RESUMO
Elucidating the mechanisms of bacterial translocation is crucial for the prevention and treatment of neonatal sepsis. In the present study, we aimed to evaluate the potential of lactoferrin to inhibit the development of late-onset blood infection in neonates. Our investigation evaluates the role of key stress factors leading to the translocation of intestinal bacteria into the bloodstream and, consequently, the development of life-threatening sepsis. Three stress factors, namely weaning, intraperitoneal administration of Gram-positive cocci and oral intake of Gram-negative rods, were found to act synergistically. We developed a novel model of rat pups sepsis induced by bacterial translocation and observed the inhibition of this process by supplementation of various forms of lactoferrin: iron-depleted (apolactoferrin), iron-saturated (hololactoferrin) and manganese-saturated lactoferrin. Additionally, lactoferrin saturated with manganese significantly increases the Lactobacillus bacterial population, which contributes to the fortification of the intestinal barrier and inhibits the translocation phenomenon. The acquired knowledge can be used to limit the development of sepsis in newborns in hospital neonatal intensive care units.
Assuntos
Translocação Bacteriana/efeitos dos fármacos , Escherichia coli , Microbioma Gastrointestinal/efeitos dos fármacos , Lactoferrina/administração & dosagem , Sepse Neonatal/prevenção & controle , Staphylococcus haemolyticus , Animais , Animais Recém-Nascidos , Apoproteínas/administração & dosagem , Infecções Transmitidas por Sangue/microbiologia , Infecções Transmitidas por Sangue/prevenção & controle , Temperatura Corporal , Peso Corporal , Infecção Hospitalar/prevenção & controle , Modelos Animais de Doenças , Esquema de Medicação , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Microbioma Gastrointestinal/fisiologia , Humanos , Recém-Nascido , Masculino , Manganês/administração & dosagem , Sepse Neonatal/diagnóstico , Sepse Neonatal/microbiologia , Permeabilidade , Distribuição Aleatória , Ratos , Ratos Wistar , Staphylococcus haemolyticus/efeitos dos fármacos , Staphylococcus haemolyticus/fisiologia , DesmameRESUMO
The surface protein SasX, has a key role in methicillin-resistant Staphylococcus aureus (MRSA) colonization and pathogenesis, and has been associated with the epidemic success of some MRSA clones. To date, only one SasX homologous protein, named SesI, has been described in Staphylococcus epidermidis. In this work, we analyze the occurrence of the sasX gene and its genetic environment in Staphylococcus haemolyticus S. haemolyticus clinical strains (n = 62) were screened for the presence of the sasX gene and its carrier, the prophage Φ SPß-like. A deep characterization was done in one strain (MD43), through which we determined the complete nucleotide sequence for the S. haemolitycus sasX-like gene. Whole genome sequencing of strain MD43 was performed, and the gene, termed here because of its unique attributes, shsA, was mapped to the Φ SPß-like prophage sequence. The shsA gene was detected in 33 out of 62 strains showing an average identity of 92 and 96% with the sasX and sesI genes and at the amino acid level, 88% identity with SasX and 92% identity with SesI. The ~124Kb Φ SPß-like prophage sequence showed a largely intact prophage compared to its counterpart in S. epidermidis strain RP62A, including the sesI insertion site. In conclusion, we identified a new sasX ortholog in S. haemolyticus (shsA). Its horizontal spread from this reservoir could represent an emergent threat in healthcare facilities since so far, no S. aureus sasX+ strains have been reported in Brazil.
Assuntos
Genoma Bacteriano , Staphylococcus haemolyticus/fisiologia , Staphylococcus haemolyticus/patogenicidade , Fatores de Virulência/genética , Brasil , Prófagos/genética , Virulência , Sequenciamento Completo do GenomaRESUMO
Bacteriocins are ribosomally-synthesized antimicrobial peptides, showing great potential as novel treatment options for multidrug-resistant pathogens. In this study, we designed a novel hybrid bacteriocin, Hybrid 1 (H1), by combing the N-terminal part and the C-terminal part of the related bacteriocins enterocin K1 (K1) and enterocin EJ97 (EJ97), respectively. Like the parental bacteriocins, H1 used the membrane-bound protease RseP as receptor, however, it differed from the others in the inhibition spectrum. Most notably, H1 showed a superior antimicrobial effect towards Staphylococcus haemolyticus-an important nosocomial pathogen. To avoid strain-dependency, we further evaluated H1 against 27 clinical and commensal S. haemolyticus strains, with H1 indeed showing high activity towards all strains. To curtail the rise of resistant mutants and further explore the potential of H1 as a therapeutic agent, we designed a bacteriocin-based formulation where H1 was used in combination with the broad-spectrum bacteriocins micrococcin P1 and garvicin KS. Unlike the individual bacteriocins, the three-component combination was highly effective against planktonic cells and completely eradicated biofilm-associated S. haemolyticus cells in vitro. Most importantly, the formulation efficiently prevented development of resistant mutants as well. These findings indicate the potential of a bacteriocins-based formulation as a treatment option for S. haemolyticus.
