Resistance profile and biofilm production capacity of Staphylococcus spp. beef slaughterhouse isolates and their sensitivity to Rosmarinus officinalis essential oil.

Microorganisms can interfere with meat quality, being a public health problem. The aim of this study was to characterize the antimicrobial susceptibility profile of Staphylococcus spp. isolated from utensils of a bovine slaughterhouse and to evaluate the antibacterial and antibiofilm activity of the essential oil of Rosmarinus officinalis L. (rosemary). Samples of surfaces and utensils used during slaughter in the northwest of the state of Paraná, Brazil were collected. After isolation and differentiation of the isolates by the coagulase test, the antimicrobial susceptibility test, Staphylococcus aureus identification and mecA gene research were performed. The study for biofilm production was carried out by the method of adhesion in borosilicate tube and by adhesion in polystyrene plate. Subsequently, the inhibitory activity of the R. officinalis essential oil and its ability to inhibit biofilm were investigated. Twenty-two of the samples collected were identified as coagulase-negative Staphylococcus and five as coagulase-positive Staphylococcus. There was resistance to all antibiotics tested, with clindamycin (33.33%) and rifampicin (29.6%) showing the highest rate. None of the samples was confirmed as Staphylococcus aureus or for the presence of the mecA resistance gene. The essential oil inhibited the growth of 48% of the isolates at a concentration of 16,000 µg/mL. Of these isolates, 33% were positive for biofilm production and this biofilm was also inhibited by the essential oil. This work revealed that multidrug-resistant Staphylococcus and biofilm producers are present in the slaughter environment and are susceptible to the essential oil of R. officinalis.

Evaluation of Polymerase Chain Reaction in the Identification and Quantification of Clinically Relevant Bacterial Species in Lower Extremity Wound Infections.

Identification of bacteria by polymerase chain reaction (PCR) is known to be more sensitive than culture, which brings to question the clinical applicability of the results. In this study, we evaluate the ability of PCR to detect clinically relevant bacterial species in lower extremity wound infections requiring operative debridement, as well as the quantitative change in biodiversity and bacterial load reflected by PCR during the course of treatment. Thirty-four infected lower extremity were examined by analysis of 16S ribosomal RNA subunit and by culture. McNemar's test was used to measure the concordance of clinically relevant bacterial species identified by PCR compared to culture during each debridement. Change in wound biodiversity from initial presentation to final closure was evaluated by Wilcoxon signed-rank test. Kaplan-Meier survival curve was used to characterize change in measured bacterial load over the course of operative debridement. A total of 15 and 12 clinically relevant bacterial species were identified by PCR and culture, respectively. The most common bacterial species identified were Coagulase-negative Staphylococcus, Staphylococcus aureus, and Enterococcus spp. PCR was less likely to detect Enterococcus spp. on initial debridement and Coagulase-negative Staphylococcus on closure in this study population. A significant decrease in mean number of clinically relevant species detected from initial debridement to closure was reflected by culture (p = .0188) but not by PCR (p = .1848). Both PCR (p = .0128) and culture (p = .0001) depicted significant reduction in mean bacterial load from initial debridement to closure. PCR is able to identify common clinically relevant bacterial species in lower extremity surgical wound infections. PCR displays increased sensitivity compared to culture with relation to detection of biodiversity, rather than bacterial load. Molecular diagnostics and conventional culture may serve a joint purpose to assist with rendering clinical judgment in complex wound infections.

Coagulase negative Staphylococcus bacteremia in hematopoietic stem cell transplant recipients: Clinical features and molecular characterization.

The purpose of our study was to investigate the epidemiology of coagulase negative staphylococci (CoNS) responsible for bacteremia in hematopoietic stem cell transplant (HSCT) recipients and to determine the prevalence and the genetic background of methicillin resistance. The prevalence of CoNS bacteremia was 7.4% (54/728), higher in allograft (10.7%) than in autograft (4.7%) recipients. A sepsis or a septic shock were observed in 9% of cases. No deaths were attributable to CoNS bacteremia. The methicillin resistance rate was 81%. All MR-CoNS, harbored mecA gene and 90% were typeable with SCCmec typing using PCR amplification. The SCCmec type IV was the most frequent (44%). Clonal dissemination of MR- Staphylococcus epidermidis strains was limited. Our study showed a low prevalence and favorable outcome of CoNS bacteremia in HSCT recipients with limited clonal diffusion. However, they were associated with a significant rate of severe infections and a high rate of methicillin resistance, mediated by SCCmec IV element in most cases.

