital das Clínicas in Botucatu, Brazil, over a 20-year period, and determine their sensitivity to other antimicrobial agents. The mecA gene was detected in 222 (74%) CoNS samples, and the four types of staphylococcal chromosomal cassette mec (SCCmec) were characterized in 19.4%, 3.6%, 54.5%, and 14.4% of specimens, respectively, for types I, II, III, and IV. Minimal inhibitory concentration (MIC) values to inhibit 50% (MIC50) and 90% (MIC90) of specimens were, respectively, 2 and >256 µL/mL for oxacillin, 1.5 and 2 µL/mL for vancomycin, 0.25 and 0.5 µL/mL for linezolid, 0.094 and 0.19 µL/mL for daptomycin, 0.19 and 0.5 µL/mL for quinupristin/dalfopristin, and 0.125 and 0.38 µL/mL for tigecycline. Resistance to oxacillin and tigecycline and intermediate resistance to quinupristin/dalfopristin were observed. Eight (2.7%) of all 300 CoNS specimens studied showed reduced susceptibility to vancomycin. Results from this study show high resistance rates of CoNS to antimicrobial agents, reflecting the necessity of using these drugs judiciously and controlling nosocomial dissemination of these pathogens.

ABSTRACT Introduction: Staphylococcus spp. – both S. aureus, including methicillin-resistant strains (MRSA) and coagulase negative staphylococci (CoNS) – are relevant agents of healthcare-associated infections. Therefore, the rapid recognition of MRSA and methicillin-resistant CoNS from blood stream infections is critically important for patient management. It is worth noting that inappropriate empiric therapy has been associated with higher in-hospital mortality. Material and methods: In this study we evaluated a multiplex polymerase chain reaction (multiplex PCR) standardized to detect Staphylococcus spp., S. aureus, and mecA gene-encoded oxacillin resistance directly from blood culture bottles. A total of 371 blood cultures with Gram-positive microorganisms confirmed by Gram-stain were analyzed. Results from multiplex PCR were compared to phenotypic characterization of isolates. Results: Staphylococcus aureus was detected in 85 (23.0%) blood cultures and CoNS in 286 (77.0%). There was 100% agreement between phenotypic and multiplex PCR identification. Forty-three (50.6%) of the 85 S. aureus carried the mecA gene and among the 286 CoNS, 225 (78.7%) were positive for the mecA gene. Conclusions: The multiplex PCR assay developed here was found to be sensitive, specific, rapid, and showed good agreement with the phenotypic results besides being less expensive. This PCR method could be used in clinical laboratories for rapid identification and initiation of specific and effective treatment, reducing patient mortality and morbidity. Furthermore, this method may reduce misuse of antimicrobial classes that are more expensive and toxic, thus contributing to the selection of antibiotic-resistant Staphylococcus spp.

Abstract INTRODUCTION: Wounds can be colonized by methicillin-resistant Staphylococcus aureus (MRSA). METHODS: We evaluated the prevalence of S. aureus and MRSA in the wounds of patients treated at Basic Health Units in Brazil and identified risk factors associated with their presence. RESULTS: The prevalence rates of S. aureus and MRSA were 51.5% and 8.7%, respectively. There was a correlation between the presence of S. aureus in wounds and nostrils (p<0.01). A positive association was detected between S. aureus infection and previous benzylpenicillin use (p=0.02). No associations were observed for MRSA. CONCLUSIONS: Multidrug-resistant pathogens are present in primary healthcare settings in Brazil.

Abstract Staphylococcus aureus and Staphylococcus saprophyticus are the most common and most important staphylococcal species associated with urinary tract infections. The objective of the present study was to compare and to evaluate the accuracy of four phenotypic methods for the detection of beta-lactamase production in Staphylococcus spp. Seventy-three strains produced a halo with a diameter ≤28 mm (penicillin resistant) and all of them were positive for the blaZ gene. Among the 28 susceptible strain (halo ≥29 mm), 23 carried the blaZ gene and five did not. The zone edge test was the most sensitive (90.3%), followed by MIC determination (85.5%), but the specificity of the former was low (40.0%). The nitrocefin test was the least sensitive (28.9%). However, the nitrocefin test together with the disk diffusion method showed the highest specificity (100%). The present results demonstrated that the zone edge test was the most sensitive phenotypic test for detection of beta-lactamase, although it is still not an ideal test to detect this type of resistance since its specificity was low. However, the inhibition halo diameter of the penicillin disk can be used together with the zone edge test since the same disk is employed in the two tests. Combined analysis of the two tests shows a sensitivity of 90.3% and specificity of 100%, proving better sensitivity, especially for S. saprophyticus. This is a low-cost test of easy application and interpretation that can be used in small and medium-sized laboratories where susceptibility testing is usually performed by the disk diffusion method.

