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.

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.

Genetic relatedness between group B streptococci originating from bovine mastitis and a human group B Streptococcus type V cluster displaying an identical pulsed-field gel electrophoresis pattern.

Twenty isolates of group B streptococcus (GBS) were recovered from the milk of cows with bovine mastitis on three farms located in the south and south-east of Brazil between 1987 and 1988. These isolates were characterised by molecular methods and compared with a collection of 103 human GBS isolates from colonised and infected patients in the same region between 1980 and 2003. Some of the bovine isolates shared identical or similar pulsed-field gel electrophoresis (PFGE) patterns with a PFGE clone of human GBS type V. In addition, these bovine and human isolates also possessed the same ribotype. Multilocus sequence typing (MLST) of representative isolates confirmed the genetic relationship between the human and bovine GBS isolates with identical PFGE patterns, which clustered in the same ST-26 clonal complex. These data support the hypothesis that some bovine GBS strains are related closely to human isolates and may infect humans, or vice versa. Further comparative genomic analyses of GBS isolates from bovine and human origins are required to investigate this hypothesis further.

Purification and molecular characterization of antibacterial compounds produced by Lactobacillus murinus strain L1.

AIMS: The aim of this work was to purify and characterize antibacterial compounds produced by Lactobacillus murinus strain L1. METHODS AND RESULTS: Antagonistic activity was observed in a deferred agar-spot assay against spoilage and pathogenic bacteria, but not against lactobacilli. The inhibitory activity occurred between pH 3.0 and 5.0, and was heat stable. The active compounds were purified by gel filtration chromatography and two peaks of antibacterial activity were observed using Bacillus cereus ATCC 11778 and Shigella sonnei ATCC 11060 as indicator strains. Two active low molecular weight compounds were responsible for this phenomenon and UV spectroscopy, gas chromatography and mass spectrometry were used to characterize them. One of them is lactic acid, while the other is a mono-substituted aromatic ring apparently constituted by group residues of m/z 192 linked in tandem to phenylalanine. CONCLUSIONS: Lactobacillus murinus produces at least two low molecular weight compounds active against B. cereus and Sh. sonnei. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first purification of a new broad-spectrum antibacterial compound from Lact. murinus which inhibits various pathogenic and food spoilage bacteria without acting on other lactobacilli. Using it as a biotechnological control agent of bacterial spoilage may be a promising possibility for the food industry.

Pulsed-field gel electrophoresis of human group B streptococci isolated in Brazil.

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.

Effect of penicillin on surface carbohydrate, hemolysin and morphology of Streptococcus pyogenes during and after the post-antibiotic phase.

The post-antibiotic effect (PAE) of penicillin was measured in vitro against a group A streptococcal strain by the kinetic growth method. The duration of the effect was 2.8 h. The bacterial morphology and some streptococcal products were analyzed during and after the PAE, after being exposed to penicillin in a concentration of 1xMIC for 2 h. Bacteria not previously exposed to penicillin were used as a control culture. Morphological changes and increases in the size of treated streptococci were observed by electronic microscope during the post-antibiotic phase. The post-penicillin effect on the production of cell-bound hemolysin and free hemolysin was examined using sheep red blood cells. Production of cell-bound hemolysin rose sharply, but was inhibited by the antimicrobial agent. The free lysin diminished significantly, and concomitantly with a higher production of free toxin by the treated cells. No effect was observed on the specific carbohydrate group when the antigen was tested with streptococcal group A antiserum.

Proteases from actinomycetes interfere in solid media plate assays of hyaluronidase activity.

Four hundred and fifteen actinomycete strains were screened for hyaluronidase activity in two plate assays media. In the first one, using hyaluronic acid as substrate and bovine serum albumin (BSA) to help precipitation of the nondegraded substrate, only strain 594 and hyaluronidase control were positive. In the second assay, plates with hyaluronic acid, but not BSA, gave the same results. For plates containing only BSA, proteinase activity was detected in strain 594. When hyaluronic acid was treated with pronase, the only clear zones, in the second assay without BSA, were those around hyaluronidase controls. Protease activity, commonly found in actinomycetes, was detected only in strain 594, among the 415 studied, when tested in hyaluronidase assay using hyaluronate plus BSA. This may be due to the composition of the growth medium, since media with different composition gave different results for protease activity in each of the 15 strains analyzed. These data suggest that proteases can affect an accurate detection of hyaluronidase in media containing proteins, not only from hyaluronate preparations, but also from other medium ingredients. Thus, for a correct interpretation of the method, they must be excluded. Commercial Hyaluronidase used as controls must be also tested for the presence of protease contamination.

