Virulence genotypes, antibiotic resistance and the phylogenetic background of extraintestinal pathogenic Escherichia coli isolated from urinary tract infections of dogs and cats in Brazil.

Urinary tract infection (UTI) is a frequent disease of humans and pets and has extra-intestinal pathogenic Escherichia coli (ExPEC) strains as one of the main etiologic agent. ExPEC are characterized by specific virulence factors and are related to a heterogeneous group of human and animal disorders, besides to be a relevant participant in the dissemination of antimicrobial resistance. The purpose of this study was to characterize E. coli strains isolated from UTI of dogs and cats for serotypes, virulence markers, phylogenetic groups and sensitivity to antimicrobial drugs. E. coli was identified as the etiologic agent of UTI in urine samples of 43 pets (7 cats and 36 dogs). Serogroups O2, O4 and O6 corresponded to more than one third of the isolates, being 62% of the total strains classified as B2, 18% as D, 16% as B1 and 4% as A. The iucD (22%), fyuA (80%), traT (51%) and cvaC (20%) genes were distributed among the four phylogenetic groups, whereas the papC/papEF (47%) and malX (67%) genes were found only in groups B2 and D. There were a high number of resistant strains, with 76% of the strains belonging to groups A, B1 and D characterized as multidrug resistant (MDR), whereas only 21% had this phenotype in the group B2. The ExPEC strains isolated in this study displayed pathotypic and phylogenetic similarities with human isolates and high percentages of drug resistance. The finding of MDR ExPEC strains suggests implications for animal and public health and deserves more investigations.

Shiga toxin-producing Escherichia coli and atypical enteropathogenic Escherichia coli strains isolated from healthy sheep of different populations in São Paulo, Brazil.

AIMS: Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent. METHODS AND RESULTS: About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx(1), stx(2), eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)-PCR was performed to investigate the variants of stx(1) and stx(2), and the flagellar antigen (fliC) genes in nonmotile isolates. Five isolates were eae(+) and stx(-), and belonged to serotypes O128:H2/beta-intimin (2), O145:H2/gamma, O153:H7/beta and O178:H7/epsilon. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx(1c)stx(2d-O118) (46.9%), stx(1c) (27.2%), stx(2d-O118) (23.4%), and stx(1c)stx(2dOX3a) (2.5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteropathogenic Escherichia coli (EPEC) isolates. CONCLUSIONS: This study demonstrated that healthy sheep in São Paulo, Brazil, can be carriers of potential human pathogenic STEC and atypical EPEC. SIGNIFICANCE AND IMPACT OF THE STUDY: As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded.

Domestic cats constitute a natural reservoir of human enteropathogenic Escherichia coli types.

Feces of 70 diarrhoeic and 230 non-diarrhoeic domestic cats from Sao Paulo, Brazil were investigated for enteropathogenic (EPEC), enterohaemorrhagic (EHEC) and enterotoxigenic (ETEC) Escherichia coli types. While ETEC and EHEC strains were not found, 15 EPEC strains were isolated from 14 cats, of which 13 were non-diarrhoeic, and one diarrhoeic. None of 15 EPEC strains carried the bfpA gene or the EPEC adherence factor plasmid, indicating atypical EPEC types. The EPEC strains were heterogeneous with regard to intimin types, such as eae-theta (three strains), eae-kappa (n = 3), eae-alpha1 (n = 2), eae-iota (n = 2), one eae-alpha2, eae-beta1 and eae-eta each, and two were not typeable. The majority of the EPEC isolates adhered to HEp-2 cells in a localized adherence-like pattern and were positive for fluorescence actin staining. The EPEC strains belonged to 12 different serotypes, including O111:H25 and O125:H6, which are known to be pathogens in humans. Multi locus sequence typing revealed a close genetic similarity between the O111:H25 and O125:H6 strains from cats, dogs and humans. Our results show that domestic cats are colonized by EPEC, including serotypes previously described as human pathogens. As these EPEC strains are also isolated from humans, a cycle of mutual infection by EPEC between cats and its households cannot be ruled out, though the transmission dynamics among the reservoirs are not yet understood clearly.

