Influence of the AgrC-AgrA Complex on the Response Time of Staphylococcus aureus Quorum Sensing

The Staphylococcus aureus agr quorum-sensing system plays a major role in the transition from the persistent to the virulent phenotype. S. aureus agr type I to IV strains are characterized by mutations in the sensor domain of the histidine kinase AgrC and differences in the sequences of the secreted autoinducing peptides (AIP). Here we demonstrate that interactions between the cytosolic domain of AgrC (AgrC(Cyto)) and the response regulator domain of AgrA (AgrA(RR)) dictate the spontaneity of the cellular response to AIP stimuli. The crystal structure of AgrC(Cyto) provided a basis for a mechanistic model of AgrC-AgrA interactions. This model enabled an analysis of the biochemical and biophysical parameters of AgrC-AgrA interactions in the context of the conformational features of the AgrC-AgrA complex. This analysis revealed distinct sequence and conformational features that determine the affinity, specificity, and kinetics of the phosphotransfer reaction. This step, which governs the response time for transcriptional reengineering triggered by an AIP stimulus, is independent of the agr type and similar for agonist and antagonist stimuli. These experimental data could serve as a basis on which to validate simulations of the quorum-sensing response and for strategies that employ the agr quorum-sensing system to combat biofilm formation in S. aureus infections.

Levels of Alpha-Toxin Correlate with Distinct Phenotypic Response Profiles of Blood Mononuclear Cells and with agr Background of Community-Associated Staphylococcus aureus Isolates

Epidemiological studies of Staphylococcus aureus have shown a relation between certain clones and the presence of specific virulence genes, but how this translates into virulence-associated functional responses is not fully elucidated. Here we addressed this issue by analyses of community-acquired S. aureus strains characterized with respect to antibiotic resistance, ST types, agr types, and virulence gene profiles. Supernatants containing exotoxins were prepared from overnight bacterial cultures, and tested in proliferation assays using human peripheral blood mononuclear cells (PBMC). The strains displayed stable phenotypic response profiles, defined by either a proliferative or cytotoxic response. Although, virtually all strains elicited superantigen-mediated proliferative responses, the strains with a cytotoxic profile induced proliferation only in cultures with the most diluted supernatants. This indicated that the superantigen-response was masked by a cytotoxic effect which was also confirmed by flow cytometry analysis. The cytotoxic supernatants contained significantly higher levels of alpha-toxin than did the proliferative supernatants. Addition of alpha-toxin to supernatants characterized as proliferative switched the response into cytotoxic profiles. In contrast, no effect of Panton Valentine Leukocidin, delta-toxin or phenol soluble modulin alpha-3 was noted in the proliferative assay. Furthermore, a significant association between agr type and phenotypic profile was found, where agrII and agrIII strains had predominantly a proliferative profile whereas agrI and IV strains had a predominantly cytotoxic profile. The differential response profiles associated with specific S. aureus strains with varying toxin production could possibly have an impact on disease manifestations, and as such may reflect specific pathotypes.

Structural Basis for Feed-Forward Transcriptional Regulation of Membrane Lipid Homeostasis in Staphylococcus aureus

11 p.

Avaliação da resposta imune a Staphylococcus aureus, Escherichia coli e Candida albicans em pacientes com neuromielite óptica e sua correlação com o grau de incapacidade neurológica

