RESUMO

The present study was conducted in order to determine the frequency of pvl gene among the pathogenic and healthy population isolates of Methicillin Resistant Staphylococcus aureus (MRSA) and Methicillin Sensitive Staphylococcus aureus (MSSA). For this purpose, nasal swab samples were collected from the healthy individuals (to be used as controls, all the samples were collected irrespective of the sex and age factors), the pathogenic samples were collected from different patients suffering from skin &soft tissue infections caused by S. aureus (to be used as test samples).Both of these population samples were analyzed for the presence of pvl gene. S.aureus were identified through conventional microbiological identification procedures. In the case of normal samples, 70 nasal swabs were collected and only 33 (47%) proved to be S. aureus while 20 pathogenic samples were collected and all (100%) were cleared as S. aureus. For further distribution of samples into MRSA and MSSA, antibiotic susceptibility pattern was checked by using the standard protocols of Kirby-Bauer disc diffusion method. Two antibiotic discs Oxacillin (OX: 1ug) and cefoxitin (FOX: 30ug) were used. Among healthy population, MRSA was found to be 79% (n=26) and MSSA were present as 21% (n= 7). Among pathogenic strains 100% MRSA was detected where n= 20. Detection of pvl gene among the MRSA and MSSA isolates was done by using the uniplex PCR followed by gel electrophoresis. MRSA and MSSA of normal healthy population carried 49% and 7% pvl gene respectively. While, pathogenic MRSA samples carried 46% pvl gene among them. Potentially alarming percentage of pvl gene is present among the normal healthy individuals which indicates a future threat and a major health concern.

O presente estudo almejou determinar a frequência do gene PVL entre os isolados patogênicos e saudáveis da população de Staphylococcus aureus resistente à meticilina (MRSA) e Staphylococcus aureus sensível à meticilina (MSSA). Para este propósito, amostras de swab nasal foram coletadas de indivíduos saudáveis (utilizadas como controle, todas as amostras foram coletadas independentemente do sexo e idade), e de diferentes pacientes com infecções de pele e tecidos moles causadas por S. aureus (utilizadas como amostras de teste). Ambas as amostras populacionais foram analisadas quanto à presença do gene PVL. S.aureus foram identificados através de procedimentos convencionais de identificação microbiológica. No caso de amostras normais, 70 swabs nasais foram coletados e apenas 33 (47%) provaram ser S. aureus, enquanto 20 amostras patogênicas foram coletadas e todas (100%) foram eliminadas como S. aureus. Para distribuição posterior de amostras em MRSA e MSSA, o padrão de suscetibilidade a antibióticos foi verificado a partir dos protocolos padrão do método de difusão de disco de Kirby-Bauer. Foram utilizados dois discos de antibióticos Oxacilina (OX: 1ug) e cefoxitina (FOX: 30ug). Entre a população saudável, MRSA foi encontrado em 79% (n = 26) e MSSA estava presente em 21% (n = 7). Entre as cepas patogênicas, 100% de MRSA foi detectado onde n = 20. A detecção do gene PVL entre os isolados de MRSA e MSSA foi feita usando a PCR uniplex seguida de eletroforese em gel. MRSA e MSSA da população saudável normal carregavam 49% e 7% do gene PVL, respectivamente. Enquanto as amostras patogênicas de MRSA carregavam 46% do gene PVL entre elas. Uma porcentagem potencialmente alarmante do gene PVL foi detectada entre os indivíduos saudáveis normais, o que indica uma ameaça futura e um grande problema de saúde.

Assuntos

RESUMO

Hybrid strains of Escherichia coli combine virulence traits of diarrheagenic (DEC) and extraintestinal pathogenic E. coli (ExPEC), but it is poorly understood whether these combined features improve the virulence potential of such strains. We have previously identified a uropathogenic E. coli (UPEC) strain (UPEC 252) harboring the eae gene that encodes the adhesin intimin and is located in the locus of enterocyte effacement (LEE) pathogenicity island. The LEE-encoded proteins allow enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) to form attaching and effacing (A/E) lesions in enterocytes. We sought to characterize UPEC 252 through whole-genome sequencing and phenotypic virulence assays. Genome analysis unveiled that this strain harbors a complete LEE region, with more than 97% of identity comparing to E2348/69 (EPEC) and O157:H7 Sakai (EHEC) prototype strains, which was functional, since UPEC 252 expressed the LEE-encoded proteins EspB and intimin and induced actin accumulation foci in HeLa cells. Phylogenetic analysis performed comparing 1,000 single-copy shared genes clustered UPEC 252 with atypical EPEC strains that belong to the sequence type 10, phylogroup A. Additionally, UPEC 252 was resistant to the bactericidal power of human serum and colonized cells of the urinary (T24 and HEK293-T) and intestinal (Caco-2 and LS174T) tracts. Our findings suggest that UPEC 252 is an atypical EPEC strain that emerges as a hybrid strain (aEPEC/UPEC), which could colonize new niches and potentially cause intestinal and extraintestinal infections.

