ABSTRACT
AIM: The occurrence of virulence markers, serotypes and invasive ability were investigated in Shiga toxin-producing Escherichia coli (STEC) isolated from faecal samples of healthy dairy cattle at Rio de Janeiro State, Brazil. METHODS AND RESULTS: From 1562 stx-positive faecal samples, 105 STEC strains were isolated by immuno-magnetic separation (IMS) or plating onto MacConkey agar (MC) followed by colony hybridisation. Fifty (47·6%) strains belonged to nine serotypes (O8:H19, O22:H8, O22:H16, O74:H42, O113:H21, O141:H21, O157:H7, O171:H2 and ONT:H21). The prevalent serotypes were O157:H7 (12·4%), O113:H21 (6·7%) and O8:H19 (5·7%). Virulence genes were identified by polymerase chain reaction (PCR). E-hlyA (77·1%) was the more prevalent virulence marker, followed by espP (64·8%), saa (39%), eae (24·8%) and astA (21·9%). All O157:H7 strains carried the γ (gamma) variant of the locus of enterocyte effacement (LEE) genes and the stx2c gene, while the stx1/stx2 genotype prevailed among the eae-negative strains. None of the eae-positive STEC produced the localized adherence (LA) phenotype in HEp-2 or Caco-2 cells. However, intimate attachment (judged by the fluorescent actin staining test) was detected in some eae-positive strains, both in HEp-2 (23·1%) and in Caco-2 cells (11·5%). Most strains (87·5%) showed 'peripheral association' (PA) adherence phenotype to undifferentiated Caco-2 cells. Twenty-five (92·6%) of 27 strains invaded Caco-2 cells. The highest average value of invasion (9·6%) was observed among the eae-negative bovine strains from serotypes described in human disease. CONCLUSION: Healthy dairy cattle is a reservoir of STEC carrying virulence genes and properties associated with human disease. SIGNIFICANCE AND IMPACT OF THE STUDY: Although reports of human disease associated with STEC are scarce in Brazil, the colonization of the animal reservoir by potentially pathogenic strains offers a significant risk to our population.
Subject(s)
Cattle/microbiology , Disease Reservoirs/microbiology , Shiga-Toxigenic Escherichia coli/isolation & purification , Animals , Brazil , Caco-2 Cells , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Feces/microbiology , Humans , Polymerase Chain Reaction , Serotyping , Shiga Toxin/metabolism , Shiga-Toxigenic Escherichia coli/classification , Shiga-Toxigenic Escherichia coli/genetics , Shiga-Toxigenic Escherichia coli/physiology , Virulence/genetics , Virulence Factors/genetics , Virulence Factors/metabolismABSTRACT
Variation in annual rainfall is considered the most important factor influencing population dynamics in dry environments. However, different factors may control population dynamics in different microhabitats. This study recognizes that microhabitat variation may attenuate the influence of climatic seasonality on the population dynamics of herbaceous species in dry forest (Caatinga) areas of Brazil. We evaluated the influence of three microhabitats (flat, rocky and riparian) on the population dynamics of four herbaceous species (Delilia biflora, Commelina obliqua, Phaseolus peduncularis and Euphorbia heterophylla) in a Caatinga (dry forest) fragment at the Experimental Station of the Agronomic Research Institute of Pernambuco in Brazil, over a period of three years. D. biflora, C. obliqua and P. peduncularis were found in all microhabitats, but they were present at low densities in the riparian microhabitat. There was no record of E. heterophylla in the riparian microhabitat. Population size, mortality rates and natality rates varied over time in each microhabitat. This study indicates that different establishment conditions influenced the population size and occurrence of the four species, and it confirms that microhabitat can attenuate the effect of drought stress on mortality during the dry season, but the strength of this attenuator role may vary with time and species.
Subject(s)
Desert Climate , Forests , Magnoliopsida/physiology , Brazil , Population Density , Seasons , Species SpecificityABSTRACT
Abstract Variation in annual rainfall is considered the most important factor influencing population dynamics in dry environments. However, different factors may control population dynamics in different microhabitats. This study recognizes that microhabitat variation may attenuate the influence of climatic seasonality on the population dynamics of herbaceous species in dry forest (Caatinga) areas of Brazil. We evaluated the influence of three microhabitats (flat, rocky and riparian) on the population dynamics of four herbaceous species (Delilia biflora, Commelina obliqua, Phaseolus peduncularis and Euphorbia heterophylla) in a Caatinga (dry forest) fragment at the Experimental Station of the Agronomic Research Institute of Pernambuco in Brazil, over a period of three years. D. biflora, C. obliqua and P. peduncularis were found in all microhabitats, but they were present at low densities in the riparian microhabitat. There was no record of E. heterophylla in the riparian microhabitat. Population size, mortality rates and natality rates varied over time in each microhabitat. This study indicates that different establishment conditions influenced the population size and occurrence of the four species, and it confirms that microhabitat can attenuate the effect of drought stress on mortality during the dry season, but the strength of this attenuator role may vary with time and species.
