Marilda Carlos Vidotto

Virulence-associated genes in Escherichia coli isolates from poultry with colibacillosis

Avian pathogenic Escherichia coli, the causative agent of colibacillosis, harbors several putative virulence genes. In this study we examined by polymerase chain reaction (PCR) the presence of 16 of those genes in 200 colibacillosis isolates from our region. The seven virulence genes iutA, iss, cvaC, tsh, papC, papG and felA were detected significantly more often amongst colibacillosis isolates than in fecal isolates from healthy birds, thereby confirming their worldwide occurrence and possible pathogenic role in colibacillosis. However, several of those genes were not detected in many colibacillosis isolates, and none of them were detected in 27.5% of those isolates, which suggests that variants of those genes and yet undetected virulence factors should be searched for.

Virulence factors of avian Escherichia coli.

A total of 45 strains of Escherichia coli isolates from chickens with colisepticemia were examined for virulence factors commonly found in pathogenic groups of E. coli. These strains were studied for the following: pathogenicity in 1-day-old chicks; toxin, hemolysin, and colicin production; cell invasiveness and adherence; hemagglutination for fimbriae detection; serum resistance; aerobactin production in iron-limited conditions; and plasmid content. The characteristics exhibited by virulent strains were invasion for HeLa and chicken fibroblast cells, serum resistance, colicin V, and aerobactin production. None of the isolates were toxigenic or positive in hemagglutination tests. The molecular genetic studies of the virulence factors by agarose electrophoresis showed that the plasmids of these strains are of high molecular weight.

Virulence factors and clonal relationships among Escherichia coli strains isolated from broiler chickens with cellulitis

ABSTRACT In this study, we compared Escherichia coli isolates from chickens with avian cellulitis with those from feces of healthy chickens. Cellulitis-derived strains presented phenotypic and genotypic characteristics of greater virulence than did the fecal isolates. Phylogenetic analysis by repetitive extragenic palindromic-PCR showed that, in agreement with their virulence characteristics, the cellulitis isolates form two clonal groups distinct from the fecal isolates.

Adherence pili of pathogenic strains of avian Escherichia coli

Fifty strains of Escherichia coli isolated from colisepticemic chickens in Londrina, Brazil, were examined for presence of gene sequences for pil and pap, hemagglutination, and adherence to chicken tracheal cells. Forty-one strains were pil+ and 22 of these showed mannose sensitive (MS) hemagglutination (MSHA) of guinea-pig erythrocytes, indicating that they possessed only type 1 pili. Seven strains were pap+ and 6 of these caused mannose resistant (MR) hemagglutination (MRHA) of human erythrocytes. Twenty-four strains (17 of which caused MSHA) showed MS-adherence to chicken tracheal cells and the remaining 26 showed MR-adherence. The former typically adhered to the mucus layer whereas the latter usually adhered to the mucosal epithelium. It is concluded that MS adherence to chicken tracheal cells is correlated with expression of type 1 fimbriae and that MR-adherence to chicken tracheal cells cannot always be attributed to P pili.

Antigenic characterization of Anaplasma marginale isolates from different regions of Brazil.

Antigenic characterization of A. marginale isolates has contributed to identifying the presence of common and restricts epitopes of major surface proteins (MSPs). The data may improve vaccine development to protect against A. marginale isolates from different regions. Brazilian A. marginale isolates were characterized antigenically by Western blot with monoclonal antibodies (MAbs) against MSPs and rabbit anti-MSP-4 from Florida strain. Six A. marginale isolates from MS, MG (AUFV1), SP, PR-L1, PR-HV, RS and Florida strain were tested with ANA22B1 to MSP-1a, AMR36A6 to MSP-1b, ANAF19E2 to MSP-2, AMG75C1 and AMG76B2 to MSP-3 and ANAF16C1 to MSP-5. ANA22B1 recognized MSP-1a epitope in all A. marginale isolates, and reacted with polypeptides of different size ranging 46-105kDa. MSP2 was not detected in MS and SP isolates by ANAF19E2, and only PR-L1 and MG (AUFV1) isolates reacted with MAbs which recognize MSP3 epitope. MSP4 and MSP5 were detected in all A. marginale isolates analyzed. The results revealed conservation of MSP-1a and MSP-5 epitopes among all Brazilian isolates, and showed antigenic variability to MSP-1b, MSP-2 and MSP-3 proteins, agreeing with recent data about the genetic diversity found in the polimorphic multigene family responsible for these proteins.

Virulence-associated factors of uropathogenic Escherichia coli strains isolated from pigs

Thirty one Escherichia coli strains isolated from pigs with urinary tract infections were investigated for presence of virulence factors and plasmid DNA profile. The most frequent virulence factors presented by these strains were mannose-resistant fimbriae, including P. fimbriae (54.8%) and aerobactin production (45.2%). The pap) operon, detected by PCR, was found in 54.8% of the strains, which is similar to its frequency in human strains. Other characteristics such as the presence of mannose-sensitive hemagglutinin (16.1%), indicative of type 1 pili, and production of hemolysin (25.8%), colicin (38.7%) and toxins (22.6% for LT and for VT) were less frequent. No strains were positive for STa production. Plasmid profiles were variable among isolates from either the same or different farms.