Resumo Staphylococcus coagulase-negativos (SCN) são os principais micro-organismos responsáveis pela mastite ovina e o tratamento ao final da lactação pode ser usado como método de controle contra a doença. Porém, o longo período seco apresentado pelas ovelhas em alguns sistemas de criação pode prejudicar os efeitos positivos do tratamento antimicrobiano. Os objetivos deste trabalho foram identificar as principais espécies de SCN na etiologia da mastite ovina antes e após o tratamento ao final da lactação das ovelhas, bem como investigar a persistência das espécies mais prevalentes na glândula mamária na lactação seguinte. Sessenta ovelhas foram divididas em dois grupos experimentais, um deles formado por animais sem tratamento antimicrobiano, enquanto o outro era composto por ovelhas cujas metades mamárias foram tratadas com cloxacilina-benzatina por via intramamária. As amostras de leite foram obtidas antes da secagem e aos 15 e 30 dias após o parto da lactação seguinte. As espécies prevalentes foram S. warneri, S. simulans e S. epidermidis. Clones das três espécies de maior ocorrência foram identificados antes e depois do tratamento, ou seja, mesmo com o extenso período seco entre as duas lactações consecutivas, os micro-organismos ainda foram identificados no interior da glândula mamária.

Abstract Coagulase-negative Staphylococci (CNS) are the main microorganisms responsible for mastitis in sheep and the treatment in the end of lactation can be used as a control method against disease. However, the long dry period presented by ewes in some breeding systems may impair the positive effects of antimicrobial treatment. The aims of this study were to identify the main CNS species in the etiology of ovine mastitis before and after treatment at dry-off of the ewes, as well as investigate the persistence of the most prevalent species in the mammary gland at the next lactation. Sixty sheep were divided into two experimental groups, one consisting of animals without antimicrobial treatment, while the other was composed by sheep whose mammary halves were treated with cloxacillin-benzathine intramammary. The milk samples were obtained before drying-off and at 15 and 30 days after calving the next lactation. The prevalent species were S. warneri, S. simulans and S. epidermidis. Clones of the three species more prevalent were identified before and after treatment, i.e., even with the extensive dry period between the two consecutive lactations, the microorganisms were still identified in the mammary gland.

The objective of this study was to characterize the clonal profile, virulence factors and antimicrobial resistance, particularly oxacillin resistance, of Staphylococcus aureus isolated from sheep milk. Milk samples were collected from all teats for the California Mastitis Test (CMT), somatic cell count, identification of S. aureus, investigation in these strains of genes encoding toxins (sea, seb, sec, sed, tst), biofilm (icaA, icaC, icaD, bap), leukocidin (luk-PV) oxacillin resistance by mecA gene detection and susceptibility testing (12 antibiotics). Messenger RNA expression was evaluated by RT-PCR in isolates carrying toxin and biofilm genes. Biofilm formation was also evaluated phenotypically by adherence to polystyrene plates. The clonal profile of S. aureus was investigated by pulsed-field gel electrophoresis. A total of 473 milk samples were collected from 242 animals on three farms and 20 S. aureus strains were isolated and none carried the mecA gene. The two sec gene-positive isolates and the isolates carrying the tst and luk-PV genes were positive by RT-PCR. Staphylococcus aureus isolated from the three flocks studied showed high susceptibility to the drugs tested and none was biofilm producer, indicating that biofilm formation was not a virulence factor causing infection by these strains. The typing of 17 S. aureus isolates revealed the presence of a common clone on the three farms studied, and the presence and expression of the sec and tst genes in one strain of this clone suggest the possible acquisition of virulence genes by this clone, a fact that is important for animal health and food hygiene.

The aim of this study was to determine the prevalence of Staphylococcus aureus and risk factors for the acquisition of MRSA (Methicillin Resistant Staphylococcus aureus) as the main cause of skin and soft tissue infections. S. aureus were characterized for the presence of PVL, TSST-1 and mecA genes. SCCmec typing was carried out in mecA positive strains and PFGE was performed only in these strains. During the study period, 127 outpatients attending a dermatology clinical the Botucatu Medical School, a regional tertiary hospital in Botucatu, Sao Paulo, Brazil, were diagnosed with active skin infections. A total 66 (56.9%) S. aureus strains were isolated. The methicillin resistance gene mecA was detected in seven (10.6%) S. aureus strains. The SCCmec types detected in the seven mecA-positive S. aureus strains were type Ia in one, type II in three, and type IV in three. The PVL gene was detected in 10 (15.1%) in sensitive strains. Pulsed field gel electrophoresis revealed non-clonal diversity among the isolates. The risk factors associated with MRSA acquisition in this study were previous ciprofloxacin use and working in a healthcare environment. The risk factors indicate plausible routes of CA-MRSA transmission among the subjects studied.

