RESUMO
BACKGROUND: Staphylococcus aureus isolates expressing the Panton-Valentine Leukocidin (PVL) have been related to a wide range of diseases. Recently, pvl-positive community-associated methicillin-resistant S. aureus belonging to USA1100 (ST30/CC30/SCCmec IV) lineage has emerged in Brazilian hospitals. OBJECTIVE: The aim of this work was to sequence the genome of a pvl-positive USA1100 Vancomycin-Intermediate-Resistant S. aureus (VISA) isolate from Rio de Janeiro, Brazil. METHODS: The 13420 genome was sequenced using the HiSeq 2500 platform. The draft genome, plasmids annotation, and genome analysis were performed using RAST. Comparison of the relative pvl gene expression of six S. aureus isolates was performed by qRT-PCR. RESULTS: The isolate presented the ÏPVL phage codifying for the H2b PVL protein isoform, and another prophage carrying a PVL variant named lukF and lukS-PV.2. The 13420 genome presented a high number of virulence determinants, such as genes codifying for serine-protease proteins, enterotoxins (egc), the immune evasion cluster (IEC), adhesion proteins, spermine/spermidine acetyltransferase gene (blt), superantigen-like proteins, as well as the ica operon. Point mutations at vraS, tcaA, and tcaB genes were detected. Moreover, the PVL mRNA relative expression of the 13420 isolate was five times higher than mRNA PVL levels of the USA300/ST8 reference strain. CONCLUSION: We described for the first time the genome sequence of a VISA isolate harboring two pvl-associated genes and other virulence factors that may improve the USA1100/ST30 lineage fitness and impact its pathogenicity and spreading at Brazilian hospitals.
RESUMO
BACKGROUND: Pseudomonas aeruginosa is an important human pathogen that causes severe infections in a wide range of immunosuppressed patients. Herein, we evaluated the proteolytic profiles of 96 Brazilian clinical isolates of P. aeruginosa recovered from diverse anatomical sites. METHODS: Cell-associated and extracellular proteases were evidenced by gelatin-SDS-PAGE and by the cleavage of soluble gelatin. Elastase was measured by using the peptide substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide. The prevalence of elastase genes (lasA and lasB) was evaluated by PCR. RESULTS: Bacterial extracts were initially applied on gelatin-SDS-PAGE and the results revealed four distinct zymographic profiles as follows: profile I (composed by bands of 145, 118 and 50kDa), profile II (118 and 50kDa), profile III (145kDa) and profile IV (118kDa). All the proteolytic enzymes were inhibited by EDTA, identifying them as metalloproteases. The profile I was the most detected in both cellular (79.2%) and extracellular (84.4%) extracts. Overall, gelatinase and elastase activities measured in the spent culture media were significantly higher (around 2-fold) compared to the cellular extracts and the production level varied according to the site of bacterial isolation. For instance, tracheal secretion isolates produced elevated amount of gelatinase and elastase measured in both cellular and extracellular extracts. The prevalence of elastase genes revealed that 100% isolates were lasB-positive and 85.42% lasA-positive. Some positive/negative correlations were showed concerning the production of gelatinase, elastase, isolation site and antimicrobial susceptibility. CONCLUSION: The protease production was highly heterogeneous in Brazilian clinical isolates of P. aeruginosa, which corroborates the genomic/metabolic versatility of this pathogen.