Catheter-related bloodstream infections with coagulase-negative staphylococci: are antibiotics necessary if the catheter is removed?

Background: Catheter-related bloodstream infections (CRBSI) with coagulase-negative Staphylococci (CoNS) are a common source of hospital-acquired bloodstream infections. The main objective of this study was to elucidate the role of systemic antibiotic therapy in the setting of catheter removal in adult patients with CoNS-CRBSI. Methods: We conducted a retrospective cohort study on patients with CoNS-CRBSI diagnosed between 2008 and 2016 with follow-up for up to 12 months. The main inclusion criterion was a removed intravascular catheter with quantitative catheter tip culture growing CoNS and the same CoNS identified in the blood culture of a given patient. Outcomes were non-resolved infection (i.e. either presence of prolonged bacteremia or symptoms attributed to CoNS-CRBSI > 2 days after catheter removal), recurrence, mortality and length of hospitalization after catheter removal. We compared outcomes between a group with antibiotic treatment prescribed according to current IDSA guidelines (≥5 days, "treatment" group) and a "no-treatment" group. Results: Our study population comprised 184 CoNS-CRBSI episodes. Seventy-six percent received antibiotic treatment ≥5 days, while 17% did not receive therapy. Non-resolved infections were absent from the patients who did not receive antibiotics. Severe neutropenia, hematologic cancer and immunosuppression were significantly more frequent in the treatment group. The subgroup analysis with 32 matched pairs showed no significant difference in frequency of non-resolved infection (0% in the no-treatment vs 15.6% in the ≥5 days treatment group, p = 0.06). The remaining outcomes were similar in the two groups. Conclusions: Our findings indicate that withholding antimicrobial therapy in CoNS-CRBSI is neither associated with short-term complications nor with long-term recurrences.

Multidrug-resistant strains of coagulase-negative staphylococci isolated from patients with chronic sinusitis - MDR, XDR, PDR strains.

INTRODUCTION: The development of resistance to multiple antimicrobial agents in pathogenic bacteria has become a threat to public health. Multidrug-resistant strains that are particularly dangerous include MDR, XDR and PDR strains. MATERIAL AND METHODS: Aspirate material from paranasal sinuses, obtained from patients with chronic sinusitis undergoing functional endoscopic sinus surgery (FESS) in Medical Center MML in Warsaw, was subjected to bacteriologic analysis. The isolated strains were identified to the species level and tested for antibiotic resistance. Then, minimal inhibitory concentration (MIC) was determined. R esults: The isolated strains of coagulase-negative staphylococci were resistant mainly to macrolides, aminoglycosides and tetracycline. Nine of the isolated strains exhibited multidrug-resistance. DISCUSSION: Bacteria causing chronic sinusitis are becoming increasingly resistant to antimicrobial agents. The diagnostic process for coagulase-negative staphylococci (CNS) is often limited to the identification of species, or even genus of the bacteria. The CNS strains are considered to be non-pathogenic and they are not subject to eradication. This may lead to erroneous therapeutic decisions and, consequently, to the development of antibiotic resistance. CNS infections are classified as nosocomial and therefore, appropriate epidemiological procedures have to be followed. The authors highlight the necessity to determine MIC values for antibiotics and to introduce personalized treatment.

RGD delivery of truncated coagulase to tumor vasculature affords local thrombotic activity to induce infarction of tumors in mice.