Assuntos
Bacteriocinas/farmacologia , Biofilmes/efeitos dos fármacos , Staphylococcus haemolyticus/fisiologia , Sequência de Aminoácidos , Anti-Infecciosos/farmacologia , Bacteriocinas/química , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Genes Bacterianos , Testes de Sensibilidade Microbiana , Modelos Biológicos , Mutação/genética , Staphylococcus haemolyticus/efeitos dos fármacos , Staphylococcus haemolyticus/genética , Sequenciamento Completo do GenomaRESUMO
OBJECTIVE: We aimed to determine factors associated with gut colonization of preterm neonates with coagulase-negative staphylococci (CoNS) from maternal milk (MM). STUDY DESIGN: CoNS isolated from weekly collected stool and MM of hospitalized preterm (n = 49) and healthy term neonates (n = 20) were genotyped. Colonization-related factors were determined by Cox proportional hazards regression. RESULT: Gut colonization with mecA-negative Staphylococcus epidermidis from MM was less prevalent (40.8% vs. 95%) and delayed (median age 15.5 vs. 2 days) in preterm compared with term neonates. Enhanced colonization was associated with higher intake of CoNS from MM (hazard ratio (95% confidence interval) 1.006 (1.00-1.01) for 106 colony-forming units), lower proportion of mecA-positive predominant NICU strains in gut (0.09 (0.01-0.49) for 1%) and lower incidence of late-onset CoNS sepsis (5% vs. 34% in those without colonization). CONCLUSION: Enteral feeding with larger proportion of unpasteurized MM and limiting spread of predominant strains may promote colonization with CoNS from MM.
Assuntos
Trato Gastrointestinal/microbiologia , Recém-Nascido Prematuro , Leite Humano/microbiologia , Staphylococcus epidermidis/fisiologia , Adulto , Aleitamento Materno , Coagulase , Fezes/microbiologia , Feminino , Humanos , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Estudos Longitudinais , Tipagem de Sequências Multilocus , Sepse Neonatal/prevenção & controle , Modelos de Riscos Proporcionais , Estudos Prospectivos , Pele/microbiologia , Staphylococcus haemolyticus/fisiologia , Nascimento a TermoRESUMO
BACKGROUND The association between Staphylococcus haemolyticus and severe nosocomial infections is increasing. However, the extent to which fomites contribute to the dissemination of this pathogen through patients and hospital wards remains unknown. OBJECTIVES In the present study, sphygmomanometers and thermometers were evaluated as potential fomites of oxacillin-resistant S. haemolyticus (ORSH). The influence of oxacillin and vancomycin on biofilm formation by ORSH strains isolated from fomites was also investigated. METHODS The presence of ORSH on swabs taken from fomite surfaces in a Brazilian hospital was assessed using standard microbiological procedures. Antibiotic susceptibility profiles were determined by the disk diffusion method, and clonal distribution was assessed in pulsed-field gel electrophoresis (PFGE) assays. Minimum inhibitory concentrations (MICs) of oxacillin and vancomycin were evaluated via the broth microdilution method. Polymerase chain reaction (PCR) assays were performed to detect the mecA and icaAD genes. ORSH strains grown in media containing 1/4 MIC of vancomycin or oxacillin were investigated for slime production and biofilm formation on glass, polystyrene and polyurethane catheter surfaces. FINDINGS ORSH strains comprising five distinct PFGE types were isolated from sphygmomanometers (n = 5) and a thermometer (n = 1) used in intensive care units and surgical wards. ORSH strains isolated from fomites showed susceptibility to only linezolid and vancomycin and were characterised as multi-drug resistant (MDR). Slime production, biofilm formation and the survival of sessile bacteria differed and were independent of the presence of the icaAD and mecA genes, PFGE type and subtype. Vancomycin and oxacillin did not inhibit biofilm formation by vancomycin-susceptible ORSH strains on abiotic surfaces, including on the catheter surface. Enhanced biofilm formation was observed in some situations. Moreover, a sub-lethal dose of vancomycin induced biofilm formation by an ORSH strain on polystyrene. MAIN CONCLUSIONS Sphygmomanometers and thermometers are fomites for the transmission of ORSH. A sub-lethal dose of vancomycin may favor biofilm formation by ORSH on fomites and catheter surfaces.
