Ana Pérez de Rozas

Role of the High-Affinity Zinc Uptake znuABC System in Salmonella enterica Serovar Typhimurium Virulence

ABSTRACT The Salmonella enterica serovar Typhimurium znuABC genes encoding a high-affinity zinc uptake system and its regulatory zur gene have been cloned. Salmonella serovar Typhimurium zur and znuC knockout mutants have been constructed by marker exchange. The 50% lethal dose of the znuC mutant increased when either orally or intraperitoneally inoculated in BALB/c mice, while virulence of the zur mutant decreased only when mice were intraperitoneally challenged.

The high‐affinity zinc‐uptake system znuACB is under control of the iron‐uptake regulator (fur) gene in the animal pathogen Pasteurella multocida

The Pasteurella multocida znuACB genes encoding a high-affinity zinc-uptake system have been identified and cloned. In contrast to what happens in Escherichia coli, znuA is not physically linked to znuCB. Through lacZ transcriptional fusions it has been demonstrated that zinc negatively regulates both znuA and znuCB operons. Nevertheless, and contrary to that determined so far for all other znuACB bacterial systems known, P. multocida znuACB genes are not under control of the zur gene, which is absent in this bacterial species, but rather are under its iron-uptake regulator (fur) gene. Furthermore, construction of defective mutants has demonstrated that P. multocida znuA and znuCB transcriptional units are required for virulence of this organism in a mouse model.

Characterization of the Pasteurella multocida hgbA Gene Encoding a Hemoglobin-Binding Protein

ABSTRACT Reverse transcriptase PCR analyses have demonstrated that open reading frames (ORFs) PM0298, PM0299, and PM0300 of the animal pathogen Pasteurella multocida constitute a single transcriptional unit. By cloning and overexpression studies in Escherichia coli cells, the product of ORF PM0300 was shown to bind hemoglobin in vitro; this ORF was therefore designated hgbA. In vitro and in vivo quantitative assays demonstrated that the P. multocida hgbA mutant bound hemoglobin to the same extent as the wild-type strain, although the adsorption kinetics was slightly slower for the hgbA cells. In agreement with this, the virulence of P. multocida hgbA cells was not affected, suggesting that other functional hemoglobin receptor proteins must be present in this organism. On the other hand, P. multocida mutants defective in PM0298 and PM0299 could be isolated only when a plasmid containing an intact copy of the gene was present in the cells, suggesting that these genes are essential for the viability of this bacterial pathogen. By adapting the recombinase-based expression technology in vivo to P. multocida, we also demonstrated that the transcriptional PM0298-PM0299-hgbA unit is iron regulated and that its expression is triggered in the first 2 h following infection in a mouse model. Furthermore, hybridization experiments showed that the hgbA gene is widespread in P. multocida strains regardless of their serotype or the animal from which they were isolated.

Virulence of Pasteurella multocida recA mutants

In order to determine the role of the RecA protein in the virulence of Pasteurella multocida, a recA mutant was constructed and used in studies of virulence and competition in relation to wild-type strain. To achieve this, firstly, the recA gene was isolated and sequenced, showing an Escherichia coli-like SOS box and encoding a protein of 354 amino acids which has the closest identity with the Haemophilus influenzae RecA protein. Further, the recA mutant was constructed, by inactivating this gene by single recombination of a suicide plasmid containing an internal region of the P. multocida recA gene, and shown to be more sensitive to UV radiation than the parental strain. The P. multocida mutant was slightly attenuated in virulence, as indicated by the LD(50), the time of death of infected animals, and a failure to compete with the wild-type strain in mixed infections. Compared to the parent strain, the mutant had a similar growth rate but a longer lag phase. These data suggest that the diminished virulence of the recA mutant as well as its failure in competition were more a consequence of the long lag phase rather than a direct effect of the inactivation of the recA gene on genes involved in virulence.