This study aimed to correlate the presence of ica genes, biofilm formation and antimicrobial resistance in 107 strains of Staphylococcus epidermidis isolated from blood cultures. The isolates were analysed to determine their methicillin resistance, staphylococcal cassette chromosome mec (SCCmec) type, ica genes and biofilm formation and the vancomycin minimum inhibitory concentration (MIC) was measured for isolates and subpopulations growing on vancomycin screen agar. The mecA gene was detected in 81.3% of the S. epidermidis isolated and 48.2% carried SCCmec type III. The complete icaADBC operon was observed in 38.3% of the isolates; of these, 58.5% produced a biofilm. Furthermore, 47.7% of the isolates grew on vancomycin screen agar, with an increase in the MIC in 75.9% of the isolates. Determination of the MIC of subpopulations revealed that 64.7% had an MIC ≥ 4 μg mL-1, including 15.7% with an MIC of 8 μg mL-1 and 2% with an MIC of 16 μg mL-1. The presence of the icaADBC operon, biofilm production and reduced susceptibility to vancomycin were associated with methicillin resistance. This study reveals a high level of methicillin resistance, biofilm formation and reduced susceptibility to vancomycin in subpopulations of S. epidermidis. These findings may explain the selection of multidrug-resistant isolates in hospital settings and the consequent failure of antimicrobial treatment.

The aim of this study was to perform SCCmec typing in Staphylococcus aureus isolates and to characterize the clonal profile of these isolates. Forty-six mecA gene-positive strains isolated between 2002 and 2006 were submitted to antimicrobial resistance testing by the E-test, SCCmec typing by multiplex PCR, and clonal profile analysis by pulsed-field gel electrophoresis. Forty-one (89.1%) isolates were typed as SCCmec III and five (10.9%) as SCCmec IV. Four circulating clones were detected, one of them comprising isolates related to the Brazilian epidemic clone. This clone was detected throughout the study period. The SCCmec III isolates were associated with a high rate of multidrug resistance and clonal dissemination of methicillin-resistant S. aureus in the wards of the University Hospital of the Botucatu School of Medicine, Universidade Estadual Paulista.

Coagulase-negative staphylococci (CoNS) are the microorganisms most frequently isolated from clinical samples and are commonly found in neonatal blood cultures. Oxacillin is an alternative treatment of choice for CoNS infections; however, resistance to oxacillin can have a substantial impact on healthcare by adversely affecting morbidity and mortality. The objective of this study was to detect and characterise oxacillin-resistant CoNS strains in blood cultures of newborns hospitalised at the neonatal ward of the University Hospital of the Faculty of Medicine of Botucatu. One hundred CoNS strains were isolated and the mecA gene was detected in 69 of the CoNS strains, including 73.2% of Staphylococcus epidermidis strains, 85.7% of Staphylococcus haemolyticus strains, 28.6% of Staphylococcus hominis strains and 50% of Staphylococcus lugdunensis strains. Among these oxacillin-resistant CoNS strains, staphylococcal cassette chromosome mec (SCCmec) type I was identified in 24.6%, type II in 4.3%, type III in 56.5% and type IV in 14.5% of the strains. The data revealed an increase in the percentage of CoNS strains isolated from blood cultures from 1991-2009. Furthermore, a predominant SCCmec profile of the oxacillin-resistant CoNS strains isolated from neonatal intensive care units was identified with a prevalence of SCCmec types found in hospital-acquired strains.

INTRODUÇÃO: O gênero Staphylococcus é responsável por um grande número de infecções bacterianas em humanos, principalmente no ambiente hospitalar. OBJETIVO: Diante dessas considerações, da importância do cuidado de enfermagem e do controle de infecções hospitalares, este trabalho verificou a taxa de portadores de S. aureus e estafilococos coagulase-negativa (ECN) resistentes a oxacilina em alunos do curso de enfermagem durante a formação profissional. MATERIAS E METÓDOS: Foram coletadas amostras das fossas nasais de alunos do curso de enfermagem da Faculdade de Medicina de Botucatu (FMB). RESULTADOS: Das 109 amostras de Staphylococcus encontradas, 30 (27,5%) foram de Staphylococcus aureus e 79 (72,5%), de ECN. Das 79 amostras de ECN submetidas às provas de identificação, 63 (79,7%) foram da espécie S. epidermidis, nove (11,4%), S. warneri, três (3,8%), S. haemolyticus, duas (2,5%), S. capitis, uma (1,3%), S. simulans e uma (1,3%) S. lugdunensis. O teste de suscetibilidade a antibióticos demonstrou 100% de sensibilidade às drogas nas amostras de S. aureus e, entre as 79 amostras de ECN, 10 (12,6%) foram resistentes a oxacilina. A técnica de reação em cadeia da polimerase (PCR) demonstrou resultado negativo para o gene mecA nas amostras de S. aureus e 11 amostras positivas entre as espécies de ECN. DISCUSSÃO: Não houve relação entre a taxa de portadores de S. aureus e o envolvimento hospitalar durante a graduação, os resultados também demonstraram maior incidência de resistência nas amostras de ECN, semelhante ao encontrado na literatura científica.