Assuntos
Proteínas de Bactérias/análise , Metaloproteases/análise , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/enzimologia , Proteínas de Bactérias/genética , Líquidos Corporais/microbiologia , Brasil , Fibrose Cística/complicações , Ácido Edético/farmacologia , Eletroforese em Gel de Poliacrilamida , Gelatinases/antagonistas & inibidores , Gelatinases/genética , Gelatinases/isolamento & purificação , Genes Bacterianos , Humanos , Metaloproteases/antagonistas & inibidores , Metaloproteases/genética , Especificidade de Órgãos , Elastase Pancreática/antagonistas & inibidores , Elastase Pancreática/genética , Elastase Pancreática/isolamento & purificação , Pneumonia Bacteriana/microbiologia , Inibidores de Proteases/farmacologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Reto/microbiologia , Sistema Respiratório/microbiologia , Virulência , Infecção dos Ferimentos/microbiologiaRESUMO
Staphylococcus aureus is an important cause of both hospital- and community-associated methicillin-resistant S. aureus (MRSA) infections worldwide. ß-Lactam antibiotics are the drugs of choice to treat S. aureus infections, but resistance to these and other antibiotics make treatment problematic. High-level ß-lactam resistance of S. aureus has always been attributed to the horizontally acquired penicillin binding protein 2a (PBP 2a) encoded by the mecA gene. Here, we show that S. aureus can also express high-level resistance to ß-lactams, including new-generation broad-spectrum cephalosporins that are active against methicillin-resistant strains, through an uncanonical core genome-encoded penicillin binding protein, PBP 4, a nonessential enzyme previously considered not to be important for staphylococcal ß-lactam resistance. Our results show that PBP 4 can mediate high-level resistance to ß-lactams.
Assuntos
Proteínas de Bactérias/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , beta-Lactamas/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Cefalosporinas/farmacologia , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Staphylococcus aureus/genética , Resistência beta-Lactâmica/genéticaRESUMO
Pseudomonas aeruginosa is an opportunistic human pathogen responsible for causing a huge variety of acute and chronic infections with significant levels of morbidity and mortality. Its success as a pathogen comes from its genetic/metabolic plasticity, intrinsic/acquired antimicrobial resistance, capacity to form biofilm and expression of numerous virulence factors. Herein, we have analyzed the genetic variability, antimicrobial susceptibility as well as the production of metallo-ß-lactamases (MBLs) and virulence attributes (elastase, pyocyanin and biofilm) in 96 strains of P. aeruginosa isolated from different anatomical sites of patients attended at Brazilian hospitals. Our results revealed a great genetic variability, in which 86 distinct RAPD types (89.6% of polymorphisms) were detected. Regarding the susceptibility profile, 48 strains (50%) were resistant to the antimicrobials, as follows: 22.92% to the three tested antibiotics, 12.5% to both imipenem and meropenem, 11.46% to ceftazidime only, 2.08% to imipenem only and 1.04% to both ceftazidime and meropenem. Out of the 34 clinical strains of P. aeruginosa resistant to both imipenem and meropenem, 25 (73.53%) were MBL producers by phenotypic method while 12 (35.29%) were PCR positive for the MBL gene SPM-1. All P. aeruginosa strains produced pyocyanin, elastase and biofilm, although in different levels. Some associations were demonstrated among the susceptibility and/or production of these virulence traits with the anatomical site of strain isolation. For instance, almost all strains isolated from urine (85.71%) were resistant to the three antibiotics, while the vast majority of strains isolated from rectum (95%) and mouth (66.67%) were susceptible to all tested antibiotics. Urine isolates produced the highest pyocyanin concentration (20.15±5.65 µg/ml), while strains isolated from pleural secretion and mouth produced elevated elastase activity (1441.43±303.08 FAU) and biofilm formation (OD590 0.676±0.32), respectively. Also, MBL-positive strains produced robust biofilm compared to MBL-negative strains. Collectively, the production of site-dependent virulence factors can be highlighted as potential therapeutic targets for the treatment of infections caused by heterogeneous and resistant strains of P. aeruginosa.
Assuntos
Variação Genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Fatores de Virulência/genética , Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Líquidos Corporais/microbiologia , Brasil , Farmacorresistência Bacteriana , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Tipagem Molecular , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/fisiologia , Técnica de Amplificação ao Acaso de DNA Polimórfico , Virulência , beta-Lactamases/metabolismoRESUMO
Staphylococcus aureus encoding Panton-Valentine leukocidin (PVL) genes has become the cause of life-threatening infections. We describe a case of carotid cavernous fistula after bacteremia in a 12-year-old male, caused by a methicillin-susceptible S. aureus isolate carrying the pvl, fnbA, and ebpS genes and related to sequence type 25 (ST25). The patient's condition was complicated by pleural empyema and osteomyelitis in the right femur. The patient was discharged in good clinical condition after 160 days of hospitalization.