Effect of marbofloxacin on Haemophilus parasuis nasal carriage

Haemophilus parasuis is a colonizer of the upper respiratory tract and the causative agent of Glässers disease in swine. This study focused on the nasal carriage of H. parasuis after treatment with marbofloxacin. Three marbofloxacin treatments (three doses of 2mg/kg body weight [bw] every 24h, two doses of 4 mg/kg bw every 48 h and 8 mg/kg bw in one single shot) were used and all of them reduce significantly (p<0.05) the nasal carriage of H. parasuis as compared to control animals. Moreover, H. parasuis was not detected in the nasal cavities of piglets after administering the highest dose. The effect of a dose of 8 mg marbofloxacin/kg bw in one shot was further studied in a farm with clinical cases of Glässers disease using a longitudinal study. Statistically significant reduction of nasal carriage of H. parasuis was detected during the first week after treatment in comparison with the control group. However, a clear relationship between the minimum inhibitory concentration (MIC) of the different strains, their putative virulence or the treatment group (antibiotic or control) from which they were isolated was not detected. Finally, the effect induced by the antibiotic treatment on the bacterial strains seemed to be transitory, since diverse H. parasuis strains (with high and low marbofloxacin MICs) were observed 7 days after finishing the treatment.

Changes in Bacterial Population of Gastrointestinal Tract of Weaned Pigs Fed with Different Additives

This study aimed to provide novel insights into the gastrointestinal microbial diversity from different gastrointestinal locations in weaning piglets using PCR-restriction fragment length polymorphism (PCR-RFLP). Additionally, the effect of different feed additives was analyzed. Thirty-two piglets were fed with four different diets: a control group and three enriched diets, with avilamycin, sodium butyrate, and a plant extract mixture. Digesta samples were collected from eight different gastrointestinal segments of each animal and the bacterial population was analysed by a PCR-RFLP technique that uses 16S rDNA gene sequences. Bacterial diversity was assessed by calculating the number of bands and the Shannon-Weaver index. Dendrograms were constructed to estimate the similarity of bacterial populations. A higher bacterial diversity was detected in large intestine compared to small intestine. Among diets, the most relevant microbial diversity differences were found between sodium butyrate and plant extract mixture. Proximal jejunum, ileum, and proximal colon were identified as those segments that could be representative of microbial diversity in pig gut. Results indicate that PCR-RFLP technique allowed detecting modifications on the gastrointestinal microbial ecology in pigs fed with different additives, such as increased biodiversity by sodium butyrate in feed.

Ultraviolet (UV-C) inactivation of Enterococcus faecium, Salmonella choleraesuis and Salmonella typhimurium in porcine plasma

The objective of this study was to assess the effectiveness of an ultraviolet (UV-C, 254 nm) irradiation system on reducing the load of Salmonella typhimurium (S. typhimurium), Salmonella choleraesuis (S. choleraesuis) resistant to streptomycin and Enterococcus faecium (E. faecium) inoculated in sterile porcine plasma and then subjected to different UV-C irradiation doses (750, 1500, 3000, 6000 and 9000 J/L) using a pilot plant UV-C device working under turbulent flow. Results indicated that UV-C treatment induced a viability reduction of 0.38, 1.18, 3.59, 4.72 and 5.06 log10 S. typhimurium when irradiated at 750, 1500, 3000, 6000 and 9000 J/L, respectively. The observed log10 reduction of S. choleraesuis was 1.44, 2.68, 5.55, 7.07 and 7.97 at 750, 1500, 3000, 6000 and 9000 J/L, respectively. The best-fit inactivation for S. choleraesuis was the Weibull distribution curve, while the best-fit curve for S. typhimurium was the Weibull plus tail model, indicating that around 102 cfu/mL resistant S. typhimurium was detected when the liquid plasma was UV-C irradiated at doses up to 9000 J/L. Viability reduction for E. faecium was 0.44, 1.01, 3.70, 5.61 and 6.22 log10 when irradiated at 750, 1500, 3000, 6000 and 9000 J/L, respectively, with no bacterial resistance observed with UV-C doses of 6000 J/L or higher. The biphasic model was the best fit model for the inactivation curve for E. faecium. For the three microorganisms tested, about a 4 log-unit reduction was achieved when the liquid plasma was irradiated at 3000J/L. Overall results demonstrate the usefulness of the UV-C system to inactivate bacteria in liquid plasma before spray-drying. We conclude that the UV-C system can provide an additional biosafety feature that can be incorporated into the manufacturing process for spray-dried animal plasma.

Authors' response: Recognition sequence for DNA uptake in Haemophilus parasuis.

We appreciate the work of Zhang et al. about sformation in Haemophilus parasuis. However we wish point out that the signal sequence for DNA uptake CGAACTC) reported by us in the rps 12 gene (Bigas et al., 5) is also present 324 bp from the translation start nt of the thy gene (accession number AY262733). cifically, the reverse complement sequence of the tif described for the rps 12 gene is at this point. Both A uptake sequences differ by only two bases and they very similar to the sequence reported by Zhang et al.