Molecular epidemiological and antibiotic susceptibility characterization of Brucella isolates from humans in Sicily, Italy.

Brucellosis is a serious problem in Sicily. Brucella melitensis was identified as the species most frequently isolated in humans in Italy. No data, however, are available about the molecular epidemiological characterization of Brucella isolates from humans. We have conducted this study to molecularly characterize clinical isolates of Brucella spp. and to evaluate their antimicrobial susceptibilities. Twenty Brucella isolates were studied. Differential growth characteristics and DNA polymorphisms such as the restriction patterns of the PCR-amplified omp2a and omp2b genes, rpoB nucleotide sequencing, and multiple-locus variable-number tandem repeat analysis of 16 loci (MLVA-16) were used to characterize the strains. In vitro antibiotic susceptibility was determined by the E-test method on two different agar media, and the results were compared. All isolates were identified as B. melitensis biovar 3. rpoB nucleotide sequence analysis allowed the identification of two different genotypes of B. melitensis biovar 3. On the other hand, the MLVA-16 typing assay recognized 17 distinct genotypes. All isolates were sensitive to all tested antibiotics (rifampin, doxycycline, ciprofloxacin, ceftriaxone, and trimethoprim-sulfamethoxazole), and the Mueller-Hinton agar plate is recommended for antibiotic susceptibility testing by the E-test method. Our findings identify B. melitensis biovar 3 as the etiological agent isolated in Sicily and encourage the use of both molecular methods, and in particular of the MLVA-16 assay, in epidemiological trace-back analysis. This study represents the first epidemiological data from molecular typing of Brucella strains circulating in Italy and, in particular, in eastern Sicily.

Molecular characterization of the rpoB gene in Brucella species: new potential molecular markers for genotyping.

The rpoB gene encoding the beta subunit of the DNA-dependent RNA polymerase was molecularly characterized by PCR amplification and DNA sequencing in 26 Brucella reference strains by using primers selected according to the B. melitensis 16 M rpoB published sequence. Comparison of the rpoB nucleotide sequence of all Brucella strains analysed revealed specific nucleotide variations associated with different Brucella species and biovars. 17 rpoB alleles were recognized and new Brucella typing is proposed. Our results suggest that the rpoB gene polymorphism can be used to identify all Brucella species and most of the biovars, offering an improvement over conventional typing methods.

Molecular study of genes involved in virulence regulatory pathways in Bacillus anthracis vaccine strain "Carbosap".

This study investigated the genetic bases of attenuation in the Bacillus anthracis vaccine strain "Carbosap" used in Italy against anthrax in cattle and sheep. Twelve genes involved in virulence regulatory pathways underwent sequence analysis in comparison with a B. anthracis virulent strain.

In vitro stimulation of murine peritoneal monocytes induced by alginates.

In this trial we assessed the effect of soluble alginates on murine cells. Mouse peritoneal monocytes were stimulated in vitro with a solution of alginate. The production of TNF-alpha and nitric oxide (NO), the expression of surface molecules CD80 and CD86, and the ability of monocytes to phagocyte bacteria were assessed, in order to evaluate the effect of alginate on cell functionality. We showed that mouse peritoneal monocytes stimulated with alginate produce NO and TNF-alpha. In addition, alginate is able also to increase their phagocytic activity and to a lesser extent also to increase the expression of CD80. Even with different degrees, it implies that alginates per se act directly on immune response, being able to effectively stimulate proinflammatory activity. These findings corroborate the idea that alginates can represent interesting adjuvants to use to increase the efficacy of antigenic stimulation.

Genetic bases of the rifampin resistance phenotype in Brucella spp.

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 beta 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.

Brucella abortus RB51 induces protection in mice orally infected with the virulent strain B. abortus 2308.

Brucellae are gram-negative, facultative intracellular bacteria which are one of the most common causes of abortion in animals. In addition, they are the source of a severe zoonosis. In this trial, we evaluated the effect of oral inoculation of Brucella abortus RB51 in mice against a challenge infection with B. abortus 2308. First, we showed that a gastric acid neutralization prior to the oral inoculation contributed to a more homogeneous and consistent infection with both vaccine strain B. abortus RB51 and virulent strain B. abortus 2308. Successively, we assessed the clearance and the immune response following an oral infection with B. abortus RB51. Oral inoculation gave a mild infection which was cleared 42 days after infection, and it induced a delayed humoral and cell-mediated immune response. Finally, we immunized mice by oral inoculation with B. abortus RB51, and we challenged them with the virulent strain B. abortus 2308 by an oral or intraperitoneal route 42 days after vaccination. Oral inoculation of B. abortus RB51 was able to give protection to mice infected with the virulent strain B. abortus 2308 by the oral route but not to mice infected intraperitoneally. Our results indicate that oral inoculation of mice with B. abortus RB51 is able to give a protective immunity against an oral infection with virulent strains, and this protection seems to rely on an immune response at the mucosal level.

Coombs antiglobulin test using Brucella abortus 99 as antigen to detect incomplete antibodies induced by B abortus RB51 vaccine in cattle.

This study showed that vaccination of cattle with Brucella abortus rough strain RB51 induces incomplete antibodies that can be detectable by a Coombs antiglobulin test using the B. abortus 99 smooth strain.

Effect of exogenous interleukin-18 (IL-18) and IL-12 in the course of Brucella abortus 2308 infection in mice.

In this study we demonstrated that combined inoculation of interleukin-12 (IL-12) and IL-18 reduced the number of bacteria in the spleens of mice infected with Brucella abortus 2308 and that the effect of the treatment was mediated by an increased capability of spleen cells to produce gamma interferon at the early phase of infection.