Assuntos
Toxinas Bacterianas/genética , Fístula Carótido-Cavernosa/diagnóstico , Infecções Comunitárias Adquiridas/microbiologia , Exotoxinas/genética , Leucocidinas/genética , Sepse/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificação , Angiografia , Antibacterianos/farmacologia , Fístula Carótido-Cavernosa/complicações , Fístula Carótido-Cavernosa/microbiologia , Fístula Carótido-Cavernosa/patologia , Criança , Infecções Comunitárias Adquiridas/complicações , Empiema/diagnóstico , Empiema/microbiologia , Genótipo , Humanos , Masculino , Meticilina/farmacologia , Tipagem Molecular , Osteomielite/diagnóstico , Osteomielite/epidemiologia , Sepse/complicações , Staphylococcus aureus/classificação , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Fatores de Virulência/genéticaRESUMO
This work characterizes MLS(b) resistance in 39 methicillin-resistant Staphylococcus aureus (MRSA) and 32 Staphylococcus epidermidis (MRSE) isolates. Of 21 erm(A) gene encoding MRSA isolates, 71.4% carried SCCmecIII, whereas of 12 isolates carrying the erm(C) gene, 83.3% carried SCCmecIV. Among the 25 MRSE isolates positive for the erm(C) gene, 80% had SCCmecIV or nontypeable cassettes. Isolates carrying these genes had MIC(90) ≥ 256 µg/mL to erythromycin and clindamycin. The msr(A) gene was associated with a low MIC(90) to these drugs. The erm(A) gene was associated with SCCmecIII in MRSA isolates, whereas the erm(C) gene was associated with SCCmecIV in both MRSA and MRSE isolates.
Assuntos
Clindamicina/farmacologia , Farmacorresistência Bacteriana Múltipla , Eritromicina/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacos , Estreptogramina B/farmacologia , Proteínas de Bactérias/genética , Brasil/epidemiologia , Cromossomos Bacterianos/genética , Genes Bacterianos , Humanos , Recém-Nascido , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Metiltransferases/genética , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas , Fenótipo , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/isolamento & purificaçãoRESUMO
Staphylococcus epidermidis is the most prevalent coagulase-negative Staphylococcus (CNS) and is a major cause of hospital bacteremia. Based on 18 reference strains and 149 Staphylococcus clinical strains, used in a novel multiplex PCR method, the aim of this study was to identify S. epidermidis with respect to the sequence of three genes: recN, which encodes a recombination/repair protein, mecA (methicillin resistance), and icaAB, which is involved in biofilm formation. Amplicons of 219 bp (S. epidermidis-recN gene), 154 bp (mecA gene), and 546 bp (icaAB genes) were obtained. Reliable results were achieved for 100% of the evaluated strains, suggesting that this new multiplex-PCR approach could be useful for the accurate identification of methicillin-resistant S. epidermidis with the potential to produce biofilm.
Assuntos
Técnicas Bacteriológicas/métodos , Biofilmes/crescimento & desenvolvimento , Resistência a Meticilina , Reação em Cadeia da Polimerase Multiplex/métodos , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/fisiologia , Amidoidrolases/genética , Proteínas de Bactérias/genética , Enzimas de Restrição do DNA/genética , Humanos , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/isolamento & purificaçãoRESUMO
Staphylococcus aureus and Staphylococcus epidermidis are among the most important bacterial species responsible for biofilm formation on indwelling medical devices, including orthopaedic implants. The increasing resistance to antimicrobials, partly attributed to the ability to form biofilms, is a challenge for the development of new antimicrobial agents. In this study, the cell-free supernatant obtained from sponge-associated Enterobacter strain 84.3 culture inhibited biofilm formation (>65%) and dissociated mature biofilm (>85%) formed by S. aureus and S. epidermidis strains. The culture supernatant was subjected to solvent partitioning and the aqueous extract presented a concentration-dependent antibiofilm activity for each strain with a minimum biofilm eradication concentration (MBEC) ranging from 16 to 256 µg/mL. The effect of the aqueous extract on mature S. aureus biofilm was analyzed by confocal scanning laser microscopy, showing a significant reduction of the biofilm layer as well as diminished interactions among the cells. This extract is not toxic for mammalian cells (L929 cell line). Studies targeting substances with antibiofilm activity gained significant attention in recent years due to difficult-to-treat biofilm infections. Here, sponge-associated Enterobacter 84.3 proved to be a source of substances capable of eradicating staphylococcal biofilm, with potential medical use in the future.