INTRODUCTION: The genus Staphylococcus is responsible for a great number of bacterial infections in human, mainly in hospital environment. OBJECTIVES: In view of these considerations and the importance of nursing care and nosocomial infection control, this study verified the rate of S. aureus and coagulase-negative Staphylococci (CNS) carriers resistant to oxacillin in nursing students during their university course. MATERIALS AND METHODS: Nasal samples were collected from Nursing students at Botucatu School of Medicine - UNESP. RESULTS: From 109 isolated samples of Staphylococcus, 30 samples (27.5%) were Staphylococcus aureus and 79 samples (72.5%) were CNS. From 79 identified CNS samples, 63 (79.7%) were S. epidermidis, nine (11.4%) S. warneri, three (3.8%) S. haemolyticus, two (2.5%) S. capitis, one (1.3%) S. simulans and one (1.3%) S. lugdunensis. The antibiotic susceptibility test showed 100% sensibility to the drugs in S. aureus samples and among 79 CNS samples, 10 (12,6%) were resistant to oxacillin. The PCR technique demonstrated negative result for mecA gene in S. aureus samples and 11 positive samples among CNS species. DISCUSSION: There was no relation between the rate of S. aureus carriers and nosocomial involvement during the course. The results also showed a higher incidence of resistance in CNS samples, which is seemingly reported in the scientific literature.

OBJETIVO: Determinar o número de unidades formadoras de colônias que melhor correlaciona com a infecção relacionada a cateter em recém-nascidos. MÉTODOS: Este foi um estudo prospectivo de culturas semiquantitativas de pontas de cateteres de recém-nascidos da unidade neonatal da Faculdade de Medicina de Botucatu. Os microrganismos isolados de cateteres e hemoculturas periféricas foram identificados e submetidos ao teste de sensibilidade a drogas. O ponto de corte ótimo foi determinado pela curva receiver operating characteristic (ROC). RESULTADOS: Foram estudados 85 cateteres de 63 recém-nascidos. A espécie Staphylococcus epidermidis foi prevalente (75%) nos cateteres. Dos 11 episódios de infecção diagnosticados, oito (72,7%) foram associados aos estafilococos coagulase-negativa, dos quais seis pertenciam à espécie S. epidermidis. Pela curva ROC, o ponto de corte ótimo para o diagnóstico de infecção relacionada a cateter foi 122 unidades formadoras de colônias. CONCLUSÃO: O ponto de corte 122 unidades formadoras de colônias melhor se correlacionou com o diagnóstico de infecção relacionada a cateter em recém-nascidos.

OBJECTIVE: To determine the number of colony-forming units (CFU) that best correlates with catheter-related infections (CRI) in newborns. METHODS: This was a prospective study of semiquantitative cultures of catheter tips obtained from newborns in the neonatal unit at Faculdade de Medicina de Botucatu, state of São Paulo, Brazil. The microorganisms isolated from catheter and peripheral blood cultures were identified and submitted to a drug susceptibility test. The optimal cutoff point was determined by the receiver operating characteristic (ROC) curve. RESULTS: A total of 85 catheters obtained from 63 newborns were studied. Staphylococcus epidermidis was the predominant species in the catheters (75%). Eight of 11 (72.7%) CRI episodes were associated with coagulase-negative staphylococci, six of which were of the S. epidermidis type. ROC curve analysis indicated that the optimal cutoff point for the diagnosis of CRI was 122 CFU. CONCLUSION: The cutoff point of 122 CFU correlated best with the diagnosis of CRI in newborns.

Coagulase-negative staphylococci (CNS) have been identified as the etiological agent in various infections and are currently the microorganisms most frequently isolated in nosocomial infections. However, little is known about the virulence factors produced by CNS that contribute to the pathogenesis of infections caused by these microorganisms. The study of CNS isolated from infectious processes of newborns hospitalized in the Neonatal Unit of the Hospital of the Botucatu Medical School, Unesp, indicated Staphylococcus epidermidis as the most frequently isolated species (77.8%), which was also associated with clinically significant situations. The analysis of virulence factors revealed the production of slime in 20 (17.1%) of all CNS samples isolated and the synthesis of a broad spectrum of enzymes and toxins, including hemolysins (19.6%), lipase (17.1%), lecithinase (3.4%), DNAse (15.4%), thermonuclease (7.7%), and enterotoxin A, B or C (37.6%). Taking into consideration that the etiological importance of CNS has often been neglected, the present investigation confirmed that these microorganisms should not be ignored or classified as mere contaminants.