Assuntos
Humanos , Oxacilina/farmacologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/transmissão , Termômetros/microbiologia , Vancomicina/farmacologia , Infecção Hospitalar/microbiologia , Biofilmes/crescimento & desenvolvimento , Esfigmomanômetros/microbiologia , Staphylococcus haemolyticus/isolamento & purificação , Staphylococcus haemolyticus/efeitos dos fármacos , Staphylococcus haemolyticus/fisiologia , Antibacterianos/farmacologia , Resistência a Medicamentos , Testes de Sensibilidade Microbiana , Infecção Hospitalar/transmissão , Eletroforese em Gel de Campo Pulsado , EletroforeseRESUMO
BACKGROUND: The association between Staphylococcus haemolyticus and severe nosocomial infections is increasing. However, the extent to which fomites contribute to the dissemination of this pathogen through patients and hospital wards remains unknown. OBJECTIVES: In the present study, sphygmomanometers and thermometers were evaluated as potential fomites of oxacillin-resistant S. haemolyticus (ORSH). The influence of oxacillin and vancomycin on biofilm formation by ORSH strains isolated from fomites was also investigated. METHODS: The presence of ORSH on swabs taken from fomite surfaces in a Brazilian hospital was assessed using standard microbiological procedures. Antibiotic susceptibility profiles were determined by the disk diffusion method, and clonal distribution was assessed in pulsed-field gel electrophoresis (PFGE) assays. Minimum inhibitory concentrations (MICs) of oxacillin and vancomycin were evaluated via the broth microdilution method. Polymerase chain reaction (PCR) assays were performed to detect the mecA and icaAD genes. ORSH strains grown in media containing 1/4 MIC of vancomycin or oxacillin were investigated for slime production and biofilm formation on glass, polystyrene and polyurethane catheter surfaces. FINDINGS: ORSH strains comprising five distinct PFGE types were isolated from sphygmomanometers (n = 5) and a thermometer (n = 1) used in intensive care units and surgical wards. ORSH strains isolated from fomites showed susceptibility to only linezolid and vancomycin and were characterised as multi-drug resistant (MDR). Slime production, biofilm formation and the survival of sessile bacteria differed and were independent of the presence of the icaAD and mecA genes, PFGE type and subtype. Vancomycin and oxacillin did not inhibit biofilm formation by vancomycin-susceptible ORSH strains on abiotic surfaces, including on the catheter surface. Enhanced biofilm formation was observed in some situations. Moreover, a sub-lethal dose of vancomycin induced biofilm formation by an ORSH strain on polystyrene. MAIN CONCLUSIONS: Sphygmomanometers and thermometers are fomites for the transmission of ORSH. A sub-lethal dose of vancomycin may favor biofilm formation by ORSH on fomites and catheter surfaces.
Assuntos
Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Fômites/microbiologia , Esfigmomanômetros/microbiologia , Staphylococcus haemolyticus/fisiologia , Termômetros/microbiologia , Infecção Hospitalar/microbiologia , Infecção Hospitalar/transmissão , Farmacorresistência Bacteriana , Eletroforese em Gel de Campo Pulsado , Humanos , Testes de Sensibilidade Microbiana , Oxacilina/farmacologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/transmissão , Staphylococcus haemolyticus/efeitos dos fármacos , Staphylococcus haemolyticus/isolamento & purificação , Vancomicina/farmacologiaRESUMO
Extracellular signal-regulated kinases (ERKs) are a group of highly conserved serine/threonine-specific protein kinases that function as important signaling intermediates in mitogen-activated protein kinase (MAPK) pathways, which are involved in a wide variety of cellular activities, including proliferation, inflammation and cytokine production. However, little is known about the roles of this kinase in mollusk immunity. In this study, we identified a molluscan ERK homolog (ChERK) in the Hong Kong oyster (Crassostrea hongkongensis) and investigated its biological functions. The open reading frame (ORF) of ChERK encoded a polypeptide of 365 amino acids, with a predicted molecular weight of 41.96 kDa and pI of 6.43. The predicted ChERK protein contained typical characteristic motifs of the ERK family, including a dual threonine-glutamate-tyrosine (TEY) phosphorylation motif and an ATRW substrate binding site. Phylogenetic analysis revealed that ChERK belonged to the mollusk cluster and shared a close evolutionary relationship with ERK from Crassostrea gigas. In addition, quantitative real-time PCR analysis revealed that ChERK expression was detected in all of the examined tissues and stages of embryonic development; its transcript level was significantly induced upon challenge with bacterial pathogens (Vibrio alginolyticus and Staphylococcus haemolyticus) in vivo and PAMPs (lipopolysaccharide and peptidoglycan) in vitro. Moreover, ChERK was mainly located in the cytoplasm of HEK293T cells. Taken together, these findings may provide novel insights into the functions of molluscan ERKs, especially their roles in response to immune challenge in oyster.