A Neuromielite Óptica (NMO), anteriormente considerada como um subtipo de Esclerose Múltipla, é uma doença autoimune, inflamatória do sistema nervoso central, na qual o sistema imune ataca a mielina dos neurônios localizados nos nervos ópticos e medula espinhal, produzindo, então, mielite e neurite óptica simultânea ou sequenciais. A patogênese da neuromielite óptica é influenciada pela combinação de fatores genéticos e ambientais, incluindo agentes infecciosos. Diferentes doenças infecciosas podem tanto desencadear como exacerbar a autoimunidade. Portanto, o objetivo do presente estudo foi de analisar a responsividade imune in vitro a Escherichia coli, Staphylococcus aureus e Candida albicans em pacientes com NMO recorrente-remitente, e a correlacionar ao nível de incapacidade neurológica. Nesse contexto, a extensão da linfoproliferação e perfil de citocinas em resposta a S. aureus e C. albicans, em culturas de células mononucleares do sangue periférico (CMSP) foram similares entre pacientes com NMO e indivíduos saudáveis. Entretanto, maior proliferação de células T associada à elevada liberação de IL-1β, IL-6 e IL-17 foi observada em culturas de células derivadas de pacientes com NMO quando estimuladas com E. coli. Ademais, nessas culturas, a produção de IL-10 foi significativamente menor quando comparada ao grupo controle. Ensaios conduzidos em culturas de CMSP depletadas de diferentes subtipos de linfócitos demonstraram que, enquanto células T CD4+ e T CD8+ produzem IL-6 em resposta a E. coli, a produção de IL-17 foi praticamente restrita às células T CD4+. Os níveis de IL-6 e IL-17 in vitro induzidos por E. coli foram correlacionados positivamente às incapacidades neurológicas. Essa maior tendência a produzir citocinas relacionadas ao perfil Th17 foi diretamente associada aos níveis de IL-23 produzidos por monócitos ativados com LPS. De modo interessante, níveis elevados de LPS foram quantificados no plasma de pacientes com NMO e estes foram correlacionados aos níveis plasmáticos de IL-6. Em conclusão, nossos resultados sugerem que uma maior responsividade a E. coli poderia estar envolvida na patogênese da NMO. Esse tipo de investigação é muito importante pois inibidores da ligação ou sinalização do TLR poderiam ser considerados terapias com grande potencial como adjuvantes no tratamento de pacientes com NMO.

Determinação do perfil fenotípico e genotípico de amostras de Staphylococus aureus resistentes à meticilina (MRSA) e sensíveis a antibióticos não ß-lactâmicos em cinco hospitais no município do Rio de Janeiro

Staphylococcus aureus resistente à meticilina (MRSA) é um dos principais microrganismos envolvidos nas Infecções relacionadas à Assistência à Saúde (IrAS). Porém, um clone de MRSA, o CA-MRSA, emergiu na comunidade e atualmente vem sendo agente de IrAS. O objetivo desta dissertação é avaliar fenotípica e genotipicamente 111 amostras de Staphylococcus aureus resistentes à meticilina e sensíveis a antibióticos não ß-lactâmicos de pacientes atendidos em cinco hospitais no município do Rio de Janeiro. Utilizando os critérios padronizados pelo CLSI 2012, foram determinadas as susceptibilidades a 11 antimicrobianos pelo método de disco difusão em ágar e concentração inibitória mínima para vancomicina e oxacilina pelo método da microdiluição em caldo. A multirresistência (resistência a 3 ou mais antimicrobianos não ß-lactâmicos) foi observada em 31,5% das amostras, sendo que 53,2% apresentaram resistência ao antimicrobiano clindamicina, uma das opções para o tratamento empírico das infecções de pele/tecidos moles. 86,4% apresentaram concentração inibitória mínima (CIM) para vancomicina ≥ 1,0 g/mL ou seja, elevado percentual de amostras associadas ao fenômeno MIC creep, o qual está associado ao insucesso na terapia antimicrobiana anti-MRSA. Não foi observado até o momento nenhuma amostra com CIM ≥ 4cg/mL para vancomicina, entretanto, já há resistência à linezolida em quatro hospitais do estudo. A tipificação do SCCmec nos permitiu classificar 4,5% das amostras em HA-MRSA e 86,5% em CA-MRSA, nas quais a resistência heterogênea típica à oxacilina foi observada em 57,2%. A toxina de Panton-Valentine (PVL) foi identificada pela metodologia de PCR em 28% das amostras com genótipo CA-MRSA. Os fatores de riscos clássicos, da literatura, relacionados à infecção por HA-MRSA foram também observados nos pacientes com infecção por CA-MRSA portadoras de SCCmec IV e V. No intuito de verificar a existência de similaridades genéticas ou a presença de clone predominante entre as amostras dos cinco hospitais, foi realizada a técnica de eletroforese em gel sob campo pulsado (PFGE) e observou-se diversidade genética assim como a presença de amostras com padrões similares aos clones OSPC (18,5%) e USA400. Não foram encontradas amostras com padrões de eletroforese similares aos clones USA300, USA800 e CEB. É essencial a vigilância da resistência aos antimicrobianos não ß-lactâmicos no CA-MRSA, em especial à vancomicina. A mudança na epidemiologia deste microrganismo vem impactando os padrões característicos dos genótipos limitando os critérios de diferenciação entre eles. Neste contexto, as técnicas moleculares atuam como excelentes ferramentas de caracterização. O conhecimento do patógeno auxilia na elaboração e implementação de medidas preventivas, contribuindo para o controle da doença tanto no ambiente hospitalar quanto na comunidade.