Induction of thrombosis in tumor vasculature represents an appealing strategy for combating cancer. Herein, we combined unique intrinsic coagulation properties of staphylocoagulase with new acquired functional potentials introduced by genetic engineering, to generate a novel bi-functional fusion protein consisting of truncated coagulase (tCoa) bearing an RGD motif on its C-terminus for cancer therapy. We demonstrated that free coagulase failed to elicit any significant thrombotic activity. Conversely, RGD delivery of coagulase retained coagulase activity and afforded favorable interaction of fusion proteins with prothrombin and αvß3 endothelial cell receptors, as verified by in silico, in vitro, and in vivo experiments. Although free coagulase elicited robust coagulase activity in vitro, only targeted coagulase (tCoa-RGD) was capable of producing extensive thrombosis, and subsequent infarction and massive necrosis of CT26 mouse colon, 4T1 mouse mammary and SKOV3 human ovarian tumors in mice. Additionally, systemic injections of lower doses of tCoa-RGD produced striking tumor growth inhibition of CT26, 4T1 and SKOV3 solid tumors in animals. Altogether, the nontoxic nature, unique shortcut mechanism, minimal effective dose, wide therapeutic window, efficient induction of thrombosis, local effects and susceptibility of human blood to coagulase suggest tCoa-RGD fusion proteins as a novel and promising anticancer therapy for human trials.

Adaptive Upregulation of Clumping Factor A (ClfA) by Staphylococcus aureus in the Obese, Type 2 Diabetic Host Mediates Increased Virulence.

Obesity and associated type 2 diabetes (T2D) are important risk factors for infection following orthopedic implant surgery. Staphylococcus aureus, the most common pathogen in bone infections, adapts to multiple environments to survive and evade host immune responses. Whether adaptation of S. aureus to the unique environment of the obese/T2D host accounts for its increased virulence and persistence in this population is unknown. Thus, we assessed implant-associated osteomyelitis in normal versus high-fat-diet obese/T2D mice and found that S. aureus infection was more severe, including increases in bone abscesses relative to nondiabetic controls. S. aureus isolated from bone of obese/T2D mice displayed marked upregulation of four adhesion genes (clfA, clfB, bbp, and sdrC), all with binding affinity for fibrin(ogen). Immunostaining of infected bone revealed increased fibrin deposition surrounding bacterial abscesses in obese/T2D mice. In vitro coagulation assays demonstrated a hypercoagulable state in obese/T2D mice that was comparable to that of diabetic patients. S. aureus with an inactivating mutation in clumping factor A (clfA) showed a reduction in bone infection severity that eliminated the effect of obesity/T2D, while infections in control mice were unchanged. In infected mice that overexpress plasminogen activator inhibitor-1 (PAI-1), S. aureusclfA expression and fibrin-encapsulated abscess communities in bone were also increased, further linking fibrin deposition to S. aureus expression of clfA and infection severity. Together, these results demonstrate an adaptation by S. aureus to obesity/T2D with increased expression of clfA that is associated with the hypercoagulable state of the host and increased virulence of S. aureus.

Antimicrobial resistance in coagulase-negative staphylococci from Nigerian traditional fermented foods.

BACKGROUND: Coagulase-negative staphylococci have become increasingly recognized as the etiological agent of some infections. A significant characteristic of coagulase-negative staphylococci especially strains isolated from animals and clinical samples is their resistance to routinely used antibiotics although, resistant strains isolated from fermented foods have not been fully reported. METHODS: A total of two hundred and fifty-five CoNS isolates were subjected to antimicrobial susceptibility test using the disc diffusion technique. The minimum inhibitory concentration of the isolates to the tested antibiotics was determined using the microbroth dilution method. Methicillin resistant strains were confirmed by detection of methicillin resistant genes (mecA) and also employing cefoxitin screening test. RESULTS: The isolates were confirmed to be methicillin resistant by the detection of mecA genes and the cefoxitin screening test. The isolates demonstrated appreciable resistance to ampicillin (86.7%), sulfomethoxazole-trimethoprim (74.9%), amoxicillin-clavulanic acid (52.5%) and oxacillin (35.7%). Methicillin resistance was exhibited by 13 out of the 255 isolates although no mecA gene was detected. It was also observed that the methicillin resistant isolates were prevalent in these traditional foods; iru, kindirmo, nono and wara. CONCLUSION: This study has ameliorated the incidence of multiple antibiotic resistant coagulase-negative staphylococci in Nigerian fermented foods and if not tackled adequately might lead to horizontal transfer of antibiotic resistance from food to man.