Assuntos
Crassostrea/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , Regulação da Expressão Gênica , Moléculas com Motivos Associados a Patógenos/farmacologia , Staphylococcus haemolyticus/fisiologia , Vibrio alginolyticus/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Crassostrea/imunologia , Crassostrea/microbiologia , DNA Complementar/genética , DNA Complementar/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/química , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Imunidade Inata , Lipopolissacarídeos/farmacologia , Peptidoglicano/farmacologia , Filogenia , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de SequênciaRESUMO
Apoptosis plays an important role in homeostasis of the immune systems. The tumor necrosis factor receptors (TNFRs) play critical roles in the extrinsic apoptosis pathways and in determining cell fate. In this study, four death receptors (DR) named ChEDAR, ChTNFR27, ChTNFR5, and ChTNFR16 were identified from the oyster Crassostrea hongkongensis. These ChDRs proteins had 382, 396, 414 and 384 amino acids, respectively, with the typical domains of death receptors, such as the signal peptide (SP), transmembrane helix region (TM) and death domains. Phylogenetic analysis showed that the ChDR proteins clustered into three distinct groups, indicating that these subfamilies had common ancestors. mRNA expression of the ChDRs were detected in all 8 of the selected oyster tissues and at different stages of development. Furthermore, expression of all the genes was increased in the hemocytes of oysters challenged with pathogens or air stress. Fluorescence microscopy revealed that the full-length proteins of the ChDRs were located in the plasma membrane of HEK293T cells. Over-expression of the ChDRs activated the NF-κB-Luc reporter in HEK293T cells in a dose-dependent manner. These results indicate that the ChDRs may play important roles in the extrinsic apoptotic pathways in oysters.
Assuntos
Crassostrea/genética , Crassostrea/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Imunidade Inata , Receptores do Fator de Necrose Tumoral/genética , Sequência de Aminoácidos , Animais , Apoptose/imunologia , Sequência de Bases , Clonagem Molecular , Crassostrea/classificação , Crassostrea/microbiologia , DNA Complementar/genética , DNA Complementar/metabolismo , Evolução Molecular , Especificidade de Órgãos , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/imunologia , Saccharomyces cerevisiae/fisiologia , Alinhamento de Sequência , Transdução de Sinais , Staphylococcus haemolyticus/fisiologia , Vibrio alginolyticus/fisiologiaRESUMO
Infections with coagulase-negative staphylococci are often related to biofilm formation. This study aimed to detect biofilm formation and biofilm-associated genes in blood culture isolates of Staphylococcus epidermidis and S. haemolyticus. Half (50.6%) of the 85 S. epidermidis isolates carried the icaAD genes and 15.3% the bhp gene, while these numbers were 42.9% and 0 for S. haemolyticus, respectively. According to the plate test, 30 S. epidermidis isolates were biofilm producers and 40% of them were strongly adherent, while only one (6%) of the 17 S. haemolyticus biofilm-producing isolates exhibited a strongly adherent biofilm. The concomitant presence of icaA and icaD was significantly associated with the plate and tube test results (P ≤ 0.0004). The higher frequency of icaA in S. epidermidis and of icaD in S. haemolyticus is correlated with the higher biofilm-producing capacity of the former since, in contrast to IcaD, IcaA activity is sufficient to produce small amounts of polysaccharide. Although this study emphasizes the importance of icaAD and bhp for biofilm formation in S. epidermidis, other mechanisms seem to be involved in S. haemolyticus.