Induction of apoptosis in Caco-2 and HT-29 human intestinal epithelial cells by enterohemolysin produced by classic enteropathogenic Escherichia coli.

AIMS: Detect the cytotoxic effects of the Enterohemolysin from enteropathogenic Escherichia coli C3888 (O 26: H-) on Caco 2 and HT-29-human epithelial intestinal cells. METHODS AND RESULTS: The Caco 2 and HT-29 cells, which were treated with Enterohemolysin (EHly) within 10-15 min, became round, lost attachment to substrate, showed extensive surface blebbing, nucleus shrank, and the chromatin became more compact. After 10 min of exposure to the EHly, the cells showed lactate dehydrogenase (LDH) leakage and reduction of mitochondrial activity. The cells showed disorganization of the actin fibers at 15 min. The death of these human epithelial intestinal cells by apoptosis was confirmed by annexin V. CONCLUSIONS: Enterohemolysin induced apoptosis on human epithelial intestinal cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The finding of EHly cytotoxic activity suggests the involvement of this hemolysin in the (Enteropathogenic Escherichia coli) EPEC infection mechanism and may facilitate the understanding of the diarrhea caused by EPEC.

Serotypes, virulence genes, and intimin types of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) isolated from calves in São Paulo, Brazil.

Shiga toxin-producing Escherichia coli (STEC), is the most important recently emerged group of foodborne pathogens. Ruminants, especially cattle, have been implicated as a principal reservoir of STEC, undercooked ground beef and raw milk being the major vehicles of foodborne outbreaks. Enteropathogenic E. coli (EPEC) strains are defined as eae-harboring diarrheagenic E. coli that possess the ability to form A/E lesions on intestinal cells and that do not possess Shiga toxin genes. In order to determine the occurrence, serotypes and virulence markers of STEC and EPEC strains, 546 fecal samples from 264 diarrheic calves and 282 healthy calves in beef farms in São Paulo, Brazil, were screened by PCR. STEC and EPEC were isolated in 10% and 2.7% of the 546 animals, respectively. Although IMS test was used, the STEC serotype O157:H7 was not detected. The most frequent serotypes among STEC strains were O7:H10, O22:H16, O111:H(-), O119:H(-) and O174:H21, whereas O26:H11, O123:H11 and O177:H11 were the most prevalent among EPEC strains. In this study, serotypes not previously reported were found among STEC strains: O7:H7, O7:H10, O48:H7, O111:H19, O123:H2, O132:H51, O173:H(-), and O175:H49. The eae gene was detected in 25% of the STEC and 100% of EPEC strains. The intimin type theta/gamma2 was the most frequent among STEC, whereas the intimin beta1 was the most frequent intimin type among EPEC strains. To our knowledge, this is the first report of the occurrence of the new intimin muB in one strain of animal origin. This new intimin was detected in one atypical EPEC strain of serotype O123:H? isolated from diarrheic cattle. The enterohemolysin (ehxA) was detected in 51% of the STEC and 80% of the EPEC strains, whereas STEC autoagglutinating adhesin (saa) virulence gene was detected only in those STEC strains negative for eae gene. All 15 bovine EPEC strains isolated in this study were negative for both eaf and bfp genes. Our data shows that in Brazil cattle are not only a reservoir of STEC and atypical EPEC, but also a potential source of infection in humans, since the important STEC serotypes previously described and associated with severe diseases in humans, such as O111:H(-), O113:H21, O118:H16, and O174:H21 were isolated.

Random amplification of polymorphic DNA reveals clonal relationships among enteropathogenic Escherichia coli isolated from non-human primates and humans.

