Virulence genes in Moraxella spp. isolates from infectious bovine keratoconjunctivitis cases.

INTRODUCTION: Infectious bovine keratoconjunctivitis (IBK) is an important ocular disease which affects cattle worldwide. To advance towards IBK effective prevention and treatment strategies, it is important to define the distribution and genetic diversity of potential virulence factors present in M. bovis and M. bovoculi. The objective of this work was to identify and to analyze Moraxella spp. potential virulence factor genes in a collection of clinical isolates. METHODOLOGY: The presence and diversity of virulence factors in a collection of Moraxella spp. strains isolated since 1983 to 2009 in Uruguay was analyzed by PCR using primers for partial amplification of tolC, omp79, plb, fur and mbxA. The selection criterion of these genes was based on the fact that they encode virulence factors which could be present and conserved within strains, an important issue for the development of vaccines. RESULTS: Differences in PCR amplification were observed within tolC (84%), omp79 (80%), plb (76%) and fur (44%) in M. bovis strains, whereas mbxA was amplified in all M. bovis and M. bovoculi strains. Regarding genetic diversity, the tolC nucleotide sequences were the less diverse within all M. bovis and mbxA were the less diverse within all M. bovis and M. bovoculi strains. CONCLUSIONS: PCR amplifications suggest the occurrence of differences between both Moraxella species, related to evaluated genes within Moraxella spp. strains and suggests that both species may have different pathogenic attributes. MbxA and the outer membrane protein TolC might be considered for future studies to develop new vaccines against IBK.

Diversity of Moraxella spp. strains recovered from infectious bovine keratoconjunctivitis cases in Uruguay.

BACKGROUND: Infectious bovine keratoconjunctivitis (IBK) is the most common ocular disease that affects cattle throughout the world and it has a very significant economic impact. IBK is caused by members of the genus Moraxella and therapeutic and preventive measures have shown limited success. Vaccines, most of them chemically inactivated bacterins, generally induce a limited protection. METHODOLOGY: In this study, the genetic diversity of Uruguayan clinical Moraxella bovis and Moraxella bovoculi isolates was assessed by RAPD-PCR, ERIC-PCR and BOX-PCR fingerprinting. Also, antibiotic resistance of the Moraxella spp. isolates was assessed utilizing the disk diffusion method. RESULTS: When interspecific molecular diversity was assessed, different bands patterns were observed even within a single outbreak of IBK, showing the coexistence of different genotypes of Moraxella spp. The high genetic diversity within M. bovis and M. bovoculi isolates did not permit to correlate isolates DNA fingerprints with geographical origins, dates or even with both different Moraxella species. Antibiotics resistance patterns showed significant differences between M. bovis and M. bovoculi. CONCLUSIONS: This is the first study of diversity that includes M. bovis and M. bovoculi associated to IBK cases. Genetic diversity did not allow to correlate DNA fingerprints of the isolates with geographical origins, isolation dates or even both different Moraxella species. Antibiotics resistance patterns showed differences between M. bovis and M. bovoculi. This remarkable variation within isolates could explain the partial protection induced by commercial vaccines. All these findings could be important for the design of prevention or treatment strategies against IBK.

Molecular and phenotypic analysis of Moraxella spp. associated with infectious bovine keratoconjunctivitis in Uruguay.

Infectious bovine keratoconjunctivitis (IBK) is a common ocular disease of cattle, which is generally thought to be caused by Moraxella bovis. However, a recently characterized Moraxella, M. bovoculi, has been isolated from animals with IBK. The aim of this study was to identify and characterize strains of Moraxella spp. obtained from IBK cases in different geographic locations within Uruguay. Ribosomal gene sequencing indicated that there were two groups of isolates that showed homology with either M. bovis or M. bovoculi. Phylogenetic analysis confirmed the presence of two species as the isolates grouped in different branches of the dendrogram. Conventional biochemical characterization did not distinguish between the species; only 9/25 isolates which had genetic homology with M. bovoculi showed any differences in biochemistry.

Assessment of effectiveness and safety of Ibicella lutea extract in the control of experimental Proteus mirabilis urinary tract infection.

INTRODUCTION: Proteus mirabilis is an important cause of complicated urinary tract infections (UTI). Like many other microorganisms, P. mirabilis has acquired resistance to many antibiotics. Due to the serious effects associated with uropathogenic P. mirabilis and the problems related to the use of antibiotics, alternative strategies for its control must be developed. Previously, we studied the effect of Ibicella lutea extract, a South American indigenous plant, on in vitro uropathogenicity of P. mirabilis. We observed that I. lutea extract had an effect on various attributes associated with P. mirabilis urovirulence. The objective of this study was to assess I. lutea extract against UTI by P. mirabilis. METHODOLOGY: This study was based on the effect of I. lutea extract to prevent or treat P. mirabilis experimental UTI in mice and the influence of this administration on the normal intestinal flora. Also, we studied the toxicity, mutagenicity, and antimutagenicity of the extract. RESULTS: In this study, while I. lutea administration showed an effect in the prevention and treatment of UTI in the mouse, the intestinal microflora did not change. The I. lutea extract was neither toxic nor mutagenic although the extract showed antimutagenic properties. CONCLUSION: These findings suggest that the administration of I. lutea extract could represent an interesting new strategy to control P. mirabilis UTI.