Assuntos
Bacteriemia/microbiologia , Biofilmes/crescimento & desenvolvimento , Genes Bacterianos , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/fisiologia , Staphylococcus haemolyticus/fisiologia , Aderência Bacteriana , Brasil , Hospitais de Ensino , Humanos , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/isolamento & purificação , Staphylococcus haemolyticus/genética , Staphylococcus haemolyticus/isolamento & purificaçãoRESUMO
Biofilm may be formed on wide variety of surfaces, including indwelling medical devices, leading to several infectious diseases, e.g., bacteremia and sepsis. The most,important pathogens related with infections associated with medical devices are coagulase-negative staphylococci, including Staphylococcus haeinolyticus - bacterial species which express quite often the multidrug resistance. The four clinical multiresistant and methicillin-resistant S. haenzolyticus were included in the present study. The evaluation of drug susceptibility was performed by using disc-diffusion method and broth microdilution method according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The biofilm formation on the Nelaton catheter and the effect of linezolid, vancomycin, tigecycline and daptomycin on the biofilm formation and disruption of mature structure was based on the method with TTC (2,3,5-triphenyltetrazolium chloride). The adhesion process of S. haenzolyticus to the Nelaton catheter was inhibited by antibiotics, as follows: line-zolid at concentration 0.25-0.5 x MIC, vancomycin - concentration 0.5 x MIC, tigecycline - concentration 0.25-4 x MIC and daptomycin - concentration 0.06-1 x MIC, depending on the isolate. Linezolid inhibited the biofilm formation at concentration between 0.5-1 x MIC, vancomycin - 1-2 x MIC, tigecycline - 0.5-4 x MIC and daptomycin - 0.06-2 x MIC. The concentration of linezolid eradicating the mature biofilm was found to be 1-2 x MIC, vancomycin - 2-8 x MIC, tigecycline - 2-4 x MIC and daptomycin - 0.06-2 x MIC. The most active antibiotic against S. haentolyticus biofilm formation and disruption of mature structure seems to be daptomycin.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Catéteres/microbiologia , Staphylococcus haemolyticus/efeitos dos fármacos , Adesividade/efeitos dos fármacos , Daptomicina/farmacologia , Farmacorresistência Bacteriana Múltipla , Linezolida/farmacologia , Testes de Sensibilidade Microbiana , Minociclina/análogos & derivados , Minociclina/farmacologia , Staphylococcus haemolyticus/fisiologia , Tigeciclina , Vancomicina/farmacologiaRESUMO
Multiple antibiotic resistance and diverse mechanisms for biofilm formation make Coagulase Negative Staphylococci (CoNS) to cause infections associated with insertion of medical devices. As the infectious life style of CoNS pose difficult to treat conditions, materials with multitargeted antimicrobial effect can offer promising ways to modify the surface of devices to limit microbial growth. The broad spectrum of antimicrobial properties shown by silver nanoparticles (AgNPs) make it as an excellent candidate to act on device surface as persistent antimicrobial structures. In the current study, AgNPs assembled by soil bacteria under visible light at room temperature were analysed for its physical properties by UV-Vis spectroscopy, FTIR, SEM, HR-TEM and EDS and they also showed significant antimicrobial and antibiofilm properties against selected members of CoNS like Staphylococcus epidermidis and Staphylococcus haemolyticus. Very interestingly, further analysis on antibacterial mechanism of AgNPs showed their remarkable ability to cause disorganization of bacterial cell membrane. Further, surface engineering application of AgNPs on urinary catheter showed its excellent potential to prevent the attachment and colonization of CoNS which make result of study with significantly novel medical applications.
Assuntos
Nanopartículas Metálicas , Prata/farmacologia , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/fisiologia , Staphylococcus haemolyticus/efeitos dos fármacos , Staphylococcus haemolyticus/fisiologia , Cateteres Urinários/microbiologia , Antibacterianos/química , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Coagulase/metabolismo , Sinergismo Farmacológico , Prata/química , Staphylococcus epidermidis/enzimologia , Staphylococcus haemolyticus/enzimologiaRESUMO
OBJECTIVE: To determine the frequency of isolation of coagulase-negative staphylococci and their resistance to methicillin over a period of time. METHODS: The descriptive cross-sectional study was carried out at Army Medical College, Rawalpindi, from June 2009 to May 2012, and comprised clinical samples mostly from patients admitted to the intensive care unit. They were inoculated onto appropriate culture media depending upon the specimen. After 24-hour incubation at 35°C, coagulase-negative staphylococci were identified on the basis of colony morphology, gram staining, a positive catalase and a negative tube coagulase test.Methicillin resistance among the isolated staphylococci was determined using a 30µg Cefoxitin disc as per the Clinical and Laboratory Standards Institute protocol. Number of coagulase-negative staphylococci for each year and their methicillin resistance rates were calculated. A comparison was made with methicillin resistant staphylococcus aureus) isolated during the same period. RESULTS: Of the total 1331 specimens studies over three years, 581(43.65%) were coagulase-negative staphylococci. The rate of coagulase-negative staphylococci and methicillin resistance was higher each year; 110(26.6%) in May 2009-Jun 2010, 134(36.5%) in 2011, and 337(61%) in 2012. Methicillin resistance rates also increased from 25(22.7%) to 46(34.3%) and then to 201(59.6%) in 2012.Maximum isolated specimens came from blood 311(53.5%), followed by pus/swabs 204(35.1%). CONCLUSIONS: The frequency of isolation of coagulase-negative staphylococci and its methicillin resistance among hospitalised patients is on the rise.