Enteropathogenic Escherichia coli (EPEC) strains are important agents of infantile diarrhea all over the world, gaining even greater importance in developing countries. EPEC have also been isolated from various animal species, but most isolates belong to serotypes that differ from those recovered from humans. However, it has been demonstrated that several isolates from non-human primates belong to the serogroups and/or serotypes related to those implicated in human disease. The objective of this study was to evaluate the genetic differences between thirteen strains isolated from non-human primates and the same number of strains isolated from human infections. Human isolates belonged to the same serogroup/serotype as the monkey strains and the evaluation was done by analysis of random amplified polymorphic DNA. Dendrogram analysis showed that there was no clustering between human and monkey strains. Human and non-human isolates of the EPEC serotypes O127:H40 and O128:H2 shared 90 and 87% of their bands, respectively, indicating strong genomic similarity between the strains, leading to the speculation that they may have arisen from the same pathogenic clone. To our knowledge, this study is the first one comparing genomic similarity between human and non-human primate strains and the results provide further evidence that monkey EPEC strains correlate with human EPEC, as suggested in a previous investigation.

Attaching and effacing Escherichia coli isolated from dogs in Brazil: characteristics and serotypic relationship to human enteropathogenic E. coli (EPEC).

Escherichia coli isolates recovered from 182 fecal specimens from dogs up to five months old from the cities of São Paulo and Campinas, SP, Brazil, were examined by polymerase chain reaction (PCR) for several virulence factors and properties. The eae gene was found in 23 isolates of E. coli from 22 dogs, 19 of 146 (13%) from dogs with diarrhea and 3 of 36 (8.3%) from dogs with no diarrhea. Two different eae+ isolates were recovered from one dog with diarrhea. Isolates from two dogs with diarrhea harbored the bfpA gene, and none of the isolates possessed genes for enterotoxins, the EAF plasmid or Shiga toxins. PCR showed that, among the 23 isolates, eight were positive for beta intimin, six for gamma, two for, one for alpha, one for kappa, and five showed no amplification with any of the nine pairs of specific intimin primers used. PCR also showed that the LEE (locus of enterocyte effacement) was inserted in selC in four isolates, likely in pheU in seven isolates, and in undetermined sites in twelve isolates. Fifteen isolates adhered to HEp-2 cells and were fluorescence actin staining (FAS) positive. The predominant adherence pattern was the localized adherence-like (LAL) pattern. The eae-positive isolates belonged to a wide diversity of serotypes, including O111:H25, O119:H2 and O142:H6, which are serotypes that are common among human EPEC. These results confirmed the presence of EPEC in dogs (DEPEC) with and without diarrhea. The virulence factors found in these strains were similar to those in human EPEC, leading to the possibility that EPEC may move back and forth among human and canine populations.

Frequency of Shiga toxin-producing Escherichia coli (STEC) isolates among diarrheic and non-diarrheic calves in Brazil.

The occurrence of Shiga toxin (Stx) gene sequences was examined in 344 fecal samples from diarrheic (n=139) and non-diarrheic (n=205) calves from 12 beef farms in São Paulo State, Brazil to study the prevalence of Shiga toxin-producing Escherichia coli (STEC) strains. Forty-four (12.7%) animals were found to be positive for stx. The frequency of carriage of stx was higher in diarrheic calves (28/139, 20%) than in non-diarrheic animals (16/205, 7.8%) (P<0.001). Among the 24 STEC strains recovered from the animals, 12 isolates carried stx1, four stx2, and 8 carried both stx1 and stx2 genes. The eae and the enterohaemolysin (Ehly) gene sequences occurred at high frequencies in these STEC strains (41.6 and 50.0%, respectively). A total of 16 serotypes were identified. The serotypes O111:NM (four isolates), O111:H8 (two) and O118:H16 (one), currently described as enterohaemorrhagic E. coli (EHEC), were isolated from cattle in Brazil for the first time. These findings reinforce the importance of cattle as a reservoir of EHEC strains in Brazil.

Properties of Shiga toxin-producing Escherichia coli (STEC) isolates from sheep in the State of São Paulo, Brazil.