Effect of Ibicella lutea on uropathogenic Proteus mirabilis growth, virulence, and biofilm formation.

BACKGROUND: Proteus mirabilis, an important uropathogen that can cause complicated urinary tract infections (UTI), has emerged as a therapeutic problem following mutations that compromise the use of antimicrobial drugs. Due to the serious effects associated with uropathogenic P. mirabilis and the problems related to the use of antibiotics, it is necessary to develop alternative strategies for its control. The objective of this study was to assess the effect of Ibicella lutea extract, a South American indigenous plant, on growth, virulence and biofilm production of uropathogenic P. mirabilis. METHODOLOGY: This study was based on the extract generation and the assessment of its effect on bacterial features related to virulence. These assays involved determination of antibacterial activity, swarming motility, Western blot to assess expression of fimbriae and flagella, biofilms formation, haemagglutination, haemolysis, and electron microscopy. RESULTS AND CONCLUSIONS: I. lutea extract had an effect on bacterial growth rate and bacterial morphology. It also affected P. mirabilis swarming differentiation, hemagglutination and biofilm formation on glass and polystyrene. These findings suggest that I. lutea may have a role as an agent for the control of P. mirabilis UTI.

Mannose-resistant Proteus-like and P. mirabilis fimbriae have specific and additive roles in P. mirabilis urinary tract infections.

Proteus mirabilis is an important uropathogen that can cause complicated urinary tract infections (UTI). It produces several types of fimbriae, including mannose-resistant Proteus-like (MR/P) fimbriae and P. mirabilis fimbriae (PMF). Previously, we determined that these fimbriae affect the ability of P. mirabilis to colonize the urinary tract. The objective of this study was to analyse the effect of the simultaneous lack of P. mirabilis MR/P and PMF fimbriae in UTI pathogenesis. A double mutant lacking both fimbriae was generated by allelic replacement mutagenesis. This mutant was characterized genetically and phenotypically, and tested using an in vitro uroepithelial cell adhesion assay and the ascending UTI murine model. In vitro adhesion to uroepithelial cells by the P. mirabilis pmfA/mrpA-D mutant was reduced when compared with the wild-type, although no significant differences were observed when it was compared with the single mrpA-D and pmfA mutants. However, in vivo assays showed that colonization of kidneys and bladders by the P. mirabilis pmfA/mrpA-D mutant was significantly reduced when compared with the wild-type and both single mutants. These results indicate that, although redundancy can occur, MR/P and PMF fimbriae have specific and additive roles in P. mirabilis UTI.

[Therapeutic assessment of emergency cerclage. Analysis of a cases series]. / Evaluación terapéutica del cerclaje de urgencia. Análisis de una serie de casos.

BACKGROUND: Cervical incompetence is the incapacity of cervix to retain a pregnancy until term or until feasibility of the fetus. Patients present cervical enlargement without pain or contractions, vaginal strange sensation and membranes protrusion through most minimum degrees of enlargement. The cervical incompetence management can be rest in bed or cerclage. The emergency cerclage is carried out in patients with enlargement > or = 2cm with or without membranes prolapsus. OBJECTIVE: To evaluate the maternal and neonatal results of emergency cerclage with Espinosa-Flores modified technique in pregnancy from 13 to 28 weeks. PATIENTS AND METHODS: This series of cases was carried out as observational and prospective study, all patients with emergency cerclage and pregnancy from 13 to 28 weeks with cervical incompetence were included, during period of January 2000 to December 2003, in Gynecology and Obstetric Hospital from Medical Center La Raza, IMSS. Variables of study were gestational age at moment of cerclage, pregnancy prolongation, and maternal and neonatal complications. RESULTS: Ten patients were included, with age of 32.1 +/- 5.1 years. It was observed a mean prolongation of pregnancy 10 weeks after cerclage. The gestation was interrupted at 31.1 +/- 5.2 weeks. The most frequently complication was premature membranes rupture. Neonatal survival was 70%. CONCLUSIONS: The placement of emergency cerclage continuous being a therapeutic procedure to improve neonatal prognostic. The shortest prolongations of pregnancy were found in patients with greater enlargement (> or = 3cm) and who had membranes protrusion.

Proteus mirabilis isolates of different origins do not show correlation with virulence attributes and can colonize the urinary tract of mice.