Assuntos
Bacteriemia/microbiologia , Bacteriúria/microbiologia , Resistência a Meticilina/fisiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus haemolyticus/fisiologia , Staphylococcus lugdunensis/fisiologia , Staphylococcus saprophyticus/fisiologia , Bacteriemia/epidemiologia , Bacteriúria/epidemiologia , Estudos Transversais , Hospitais Militares , Humanos , Unidades de Terapia Intensiva , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Staphylococcus aureus Resistente à Meticilina/fisiologia , Testes de Sensibilidade Microbiana , Paquistão/epidemiologia , Infecções Estafilocócicas/epidemiologia , Staphylococcus/isolamento & purificação , Staphylococcus/fisiologia , Staphylococcus haemolyticus/isolamento & purificação , Staphylococcus lugdunensis/isolamento & purificação , Staphylococcus saprophyticus/isolamento & purificação , Supuração/epidemiologia , Supuração/microbiologia , Centros de Atenção TerciáriaRESUMO
Staphylococcus haemolyticus is of increasing concern as a cause of several biofilm-associated infections, and today, it represents the second most common organism among clinical isolates of coagulase-negative staphylococci. However, little is known regarding the treatment of infections caused by these bacteria. In this study, we characterize the biofilm formed by S. haemolyticus strains isolated from bloodstream infections and assess in vitro the activity of rifampicin combined with daptomycin or tigecycline against bacteria growing in a biofilm. The results of our studies indicated that the majority (78 %) of methicillin-resistant Staphylococcus haemolyticus strains have the ability to form a biofilm in vitro. None of these strains carried icaADBC genes indicating that they form biofilm via ica-independent mechanisms. The molecular characterization of the biofilm showed that proteins are the predominant matrix component and play a major role in biofilm structure. Extracellular DNA and polysaccharides, other than polysaccharide intercellular adhesin, are also present in the biofilm matrix, but they play a minor role. The images obtained by confocal laser scanning microscopy showed that most S. haemolyticus strains formed a dense biofilm with a low number of dead cells. In vitro study demonstrated excellent activity of tigecycline in combination with rifampicin against cell growth in the proteinous biofilm. The BIC (biofilm inhibitory concentration) value for tigecycline/rifampicin ranged from 0.062 to 1 µg/ml, whereas for daptomycin/rifampicin from 0.125 to 2 µg/ml. These results indicated that the tigecycline/rifampicin combination was more effective against ica-independent biofilm, formed by S. haemolyticus strains, than the daptomycin/rifampicin combination.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Minociclina/análogos & derivados , Rifampina/farmacologia , Staphylococcus haemolyticus/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sinergismo Farmacológico , Humanos , Testes de Sensibilidade Microbiana , Minociclina/farmacologia , Infecções Estafilocócicas/microbiologia , Staphylococcus haemolyticus/genética , Staphylococcus haemolyticus/fisiologia , TigeciclinaRESUMO
Staphylococcus haemolyticus is one of the most frequently isolated coagulase-negative staphylococci. The ability to produce biofilm has contributed to its emergence as a nosocomial pathogen. In this study, some growth conditions were tested to determine their influence on biofilm formation. Brain-heart infusion (BHI) broth containing glucose was used to screen 64 clinical strains. A strong biofilm producer strain showed cells surrounded by a thick layer of extracellular matrix. The presence of atlE, fbp, bap, and icaA genes was analyzed. We concluded that S. haemolyticus biofilm production can be increased with cells grown in BHI, and highlighted that it could be an ica-independent process.
Assuntos
Técnicas Bacteriológicas , Biofilmes/crescimento & desenvolvimento , Staphylococcus haemolyticus/genética , Staphylococcus haemolyticus/fisiologia , Biopolímeros/metabolismo , Meios de Cultura/química , Genes Bacterianos , Genótipo , Humanos , Fenótipo , Infecções Estafilocócicas/microbiologia , Staphylococcus haemolyticus/crescimento & desenvolvimento , Staphylococcus haemolyticus/isolamento & purificaçãoRESUMO
Prostaglandin E receptor 4 (PTGER4) is an essential receptor that can detect various physiological and pathological stimuli and has been implicated in a wide variety of biological processes, including the regulation of immune responses, cytokine production, and apoptosis. In this report, the first mollusk PTGER4, referred to as ChPTGER4, was cloned and characterized from the Hong Kong oyster Crassostrea hongkongensis. Its full-length cDNA is 1734 bp in length, including 5'- and 3'-untranslated region (UTRs) of 354 bp and 306 bp, respectively, and an open reading frame (ORF) of 1074 bp. ChPTGER4 comprises 357 amino acids and shares significant homology with its vertebrate homologs. The results of phylogenetic analysis revealed that ChPTGER4 clusters with PTGER4 from the Pacific oyster. In addition, quantitative real-time PCR analysis revealed that ChPTGER4 was constitutively expressed in all tissues examined and that its expression was significantly up-regulated in hemocytes and gills following challenge by pathogens (Vibrio alginolyticus, Staphylococcus haemolyticus and Saccharomyces cerevisiae) and pathogen-associated molecular patterns (PAMPs: lipopolysaccharide (LPS) and peptidoglycan (PGN). Moreover, fluorescence microscopy analysis revealed that ChPTGER4 localized to the membrane, and its overexpression significantly enhanced NF-κB reporter gene activation in the HEK293T cell line. In summary, this study provides the first experimental evidence of a functional PTGER4 in mollusks, which suggests its involvement in the innate immune response in oyster.