Assuntos
Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Extratos Celulares/farmacologia , Enterobacter/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacos , Animais , Infecções Relacionadas a Cateter/tratamento farmacológico , Infecções Relacionadas a Cateter/microbiologia , Cateteres de Demora/microbiologia , Linhagem Celular , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/microbiologia , Células L , Camundongos , Testes de Sensibilidade Microbiana , Poríferos/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/prevenção & controleRESUMO
BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) colonization in Atopic Dermatitis (AD) patients can contribute to worsening their clinical condition. OBJECTIVE: A cohort study was carried out to determine the incidence of MRSA acquisition and its risk factors in AD children. METHODS: Patients with AD (2 months-14 years old) were followed up for about 1 year at a reference center for AD treatment in Rio de Janeiro, Brazil, from September 2011 to February 2014. Nasal swabs from patients and contacts were collected every 2 months. The SCORAD system assessed the severity of the AD. S. aureus isolates were evaluated to determine the methicillin resistance and the clonal lineages. RESULTS: Among 117 AD patients, 97 (82.9%) were already colonized with S. aureus and 26 (22.2%) had MRSA at the first evaluation. The incidence of MRSA acquisition in the cohort study was 27.47% (n = 25). The SCORAD assessments were: mild (46.15%), moderate (37.36%) or severe (16.48%). Risk factors were: colonized MRSA contacts (HR = 2.27; 95% CI: 1.16-7.54), use of cyclosporine (HR = 5.84; 95% CI: 1.70-19.98), moderate or severe AD (HR = 3.26; 95% CI: 1.13-9.37). Protective factors were: availability of running water (HR = 0.21; 95% CI: 0.049-0.96) and use of antihistamines (HR = 0.21; 95% IC: 0.64-0.75). MRSA isolates carried the SCCmec type IV and most of them were typed as USA800/ST5. CONCLUSIONS: The high incidence of MRSA acquisition found among AD patients and the risk factors associated show that an effective surveillance of MRSA colonization in these patients is needed.
Assuntos
Dermatite Atópica/epidemiologia , Dermatite Atópica/microbiologia , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Adolescente , Antibacterianos/farmacologia , Brasil/epidemiologia , Criança , Pré-Escolar , Estudos de Coortes , Ciclosporina , Feminino , Antagonistas dos Receptores Histamínicos , Humanos , Incidência , Lactente , Masculino , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Análise Multivariada , Estudos Prospectivos , Fatores de Proteção , Fatores de RiscoRESUMO
Staphylococcus epidermidis is one of the leading causes of bloodstream infections, particularly in premature neonates, and biofilm formation is a major virulence factor. We characterized biofilm formation by 50â¯S. epidermidis neonatal isolates under osmotic stress and evaluated the expression of biofilm-associated genes. Phenotypical analyses of biofilm production were performed in culture medium with or without addition of NaCl or glucose. In control medium (no additions), most isolates (84%) were nonproducers or weak biofilm producers. Growth in NaCl-containing medium increased the number of moderate/strong producers, and this increase was even greater in medium containing glucose. Most of the protein-enriched biofilms (60%) could be observed only during growth in glucose, whereas 50% of the polysaccharide-enriched biofilms were observed during growth in NaCl. Studies that evaluate the conditions used to characterize biofilm production are important to help us understand the dynamics of this important virulence factor in S. epidermidis and their impact on neonatal infections.