Molecular epidemiology of coagulase-negative bloodstream isolates: detection of Staphylococcus epidermidis ST2, ST7 and linezolid-resistant ST23

Abstract The mechanisms contributing to persistence of coagulase-negative staphylococci are diverse; to better understanding of their dynamics, the characterization of nosocomial isolates is needed. Our aim was to characterize phenotypic and molecular characteristics of Staphylococcus epidermidis and Staphylococcus haemolyticus human blood isolates from two tertiary care hospitals in Mexico, the Hospital Universitario in Monterrey and the Hospital Civil in Guadalajara. Antimicrobial susceptibility was determined. Biofilm formation was assessed by crystal violet staining. Detection of the ica operon and Staphylococcal Cassette Chromosome mec typing were performed by PCR. Clonal relatedness was determined by Pulsed-fiel gel electrophoresis and Multi locus sequence typing. Methicillin-resistance was 85.5% and 93.2% for S. epidermidis and S. haemolyticus, respectively. Both species showed resistance >70% to norfloxacin, clindamycin, levofloxacin, trimethoprim/sulfamethoxazole, and erythromycin. Three S. epidermidis and two S. haemolyticus isolates were linezolid-resistant (one isolate of each species was cfr+). Most isolates of both species were strong biofilm producers (92.8% of S. epidermidis and 72.9% of S. haemolyticus). The ica operon was amplified in 36 (43.4%) S. epidermidis isolates. SCCmec type IV was found in 47.2% of the S. epidermidis isolates and SCCmec type V in 14.5% of S. haemolyticus isolates. No clonal relatedness was found in either species. Resistance to clindamycin, levofloxacin, erythromycin, oxacillin, and cefoxitin was associated with biofilm production for both species (p < 0.05). A G2576T mutation in 23S rRNA gene was detected in an S. haemolyticus linezolid-resistant isolate. All linezolid-resistant S. epidermidis isolates belonged to ST23; isolate with SCCmec type IV belonged to ST7, and isolate with SCCmec type III belonged to ST2. This is the first report of ST7 in Mexico. There was a high genetic diversity in both species, though both species shared characteristics that may contibute to virulence.

Eventos estressores na família e indicativos de problemas de saúde mental em crianças com idade escolar / Stressor events in the family environment that are indicative of mental health problems in children of school age

Resumo O objetivo deste artigo é avaliar a relação entre eventos estressores ocorridos no último ano na família de crianças e adolescentes com indicativos de problemas de saúde mental em uma amostra de estudantes de duas escolas de uma cidade no sul do Brasil. Estudo transversal com 1.075 estudantes matriculados em duas escolas públicas de ensino fundamental (uma estadual e outra municipal). Foi utilizado o Strengths and Difficulties Questionnaire para avaliação de fatores emocionais e comportamentais da criança, e a Escala de Avaliação de Reajustamento Social de Holmes e Rahe (1967) para avaliar os eventos estressores. Foram utilizados o teste qui-quadrado e a regressão de Poisson, com ajuste robusto para variância, expressando os resultados em razão de prevalências (RP) e intervalos de confiança de 95%. As chances de apresentar problemas de hiperatividade foram 1,42 (IC 95% 1,10-1,83) vezes maiores no tercil intermediário e 1,37 (IC 95% 1,06-1,78) no tercil superior, quando comparados ao tercil inferior. Quanto aos problemas de relacionamento, as chances foram de 1,49 (IC 95% 1,15-1,93) vezes maiores no tercil superior ao serem comparados com o tercil inferior. Os resultados sugerem que fatores ambientais podem ser fortemente relacionados à etiologia dos transtornos mentais na infância e adolescência.

Abstract The scope of this article is to evaluate the relationship between stressor events that occurred last year in the family of children and adolescents that are indicative of mental health problems in a sample of students from two schools in a city in southern Brazil. It involved a cross-sectional study with 1,075 students enrolled in two public elementary schools. The Strengths and Difficulties Questionnaire was used to assess emotional and behavioral factors of the child and the Social Readjustment Rating Scale (SRRS) of Holmes and Rahe (1967) to assess stressor events. The chi-square and Poisson regression test with robust variance adjustment for expressing the results in the prevalence ratio (PR) and confidence intervals of 95% were used. The chances of presenting problems of hyperactivity were 1.42 (95% CI 1.10 to 1.83) times higher in the intermediate tercile and 1.37 (95% CI 1.06-1.78) in the higher tercile compared with the lower tercile. With respect to relationship problems the chances were of 1.49 (95% CI 1.15 to 1.93) times higher in the higher tercile when compared with the lower tercile. The results suggest that environmental factors may be strongly related to the etiology of mental disorders in childhood and adolescence.