Postantibiotic effect of penicillin on Streptococcus anginosus.

We studied the postantibiotic effect of penicillin G on bacterial growth of two strains of Streptococcus anginosus by optical density readings of the cultures and by counting the numbers of viable cells. Duration of the effect of the drug in concentrations equivalent to the MICs after exposure for 2 h was 3.4 and 3.5 h. The production of streptococcal substances was examined during the postantibiotic phase. The antibiotic caused an increase in deoxyribonuclease and a decrease in both free and cell-bound hemolysin activities of one strain. The other strain displayed an increase in hyaluronidase and both free and bound hemolysin production.

Changes in the surface carbohydrate composition and exposure of anionic groups caused by beta-lactam antibiotics in streptococci.

Upon the addition of beta-lactam antibiotics at concentrations that caused a 50% reduction in the dry weight, beta-haemolytic streptococci produced increased amount of rhamnose, though the hexosamine content remained unchanged. These sugars are components of C-carbohydrate. Sialic acid content also increased in group B streptococcal surfaces and penicillin treatment generated new accessible surface sialic acid residues.

Penicillin post-antibiotic effects on the biology of group A streptococci.

Microbial growth in a Todd-Hewitt broth has been followed to determine the in-vitro post-antibiotic effects of penicillin in a Lancefield group A streptococcal strain. Bacteria were exposed for 2 h at 37 degrees C to 1 x MIC of penicillin. Following antibiotic removal, inactivation with penicillinase and regrowth in a drug-free broth, the duration of the effect was found to be 2.8 h. By studying the affinity of streptococci for xylene in the post-antibiotic phase we observed that the penicillin treatment had no effect on the cell surface hydrophobicity. The ability of the same streptococci to adhere to human buccal epithelial cells was greatly reduced. Streptococci exposed to penicillin produced much more deoxyribonuclease and hyaluronidase than control bacteria.

Penicillin tolerance among beta-hemolytic streptococci and production of the group carbohydrates, hemolysins, hyaluronidases and deoxyribonucleases.

Penicillin tolerance among 67 strains of beta-hemolytic streptococci was examined by determining the ratio of the minimal bactericidal concentration to the minimal inhibitory concentration as 32 or greater. Tolerance was demonstrated in 15 group A strains and in 11.7, and 4 of groups B, C and G, respectively. Thereafter the effects of a subminimal inhibitory concentration (1/2 MIC) of penicillin on the bacterial products of four tolerant and four nontolerant strains (two of each Lancefield group) were analyzed and compared. The antibiotic caused a marked increase in the expression of the group carbohydrates for strains of group B. Penicillin was found to reduce the cell-bound hemolysin activities of the four tolerant strains and to increase the activity of the other (free) form of nontolerant groups A, C and G hemolysins. Penicillin caused an increase in the extracellular hyaluronidase activities of one group A and groups B, C and G streptococci. With added antibiotic the production of deoxyribonuclease by tolerant groups A, C and G was greatly enhanced and that of the group B streptococcus was arrested.

Antibacterial activity of a substance produced by the fungus Pycnoporus sanguineus (Fr.) Murr.

A fraction obtained from the culture fluids of Pycnoporus sanguineus fungus was shown to contain a compound with biological activity against strains of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus and members of the genus Streptococcus. The fraction was clearly more active on Gram-positive cocci than on Gram-negative bacilli.

Cell surface hydrophobicity and the net electric surface charge of group B streptococci: the role played in the micro-organism-host cell interaction.

The cell surface hydrophobicity, net electric surface charge and cell adhesion of six group B streptococci strains were assessed. Treatment with trypsin reduced cytoadhesion of the six strains (80340, 90356, 85147, 90222, 90186 and 88641) and induced loss of surface negative charge in the other four strains (80340, 85147, 90222 and 90186). The same treatment increased the surface hydrophobicity of three strains (90356, 90222 and 88641). Neuraminidase treatment caused a decrease in the negative surface charge of all the strains resulting in significant increases in both cytoadhesion and surface hydrophobicity of five (80340, 90356, 85147, 90222 and 88641) and four (90356, 85147, 90222 and 88641) strains, respectively. This indicates that sialic acid residues are important anionogenic groups exposed on the streptococcal cell surface. Treatment of buccal epithelial cells with N-acetyl-beta-D-glucosaminidase made them less adherent for most of the strains (80340, 85147, 90222, 90186 and 88641) assayed.

