Virulence factors in vancomycin-resistant and vancomycin- susceptible Enterococcus faecalis from Brazil

Enterococci are members of commensal flora of animals and insects, but are also important opportunistic pathogens. Our objective was to observe if there was any difference of virulence in several groups of E. faecalis, mainly between vancomycin-resistant E. faecalis (VREFS) of colonization and infection. VREFS and vancomycin-sensitive E. faecalis from Brazil were screened for the presence of virulence factor genes. Phenotypic assays were used to assess in vitro expression, to understand the pathogenic potential of these isolates and to determine whether a correlation exists between virulence and antibiotic resistance. Different virulence profiles were found suggesting that the disseminating clone may have generated several variations. However, our study showed that one constellation of traits appeared most commonly: gelatinase, aggregation substance and esp (GEA). These factors are important because they have been implicated in cell aggregation and biofilm formation. Biofilm formation may promote the conjugation of plasmids harboring resistance and virulence genes, enhancing the probability of entry of new resistance genes into species. Curiously, the profile GEA was not exclusive to VREFS, it was the second most observed in VSEFS isolates from colonization and infection in hospitalized patients and also from rectal swabs of healthy volunteers. Such strains appear to represent the entry gateway to new resistance genes into E. faecalis and may contribute to the spreading of E. faecalis mainly in hospitals

Two outbreaks of mixed etiology associated with central venous catheters inserted by phlebotomy in critical neonates

Staphylococcus aureus and coagulase-negative staphylococci are the main cause of sepsis in Neonatal Intensive Care Unit (NICU). Central venous catheters (CVCs) are an important part of critical neonates' treatment and are associated with sepsis. The aim of this study was to investigate two outbreaks caused by Staphylococcus aureus and Staphylococcus epidermidis associated with CVC inserted by phlebotomy in critical neonates. The surveillance was performed from January 2001 to December 2005 at the Brazilian NICU. The genotypic analysis of oxacillin susceptible S. aureus (OSSA) and oxacillin resistant S. epidermidis (ORSE) was performed based on pulsed-field gel electrophoresis (PFGE). Staphylococcus was the most frequent pathogen (65.8%) with highest incidence of CoNS (59.9%) followed by S. aureus (40.1%). During the five years of surveillance, there were two outbreaks detected, occurred in January-February/02 and August/02 and confirmed by PFGE analysis. The predisposing factors for infection corresponding to both outbreaks were: age <7 days, hospitalization > 7 days, and use of polyethylene CVC through dissection of vein (phlebotomy). This is the first relate of staphylococcal outbreaks associated with CVC inserted by phlebotomy in NICU. PFGE showed polyclonal spread of OSSA during both epidemic and endemic period, and two monoclonal outbreaks of ORSE in the same epidemic period of OSSA

Predominance of CTX-M–type extended-spectrum β-lactamase genes among enterobacterial isolates from outpatients in Brazil

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Mutations in the quinolone resistance-determining regions of gyrA and parC in Enterobacteriaceae isolates from Brazil

Mutations in the quinolone resistance-determining regions (QRDR) in chromosomal gyrA and parC genes and fluoroquinolone susceptibility profiles were investigated in quinolone-resistant Enterobacteriaceae isolated from community and hospitalized patientsin the Brazilian Southeast region. A total of 112 nalidixic acid-resistant enterobacterial isolates collected from 2000 to 2005 were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the QRDR regions. Susceptibility to fluoroquinolones was tested by the agar dilution method. Amongst the 112 enterobacterial isolates, 81 (72.3%) were resistant to ciprofloxacin and 5 (4.5%) showed reduced susceptibility. Twenty-six (23.2%) were susceptible to ciprofloxacin. Several alterations were detected in gyrA and parC genes. Escherichia coli isolates (47.7%) showed double mutations in the gyrA gene and a single one in the parC gene. Two unusual aminoacid substitutions are reported, an Asp87-Asn in a Citrobacter freundii isolate with reduced susceptibility to fluoroquinolones and a Glu84-Ala in one E. coli isolate.Only a parC gene mutation was found in fluoroquinolone-susceptible Enterobacter aerogenes. None of the isolates susceptible to ciprofloxacin presented mutations in topoisomerase genes. This comprehensive analysis of QRDRs in gyrA and parC genes, covering commonly isolated Enterobacteriaceae in Brazil is the largest reported up to now

Biological Activity Of Serratia Marcescens Cytotoxin.