An adaptive biointerface from self-assembled functional peptides for tissue engineering.

A self-assembled peptide-based biointerface is demonstrated with triple functional layers that can significantly improve the tissue self-healing process or prevent biofilm-mediated chronic inflammation. This smart biointerface is composed of three functional moieties (i.e., a cell-adhesive peptide, an infectious environment-responsive peptide, and an antifouling hexaethylene glycol (HEG) layer), and the resulting interface coated onto prosthetic replacements can smartly respond to the surrounding physiological or pathological microenvironment.

Technical note: a pilot study using a mouse mastitis model to study differences between bovine associated coagulase-negative staphylococci.

Coagulase-negative staphylococci (CNS) are a group of bacteria classified as either minor mastitis pathogens or commensal microbiota. Recent research suggests species- and even strain-related epidemiological and genetic differences within the large CNS group. The current pilot study investigated in 2 experiments whether a mouse mastitis model validated for bovine Staphylococcus aureus can be used to explore further differences between CNS species and strains. In a first dose titration experiment, a low inoculum dose of S. aureus Newbould 305 (positive control) was compared with increasing inoculum doses of a Staphylococcus chromogenes strain originating from a chronic bovine intramammary infection to a sham-inoculated mammary glands (negative control). In contrast to the high bacterial growth following inoculation with S. aureus, S. chromogenes was retrieved in very low levels at 24 h postinduction (p.i.). In a second experiment, the inflammation inflicted by 3 CNS strains was studied in mice. The host immune response induced by the S. chromogenes intramammary strain was compared with the one induced by a Staphylococcus fleurettii strain originating from cow bedding sawdust and by a S. chromogenes strain originating from a teat apex of a heifer. As expected, at 28 and 48 h p.i., low bacterial growth and local neutrophil influx in the mammary gland were induced by all CNS strains. As hypothesized, bacterial growth p.i. was the lowest for S. fleurettii compared with that induced by the 2 S. chromogenes strains, and the overall immune response established by the 3 CNS strains was less pronounced compared with the one induced by S. aureus. Proinflammatory cytokine profiling revealed that S. aureus locally induced IL-6 and IL-1ß but not TNF-α, whereas, overall, CNS-inoculated glands lacked a strong cytokine host response but also induced IL-1ß locally. Compared with both other CNS strains, S. chromogenes from the teat apex inflicted a more variable IL-1ß response characterized by a more intense local reaction in several mice. This pilot study suggests that an intraductal mouse model can mimic bovine CNS mastitis and has potential as a complementary in vivo tool for future CNS mastitis research. Furthermore, it indicates that epidemiologically different bovine CNS species or strains induce a differential host innate immune response in the murine mammary gland.

Development and evaluation of hexaplex PCR for rapid detection of methicillin, cadmium/zinc and antiseptic-resistant staphylococci, with simultaneous identification of PVL-positive and -negative Staphylococcus aureus and coagulase negative staphylococci.

We developed a multiplex PCR to detect the presence of methicillin- (mecA), cadmium/zinc-(czrC) and antiseptic-resistant (qacA/B) staphylococci and to identify Panton-Valentine leukocidin (PVL)-positive and -negative Staphylococcus aureus and coagulase-negative staphylococci (CoNS) from infected and healthy eyes. The assay was validated on 177 staphylococci comprising of 55 each of S. aureus and CoNS isolated from infected eyes and five S. aureus and 62 CoNS isolated from healthy eyes and nine direct ocular samples. Nine direct ocular samples for in situ testing consisted of corneal scrapings (4), conjunctiva swabs (2) and others (3). Multiplex PCR result was correlated with genotype data obtained with single PCR and dot-blot assay. The control strains that were positive in multiplex PCR for 16S rRNA, nuc, mecA, pvl, czrC and qacA/B genes were also positive in the dot-blot assay. The specificity of amplified genes obtained with reference strains was further confirmed by DNA sequencing. The single step-hexaplex PCR method can be used for rapid detection of mecA, nuc, pvl, czrC and qacA/B genes in staphylococci with simultaneous identification of PVL-positive and -negative S. aureus and CoNS from a variety of ocular samples.

