Michela Tarantino

Genetic Bases of the Rifampin Resistance Phenotype in Brucella spp.

ABSTRACT 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 β subunit of the DNA-dependent RNA polymerase encoded by the rpoB gene. Mutations of the rpoB gene have been characterized in rifampin-resistant (Rifr) strains of Escherichia coli and Mycobacterium tuberculosis. The genetic bases of Rifr in Brucella spp. are still unknown. In the present study, the nucleotide sequences of the rpoB gene of the Rifr vaccine strain Brucella abortus RB51 and of 20 Rifr clones derived in our laboratory from two Brucella melitensis isolates were determined. These sequences were then compared to those of the respective rifampin-susceptible (Rifs) parental strains and to the published B. melitensis strain 16M. All Rifr strains carried one or more missense mutations mapping in two regions of the rpoB gene. These two “hot” regions were investigated in eight additional RifrBrucella laboratory mutants and in 20 reference RifsBrucella strains. rpoB mutations were found in all Rifr mutants. In contrast, no missense mutations were found in any analyzed Rifs 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 Rifr phenotype in prokaryotes. In addition, because of the relationship between Rifr 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.

Attenuated Salmonella enterica serovar Typhimurium lacking the ZnuABC transporter confers immune-based protection against challenge infections in mice

Salmonella enterica has long been recognised as an important zoonotic pathogen of economic significance, both in animals and humans. We have recently shown that inactivation of the ZnuABC high affinity zinc transporter significantly affects the pathogenicity of S. enterica, likely due to zinc shortage in the eukaryotic tissues. Here, we demonstrate that a S. enterica serovar Typhimurium znuABC deleted strain is able to induce a short lasting infection in mice. On the same time, it primes a cell-mediated immune response, which confers a solid and durable immune-based protection against challenge infections with virulent strains of S. Typhimurium. These findings suggest the possibility to explore the use of S. enterica ZnuABC deleted mutants for the production on novel vaccines.

Experimental Infection of Calves with Bovine Viral Diarrhoea Virus Type-2 (BVDV-2) Isolated from a Contaminated Vaccine

A non-cytopathic strain of BVDV-2 was isolated from a batch of live infectious bovine rhinotracheitis (IBR) vaccine, and inoculated intranasally into four 3-month-old calves. Severe signs of disease developed by days 4 and 6 in three of the calves, free of BVDV and antibodies to BVDV, that had been exposed to the virus. These calves survived the acute phase of the infection and progressively recovered. BVDV was consistently isolated, or the respective viral RNA was detected, in the buffy coats from blood samples collected starting from days 2 or 4 up to days 11 or 14 after the experimental infection. Viral RNA was also detected in sera from these infected calves until the presence in the serum of virus neutralizing antibodies was demonstrated. By contrast, the only calf having pre-existing neutralizing antibodies to BVDV at the start of the study was protected from the disease. No virus was detected at any time after experimental inoculation of this calf. Genomic characterization of the BVDV-2 isolated in cell cultures, or detected in sera from the experimentally infected animals, revealed 100% homology in the nucleotide sequence with the BVDV-2 detected as a contaminant of the live IBR virus vaccine. These findings provided evidence of the infective nature of the contaminant BVDV-2 and of its potential to generate disease outbreaks when inoculated into susceptible animals.

Genetic heterogeneity of bovine viral diarrhoea virus in Italy

The genetic characteristics, of 38 field isolates of bovine viral diarrhoea virus (BVDV) collected in 1999 from sick or healthy and persistently infected cattle of dairy farms situated in northern Italy, were investigated. A partial 5′-untranslated region (5′-UTR) sequence of each isolate was determined and a phylogenetic analysis was performed. All the isolates were classified as belonging to the BVDV-1 genotype and could be assigned to different BVDV-1 groups, namely BVDV-1b (n = 20), BVDV-1d (n = 6) and BVDV-1e (n = 10). Two remaining isolates could be classified as BVDV-1f and BVDV-1h, respectively. These results provided evidence for genetic heterogeneity of BVDV in Italy, and contribute to a better knowledge of the circulation of BVDV strains, and to their classification.

An attenuated Salmonella enterica serovar Typhimurium strain lacking the ZnuABC transporter induces protection in a mouse intestinal model of Salmonella infection

Salmonella enterica serovar Typhimurium has long been recognised as a zoonotic pathogen of economic significance in animals and humans. Attempts to protect humans and livestock may be based on immunization with vaccines aimed to induce a protective response. We recently demonstrated that the oral administration of a Salmonella enterica serovar Typhimurium strain unable to synthesize the zinc transporter ZnuABC is able to protect mice against systemic salmonellosis induced by a virulent homologous challenge. This finding suggested that this mutant strain could represent an interesting candidate vaccine for mucosal delivery. In this study, the protective effect of this Salmonella strain was tested in a streptomycin-pretreated mouse model of salmonellosis that is distinguished by the capability of evoking typhlitis and colitis. The here reported results demonstrate that mice immunized with Salmonella enterica serovar Typhimurium (S. Typhimurium) SA186 survive to the intestinal challenge and, compared to control mice, show a reduced number of virulent bacteria in the gut, with milder signs of inflammation. This study demonstrates that the oral administration a of S. Typhimurium strain lacking ZnuABC is able to elicit an effective immune response which protects mice against intestinal S. Typhimurium infection. These results, collectively, suggest that the streptomycin-pretreated mouse model of S. typhimurium infection can represent a valuable tool to screen S. typhimurium attenuated mutant strains and potentially help to assess their protective efficacy as potential live vaccines.

