Heterogeneity of Moraxella isolates found in the nasal cavities of piglets.

BACKGROUND: Previous studies have shown that the genus Moraxella is commonly present in the nasal microbiota of swine. RESULTS: In this study, 51 isolates of Moraxella were obtained from nasal swabs from 3 to 4 week old piglets, which represented 26 different fingerprintings by enterobacterial repetitive intergenic consensus (ERIC)-PCR. Whole 16S rRNA gene sequencing allowed the identification at species level of the Moraxella spp. isolates. The majority of the field strains were identified as Moraxella pluranimalium, but Moraxella porci was also detected. In addition, a cluster of 7 strains did not group with any described Moraxella species, probably representing a new species. Subsequent phenotypic characterization indicated that strains of Moraxella pluranimalium were mainly sensitive to serum complement, while the cluster representing the putative new species was highly resistant. Biofilm formation capacity was very variable among the Moraxella spp. isolates, while adherence to epithelial cell lines was similar among selected strains. Additionally, variability was also observed in the association of selected strains to porcine alveolar macrophages. Antimicrobial tests evidenced the existence of multidrug-resistance in the strains. CONCLUSIONS: In summary, phenotypic characterization revealed heterogeneity among Moraxella strains from the nasal cavity of piglets. Strains with pathogenic potential were detected as well as those that may be commensal members of the nasal microbiota. However, the role of Moraxella in porcine diseases and health should be further evaluated.

Pasteurella multocida contains multiple immunogenic haemin- and haemoglobin-binding proteins.

Iron-dependent outer membrane proteins (IROMPs) play an important role in bacterial pathogenesis and present several attributes of potential vaccine candidates. TBLASTN analysis of the Pasteurella multocida Pm70 genome using the same molecules of other bacterial pathogens as a query identified eight putative haemin and haemoglobin receptors for this organism. Quantitative binding assays have demonstrated that the proteins PM0040, PM0236, PM0741, PM1081, PM1428, PM0592 and HgbA bind both haemin and haemoglobin, whereas PM0576 and PM1282 ORFs only bind either haemoglobin or haemin, respectively. Furthermore, Western blot analysis showed that P. multocida-infected mice generate specific antibodies against PM0040, PM0236, PM0741, PM1081, PM1428, PM0592 and HgbA proteins. Nevertheless, inoculation of mice with any single one of these receptors alone did not protect against P. multocida infection.

fur-independent regulation of the Pasteurella multocida hbpA gene encoding a haemin-binding protein.

Treatment of bacterial cultures with chelating agents such as 2,2'-dipyridyl (DPD) induces expression of iron-regulated genes. It is known that in the gamma-Proteobacteria, the Fur protein is the major regulator of genes encoding haem- or haemoglobin-binding proteins. Electrophoretic analysis of outer-membrane proteins of the gamma-proteobacterium Pasteurella multocida has revealed the induction of two proteins of 60 and 40 kDa in DPD-treated cultures in both wild-type and fur-defective strains. These two proteins have the same N-terminal amino acid sequence, which identifies this protein as the product of the PM0592 ORF. Analysis of the sequence of this ORF, which encodes a protein of 60 kDa, revealed the presence of a hexanucleotide (AAAAAA) at which a programmed translational frameshift can occur giving rise to a 40 kDa protein. Analyses conducted in Escherichia coli, using the complete PM0592 ORF and a derivative truncated at the hexanucleotide position, have shown that both polypeptides bind haemin. For this reason, the PM0592 ORF product has been designated HbpA (for haemin-binding protein). Expression studies using both RT-PCR and lacZ fusions, as well as electrophoretic profiles of outer-membrane protein composition, have demonstrated that the hbpA gene is negatively regulated by iron, manganese and haemin through a fur-independent pathway. Despite the fact that serum of mice infected with P. multocida contained antibodies that reacted with both the 60 and 40 kDa products of the hbpA gene, these proteins did not offer protection when used in immunization assays against this micro-organism.

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.

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.

Role of the high-affinity zinc uptake znuABC system in Salmonella enterica serovar typhimurium virulence.

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.

Pasteurella multocida exbB, exbD and tonB genes are physically linked but independently transcribed.

The exbB, exbD and tonB genes of the Pasteurella multocida animal pathogen have been cloned by complementation of an Escherichia coli tonB mutant. Despite these three genes being physically linked, RT-PCR analysis, lacZ transcriptional fusions and construction of insertional mutants have demonstrated that they do not constitute an operon, but rather are transcribed independently from each other. Furthermore, expression of these three genes is under iron control as revealed by lacZ fusions and Fur titration assay analysis. Moreover, each of these three genes is necessary for the virulence of P. multocida cells and all of them contribute equally to the infectious process of this microorganism.