Assuntos
Biofilmes/crescimento & desenvolvimento , Pressão Osmótica , Staphylococcus epidermidis/fisiologia , Biofilmes/efeitos dos fármacos , Meios de Cultura/química , DNA Bacteriano/genética , Expressão Gênica , Glucose/farmacologia , Humanos , Recém-Nascido , Fenótipo , Cloreto de Sódio/farmacologia , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/efeitos dos fármacosRESUMO
Staphylococcus haemolyticus is the most frequently coagulase-negative Staphylococcus species associated with antimicrobial resistance isolated from nosocomial infections. We developed an accurate and simple multiplex PCR assay to identify methicillin-resistant S. haemolyticus (MRSH) isolates. We designed species-specific primers of the mvaA gene that encodes a 3-hydroxy-3-methylglutaryl coenzyme A involved in the mevalonate pathway of the microorganism. Simultaneously, mecA gene primers of methicillin resistance were also used. The PCR assay was established using 16 strains of different reference Staphylococcus species and validated with a collection of 147 clinical staphylococcal isolates that were also phenotypically characterized. Reliable results for the detection of MRSH isolates were obtained for 100% of the strains evaluated, showing that this PCR assay can be used for the routine microbiology laboratories. This is the first report using species-specific multiplex PCR to detect a single segment of S. haemolyticus associated with a segment of mecA gene.
Assuntos
Resistência a Meticilina , Reação em Cadeia da Polimerase/métodos , Staphylococcus haemolyticus/classificação , Primers do DNA , DNA Bacteriano/análise , Meticilina/farmacologia , Sensibilidade e Especificidade , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/microbiologia , Staphylococcus haemolyticus/efeitos dos fármacos , Staphylococcus haemolyticus/genética , Staphylococcus haemolyticus/isolamento & purificaçãoRESUMO
Previous analysis of Pseudomonas aeruginosa class-1 integrons from Rio de Janeiro, Brazil, revealed the blaGES gene in one isolate. We screened isolates of two widespread PFGE genotypes, A and B, at a public hospital in Rio, for the presence of blaGES. The gene was detected in all seven P. aeruginosa isolates belonging to genotype B. Three of the seven genotype-B isolates were resistant to amikacin, aztreonam, ceftazidime, cefepime, ciprofloxacin, gentamicin, imipenem, meropenem, piperacillin-tazobactam and ticarcillin-clavulanic acid. The other four isolates were resistant to all these agents, except gentamicin, imipenem, meropenem and piperacillin-tazobactam. A synergistic effect between ceftazidime and imipenem or clavulanic acid suggested the production of GES-type ESBL.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Pseudomonas aeruginosa/genética , Antibacterianos/farmacologia , Brasil , Sinergismo Farmacológico , Genótipo , Hospitais Públicos , Humanos , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , beta-Lactamases/biossínteseRESUMO
AIMS: This cross-sectional study investigated the association between eight herpesviruses and the bacterial community profiles from the oral cavity of children with and without leukaemia. METHODS: Sixty participants (aged 3-13), divided into the leukaemia group (LG) and healthy group (HG), were evaluated. Collection of medical data, intraoral examination and collection of clinical specimens were carried out. Single PCR and nested-PCR techniques were used to identify the viral types; denaturing gradient gel electrophoresis (DGGE) and real-time PCR techniques were used to evaluate the profile and abundance of bacterial communities. RESULTS: All the children with leukaemia were positive for at least one type of herpesvirus, compared with healthy participants (33.3%; p<0.000). Human cytomegalovirus (HCMV; 46.7%), human herpesvirus-7 (HHV-7; 20%) and HHV-8 (77.3%) were in higher prevalence in the LG (p ≤ 0.01). Children with leukaemia had positive associations with the presence of HCMV, HHV-7 and HHV-8 in the oral cavity when under chemotherapy (p<0.05). There was a qualitative (means of DGGE bands) and quantitative (means of 16S rRNA gene abundance) difference in relation to the bacterial community between the two groups (p<0.05). CONCLUSIONS: Based on the results, the prevalence of herpesviruses and the qualitative bacterial profiles was higher in children with leukaemia and HCMV, HHV-7 and HHV-8 were related to the use of chemotherapy. Moreover, HHV-6 was correlated with an increased bacterial community profile in patients with leukaemia (p<0.05). More attention should be paid to the oral health of these individuals, mainly those under chemotherapy, in order to prevent infections by opportunistic pathogens.