Virulence properties of Escherichia coli strains belonging to enteropathogenic (EPEC) serogroups isolated from calves with diarrhea

Nineteen Escherichia coli strains belonging to enteropathogenic (EPEC) serogroups were isolated from calves with diarrhea in Paraná State. Brazil, and studied for virulence markers associated with EPEC or enterohemorrhagic E. coli (EHEC). The 19 isolates belonged to 12 serotypes with isolates of O26:H11, O119:H25 and O114:H- being the most prevalent Localized adherence (LA) was demonstrated for 37% of the isolates, consisting of all four O26:H11, both O114:H- and one O114:H40 isolates. All the LA strains were positive in the fluorescent-actin staining (FAS) test and possessed attaching-effacing E. coli (eae) sequences, but only O114 strains hybridized with the EPEC adherence factor (EAF) probe. None of the strains produced Shiga-like toxins (Verotoxin). Only the O26:H11 strains hybridized with the EHEC plasmid specific (CVD419) probe and were enterohemolytic, properties associated with EHEC strains. This investigation demonstrates that among the bovine strains isolated only those of serogroup O114 behaved as typical EPEC.

Occurrence of Ehrlichia canis and Anaplasma platys in household dogs from northern Parana

Canine monocytic ehrlichiosis caused primarily by Ehrlichia canis and canine thrombocytic anaplasmosis induced by Anaplasma platys are important emerging zoonotic tick-borne diseases of dogs. There is evidence that these pathogens can also affect humans. This study evaluated the presence of E. canis and A. platys in blood samples collected from 256 domiciled dogs in the municipality of Jataizinho, located in north region of the State of Parana, Brazil, by PCR assay. The occurrence of E. canis and A. platys was 16.4% (42/256) and 19.4% (49/256), respectively; while 5.47% (14/256) of the dogs evaluated were co-infected by these two organisms. The presence of E. canis and A. platys was not significantly associated with the variables evaluated (sex, age, outdoor access, and presence of ticks during blood collection). Infection of dogs by E. canis was associated with anemia and thrombocytopenia, while infection induced by A. platys was related only to thrombocytopenia. Canine monocytic ehrlichiosis and canine thrombocytic anaplasmosis should be included in the differential diagnoses when these hematological alterations are observed during routine laboratory evaluation of dogs.

Molecular Screening of Virulence Genes in Extraintestinal Pathogenic Escherichia coli Isolated from Human Blood Culture in Brazil

Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the main etiological agents of bloodstream infections caused by Gram-negative bacilli. In the present study, 20 E. coli isolates from human hemocultures were characterized to identify genetic features associated with virulence (pathogenicity islands markers, phylogenetic group, virulence genes, plasmid profiles, and conjugative plasmids) and these results were compared with commensal isolates. The most prevalent pathogenicity island, in strains from hemoculture, were PAI IV536, described by many researchers as a stable island in enterobacteria. Among virulence genes, iutA gene was found more frequently and this gene enconding the aerobactin siderophore receptor. According to the phylogenetic classification, group B2 was the most commonly found. Additionally, through plasmid analysis, 14 isolates showed plasmids and 3 of these were shown to be conjugative. Although in stool samples of healthy people the presence of commensal strains is common, human intestinal tract may serve as a reservoir for ExPEC.

Evaluation of adherence, hemagglutination, and presence of genes codifying for virulence factors of Acinetobacter baumannii causing urinary tract infection

Acinetobacter baumannii is a strictly aerobic bacterium which causes severe infections, however its pathogenic characteristics are not well defined. Thirteen A. baumannii strains isolated from urine of hospitalized and nonhospitalized patients with different ages were investigated for the presence of virulence factors. The isolates belonged to biotypes 2, 6, and 9 and were sensitive to imipenem. The majority of them showed resistance to amikacin, ceftazidime, ceftriaxone, ciprofloxacin, gentamicin, norfloxacin, and trimethoprim-sulfamethoxazole. None of A. baumannii strains presented genes codifying for 17 different virulence factors previously described in uropathogenic Escherichia coli, when tested by polymerase chain reaction (PCR). Nine isolates agglutinated human group AB erythrocytes, in presence of mannose, but none of them agglutinated group O erythrocytes. Adherence to polystyrene was observed in 7 isolates, and this result did not correlate with that obtained in hemagglutination assay. All the isolates were able to grow in iron-limiting conditions, showing that A. baumannii produces some type of siderophore. However, the genes iutA and fyuA, from iron uptake system of E. coli and Yersinia sp., respectively, were not present in the isolates, suggesting the presence of a different type of siderophore. The fimbriae of A. baumannii strains that mediates the adherence are possibly mannose-resistant, even though the mechanism of adherence to human epithelial cells still remains to be elucidated.