Serratia marcescens cytotoxin was purified to homogeneity by ion-exchange chromatography on a DEAE Sepharose Fast Flow column, followed by gel filtration chromatography on a Sephadex G100 column. The molecular mass of the cytotoxin was estimated to be about 50 kDa. Some biological properties of the cytotoxin were analyzed and compared with well-characterized toxins, such as VT1, VT2 and CNF from Escherichia coli and hemolysin produced by S. marcescens. The sensitivity of the cell lines CHO, HeLa, HEp-2, Vero, BHK-21, MA 104 and J774 to the cytotoxin was determined by the cell viability assay using neutral red. CHO and HEp-2 were highly sensitive, with massive cellular death after 1 h of treatment, followed by BHK-21, HeLa, Vero and J774 cells, while MA 104 was insensitive to the toxin. Cytotoxin induced morphological changes such as cell rounding with cytoplasmic retraction and nuclear compactation which were evident 15 min after the addition of cytotoxin. The cytotoxic assays show that 15 min of treatment with the cytotoxin induced irreversible intoxication of the cells, determined by loss of cell viability. Concentrations of 2 CD50 (0.56 g/ml) of purified cytotoxin did not present any hemolytic activity, showing that the cytotoxin is distinct from S. marcescens hemolysin. Antisera prepared against S. marcescens cytotoxin did not neutralize the cytotoxic activity of VT1, VT2 or CNF toxin, indicating that these toxins do not share antigenic determinants with cytotoxin. Moreover, we did not detect gene sequences for any of these toxins in S. marcescens by PCR assay. These results suggest that S. marcescens cytotoxin is not related to any of these toxins from E. coli.36351-

Ribotyping on molecular epidemiology of bacterial infections

A tipagem molecular do genoma bacteriano, na maioria das vezes, envolve a análise de fragmentos de restrição do DNA cromossômico. Desde que a ribotipagem foi descrita, em 1986, tem sido amplamente utilizada para analisar relações taxonômicas e/ou epidemiológicas entre os diferentes grupos de organismos. A ribotipagem usa o padrão de restrição do opéron de RNA ribossômico (rrn) como ferramenta epidemiológica e tem fornecido ótimos resultados para a detecção de polimorfismo do comprimento dos fragmentos de restrição (RFLPs). O número de opérons rrn da bactéria está diretamente relacionado ao potencial discriminatório da técnica, fornecendo um maior ou menor número de bandas. &nbsp; &nbsp;Molecular typing usually involves restriction fragment analysis of chromossomal DNA. Since 1986, ribotyping has been used to analyse taxonomic and/or epidemiological relationship among microrganisms. Ribotyping uses, as an epidemiological tool, the restriction profile of the ribosomal RNA operons and it has provided good results in RFLP detection. The number of bacterial rrn operons correlates with the discriminatory potential of the technique