Assuntos
Antineoplásicos/uso terapêutico , Bactérias/efeitos dos fármacos , Infecções por Herpesviridae/virologia , Herpesviridae/efeitos dos fármacos , Leucemia/tratamento farmacológico , Boca/efeitos dos fármacos , Saúde Bucal , Adolescente , Antineoplásicos/efeitos adversos , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Estudos de Casos e Controles , Criança , Pré-Escolar , Estudos Transversais , DNA Bacteriano/isolamento & purificação , DNA Viral/isolamento & purificação , Eletroforese em Gel de Gradiente Desnaturante , Feminino , Herpesviridae/classificação , Herpesviridae/genética , Herpesviridae/isolamento & purificação , Infecções por Herpesviridae/diagnóstico , Humanos , Leucemia/diagnóstico , Masculino , Boca/microbiologia , Boca/virologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Daptomycin is an alternative option for the treatment of catheter-related bloodstream-infections caused by methicillin-resistant Staphylococcus aureus. This study reports a case of a daptomycin and methicillin-resistant Staphylococcus aureus isolate recovered from the blood of a Brazilian patient undergoing hemodialysis. CASE PRESENTATION: A 64-year-old white male patient suffering from diabetes mellitus, systolic hypertension, heart disease with a coronary stent, obesity and chronic renal failure and on use of permcath catheter developed a catheter-related bloodstream-infection by a daptomycin-methicillin-resistant Staphylococcus aureus isolate after one month of daptomycin therapy. The isolate was identified as the SCCmec II/USA100/sequence type 5 lineage by molecular techniques. CONCLUSIONS: In this work we described a Brazilian patient with bloodstream infection caused by a daptomycin and methicillin-resistant Staphylococcus aureus belonging to the lineage USA100/sequence type 5. Our case highlights the careful management of bloodstream infections and the importance of the judicious use of antimicrobials due the possibility of daptomycin-resistance developing among S. aureus isolates, especially in patients under hemodialysis, which are frequently exposed to vancomycin and daptomycin therapy.
Assuntos
Infecções Relacionadas a Cateter/sangue , Infecções Relacionadas a Cateter/microbiologia , Daptomicina/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Evolução Fatal , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Vancomicina/farmacologiaRESUMO
Staphylococcus lugdunensis is an unusually virulent coagulase-negative species, which causes serious infection similar to S. aureus. We evaluated the expression of virulence factors such as S. lugdunensis synergistic haemolysin (SLUSH), fibrinogen-binding protein (Fbl), biofilm production and biofilm-production-related genes in 23 S. lugdunensis clinical isolates and one type strain that had been previously characterized for their genotypes. In addition, the biofilm composition and the ability of isolates to adhere to and invade human epithelial lung cells were also investigated. The PCR method used detected the presence of slush and intercellular adhesin (ica) virulence genes in all isolates. All isolates produced the Fbl protein and, with the exception of the type strain, all isolates produced the SLUSH haemolysin. Fourteen (60.9â%) isolates produced biofilms. The detachment assay, using sodium metaperiodate or proteolytic enzymes to analyse the biofilm composition, showed protein-mediated biofilms in two representative isolates, one for each colony type (rough and smooth). All strongly biofilm-producing isolates, including three with rough colony morphology, had the same prevalent PFGE pattern. However, among the representative strains tested, only the S. lugdunensis isolate that formed rough colonies was able to adhere to and invade A549 cell monolayers in the same quantities as those observed with S. aureus isolates (Pâ=â1.000). No significant adhesion or invasion was observed for the other isolates in comparison with the S. aureus isolate, independent of biofilm production or clonality. Our results could explain the incredible ability of this pathogen to cause infections that are as aggressive as S. aureus. In addition, the ability of S. lugdunensis to adhere to and invade eukaryotic cells was also noticed for isolates with rough colony morphology, reinforcing the increased virulence in this species.
