Leslie C Benchetrit

Genotyping of Streptococcus agalactiae strains isolated from fish, human and cattle and their virulence potential in Nile tilapia.

Streptococcus agalactiae (Lancefield group B; GBS) is a pathogen that causes meningoencephalitis in fish, mastitis in cows, and neonatal sepsis in humans. The objective of this study was to characterize S. agalactiae isolated from fish (n=27), cows (n=9), and humans (n=10) using pulsed-field gel electrophoresis (PFGE) and to investigate the virulence of the identified strains in Nile tilapia (Oreochromis niloticus). The PFGE types were determined by dendogram analyses and the in vivo virulence was evaluated by experimental infection (using i.p. and immersion routes) of Nile tilapia. Among the fish strains, 5 different PFGE patterns were observed and 21 strains showed the same genetic pattern. In some farms two or three profiles occurred simultaneously. The bovine and human strains exhibited high genetic diversity and few relationships were established among S. agalactiae strains from the three host origins analyzed. Eight S. agalactiae strains from fish caused high mortality of Nile tilapia. Three bovine strains infected Nile tilapia (by i.p. route) and two of those strains caused clinical signs of meningoencephalitis. All human strains (n=5) infected Nile tilapia (by i.p. route) and meningoencephalitis was induced by one strain (by both i.p. and immersion routes). In conclusion, the analyzed strains from the three natural hosts did not show genetic relatedness, yet some of the bovine and human strains were able to infect fish and cause meningoencephalitis. We suggest that genetic linkage is not a prerequisite for S. agalactiae to cross the host-specific barrier.

Pulsed-Field Gel Electrophoresis of Human Group B Streptococci Isolated in Brazil

Abstract The present study addresses epidemiological aspects of Brazilian human group B streptococci (GBS). GBS (103 isolates) were serotyped with specific rabbit anticapsular antibodies by double diffusion in agarose gels. They represented 3 serotypes: 26 II, 41 III, and 36 V. Thereafter, the strains were characterized by pulsed-field gel electrophoresis (PFGE) of DNA treated with SmaI. DNA restriction band sizes were compared and displayed 54 PFGE profiles that were arranged into 18 patterns. Of the predominant patterns detected for the 41 type III isolates 4 were observed in 15 strains from individuals with infections whereas only 3 were identified in 22 streptococci from healthy carriers. Such differences did not separate types II and V streptococci from carriers and patients. The PFGE method is a sensitive, precise, and powerful tool for discriminating streptococcal strains for epidemiological purposes.

Distribution of serotypes and evaluation of antimicrobial susceptibility among human and bovine Streptococcus agalactiae strains isolated in Brazil between 1980 and 2006

Streptococcus agalactiae is a common agent of clinical and subclinical bovine mastitis and an important cause of human infections, mainly among pregnant women, neonates and nonpregnant adults with underlying diseases. The present study describes the genetic and phenotypic diversity among 392 S. agalactiae human and bovine strains isolated between 1980 and 2006 in Brazil. The most prevalent serotypes were Ia, II, III and V and all the strains were susceptible to penicillin, vancomycin and levofloxacin. Resistance to clindamycin, chloramphenicol, erythromycin, rifampicin and tetracycline was observed. Among the erythromycin resistant strains, mefA/E, ermA and, mainly, ermB gene were detected, and a shift of prevalence from the macrolide resistance phenotype to the macrolide-lincosamide-streptogramin B resistance phenotype over the years was observed. The 23 macrolide-resistant strains showed 19 different pulsed-field gel electrophoresis profiles. Regarding macrolide resistance, a major concern in S. agalactiae epidemiology, the present study describes an increase in erythromycin resistance from the 80s to the 90s followed by a decrease in the 2000-2006 period. Also, the genetic heterogeneity described points out that erythromycin resistance in Brazil is rather due to horizontal gene transmission than to spreading of specific macrolide-resistant clones.

Pulsed-field gel electrophoresis, virulence determinants and antimicrobial susceptibility profiles of type Ia group B streptococci isolated from humans in Brazil

Group B streptococci (GBS) infections occur worldwide. Although serotyping has been used for epidemiologic purposes, this does not accurately characterize enough members of a genetically heterogeneous bacterial population. The aims of this work were to evaluate the genetic diversity of 45 type Ia GBS strains isolated in Brazil by pulsed-field gel electrophoresis as well as to evaluate antimicrobial susceptibility profiles and identify virulence genes. Twenty-four strains were assigned to cluster A. All strains under study contained the hylB and scpB genes. The bca gene was detected in only 10 strains and none of the streptococci carried the bac gene. Thirty-nine strains were resistant to tetracycline.

Extracellular deoxyribonucleases of streptococci: a comparison of their occurrence and levels of production Smong beta-hemolytic strains of various serological groups

Production of extracellular deoxyribonuclease by 394 strains of beta hemolytic streptococci was examined employing a deoxyribonucleic acid-methyl green assay. Enzymatic activities were measured in supernatants of bacterial cultures. Of the strains tested, 316 (80%) produced the enzyme. Nuclease production was demonstrated in 100% of group A strains and in 85, 74 and 58% of groups B, C and G, respectively. Levels of nuclease activity were then evaluated statistically. The analysis of variance showed that group A strains produced more enzyme than did streptococci of groups B, C or G. Group B strains produced less nuclease than did isolates of groups C or G. There was no significant difference in the levels of nuclease produced by groups C and G or by the various serological types of group B streptococci. Human group C strains produced more enzyme than animal strains.