Prevalence of enterotoxin-encoding genes and antimicrobial resistance in coagulase-negative and coagulase-positive Staphylococcus isolates from black pudding / Prevalência de genes codificadores de enterotoxinas e resistência antimicrobiana em estafilococos coagulase-negativo e coagulase-positivo isolados de morcilhas no sul do Brasil

INTRODUCTION: Staphylococcal species are pathogens that are responsible for outbreaks of foodborne diseases. The aim of this study was to investigate the prevalence of enterotoxin-genes and the antimicrobial resistance profile in staphylococcus coagulase-negative (CoNS) and coagulasepositive (CoPS) isolates from black pudding in southern Brazil. METHODS: Two hundred typical and atypical colonies from Baird-Parker agar were inoculated on mannitol salt agar. Eighty-two mannitol-positive staphylococci were submitted to conventional biochemical tests and antimicrobial susceptibility profiling. The presence of coagulase (coa) and enterotoxin (se) genes was investigated by polymerase chain reaction. RESULTS: The isolates were divided into 2 groups: 75.6% (62/82) were CoNS and 24.4% (20/82) were CoPS. The biochemical tests identified 9 species, of which Staphylococcus saprophyticus (37.8%) and Staphylococcus carnosus (15.9%) were the most prevalent. Antimicrobial susceptibility tests showed resistance phenotypes to antibiotics widely administered in humans, such as gentamicin, tetracycline, chloramphenicol, and erythromycin. The coa gene was detected in 19.5% (16/82) of the strains and 4 polymorphic DNA fragments were observed. Five CoNS isolates carrying the coa gene were submitted for 16S rRNA sequencing and 3 showed similarity with CoNS. Forty strains were positive for at least 1 enterotoxin-encoding gene, the genes most frequently detected were sea (28.6%) and seb (27.5%). CONCLUSIONS: The presence of antimicrobial resistant and enterotoxin-encoding genes in staphylococci isolates from black pudding indicated that this fermented food may represent a potential health risk, since staphylococci present in food could cause foodborne diseases or be a possible route for the transfer of antimicrobial resistance to humans.

INTRODUÇÃO: Estafilococos são patógenos responsáveis por surtos de doenças transmitidas por alimentos. O estudo investigou a prevalência de genes de enterotoxinas e o perfil de resistência aos antimicrobianos em estafilococos coagulase-negativo (CoNS) e estafilococos coagulase-positivo (CoPS) isolados de morcilhas no sul do Brasil. MÉTODOS: Duzentas colônias típicas e atípicas do ágar Baird-Parker foram inoculadas em ágar sal-manitol. Oitenta e dois estafilococos manitol-positivos foram submetidos a testes bioquímicos e perfil de susceptibilidade antimicrobiana. A presença dos genes da coagulase (coa) e enterotoxinas (se) foi investigada por reação em cadeia da polimerase (PCR). RESULTADOS: Os isolados foram divididos em dois grupos: 75,6% (62/82) CoNS e 24,4% (20/82) CoPS. Através dos testes bioquímicos, 9 espécies foram determinadas, Staphylococcus saprophyticus (37,8%) e Staphylococcus carnosus (15,9%) foram as mais prevalentes. Testes de susceptibilidade demostraram fenótipos de resistência aos antibióticos administrados em humanos, como gentamicina, tetraciclina, cloranfenicol e eritromicina. O gene coa foi detectado em 19,5% (16/82) das cepas e quatro fragmentos de DNA polimórficos foram observados. Cinco CoNS contendo o gene coa foram submetidos ao sequenciamento do 16S rRNA e três mostraram similaridade com CoNS. Quarenta amostras foram positivas para pelo menos um gene se, os mais frequentes foram sea (28,6%) e seb (27,5%). CONCLUSÕES: A presença de resistência aos antimicrobianos e de genes se nos isolados de morcilha indicou que este alimento pode representar um risco potencial à saúde, já que a presença nos alimentos pode causar doenças de origem alimentar ou ser uma possível rota de transferência de estafilococos resistentes aos humanos.