Evaluation of Molecular Methods for the Detection of Brucella Species in Water Buffalo Milk

Brucellosis is a highly infectious disease affecting both animals and humans. The current standard tools for the diagnosis of this bacterial infection are serological and microbiological. In this study, we evaluated the feasibility of molecular assays as diagnostic tools for the detection of Brucella spp. in water buffalo milk. For this purpose, we first compared different DNA extraction protocols and PCR methods on artificially spiked milk samples. The most sensitive methods were then used to examine milk from serologically positive and negative water buffaloes. Molecular results were compared with serological and bacteriological test results. Milk samples from 53 Brucella seropositive buffaloes (by either rose Bengal or complement fixation test) were positive by ELISA, 37 were positive by culture, 33 were positive by PCR, and 35 were positive by real-time PCR. Of the 37 culture-positive samples, a total of 25 and 26 were positive by PCR and real-time PCR, respectively. Of the 16 culture-negative samples, 8 were positive by PCR and 9 by real-time PCR. Thus, although culture showed greater sensitivity than PCR, some animals found positive by serological methods and PCR tested negative by milk culture. The combined use of bacteriological and molecular tools increased the number of positive samples to 46. In conclusion, these results suggest that the simultaneous application of these 2 direct detection methods (culture and PCR) could be more useful than one test alone for the diagnosis of Brucella spp. in buffalo milk.

Salmonella enterica Serovar Typhimurium Exploits Inflammation to Modify Swine Intestinal Microbiota

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.

CD4+CD25+ T regulatory cells limit effector T cells and favor the progression of brucellosis in BALB/c mice.

Brucellosis is one of the most common bacterial zoonoses worldwide. Infection is usually chronic and sometimes lifelong. Different mechanisms can be postulated as to the basis for the induction of the chronic status of brucellosis, but a comprehensive knowledge is still lacking. Here, we carried out a series of experiments in order to assess if the persistence of Brucella abortus could be ascribed to the effect of a down regulation of the immune response due to activity of regulatory T cells. We demonstrate that CD4+CD25+T regulatory cells are able to limit the effectiveness of CD4+T cells and are able to favor the maintenance and the progression of B. abortus infection.

Antigenic, Immunologic and Genetic Characterization of Rough Strains B.abortus RB51, B.melitensis B115 and B.melitensis B18

The lipopolysaccharide (LPS) is considered the major virulent factor in Brucella spp. Several genes have been identified involved in the synthesis of the three LPS components: lipid A, core and O-PS. Usually, Brucella strains devoid of O-PS (rough mutants) are less virulent than the wild type and do not induce undesirable interfering antibodies. Such of them proved to be protective against brucellosis in mice. Because of these favorable features, rough strains have been considered potential brucellosis vaccines. In this study, we evaluated the antigenic, immunologic and genetic characteristics of rough strains B.abortus RB51, B.melitensis B115 and B.melitensis B18. RB51 derived from B.abortus 2308 virulent strain and B115 is a natural rough strain in which the O-PS is present in the cytoplasm. B18 is a rough rifampin-resistan mutant isolated in our laboratory. The surface antigenicity of RB51, B115 and B18 was evaluated by testing their ability to bind antibodies induced by rough or smooth Brucella strains. The antibody response induced by each strain was evaluated in rabbits. Twenty-one genes, involved in the LPS-synthesis, were sequenced and compared with the B.melitensis 16M strain. The results indicated that RB51, B115 and B18 have differences in antigenicity, immunologic and genetic properties. Particularly, in B115 a nonsense mutation was detected in wzm gene, which could explain the intracellular localization of O-PS in this strain. Complementation studies to evaluate the precise role of each mutation in affecting Brucella morphology and its virulence, could provide useful information for the assessment of new, attenuated vaccines for brucellosis.

Identification of single nucleotide polymorphisms in Toll-like receptor candidate genes associated with tuberculosis infection in water buffalo (Bubalus bubalis)

BackgroundToll-like receptors play a key role in innate immunity by recognizing pathogens and activating appropriate responses. Pathogens express several signal molecules (pathogen-associated molecular patterns, PAMPs) essential for survival and pathogenicity. Recognition of PAMPs triggers an array of anti-microbial immune responses through the induction of various inflammatory cytokines. The objective of this work was to perform a case-control study to characterize the distribution of polymorphisms in three candidate genes (toll-like receptor 2, toll-like receptor 4, toll-like receptor 9) and to test their role as potential risk factors for tuberculosis infection in water buffalo (Bubalus bubalis).ResultsThe case-control study included 184 subjects, 59 of which resulted positive to both intradermal TB test and Mycobacterium bovis isolation (cases) and 125 resulted negative to at least three consecutive intradermal TB tests. The statistical analysis indicated that two polymorphisms exhibited significant differences in allelic frequencies between cases and controls. Indeed, the TT genotype at TLR9 2340 C > T locus resulted significantly associated with susceptibility to bovine tuberculosis (P = 0.030, OR = 3.31, 95% CI = 1.05-10.40). One polymorphism resulted significantly associated with resistance to the disease, and included the CC genotype, at the TLR4 672 A > C locus (P = 0.01, OR = 0.26, 95% CI = 0.08-0.80). Haplotype reconstruction of the TLR2 gene revealed one haplotype (CTTACCAGCGGCCAGTCCC) associated with disease resistance (P = 0.04, OR = 0.51, 95% CI = 0.27–0.96), including the allelic variant associated with disease resistance.ConclusionsThe work describes novel mutations in bubaline TLR2, TLR4 and TLR9 genes and presents their association with M. bovis infection. These results will enhance our ability to determine the risk of developing the disease by improving the knowledge of the immune mechanisms involved in host response to mycobacterial infection, and will allow the creation of multiple layers of disease resistance in herds by selective breeding.