Fecal samples from 48 sheep from two farms in São Paulo, SP, Brazil, were examined to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC). Forty-two STEC strains were isolated from 25 (52.1%) of 48 sheep feces, and were examined for the presence of genes encoding STEC-related virulence factors. Twenty-one (50.0%) of the 42 STEC isolates were positive for stx(1) and stx(2), 16 isolates (38.1%) were stx(1), and five (11.9%) were stx(2). Expression of Shiga toxins was demonstrated by the Vero cell toxicity test for all the strains carrying stx. Fourteen of the STEC strains (33.3%) carried the enterohemolysin gene (ehly) and presented the enterohemolytic phenotype, and five (11.9%) were positive for the plasmid encoded katP gene. The eae gene was not present in any of the isolates. STEC strains presenting stx(1), stx(2) and ehly were most commonly (23.8%) recovered from these sheep. The predominant STEC serotype found was ONT:H8, and others included O5:H-, O16:H-, O75:H-, O75:H8, O87:H16, O91:H-, O146:H21, O172:H-, OR:H-, ONT:H- and ONT:H16. This is the first report on ovine STEC in South America, and identifies a number of ovine non-O157 STEC that belong to serotypes implicated in human disease.

Use of single-enzyme amplified fragment length polymorphism for typing Pasteurella multocida subsp. multocida isolates from pigs.

Single-enzyme amplified fragment length polymorphism (SE-AFLP) analyses were used to differentiate 97 isolates of porcine Pasteurella multocida subsp. multocida. The strains, isolated from animals with pneumonia, rhinitis, and septicemia, were classified as capsular types A, D, and F. SE-AFLP showed a discriminatory index of 0.87 and identified 18 different profiles.

Determination of the clonal structure of avian Escherichia coli strains by isoenzyme and ribotyping analysis.

Forty-nine avian Escherichia coli strains isolated from different outbreak cases of septicemia (24), swollen head syndrome (14) and omphalitis (11), and 20 strains isolated from poultry with no signs of the mentioned illnesses, for a total of 69 strains, were typed by isoenzyme profile and ribotyping analysis by restriction fragment length polymorphism (RFLP). Isoenzyme analysis discriminated better among strains (0-0.07 degree of genetic dissimilarity) than ribotyping analysis (0- 0.02 degree of genetic dissimilarity). The enzyme profiles of the E. coli isolates allowed the identification of 33 clones that were organized into six main clusters (A-F). Cluster A comprised 87% of the pathogenic strains and had no commensal strains, while commensal strains were assigned to clusters B-F. The ribotyping analysis resulted in a more heterogenous distribution of strains but most of those that cause the same type of infection were kept close together. Taken as a whole, these results demonstrate that pathogenic clones are more similar to one another when compared with commensal strains and suggest a correlation between the genetic background and the pathogenic characteristics of avian pathogenic E. coli strains.

Clonal distribution of an atypical MRP+, EF*, and suilysin+ phenotype of virulent Streptococcus suis serotype 2 strains in Brazil.

Streptococcus suis is considered one of the most important bacterial swine pathogens worldwide. The distribution of the 35 described serotypes in diseased animals may vary in different regions. Data regarding S. suis isolation from pigs in South America is not available. In the present study, 51 isolates of S. suis recovered in pure culture or as the predominant species from diseased animals in Brazil, were analyzed. These isolates were classified as serotypes 2 (58.8%), 3 (21.5%), 7 (13.7%), 1 (3.9%), and 14 (2%). Serotype 2 isolates were further studied for their production of virulence-related proteins muramidase-released protein (MRP), extracellular factor (EF), and suilysin. In addition, the genetic diversity was studied by randomly amplified polymorphic DNA. All but 1 of the serotype 2 isolates showed a clonal distribution of an atypical phenotype (MRP+, EF*, suilysin+), different from the known European (MRP+, EF+, suilysin+), and North American (MRPv, EF-, suilysin-), phenotypes.

Microbiological comparative study of isolates of Edwardsiella tarda isolated in different countries from fish and humans.