Antibiotic sensitivity of Staphylococcus aureus strains isolated from fishery products

During the course of an investigation on the incidence of Staphylococcus aureus contamination in frozen crab meat, frozen prawns and dried fishes from Cochin, 116 strains of S. aureus were isolated. The sensitivity of the isolated strains towards nine antibiotics showed that all the S. aureus strains were sensitive to kanamycin and streptomycin (100%). Sensitivity to other antibiotics like chloramphenicol, polymyxin-B, erythromycin, tetracycline, neomycin, penicillin and ampicillin were shown by 98.28, 93.10, 87.93, 68.10, 67.24, 56.90 and 55.17 percent of the isolates respectively.

Effect of liquid nitrogen freezing and subsequent storage on survival of Staphylococcus aureus and Streptococcus pyogenes in treated prawn meat

Prawn meat treated with Streptococcus pyogenes B-49-2 culture and Staphylococcus aureus ATCC-12598 culture were frozen in conventional plate freezer at -40°C and by spray type liquid nitrogen freezer. The frozen products were stored at -18°C. Streptococcus pyogenes B-49-2 showed low sensitivity to cold injury during freezing and frozen storage. Staphylococcus aureus ATCC-12598 survived during the entire storage period of 240 days. Total bacterial count of untreated prawn meat was found to be always lesser in liquid nitrogen frozen products than that in plate frozen products.

Antibiotic resistance of Staphylococcus aureus strains isolated from fish processing factory workers

One hundred and twenty two strains of Staphylococcus aureus isolated from throats and palms of 39 workers from 6 fish processing factories situated in and around Cochin were tested for their sensitivity to nine commonly used antibiotics-ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin, penicillin, polymyxin-B, streptomycin and tetracycline. Highest percentage of resistance was observed towards ampicillin followed by penicillin i.e. 64.75% and 59.84%. Resistance towards other antibiotics like tetracycline, polymyxin-B, erythromycin, kanamycin, neomycin, chloramphenicol and streptomycin were shown by 22.95, 16.39, 7.38, 5.74, 3.28 and 1.64% of the isolates respectively.

Application of encapsulated nisin and sodium citrate for shelf life increasing of Rainbow trout (Onchorhynchus mykiss) fillet packaged with MAP and their effect on Staphylococcus aureus

Nisin is a widely used naturally occurring antimicrobial effective against many pathogenic and spoilage microorganisms. It has been proposed that reduced efficacy of nisin in foods can be improved by technologies such as encapsulation to protect it from interferences by food matrix components. The aim of this study was using of spray dried encapsulated nisin with zein in concentration of (0.15 and 0.25 g/kg) and sodium citrate (1.5 and 2.5%) and treatments with both of them to extent the shelf life of filleted trouts packaged by Modified Atmosphere Packaging (45% CO2, 50% N2 ,5% O2) and stored at 4±1 °C for 20 days. Furthermore, to evaluate the antimicrobial efficiency of encapsulated nisin and soudium citrate the trouts fillets was inoculated with Staphylococcus aureus as an index pathogenic bacteria. Assessment of chemical spoilage indexes such as (Proxide value, Thiobarbituric acid, total volatile base nitrogen and pH) , microbial parameters (Total Plate Count, Psychrotrophic count, Lactic acid bacteria count), Staphylococcus aureus cont in treatments which were inoculated with 5 logcfu/g of this bacteria and sensory evaluation of fillets including (smell, color, texture and total acceptability) was carried out in days of 0, 4, 8, 12, 16 and 20. The results revealed that treatment with both exposure of nisin and sodium citrate showed significantly lower chemical spoilage indexes in comparison with controls (vaccum packed and MAP) (P<0.05). Furthermore, (nisin 0.25 g/kg sodium citrate 2.5%) treatment which was exposed to the maximal level used of both materials was significantly the lowest treatment with (Proxide value, Thiobarbituric acid, total volatile base nitrogen and pH) of 9.95 (meq O2/kg) , 1.55 (mgMA/kg), 29.65 (mgN/100g) and 6.65 , respectively and according to the maximal recommended level of this indices , shelf life of fillets in this treatment was esstimated 20 days.The control (vaccum packed) treatment was significantly the highest treatment with (Proxide value, Thiobarbituric acid, total volatile base nitrogen and pH) of 15.17 (meq O2/kg), 3.03 (mgMA/kg), 38.4 (mgN/100g) and 6.95 , respectively and according to the maximal recommended level of this indices , shelf life of fillets in this treatment was estimated 11 days. Also, in microbial point of view (nisin 0.25 g/kg- sodium citrate 2.5%) treatment was the lowest treatment with Total Plate Count, Psychrotrophic count, Lactic acid bacteria count and Staphylococcus aureus count of 6.7, 6.83, 5.25 and 6.04 logcfu/g respectively, and conrol (vaccum packed) treatment was the highest treatment with 9.15, 9.41, 7.7 and 9.01 logcfu/g respectively. According to the lower results of chemical and microbial indices and higher sensory evaluated scores assessed in this research for encapsulated nisin in comparison with free nisin , it was concluded that encapsulation of nisin with zein capsules may improve the efficiency of nisin. The measuremented values of Mass yield, Total solids content of capsules, Encapsulation efficiency, In vitro release kinetics in 200 hour for encapsulated nisin in this study was 49.89, 62, 98.31 and 69% respectively and Encapsulated particle size was lower than 674.21 μm for 90% of particles. As a consequence, nisin , in particular encapsulated nisin, and sodium citrate alone or together with and Modified Atmosphere packaging might be considered as effective tools in preventing the quality degradation of the fillets, resulting in an extension of their shelf life.