Bacteremias por Staphylococcus coagulase negativos oxacilina resistentes em um hospital escola na cidade de Santa Maria, Estado do Rio Grande do Sul / Oxacillin-resistant coagulase-negative Staphylococci bacteremia at a teaching hospital in Santa Maria, State of Rio Grande do Sul, Brazil

INTRODUÇÃO: Neste estudo, objetivou-se caracterizar a prevalência e o perfil de suscetibilidade de cepas de Staphylococcus coagulase negatives resistentes à oxacilina isoladas de culturas de sangue, em um hospital escola, localizado na Cidade de Santa Maria. Além disso, buscou-se comparar ao teste genotípico de referência, diferentes metodologias fenotípicas para a caracterização da resistência mediada pelo gene mecA. MÉTODOS: Após identificação (MicroScan® - Siemens), os isolados foram submetidos a testes de sensibilidade antimicrobiana a partir da difusão do disco e automação (MicroScan® - Siemens). A presença do gene mecA foi evidenciada através da técnica molecular de reação em cadeia da polimerase. RESULTADOS: A espécie prevalente foi Staphylococcus epidermidis (67 por cento). O gene mecA foi detectado em 90 por cento das cepas e conforme análise dos perfis de sensibilidade, observou-se um índice elevado de resistência a várias classes de antimicrobianos. Contudo, todos os isolados mostraram-se uniformemente sensíveis à vancomicina e tigeciclina. O disco de cefoxitina foi a metodologia fenotípica que melhor correlacionou-se com o padrão ouro. CONCLUSÕES: A análise da significância clínica de SCN isolados de hemoculturas e a detecção precisa da resistência à oxacilina representam fatores decisivos para a instituição correta da antibioticoterapia. Apesar da vancomicina constituir o tratamento usual na maioria dos hospitais brasileiros, tem a redução de seu emprego recomendada.

INTRODUCTION: This study aimed to characterize the prevalence and susceptibility profile to oxacillin-resistant Coagulase-negative Staphylococci strains isolated from blood cultures in a teaching hospital, located in Santa Maria, RS. In addition, different methodologies for phenotypic characterization of mecA-mediated oxacillin resistance were compared with genotypic reference testing. METHODS: After identification (MicroScan® - Siemens), the isolates were tested for antimicrobial sensitivity using disk diffusion and automation (MicroScan® - Siemens). The presence of mecA gene was identified by the polymerase chain reaction molecular technique. RESULTS: The most common species was Staphylococcus epidermidis (n=40, 67 percent). The mecA gene was detected in 54 (90 percent) strains, while analysis of the sensitivity profiles revealed a high rate of resistance to multiple classes of antimicrobial drugs. However, all isolates were uniformly sensitive to vancomycin and tigecycline. The cefoxitin disk was the phenotypic method that best correlated with the gold standard. CONCLUSIONS: Analysis of the clinical significance of CoNS isolated from hemocultures and the precise detection of oxacillin resistance represent decisive factors for the correct choice of antibiotic therapy. Although vancomycin constitutes the normal treatment in most Brazilian hospitals, reduction in its use is recommended.

Glutathione-bound gold nanoclusters for selective-binding and detection of glutathione S-transferase-fusion proteins from cell lysates.

A straightforward method for the rapid detection of the presence of glutathione S-transferase (GST)-tagged proteins from sample solutions using glutathione (GSH)-bound gold nanoclusters (Au@GSH NCs) with luminescence properties as the detection probes by simple observation with the naked eye was proposed in this study.

[Detection of inducible resistance to clindamycin in cutaneous isolates of Staphylococcus spp. by phenotypic and genotypic methods]. / Detección de resistencia inducible a clindamicina en aislados cutáneos de Staphylococcus spp. por métodos fenotípicos y genotípicos.

INTRODUCTION: Resistance to macrolides, lincosamides and type B streptogramins (MLSB) in Staphylococcus isolates can be due to several mechanisms. The most important are an active efflux mechanism (MSB phenotype) and ribosomal target modification (MLSB phenotype); this latter mechanism confers resistance to all three groups of antimicrobials (MLSB resistance). Expression of MLSB resistance can be constitutive (cMLSB) or inducible (iMLSB). METHODS: A group of 117 erythromycin-resistant Staphylococcus spp. clinical isolates from cutaneous samples were selected from 536 recent clinical isolates of this microorganism. Resistance phenotypes were determined by the double disk diffusion test. Presence of the ermA, ermC, ermB and msrA genes was detected by real time PCR. RESULTS: The MSB phenotype was the most common, comprising 11.2% (7.2% in S. aureus and 23% in CoNS) of the erythromycin-resistant strains. The rate of iMLSB resistance was significantly higher, 7.4% (5.2% in S. aureus and 14% in CoNS), than the rate of cMLSB resistance, 3.2% (1.7% in S. aureus and 7.4% in CoNS). The msrA gene was present in all isolates with the MSB phenotype, and the ermC gene was the most common among clindamycin-resistant strains with the MLSB phenotype (constitutive or inducible). CONCLUSION: The good correlation between the phenotypic (disk-diffusion) and genotypic (real time PCR) methods used allows prediction of the mechanisms of erythromycin and clindamycin resistance, provides insight into the epidemiological differences in their distribution, and is an aid to selecting the most appropriate antimicrobial therapy.