It is difficult to use tissue culture assays to investigate adherence and other properties of Edwardsiella tarda because the organism is invasive and produces a potent hemolysin. We therefore relied on polymerase chain reaction (PCR) to determine the occurrence of genes for enterotoxins (LT-I, EAST-1), Shiga toxin (Stx-1, Stx-2), cytotoxic necrotizing factors (CNF-1, CNF-2), aerobactin, invasion plasmid of enteroinvasive Escherichia coli, EPEC adherence factor (EAF), intimin (Eae), enterohemolysin (EntHly) and hemolysin (Hly) in 53 isolates of E. tarda from humans and fish from several countries. All isolates were negative for all genes investigated by PCR. Adhesion to and invasion of HeLa cells were determined by using the unusually short incubation time of 1h or 30 min. All isolates adhered and invaded in these tests. Finally, a random amplified polymorphic DNA (RAPD) test distinguished, with a few exceptions, isolates of human and fish origin.

Serobiotypes and virulence genes of Escherichia coli strains isolated from diarrheic and healthy rabbits in Brazil.

A total of 178 Escherichia coli isolates from diarrheic and healthy rabbits in the São Paulo State (Brazil) were serobiotyped and investigated by PCR for the presence of virulence genes. Among the 90 (50.6%) isolates which possessed the eae gene, 74 were from diarrheic animals and all but one encoded intimin beta. Sixty five (72.2%) of the eae+ isolates had insertion of the locus of enterocyte effacement locus in the pheU locus, 11 (12.2%) in the selC and 14 (15.6%) did not insert in either of these loci. All isolates were negative for genes of the E. coli enterotoxins, Stx1, Stx2, CNF1, CNF2 and EHEC hemolysin. The O132:H2 serotype was dominant, being present in 63 isolates (70%) of the 90 eae+ isolates, and 57 of the 63 isolates of this serotype belonged to biotype 30. PCR detected the gene for AF/R2 fimbriae in 75 (83.3%) of the 90 eae+ isolates. Adherence to HeLa cells was best detected following 6h incubation and a positive fluorescence actin staining (FAS) test was given by 52 isolates. These data show that isolates of E. coli associated with diarrhea in rabbits in Brazil possess the genotype and phenotype typically associated with rabbit enteropathogenic E. coli (EPEC). We conclude that EPEC that possess the eae gene are a common cause of diarrhea in Brazilian rabbit farms and that the pathogenic eae+ AF/R2+ isolates of O132:H2:B30 serobiotype are especially predominant.

eae-negative attaching and effacing Escherichia coli from piglets with diarrhea.

One hundred and ninety strains of Escherichia coli that were isolated from pigs with diarrhea in the state of São Paulo, Brazil, and that were negative for enterotoxins and cytotoxins were investigated. Strains which adhered to HeLa cells were examined for fluorescence actin staining (FAS), the ability to induce attaching and effacing (A/E) lesions on HEp-2 cells detectable by transmission electron microscopy and the presence of eae gene sequences detected by PCR. Intimin production was detected by western blot and serogrouping was performed. Forty-seven isolates adhered to HeLa cells in several patterns, but none adhered in a localized adherence pattern. However, seven of the 47 adherent strains were positive for the FAS reaction, although the reactions were usually weak or atypical. One FAS-negative and three FAS-positive strains, which were examined for their ability to induce A/E lesions, were all positive. Subsequently, testing of these strains for the eae gene showed that they all lacked this gene. These findings, along with earlier reports of eae-negative A/E E. coli, suggest that higher quantities of E. coli in this category might be detected if more reliance were placed on phenotypic tests rather than on gene detection tests alone.

Subtypes of intimin among non-toxigenic Escherichia coli from diarrheic calves in Brazil.