Assuntos
Crassostrea/imunologia , Crassostrea/microbiologia , Imunidade Inata , Receptores de Prostaglandina E Subtipo EP4/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Crassostrea/genética , Crassostrea/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Peptidoglicano/farmacologia , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Aleatória , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Saccharomyces cerevisiae/fisiologia , Alinhamento de Sequência , Staphylococcus haemolyticus/fisiologia , Vibrio alginolyticus/fisiologiaRESUMO
Oxacillin-resistant Staphylococcus haemolyticus (ORSH) was found as the most prevalent (77.5%) species of coagulase-negative staphylococci associated with bacteremia in neonates making use of intravenous catheters in an intensive care unit of a Brazilian teaching hospital. Thirty-one blood isolates were confirmed as S. haemolyticus by sequencing of the 16S and clustered in 6 pulsed-field gel electrophoresis types (with 58% of the strains belonging to 2 predominant types B and D). S. haemolyticus was mostly oxacillin-resistant (90.3%) displaying multiresistance profiles (70.4%). However, the mecA gene was undetected in 22.6% strains. ORSH exhibited slime production on Congo-Red agar (67.7%), adherence to polystyrene (96.7%), and glass (87%) surfaces. Interestingly, ica-operon was detected in 58% strains, mostly belonging to the B, D, and F genotypes, which is a significantly higher percentage when compared to other studies conducted at different parts of the globe. Data indicated that ica operon and biofilm-forming ORSH are endemic in Brazilian nosocomial environment.
Assuntos
Bacteriemia/epidemiologia , Infecção Hospitalar/epidemiologia , Farmacorresistência Bacteriana Múltipla , Infecções Estafilocócicas/epidemiologia , Staphylococcus haemolyticus/isolamento & purificação , Bacteriemia/microbiologia , Aderência Bacteriana , Brasil/epidemiologia , Análise por Conglomerados , Infecção Hospitalar/microbiologia , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Genótipo , Hospitais de Ensino , Humanos , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Dados de Sequência Molecular , Polissacarídeos Bacterianos/metabolismo , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Infecções Estafilocócicas/microbiologia , Staphylococcus haemolyticus/classificação , Staphylococcus haemolyticus/genética , Staphylococcus haemolyticus/fisiologiaRESUMO
AIM: In vitro efficacy evaluation of eleven topical antimicrobials against multidrug-resistant (MDR) bacteria isolated from burn wounds of our patients. MATERIAL AND METHODS: Growth of six MDR bacterial strains: Pseudomonas aeruginosa (2 strains), Staphylococcus aureus, Staphylococcus haemolyticus, Enterococcus faecalis and Escherichia coli in burn-wound models was evaluated 24-h after application of the tested agents. Four different wound models were created to investigate the role of time elapsed between inoculation of bacteria and application of the agents on their antimicrobial activity and efficacy. RESULTS: The efficacy against all the 6 bacteria in freshly contaminated wounds was excellent in majority of the tested agents. The longer was the time interval between inoculation and application of the topical antimicrobial agents, the higher failure of the agents was observed. CONCLUSIONS: Topical antimicrobials play an important role in treatment of burn wounds, but they should be used according to their efficacy against bacterial strains present in patients' wounds. In cases where topical agents have been applied after 24 h, when formation of mature biofilm in the wound could be expected, it would probably not be possible to kill all the bacteria using topical antimicrobial therapy only.