Assuntos
Aderência Bacteriana/fisiologia , Células Epiteliais/microbiologia , Mucosa Respiratória/citologia , Staphylococcus lugdunensis/citologia , Staphylococcus lugdunensis/genética , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Brasil/epidemiologia , Linhagem Celular Tumoral , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Pulmão/citologia , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus lugdunensis/fisiologiaRESUMO
Staphylococcus lugdunensis are unusually virulent coagulase-negative staphylococci associated with skin infections and endocarditis. We developed an accurate and simple PCR assay to identify S. lugdunensis isolates based on detection of the fbl gene, which encodes a fibrinogen-binding protein involved in pathogen adhesion. The PCR assay was established using 16 reference strains of different Staphylococcus species and further validated with a collection of 63 clinical staphylococcal isolates that were also phenotypically characterized. Reliable results for the detection of S. lugdunensis isolates were obtained for 100% of the strains evaluated, indicating that this PCR assay can be used in the routine of microbiology laboratories as one more tool for correct species differentiation.
Assuntos
Técnicas Bacteriológicas/métodos , Reação em Cadeia da Polimerase/métodos , Infecções Estafilocócicas/diagnóstico , Staphylococcus/isolamento & purificação , Adesinas Bacterianas/genética , Genes Bacterianos , Humanos , Sensibilidade e EspecificidadeRESUMO
In this study, we standardized and evaluated a multiplex-PCR methodology using specific primers to identify Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus and their methicillin-resistance directly from blood cultures. Staphylococci clinical isolates (149) and control strains (16) previously identified by conventional methods were used to establish the multiplex PCR protocol. Subsequently, this methodology was evaluated using a fast and cheap DNA extraction protocol from 25 staphylococci positive blood cultures. A wash step of the pellet with 0.1% bovine serum albumin (BSA) solution was performed to reduce PCR inhibitors. Amplicons of 154bp (mecA gene), 271bp (S. haemolyticus mvaA gene) and 108 and 124bp (S. aureus and S. epidermidis species-specific fragments, respectively) were observed. Reliable results were obtained for 100% of the evaluated strains, suggesting that this new multiplex-PCR combined with an appropriate DNA-extraction method could be useful in the laboratory for fast and accurate identification of three staphylococci species and simultaneously their methicillin resistance directly in blood cultures.
Assuntos
Técnicas Bacteriológicas/métodos , Sangue/microbiologia , Resistência a Meticilina , Reação em Cadeia da Polimerase/métodos , Staphylococcus aureus/isolamento & purificação , Staphylococcus epidermidis/isolamento & purificação , Staphylococcus haemolyticus/isolamento & purificação , Técnicas Bacteriológicas/normas , Primers do DNA/genética , Humanos , Reação em Cadeia da Polimerase/normas , Sensibilidade e Especificidade , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/genética , Staphylococcus haemolyticus/efeitos dos fármacos , Staphylococcus haemolyticus/genéticaRESUMO
Previous analysis of Pseudomonas aeruginosa class-1 integrons from Rio de Janeiro, Brazil, revealed the blaGES gene in one isolate. We screened isolates of two widespread PFGE genotypes, A and B, at a public hospital in Rio, for the presence of blaGES. The gene was detected in all seven P. aeruginosa isolates belonging to genotype B. Three of the seven genotype-B isolates were resistant to amikacin, aztreonam, ceftazidime, cefepime, ciprofloxacin, gentamicin, imipenem, meropenem, piperacillin-tazobactam and ticarcillin-clavulanic acid. The other four isolates were resistant to all these agents, except gentamicin, imipenem, meropenem and piperacillin-tazobactam. A synergistic effect between ceftazidime and imipenem or clavulanic acid suggested the production of GES-type ESBL.