Protective immune responses to a multi-gene DNA vaccine against Staphylococcus aureus.

To investigate the strategy of using a multivalent polyprotein DNA vaccine against Staphylococcus aureus, a series of plasmids was used to immunize mice followed by infectious challenge. The plasmid vaccines expressed Clumping factor A (Clfa), fibronectin binding protein A (FnBPA) and the enzyme Sortase (Srt) as single proteins or combined as a polyprotein. All animals produced a mixed Th1 and Th2 response including functional antigen-specific, mostly IgG2a antibodies, sustained production of IFN-gamma and a predominantly CD8+ T-cell response. Upon challenge with a virulent S. aureus isolate (Sa042), after 21 days, 55% of the multi-gene vaccinated mice survived infection compared to only 15% of the control groups. Vaccinated mice showed no signs of arthritis when challenged with the less virulent "Newman" strain that caused reactive arthritis in the controls. The results suggest that a multi-gene polyprotein-expressing nucleic acid vaccine alone produces a combined Th1 and Th2 response that can contribute to protection against the complex pathogenesis of S. aureus.

Prevalence of macrolide resistance genes among staphylococci in Cyprus.

The aims of the present study were to evaluate the frequency of macrolide-resistant staphylococci in Cyprus and to examine the phenotypic and genotypic characteristics of these isolates. Antimicrobial susceptibility testing was performed by broth microdilution method and the macrolide resistance determinants were detected by PCR. The relatedness among the isolates was examined by pulsed-field gel electrophoresis. Ninety-six (67.61%) of the 142 Staphylococcus aureus and 19 (59.4%) of the 32 coagulase-negative staphylococci were resistant to erythromycin. Among the 115 erythromycin-resistant staphylococci, 70 expressed the MLSB-inducible phenotype, 38 the MLSB-constitutive, and 7 the MS. The predominant genes associated with macrolide resistance were the ermA for S. aureus and the ermC for coagulase-negative staphylococci, detected in 90.62% and 47.37% of the isolates respectively. Dissemination of one clone carrying the ermA gene accounted for macrolide resistance in the majority of S. aureus isolates.

RAPD for the typing of coagulase-negative staphylococci implicated in catheter-related bloodstream infection.

OBJECTIVES: A rapid random amplification of polymorphic DNA (RAPD) technique was developed to distinguish between strains of coagulase-negative staphylococci (CoNS) involved in central venous catheter (CVC)-related bloodstream infection. Its performance was compared with that of pulsed-field gel electrophoresis (PFGE). METHODS: Patients at the University Hospital Birmingham NHS Foundation Trust, U.K. who underwent stem cell transplantation and were diagnosed with CVC-related bloodstream infection due to CoNS whilst on the bone marrow transplant unit were studied. Isolates of CoNS were genotyped by PFGE and RAPD, the latter employing a single primer and a simple DNA extraction method. RESULTS: Both RAPD and PFGE were highly discriminatory (Simpson's diversity index, 0.96 and 0.99, respectively). Within the 49 isolates obtained from blood cultures of 33 patients, 20 distinct strains were identified by PFGE and 25 by RAPD. Of the 25 strains identified by RAPD, nine clusters of CoNS contained isolates from multiple patients, suggesting limited nosocomial spread. However, there was no significant association between time of inpatient stay and infection due to any particular strain. CONCLUSION: The RAPD technique presented allows CoNS strains to be genotyped with high discrimination within 4h, facilitating real-time epidemiological investigations. In this study, no single strain of CoNS was associated with a significant number of CVC-related bloodstream infections.