Effects of Zataria multiflora Boiss essential oil and nisin on the growth of Staphylococcus aureus in light salted silver carp fillet (Hypophthalmichthys molitrix)

There is an increasing demand in developing newer and safer methods in preserving food products.Among which herbal additives seem to attract evermore attention recently.the major advantage of herbal additives is due to their favorable aroma besides their antimicrobial effects and less expensive than chemical additives. Zataria multiflora Boiss is a native Iranian herb which is used vastly as a food preserver essential oils and also medical usage. Metabolites of harmless bacteria, such as Nisin are also known to be safe preservatives that have antimicrobial activity. However to establish the usefulness of natural antimicrobial preservatives, they must be evaluated alone and in combination with other preservation factors to determine whether there are synergistic effects in rigid media . In this study were evaluated the effects of different concentrations of Zataria multiflora (EO 0, 0.005, 0.015, 0.045, 0.135, 0.405 ,0.810 %) and Nisin(0, 0.15, 0.25, 0.75 μg/ml) and Storage time (up to 21 days) on growth of Staphylococcus aureus ATCC 6538 in a food model system(light salted fish of silver carp, Hypophthalmichthys molitrix). The results on growth of S. aureus were evaluated using SPSS 15.0 statistical software (SPSS 15.0 for windows, SPSS Inc.) and analyzed the logarithm of total count of the bacteria by Tukey Test. Results were considered statistically significant when P≤0.05. The growth of Staphylococcus aureus was affected significantly(P<0.05) by EO and Nisin and also combinations of EO and Nisin. Samples treated with 0.135, 0.405 and 0.810% of thyme essential oil showed a significant decrease on the growth of the bacteria compared with an treated samples(P<0.05). No significant difference was seen on the growth of S.aureus in samples treated with lower concentrations of Z.multiflora(below 0.045%) and untreated group(P>0.05). The most inhibitory effects were seen in samples treated with 0.405% and 0.810% of thyme essential oil until 9 and 12 days after storage,respectively. Also there was significant inhibtory effect(P<0.05) in different concentration of nisin on the organism compared with an treated samples. The synergism effects of the Eo and nisin on the growth rate of the bacteria was significant (P<0.05) compared with untreated samples and samples treated with the Eo or nisin, only. Synergismic effects was observed at concentration of 0.405 and 0.810% of Z. multiflora essential oil with 0.25 μg/ml Nisin, respectively until 15 days after storage. As expected it is preferred to apply the least possible amounts of additives in food preserving that not only are effective and safe but are economically justifiable.

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2

Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-kappa B, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-kappa B activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but not TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1). antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and triggers innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules. (C) 2007 Elsevier Ltd. All rights reserved.

Qualidade microbiológica de salames tipo colonial comercializados na cidade de Concórdia-SC: análise de Staphylococcus aureus e Toxoplasma gondii.

2006

Base pairing interaction between 5′- and 3′-UTRs controls icaR mRNA translation in Staphylococcus aureus

UPNa. Instituto de Agrobiotecnología. Laboratorio de Biofilms Microbianos.