Bacteriological diagnostic of urinary tract infections: A technical revision

Urinary tract infection is one of the most common diseases and it can affect more than one site or one single place like urethra (urethritis), prostate (prostatitis), urinary bladder (cystitis) or kidney (pielonephritis). Urine is considered sterile and may suffer bacterial contamination from skin, clothes or external genitals, so it should be collected, kept and transported appropriately, to avoid false results in laboratory analysis. Urethritis and cistitis non gonococcal are commonly caused by members of Enterobacteriacea family but Escherichia coli is the casual agent of approximately 80% of the cases between fertile age women without urinary tract leison. Other microrganisms including Klebsiella sp., Enterobacter sp., Proteus sp., Pseudomonas sp and the Enterococcus sp are frequently found in patients with obstructive leisons or paralytical diseases affecting the renal function. Staphylococcus saprophyticus is an important opportunistic pathogenin human urinary tract infections, especially in young, sexually active females. The patient must be informed about the recommended procedures related to the collect time, way of obyaining and all the necessary asepsis, such as the professional must be up to date about the techniques utilized for the isolation, identification and susceptibility test of the microrganism. Currently, there are chemical and authomatical methods and excelent kits for laboratorial diagnosis of urinary infections, helping and accelerating the process of identification and efficient treatment to the infected patient.Entre as doenças mais comuns está a infecção do trato urinário, afetando mais de um sítio ou um único local como a uretra (uretrite), próstata (prostatite), bexiga (cistite) ou rins (pielonefrite). A urina é considerada estéril e pode sofrer contaminação de bactérias da pele, da roupa ou da genitália. Por isso, se não colhida, armazenada e transportada adequadamente, pode-se obter falsos resultados em exames bacteriológicos. Bactérias da família Enterobacteriacea estão envolvidas em quase todas as uretrocistites não gonocócicas, sendo a Escherichia coli o agente causal de aproximadamente 80% dos casos entre mulheres na idade fértil, sem lesões do trato urinário. Outros microrganismos, incluindo Klebsiella sp., Enterobacter sp., Proteus sp., Pseudomonas sp. e Enterococcus sp., são freqüentemente encontrados em pacientes com lesões obstrutivas ou doenças paralíticas, afetando a função renal. Staphylococcus saprophyticus é um importante patógeno oportunista na infecção do trato urinário em humanos, especialmente em mulheres jovens, sexualmente ativas. O paciente deve ser informado quanto aos procedimentos recomendados, relacionados com o horário da colheita, modo de obtenção e toda a assepsia necessária, assim como o profissional deve estar bem atualizado quanto às técnicas utilizadas para o isolamento, identificação e antibiograma. Atualmente, existem métodos químicos automatizados e kits excelentes para o diagnóstico presuntivo de infecções urinárias, auxiliando e agilizando os processos de identificação e de tratamento eficaz ao paciente infectado

Sequence Analysis of Enterococcus faecium Strain 10/96A (VanD4), the Original Vancomycin-Resistant E. faecium Strain in Brazil

Enterococcus faecium strain 10/96A (VanD4) was the first vancomycin-resistant enterococcus (VRE) isolated in Brazil. Subsequent Brazilian VRE strains have all had the VanA phenotype. Multilocus sequence typing showed that strain 10/96A was isolated sporadically, has a unique sequence type (ST 281), and was not the progenitor of the VRE strains isolated from hospital outbreaks in Brazil

Disentangling the initiation from the response in joint attention: An eye-tracking study in toddlers with autism spectrum disorders

Joint attention (JA), whose deficit is an early risk marker for autism spectrum disorder (ASD), has two dimensions: (1) responding to JA and (2) initiating JA. Eye-tracking technology has largely been used to investigate responding JA, but rarely to study initiating JA especially in young children with ASD. The aim of this study was to describe the differences in the visual patterns of toddlers with ASD and those with typical development (TD) during both responding JA and initiating JA tasks. Eye-tracking technology was used to monitor the gaze of 17 children with ASD and 15 age-matched children with TD during the presentation of short video sequences involving one responding JA and two initiating JA tasks (initiating JA-1 and initiating JA-2). Gaze accuracy, transitions and fixations were analyzed. No differences were found in the responding JA task between children with ASD and those with TD, whereas, in the initiating JA tasks, different patterns of fixation and transitions were shown between the groups. These results suggest that children with ASD and those with TD show different visual patterns when they are expected to initiate joint attention but not when they respond to joint attention. We hypothesized that differences in transitions and fixations are linked to ASD impairments in visual disengagement from face, in global scanning of the scene and in the ability to anticipate object's action