Rifampin is one of the most potent and broad-spectrum antibiotics against bacterial pathogens. Its bactericidal activity is due to its ability to bind to the ␤ subunit of the DNA-dependent RNA polymerase encoded by the rpoB gene. Mutations of the rpoB gene have been characterized in rifampin-resistant (Rif r ) strains of Escherichia coli and Mycobacterium tuberculosis. The genetic bases of Rif r in Brucella spp. are still unknown. In the present study, the nucleotide sequences of the rpoB gene of the Rif r vaccine strain Brucella abortus RB51 and of 20 Rif r clones derived in our laboratory from two Brucella melitensis isolates were determined. These sequences were then compared to those of the respective rifampin-susceptible (Rif s ) parental strains and to the published B. melitensis strain 16M. All Rif r strains carried one or more missense mutations mapping in two regions of the rpoB gene. These two "hot" regions were investigated in eight additional Rif r Brucella laboratory mutants and in 20 reference Rif s Brucella strains. rpoB mutations were found in all Rif r mutants. In contrast, no missense mutations were found in any analyzed Rif s strains. Our results represent the first from a study of the molecular characterization of rpoB mutations in resistant Brucella strains and provide an additional proof of the association of specific rpoB mutations with the development of the Rif r phenotype in prokaryotes. In addition, because of the relationship between Rif r and the attenuation of virulence in Brucella spp., studies of virulence in these mutants may provide useful information about the genetic basis of pathogenesis in Brucella.Rifampin is one of the most potent and broad-spectrum antibiotics against bacterial pathogens and is an important component of effective multidrug therapies for the treatment of brucellosis in humans (2,5,26). Previously reported data have indicated that the addition of rifampin to media tends to turn Brucella abortus cultures rough and that organisms with rifampin resistance (Rif r ) are less virulent than rifampin-susceptible (Rif s ) strains (17, 23). Rifampin was therefore utilized to obtain the stable, rough, and attenuated B. abortus strain RB51, currently used in the United States as the official vaccine for brucellosis eradication in cattle. The roughness and attenuation are two important characteristics for the vaccine strain RB51, which is able to induce an adequate, protective cell-mediated response against infection with virulent Brucella strains without interfering with standard serological tests for brucellosis diagnosis (7,15,16,18,19,20,22,27). Strain RB51 was derived by repeated passaging of the smooth and virulent B. abortus strain 2308 on media supplemented with rifampin (23). Its high level of Rif r constitutes a useful biochemical marker for its identification. This characteristic was used to develop a selective medium to assist in the recovery of the vaccine strain RB51 from experimental and field samples for its isolation (10).The genetic bases for Rif r in ...

Salmonella enterica serovar Typhimurium is an important zoonotic gastrointestinal pathogen responsible for foodborne disease worldwide. It is a successful enteric pathogen because it has developed virulence strategies allowing it to survive in a highly inflamed intestinal environment exploiting inflammation to overcome colonization resistance provided by intestinal microbiota. In this study, we used piglets featuring an intact microbiota, which naturally develop gastroenteritis, as model for salmonellosis. We compared the effects on the intestinal microbiota induced by a wild type and an attenuated S. Typhimurium in order to evaluate whether the modifications are correlated with the virulence of the strain. This study showed that Salmonella alters microbiota in a virulence-dependent manner. We found that the wild type S. Typhimurium induced inflammation and a reduction of specific protecting microbiota species (SCFA-producing bacteria) normally involved in providing a barrier against pathogens. Both these effects could contribute to impair colonization resistance, increasing the host susceptibility to wild type S. Typhimurium colonization. In contrast, the attenuated S. Typhimurium, which is characterized by a reduced ability to colonize the intestine, and by a very mild inflammatory response, was unable to successfully sustain competition with the microbiota.

Determination of the G and P serotypes of group A bovine rotaviruses from 149 samples of feces or intestinal contents collected from calves showing clinical signs of neonatal diarrhea was performed by a nested reverse transcription-PCR typing assay. The G6 serotype was the most prevalent, accounting for viruses in 55.7% of the samples; viruses of the G10 and G8 serotypes were found in 34.9 and 4.7% of the samples, respectively. The virus in one sample (0.7%) was not classified due to concomitant infection with G6 and G8 strains, whereas viruses in six samples (4.0%) could not be characterized with any of the three G serotype-specific primers selected for the present study. When examined for their P-serotype specificities, viruses in 55 and 42.3% of the samples were characterized as P[11] and P[5], respectively, no P[1] serotype was identified, and viruses in 2.7% of the samples could not be classified due to multiple reactivity with both P[5]- and P[11]-specific primers. Various combinations of G and P serotypes were observed, the most frequent being G6,P[5] (38.3%), G10,P[11] (31.5%), and G6,P[11] (15.4%). The results of the present study, while contributing to a better understanding of the epidemiology of bovine rotaviruses in Italy, address the relevance of serotype specificity with regard to the constancy of the quality of bovine rotavirus vaccines under different field conditions.

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Vaccination against Brucella infections in animals is usually performed by administration of live attenuated smooth B. abortus strain S19 and B. melitensis strain Rev1. They are proven effective vaccines against B. abortus in cattle and against B. melitensis and B. ovis in sheep and goats, respectively. However, both vaccines have the main drawback of inducing O-polysaccharide-specific antibodies that interfere with serologic diagnosis of disease. In addition, they retain residual virulence, being a cause of abortion in pregnant animals and infection in humans. To overcome these problems, one approach is to develop defined rough mutant Brucella strains lacking O antigen of lipopolysaccharide. B. abortus rough strain RB51, a rifampin-resistant mutant of virulent strain B. abortus 2308, is used as a vaccine against B. abortus infection in cattle in some countries. However, RB51 is not effective in sheep, and there is only preliminary evidence that it is effective in goats. In this study, we tested the efficacies of six rifampin-resistant rough strains of B. melitensis in protecting BALB/c mice exposed to B. melitensis infection. The protective properties, as well as both humoral and cellular immune responses, were assessed in comparison with those provided by B. melitensis Rev1 and B. abortus RB51 vaccines. The results indicated that these rough mutants were able to induce a very good level of protection against B. melitensis infection, similar to that provided by Rev1 and superior to that of RB51, without inducing antibodies to O antigen. In addition, all B. melitensis mutants were able to stimulate good production of gamma interferon. The characteristics of these strains encourage further evaluation of them as alternative vaccines to Rev1 in primary host species.Brucellosis is a major zoonotic disease, widely distributed in humans and domestic and wild animals, especially in developing countries. Among the different species of the Brucella genus, B. abortus and B. melitensis are the most pathogenic and virulent, not only for cattle or for sheep and goats, respectively, but also for other animal species. The occurrence of the disease in humans is largely dependent on the animal reservoir, with the highest rate of human infection in areas where rates of brucellosis in sheep and goats are high (6,35).Brucellosis vaccines are essential elements in control programs. Attenuated B. abortus strain 19 and B. melitensis strain Rev1 are proven effective vaccines; they induce good levels of protection against B. abortus in cattle and against B. melitensis in sheep and goats, preventing premature abortions (7, 30, 31). However, both vaccines have the drawback of inducing Opolysaccharide-specific antibodies that interfere with the discrimination between vaccinated and infected animals during serological screening (7, 31). In addition, they retain pathogenicity and sometimes cause abortion in vaccinated animals (10, 18, 41) and remain infectious for humans (3,5,26). The use of the conjunctival route when administering B. melit...