Assuntos
Anti-Infecciosos Locais/farmacologia , Queimaduras/microbiologia , Farmacorresistência Bacteriana Múltipla/fisiologia , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus haemolyticus/efeitos dos fármacos , Ácido Acético/farmacologia , Enterococcus faecalis/isolamento & purificação , Enterococcus faecalis/fisiologia , Escherichia coli/isolamento & purificação , Escherichia coli/fisiologia , Mel , Humanos , Iminas , Testes de Sensibilidade Microbiana , Nitrofurazona/farmacologia , Povidona-Iodo/farmacologia , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/fisiologia , Piridinas/farmacologia , Sulfadiazina de Prata/farmacologia , Staphylococcus haemolyticus/isolamento & purificação , Staphylococcus haemolyticus/fisiologiaRESUMO
We analysed the antimicrobial susceptibility, biofilm formation and genotypic profiles of 27 isolates of Staphylococcus haemolyticus obtained from the blood of 19 patients admitted to a hospital in Rio de Janeiro, Brazil. Our analysis revealed a clinical significance of 36.8% and a multi-resistance rate of 92.6% among these isolates. All but one isolate carried the mecA gene. The staphylococcal cassette chromosome mec type I was the most prevalent mec element detected (67%). Nevertheless, the isolates showed clonal diversity based on pulsed-field gel electrophoresis analysis. The ability to form biofilms was detected in 66% of the isolates studied. Surprisingly, no icaAD genes were found among the biofilm-producing isolates.
Assuntos
Antibacterianos/uso terapêutico , Bacteriemia , Biofilmes/crescimento & desenvolvimento , Resistência Microbiana a Medicamentos/fisiologia , Staphylococcus haemolyticus , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Criança , Pré-Escolar , Eletroforese em Gel de Campo Pulsado , Feminino , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Fatores de Risco , Staphylococcus haemolyticus/efeitos dos fármacos , Staphylococcus haemolyticus/genética , Staphylococcus haemolyticus/fisiologia , Adulto JovemRESUMO
Infections due to coagulase-negative staphylococci (CoNS) most frequently occur after the implantation of medical devices and are attributed to the biofilm-forming potential of CoNS. Staphylococcus haemolyticus is the second most frequently isolated CoNS from patients with hospital-acquired infections. There is only limited knowledge of the nature of S. haemolyticus biofilms. The aim of this study was to characterize S. haemolyticus biofilm formation. We analyzed the biofilm-forming capacities of 72 clinical S. haemolyticus isolates. A detachment assay with NaIO(4), proteinase K, or DNase was used to determine the main biofilm components. Biofilm-associated genes, including the ica operon, were analyzed by PCR, and the gene products were sequenced. Confocal laser scanning microscopy (CLSM) was used to elucidate the biofilm structure. Fifty-three isolates (74%) produced biofilms after growth in Trypticase soy broth (TSB) with glucose, but only 22 (31%) produced biofilms after growth in TSB with NaCl. It was necessary to dissolve the biofilm in ethanol-acetone to measure the optical density of the full biofilm mass. DNase, proteinase K, and NaIO(4) caused biofilm detachment for 100%, 98%, and 38% of the isolates, respectively. icaRADBC and polysaccharide intercellular adhesin (PIA) production were found in only two isolates. CLSM indicated that the biofilm structure of S. haemolyticus clearly differs from that of S. epidermidis. We conclude that biofilm formation is a common phenotype in clinical S. haemolyticus isolates. In contrast to S. epidermidis, proteins and extracellular DNA are of functional relevance for biofilm accumulation, whereas PIA plays only a minor role. The induction of biofilm formation and determination of the biofilm mass also needed to be optimized for S. haemolyticus.
Assuntos
Biofilmes/crescimento & desenvolvimento , Staphylococcus haemolyticus/fisiologia , Adulto , Proteínas de Bactérias/metabolismo , Análise por Conglomerados , Impressões Digitais de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Eletroforese em Gel de Campo Pulsado , Genes Bacterianos , Humanos , Recém-Nascido , Microscopia Confocal , Dados de Sequência Molecular , Filogenia , Polissacarídeos Bacterianos/metabolismo , Análise de Sequência de DNA , Homologia de Sequência , Infecções Estafilocócicas/microbiologia , Staphylococcus haemolyticus/isolamento & purificação , Adulto JovemRESUMO
Staphylococcus haemolyticus is a pathogen frequently isolated from dairy cows and small ruminants. However, it always appears in only a few animals and not as a major pathogen. Recently, in a dairy goat herd of approximately 250 milking animals, 25.6% (46/180 goats) had milk cultures with atypical highly mucoid colonies accompanied by elevated somatic cell counts. The isolates were identified as Staph. haemolyticus. The present study describes the steps used in an attempt to identify the bacterium and to compare it with other coagulase-negative staphylococci (CNS) including Staph. haemolyticus. Species identification performed with the API STAPH-IDENT 32 kit showed >99.4% identity confirmed by 16S rDNA sequencing tests. Microscopically the atypical Staph. haemolyticus strains showed unique cuboidal tetrad clusters reminiscent of those of the genus Sarcina. The outbreak caused by an atypical CNS underlines the need for accurate biochemical and genetic methods for ultimate identification of CNS to the species level.