One hundred and five strains of Escherichia coli that were isolated from calves with diarrhea in the state of São Paulo, Brazil, and were negative for enterotoxins and cytotoxins, were examined for the eae gene. Four (3.8%) strains were positive by polymerase chain reaction (PCR) and were shown to produce intimin by using Western blot with specific antiserum against the conserved N-terminal region of intimin. Subtyping of the intimins was done by PCR with specific primers and by Western blot with specific antisera against the C-terminal variable region of the protein. Three of these isolates (O?:H11, O26:H-, O123:H1) produced the beta subtype of intimin, and the 4th (0103:H2) produced intimin that was not typable. The 0103:H2 and the O26:H-isolates adhered to HEp-2 cells with diffuse adherence and localized-like adherence patterns, respectively. The other strains did not adhere to HEp-2 cells. To our knowledge, this is the first report of the occurrence of a subtype of intimin described for human enteropathogenic E. coli among bovine diarrheogenic E. coli. It is also the first report from Brazil demonstrating the presence of bovine E. coli harboring the eae gene.

Research of antigen and antibodies from retroviruses, CMV and HBV among prisoners of the penitentiary complex of the region of Campinas, SP, Brazil.

Some viruses of the families Retroviridae, such as Human T Lymphotropic Virus (HTLV); Herpesviridae as the Cytomegalovirus (CMV) and Hepadnaviridae such as the Hepatitis B Virus (HBV) are liable to be co-transmitted with the Human Immunodeficiency Virus (HIV). Since prisoners are exposed to several and important risk factors involved in the transmission of HIV and the above mentioned viruses, male inmates from the penitentiary complex of Campinas, SP, Brazil, including HIV+ and HIV- ones, were examined for the presence of HTLV-I and/or II antibodies; IgG and IgM anti-CMV antibodies, and the research of the superficial hepatitis B antigen (HBsAg). The presence of anti-HTLV-I and/or II was determined by the Western Blot (WB) technique, whereas IgG and IgM anti-CMV and the search of HBsAg were carried out by the Microparticle Enzyme Immunoassay (MEIA-Abbott Lab). With regard to anti-HTLV-I and/or II, 58.3% (14/24-Number of positive reactions/number of sera examined) were reactive among the anti-HIV positive sera. Conversely, only 12.5% (3/24) among the HIV- negative sera showed positive reactions to HTLV-I and/or II antibodies. When looking for IgG anti-CMV percentages of 97.7% (43/44) and 95% (38/40) were obtained for anti-HIV positive and negative sera, respectively. As to IgM anti-CMV antibodies 11.36% (5/44) and 2.5% (1/40) of reactive sera were found for anti-HIV positive and negative, respectively. The HBsAg was found in 12.8% (5/39) of the sera which were anti-HIV positive.

Receptors on chicken erythrocytes for F42 fimbriae of Escherichia coli isolated from pigs.

Haemagglutination of purified F42 fimbriae was found to be inhibited by N-acetyl-galactosamine. Purified F42 fimbrial adhesin reacted with distinct membrane components from chicken erythrocytes (35, 37 and 40 kDa) in immunoblot analysis, suggesting that the binding occurred to proteins or glycoproteins.

Determination of the efficiency of K99-F41 fimbrial antigen vaccine in newborn calves.

Semipurified K99 and F41 fimbrial antigens were used to prepare an oil-emulsified vaccine against bovine enterotoxigenic colibacillosis. Nine Nelore cows about 7 months pregnant were divided into 3 groups (A, B and C) of 3 animals each, which received different doses of vaccine (1,500 HU, 750 HU and 380 HU, respectively) 8 and 2 weeks before delivery, in the neck by the subcutaneous route. As a control (group D), 3 pregnant cows of the same breed were not vaccinated for later challange of their calves. Vaccine efficiency was measured by the serological tests double diffusion and ELISA. Challenge of calves from the vaccinated and from the three control unvaccinated cows was carried out with the virulent Escherichia coli B41 strain (0101, STa+, K99+, F41+). Two of the 3 calves from the unvaccinated cows died within 48 h with acute diarrhea. E. coli B41 was recovered as pure culture from their stools. In contrast, none of the calves born from vaccinated cows presented diarrhea. These data suggest that the antibody transfer to calves through colostrum gave full protection against the challenge. This semipurified fimbrial vaccine against K99-F41-harboring strains is the first oil-emulsified immunogen prepared in Brazil, which was not only efficient, but also had no adverse effects on vaccinated pregnant cows.