The genetic diversity and phenotypic characterisation of Streptococcus agalactiae isolates from Rio de Janeiro, Brazil

Streptococcus agalactiae isolates are more common among pregnant women, neonates and nonpregnant adults with underlying diseases compared to other demographic groups. In this study, we evaluate the genetic and phenotypic diversity in S. agalactiae strains from Rio de Janeiro (RJ) that were isolated from asymptomatic carriers. We analysed these S. agalactiae strains using pulsed-field gel electrophoresis (PFGE), serotyping and antimicrobial susceptibility testing, as well as by determining the macrolide resistance phenotype, and detecting the presence of the ermA/B, mefA/E and lnuB genes. The serotypes Ia, II, III and V were the most prevalent serotypes observed. The 60 strains analysed were susceptible to penicillin, vancomycin and levofloxacin. Resistance to clindamycin, chloramphenicol, erythromycin, rifampin and tetracycline was observed. Among the erythromycin and/or clindamycin resistant strains, the ermA, ermB and mefA/E genes were detected and the constitutive macrolides, lincosamides and streptogramin B-type resistance was the most prevalent phenotype observed. The lnuB gene was not detected in any of the strains studied. We found 56 PFGE electrophoretic profiles and only 22 of them were allocated in polymorphism patterns. This work presents data on the genetic diversity and prevalent capsular serotypes among RJ isolates. Approximately 85% of these strains came from pregnant women; therefore, these data may be helpful in developing future prophylaxis and treatment strategies for neonatal syndromes in RJ.

Virulence characteristics of genetically related isolates of group B streptococci from bovines and humans.

The present study had the objective of evaluating the pathogenic potential of the genetically related strains of Streptococcus agalactiae no. 80427 (human origin) and no. 87159 (bovine origin), and comparing the results with two other strains isolated from bovine mastitis (no. 87244) and invasive human infection (no. 90356), with no genetic or epidemiologic relationship between them or with the first 2 isolates. Virulence genes hylB (hyaluronidase) and lmb (laminin-binding protein) were detected in the 4 strains, and genes bac (beta protein) and bca (alpha protein) were only detected in human strains. The protein profile obtained using SDS-PAGE did not indicate any differences between the 4 strains. No significant difference was detected between human and bovine strains in the assays of adherence to and invasion of 16HBe cells, as well as in the resistance assay for intracellular bacterial survival in macrophages. However, the strain 87159 exhibited a greater survival in the killing test with whole human blood and was more virulent in newborn mice than the 80427 strain. The strain 87244 was not virulent in mice. These data suggest that isolates of human and bovine origins may express similar virulence attributes, leading to a possible, however limited, dissemination.

Evaluation of antimicrobial, antioxidant and phototoxic activities of extracts and isolated compounds from Stachytarpheta cayennensis (Rich.) Vahl, Verbenaceae

Stachytarpheta cayennensis (Rich.) Vahl, Verbenaceae, plant extract, is a Brazilian medicinal plant externally used in folk medicine for purulent ulcers, skin lesions and internally for inflammations, fever, renal disorders and atherosclerosis. S. cayennesis was studied to identify potential bioactive compounds that may justify their therapeutic use against skin lesions and atherosclerosis. The antioxidant, antimicrobial and phototoxicity capacities of the crude ethanolic extract, fractions and isolated compounds from roots of S. cayennesis were evaluated through in vivo and in vitro tests. Strains of Saccharomyces cerevisiae, an eukaryotic cell model, were used to assess both the phototoxicity and the capacity to protect against the lethal oxidative stress caused by menadione and hydrogen peroxide. The extract, fractions and the two major isolated compounds, verbascoside and betulinic acid, of S. cayennensis were able to increase the tolerance and decrease the lipid peroxidation of S. cerevisiae to reactive oxygen species (ROS). The antioxidant activity was also evaluated by scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH•). Verbascoside exhibited a moderate antimicrobial activity against Streptococcus pyogenes, S.epidermidis and Staphylococcus aureus. Neither the ethanolic extract nor fractions showed phototoxicity, indicating that the S. cayennensis extract is safe for use in the treatment of skin lesions and as an active cosmetic ingredient.

Cell surface hydrophobicity and adherence of a strain of group B streptococci during the post-antibiotic effect of penicillin

The minimum inhibitory concentration and post-antibiotic effects of an antimicrobial agent are parameters to be taken into consideration when determining its dosage schedules. The in vitro post-antibiotic effects on cell surface hydrophobicity and bacterial adherence were examined in one strain of group B streptococci. Exposure of the microorganism for 2 h at 37 degrees C to 1 x MIC of penicillin induced a PAE of 1.1 h. The cell surface charge of the Streptococcus was altered significantly during the post-antibiotic phase as shown by its ability to bind to xylene: hydrophobicity was decreased. Bacterial adherence to human buccal epithelial cells was also reduced. The results of the present investigation indicate that studies designed to determine therapeutic regimens should evaluate the clinical significance of aspects of bacterial physiology during the post-antibiotic period.

Hyaluronidase Production by Groups A, B, C, and G Streptococci: A Statistical Analysis

Production of hyaluronidase by 147 strains of beta hemolytic streptococci was studied employing a sensitive dye-binding assay and levels of enzyme activity were evaluated statistically. The analysis of variance showed that group A strains isolated in Rio de Janeiro produced significantly smaller amounts of enzyme than group A strains from a Minneapolis (USA) collection or than groups B and G brazilian isolates. The t test revealed that M not typable nose/throat group A strains produced more hyaluronidase than skin isolates. The analysis of variance did not show a significant difference in the enzyme production between M typable and not typable brazilian strains of group A streptococci. High enzyme--producing strains were not restricted to a few serotypes of group B streptococci.