Sialic acid content and surface hydrophobicity of group B streptococci.

The sialic acid content and the cell-surface hydrophobicity index of 40 group B streptococci (GBS) strains were assessed. GBS isolated from invasive infections (virulent strains) presented an increased level of sialic acid content (1.4%) when compared with GBS isolated from asymptomatic patients (0.53%). Treatment of GBS strain 85634 with neuraminidase resulted in a decrease (about 25%) in the net negative surface charge as assessed by cell electrophoresis. This finding suggests that sialic acid residues are important anionogenic groups exposed on GBS cell surface. N-acetylneuraminic acid was the only sialic acid derivative characterized in the strain 85634 as evaluated by gas-liquid chromatography. GBS from different serotypes presented a hydrophobic index mean value of 0.9. Even though the sialic acid contributed effectively to the negative charge on GBS cell surface, no difference was observed in the hydrophobic index when virulent and avirulent strains were compared.

Estreptococos do grupo C isolados no Brasil: incidência das espécies, perfil de seuscetibilidade à penicilina e eritromicina e comportamento frente ao teste da bacitracina / Group C streptococci: susceptilities to penicilin, erythromycin and bacitracin

Uma coleção de 27 amostras de estreptococos do grupo C de Lancefield tiveram suas espécies determinadas através de testes bioquímicos. As amostras foram também examinadas quanto à suscetibilidade à penicilina, eritromicina e bacitracina (discos contendo 0,04UI). Uma das amostras era padrão do sorogrupo e as demais foram isoladas no Brasil. Das 1127 amostras estudadas, 112 foram obtidas de material clínico humano, sendo 105 classificadas como S. equisimilis, 3 como S. anginosus, 3 como S. zooepidemicus e 1 como S. dysgalactiae. Dentre as 15 amostras de origem animal, 6 amostras eram de S. equisimilis e 9 de S. zoopidemicus. Quinze das amostras humanas foram sensíveis à bacitracina pelo método de difusão em disco. A concentração mínima inibitória (CMI) da penicilina para as 112 amostras humanas variou de 0,0075 a 0,12 ug/ml e de 0,0075 a 0,03 ug/ml para as 15 amostras animais. O valor da CMI mais encontrado nas amostras de ambas as origens foi de 0,015 ug/ml. Quatorze das 127 amostras foram resistentes à eritromicina sendo que 10 destas eram humanas (S. equisimilis) e 4 animais (3 de S. equisimilis e 1 de S. zooepidemicus). Até 6% dos estreptococos do grupo C são sensíveis à bacitracina (resiltados falso-positivos) e isto representa uma percentagem aceitável. Se estudos adicionais confirmarem nossas observações, esforços para melhorar a identificação de amostras de grupo C deveriam ser feitos para evitar uma incorreta identificação presuntiva como estreptococos do grupo A nos laboratórios de rotina. Mais atenção deveria ser dada para a suscetibilidade à eritromicina uma vez que 11% das nossas amostras foram resistentes a este antimicrobiano, enquanto que os dados da literatura relatam uma resistência em torno de 2%.

Extracellular deoxyribonucleases of streptococci: a comparison of their occurrence and levels of production among 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.

Liquid medium for rapid presumptive identification of group B streptococci.

A suitable test was developed for distinguishing group B streptococci from other beta-hemolytic streptococci during growth in liquid medium. One hundred and sixty of 161 human group B strains tested yielded positive reactions within a 5 h incubation. The dye medium tested is a reliable substitute for more expensive serological procedures.

Correlation between growth in antibiotic-medium and hemolytic activity of group C and G streptococci.

The effect of a subminimal inhibitory concentration of penicillin on the production of bound and free hemolysins by streptococci was examined using sheep red blood cells. A marked decrease of a group C cell-free and bound activities was observed with penicillin at a concentration of 1/3 of the MIC whereas an increase was observed with those of a group G strain. Potassium ferricyanide and anti-streptolysin O (group A streptococcus) were strongly inhibitory for the free activities of both strains. The cell-bound activities were stimulated by addition of RNA during bacterial growth in control cultures and also in drug-containing media.

Penicillin and clindamycin alter some group A streptococcal products.

The effects of subMICs of penicillin and clindamycin on group A streptococcal products were analyzed. Penicillin caused an increase in the expression of the group carbohydrate for the three strains tested; in contrast, clindamycin inhibited the expression in two strains. The M and T proteins were not affected by the drugs. Clindamycin caused an increase in the hyaluronidase activity and both antimicrobial agents inhibited the activity of streptolysin at 1/2 MIC.