Proteus mirabilis has been described as an aetiological agent in a wide range of infections, playing an important role in urinary tract infections (UTIs). In this study, a collection of P. mirabilis isolates obtained from clinical and non-clinical sources was analysed in order to determine a possible correlation between origin, virulence factors and in vivo infectivity. Isolates were characterized in vitro, assessing several virulence properties that had been previously associated with P. mirabilis uropathogenicity. Swarming motility, urease production, growth in urine, outer-membrane protein patterns, ability to grow in the presence of different iron sources, haemolysin and haemagglutinin production, and the presence and expression of diverse fimbrial genes, were analysed. In order to evaluate the infectivity of the different isolates, the experimental ascending UTI model in mice was used. Additionally, the Dienes test and the enterobacterial repetitive intergenic consensus (ERIC)-PCR assay were performed to assess the genetic diversity of the isolates. The results of the present study did not show any correlation between distribution of the diverse potential urovirulence factors and isolate source. No significant correlation was observed between infectivity and the origin of the isolates, since they all similarly colonized the urinary tract of the challenged mice. Finally, all isolates showed unique ERIC-PCR patterns, indicating that the isolates were genetically diverse. The results obtained in this study suggest that the source of P. mirabilis strains cannot be correlated with pathogenic attributes, and that the distribution of virulence factors between isolates of different origins may correspond to the opportunistic nature of the organism.

Mucosal vaccination of mice with recombinant Proteus mirabilis structural fimbrial proteins.

Proteus mirabilis, a common cause of urinary tract infection (UTI), expresses several types of fimbria including mannose-resistant/Proteus-like fimbriae (MRP), uroepithelial cell adhesin (UCA), renamed non-agglutinating fimbriae (NAF) by some authors, and P. mirabilis fimbriae (PMF), which are potentially involved in adhesion to the uroepithelium. In this study, we immunised different groups of mice with recombinant structural subunits of these fimbriae (MrpA, UcaA and PmfA) using two mucosal routes (nasal and transurethral) and we transurethrally challenged the animals with a P. mirabilis uropathogenic isolate. Induction of specific serum and urine IgG and IgA was measured to assess the potential role of the humoral immune response in protection against experimental ascending P. mirabilis UTI. Intranasally MrpA- and UcaA-immunised mice were protected against P. mirabilis ascending UTI, since recovery of bacteria from kidneys and bladders was significantly lower than in PBS-treated mice, and both fimbrial subunits significantly induced specific serum and urine antibodies. Only MrpA and PmfA transurethrally immunised animals were protected only at the kidney level, and in this case only MrpA-immunised mice exhibited significant serum IgG induction. Correlation analysis did not show a significant relationship between serum and urine specific antibody response and protection observed against infection. Our results suggest that an immunisation strategy based on structural fimbrial proteins may be useful to prevent P. mirabilis UTI. Further studies are being carried out to characterise the immune and inflammatory response induced by P. mirabilis recombinant fimbrial subunits.

Proteus mirabilis fimbriae (PMF) are important for both bladder and kidney colonization in mice.

Proteus mirabilis expresses different types of fimbriae simultaneously. Several fimbrial types have been described and their role in the colonization of the urinary tract is under study. Previously, P. mirabilis fimbriae (PMF) have been shown to be associated with bacterial colonization of the lower urinary tract but not of the kidneys. In this study, a pmfA mutant was generated and used in several in vivo and in vitro studies. Two different urinary tract infection models in the mouse and two in vitro assays of bacterial adhesion to uroepithelial cells were performed. Expression of PmfA in a collection of P. mirabilis strains of different sources was also assessed. The results shown here indicate that PMF are involved in both bladder and kidney colonization by P. mirabilis and that these fimbriae are widely distributed among P. mirabilis isolates from different origins since all strains tested expressed PmfA.

Evaluation of Proteus mirabilis structural fimbrial proteins as antigens against urinary tract infections.

Proteus mirabilis is a common cause of urinary tract infection (UTI) and produce several types of different fimbriae, including mannose-resistant/Proteus-like fimbriae, uroepithelial cell adhesin (UCA), and P. mirabilis fimbriae (PMF). Different authors have related these fimbriae with different aspects of P. mirabilis pathogenesis, although the precise role of fimbriae in UTI has not yet been elucidated. In this work we expressed and purified recombinant structural fimbrial proteins of these fimbriae (MrpA, UcaA, and PmfA) and assessed their role as protective antigens using an ascending and a haematogenous model of UTI in the mouse. MrpA protected subcutaneously immunised mice in both models, suggesting that it could be taken into account as a promising vaccine candidate against P. mirabilis UTI. UcaA could also be an interesting subunit to be studied although it only protected mice that were challenged intravenously. All subunits elicited a strong specific serum IgG response but there was no significant correlation between antibody levels and protection. Only PmfA-immunised mice elicited a significant urinary antibody response but this protein was unable to confer protection against P. mirabilis experimental challenges. These results may contribute to the development of vaccines against P. mirabilis, an important cause of complicated UTI.