Assuntos

RESUMO

Hybrid strains of Escherichia coli combine virulence traits of diarrheagenic (DEC) and extraintestinal pathogenic E. coli (ExPEC), but it is poorly understood whether these combined features improve the virulence potential of such strains. We have previously identified a uropathogenic E. coli (UPEC) strain (UPEC 252) harboring the eae gene that encodes the adhesin intimin and is located in the locus of enterocyte effacement (LEE) pathogenicity island. The LEE-encoded proteins allow enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) to form attaching and effacing (A/E) lesions in enterocytes. We sought to characterize UPEC 252 through whole-genome sequencing and phenotypic virulence assays. Genome analysis unveiled that this strain harbors a complete LEE region, with more than 97% of identity comparing to E2348/69 (EPEC) and O157:H7 Sakai (EHEC) prototype strains, which was functional, since UPEC 252 expressed the LEE-encoded proteins EspB and intimin and induced actin accumulation foci in HeLa cells. Phylogenetic analysis performed comparing 1,000 single-copy shared genes clustered UPEC 252 with atypical EPEC strains that belong to the sequence type 10, phylogroup A. Additionally, UPEC 252 was resistant to the bactericidal power of human serum and colonized cells of the urinary (T24 and HEK293-T) and intestinal (Caco-2 and LS174T) tracts. Our findings suggest that UPEC 252 is an atypical EPEC strain that emerges as a hybrid strain (aEPEC/UPEC), which could colonize new niches and potentially cause intestinal and extraintestinal infections.

RESUMO

RESUMEN Debido a las propiedades probióticas características de las bacterias ácido lácticas, tales como generar compuestos derivados de su fermentación capaces de inhibir múltiples organismos patógenos, hasta crear un ambiente desfavorable para los mismos y finalmente ser usadas como alternativas al uso de medicamentos para tratar y prevenir diversas patologías, en el presente estudio, se buscó evaluar las características probióticas de L. gasseri sobre S. epidermidis en condiciones in vitro. Se determinó la susceptibilidad de las dos cepas a diferentes antibióticos; el efecto de inhibición de L. gasseri y su sobrenadante sobre S. epidermidis; crecimiento de la cepa láctica a diferentes pH, temperatura, sales biliares y bilis bovina; también se estableció la cinética de fermentación y en ella se determinó conteo de microorganismos viables en placa, pH, consumo de azúcar, consumo de proteína y porcentaje de ácido láctico; finalmente mediante HPLC-DAD para L. gasseri se determinó péptidos y ácido láctico, y en el caso de aminoácidos en el sobrenadante se determinó para las dos cepas mediante HPLC-PDA. Se encontró resistencia de ambas cepas a los antibióticos gentamicina y dicloxacilina. La cepa láctica y el sobrenadante inhibieron el crecimiento de S. epidermidis. El crecimiento fue adecuado para las diferentes variables con valores entre 1,8 x 109 a 3,0 x 1012 UFC/150 μl. Se observó la fase exponencial a las 12 horas con un valor de 3 x 1011 UFC/150 μl, con valores de 4,296, 1,26%, 2,032 mg/l y 0,65 mg/l para pH, ácido láctico, consumo de azúcar y consumo de proteína respectivamente. Por último, se identificaron en el sobrenadante de L. gasseri mediante HPLC-DAD el péptido VAL-TIR-VAL con un valor de 0,73 mg/ml, 11,70 g/l de ácido láctico. Los resultados demuestran que Lactobacillus gasseri posee características probióticas sobre S. epidermidis en condiciones in vitro.

ABSTRACT Due to the characteristic probiotic properties of lactic acid bacteria such as the generation of compounds derived from fermentation, which can inhibit multiple pathogenic organisms to create an unfavorable environment for them and finally to be used as an alternative to the use of drugs to treat and prevent various diseases. The present study sought to assess probiotic characteristics of L. gasseri on S. epidermidis under in vitro conditions. The susceptibility of both strains to different antibiotics, the inhibitory effect of L. gasseri and supernatant on S. epidermidis, and the growth of the lactic strain at different pH, temperature, bile salts and bovine bile were determined. The fermentation kinetics was established, and the count of viable microorganisms in plaque, pH, sugar consumption, consumption of protein and percentage of lactic acid was defined. Finally, peptides and lactic acid were determined using HPLC-DAD for L. gasseri, and in the case of amino acids in the supernatant, these were determined with HPLC-PDA for the two strains. The resistance of both strains to the antibiotics gentamicin and dicloxacillin was found. The lactic strain and the supernatant inhibited the growth of S. epidermidis. The growth was suitable for the different variables with values between 1.8 x 109 and 3.0 x 1012 CFU/150 μl. The exponential phase was observed at 12 hours with a value of 3 x 1011 CFU/150 μl, with values of 4.296, 1.26%, 2.032 mg/l and 0.65 mg/l for pH, lactic acid, sugar consumption and protein consumption, respectively. Finally, the peptide VAL-TIR-VAL with a value of 0.73 mg/ml, 11.7 g/l of lactic acid, and the amino acid tyrosine were identified in the supernatant of L. gasseri by HPLC-DAD. The results show that Lactobacillus gasseri have probiotic characteristics on S. epidermidis under in vitro conditions.