Resumo A variação anual na precipitação é considerada o fator mais importante que influencia a dinâmica populacional em ambientes secos. No entanto, diferentes fatores podem controlar a dinâmica populacional em diferentes microhabitats. A hipótese deste estudo é que as variações de microhabitats podem atenuar a influência da sazonalidade climática sobre a dinâmica populacional de espécies herbáceas em uma áreas floresta seca (Caatinga) no Brasil. Então, objetivou-se avaliar a influência de três microhabitats (plano, rochoso e ciliar) sobre a dinâmica das populações de quatro espécies herbáceas (Delilia biflora, Commelina obliqua, Phaseolus peduncularis e Euphorbia heterophylla) em um fragmento de Caatinga da Estação Experimental do Instituto de Pesquisas Agronômicas de Pernambuco, no Brasil, durante um período de três anos. D. biflora, C. obliqua e P. peduncularis foram encontrados em todos os microhabitats, mas elas estavam presentes em baixas densidades no microhabitat ciliar. Não houve registro de E. heterophylla no microhabitat ciliar. O tamanho das populações, as taxas de mortalidade e de natalidade variaram ao longo do tempo em cada microhabitat. Este estudo indica que diferentes as condições de estabelecimento influenciaram o tamanho das populações e a ocorrência das quatro espécies e confirma que microhabitat pode atenuar o efeito do estresse hídrico sobre a mortalidade durante a estação seca, mas a força desse papel atenuador pode variar com o tempo e a espécie considerada.
Subject(s)
Magnoliopsida/physiology , Desert Climate , Forests , Brazil , Population Density , Seasons , Species SpecificityABSTRACT
Abstract Variation in annual rainfall is considered the most important factor influencing population dynamics in dry environments. However, different factors may control population dynamics in different microhabitats. This study recognizes that microhabitat variation may attenuate the influence of climatic seasonality on the population dynamics of herbaceous species in dry forest (Caatinga) areas of Brazil. We evaluated the influence of three microhabitats (flat, rocky and riparian) on the population dynamics of four herbaceous species (Delilia biflora, Commelina obliqua, Phaseolus peduncularis and Euphorbia heterophylla) in a Caatinga (dry forest) fragment at the Experimental Station of the Agronomic Research Institute of Pernambuco in Brazil, over a period of three years. D. biflora, C. obliqua and P. peduncularis were found in all microhabitats, but they were present at low densities in the riparian microhabitat. There was no record of E. heterophylla in the riparian microhabitat. Population size, mortality rates and natality rates varied over time in each microhabitat. This study indicates that different establishment conditions influenced the population size and occurrence of the four species, and it confirms that microhabitat can attenuate the effect of drought stress on mortality during the dry season, but the strength of this attenuator role may vary with time and species.(AU)
Resumo A variação anual na precipitação é considerada o fator mais importante que influencia a dinâmica populacional em ambientes secos. No entanto, diferentes fatores podem controlar a dinâmica populacional em diferentes microhabitats. A hipótese deste estudo é que as variações de microhabitats podem atenuar a influência da sazonalidade climática sobre a dinâmica populacional de espécies herbáceas em uma áreas floresta seca (Caatinga) no Brasil. Então, objetivou-se avaliar a influência de três microhabitats (plano, rochoso e ciliar) sobre a dinâmica das populações de quatro espécies herbáceas (Delilia biflora, Commelina obliqua, Phaseolus peduncularis e Euphorbia heterophylla) em um fragmento de Caatinga da Estação Experimental do Instituto de Pesquisas Agronômicas de Pernambuco, no Brasil, durante um período de três anos. D. biflora, C. obliqua e P. peduncularis foram encontrados em todos os microhabitats, mas elas estavam presentes em baixas densidades no microhabitat ciliar. Não houve registro de E. heterophylla no microhabitat ciliar. O tamanho das populações, as taxas de mortalidade e de natalidade variaram ao longo do tempo em cada microhabitat. Este estudo indica que diferentes as condições de estabelecimento influenciaram o tamanho das populações e a ocorrência das quatro espécies e confirma que microhabitat pode atenuar o efeito do estresse hídrico sobre a mortalidade durante a estação seca, mas a força desse papel atenuador pode variar com o tempo e a espécie considerada.(AU)
Subject(s)
Ecosystem , Spatial Analysis , Semi-Arid Zone , DemographyABSTRACT
Abstract Variation in annual rainfall is considered the most important factor influencing population dynamics in dry environments. However, different factors may control population dynamics in different microhabitats. This study recognizes that microhabitat variation may attenuate the influence of climatic seasonality on the population dynamics of herbaceous species in dry forest (Caatinga) areas of Brazil. We evaluated the influence of three microhabitats (flat, rocky and riparian) on the population dynamics of four herbaceous species (Delilia biflora, Commelina obliqua, Phaseolus peduncularis and Euphorbia heterophylla) in a Caatinga (dry forest) fragment at the Experimental Station of the Agronomic Research Institute of Pernambuco in Brazil, over a period of three years. D. biflora, C. obliqua and P. peduncularis were found in all microhabitats, but they were present at low densities in the riparian microhabitat. There was no record of E. heterophylla in the riparian microhabitat. Population size, mortality rates and natality rates varied over time in each microhabitat. This study indicates that different establishment conditions influenced the population size and occurrence of the four species, and it confirms that microhabitat can attenuate the effect of drought stress on mortality during the dry season, but the strength of this attenuator role may vary with time and species.
Resumo A variação anual na precipitação é considerada o fator mais importante que influencia a dinâmica populacional em ambientes secos. No entanto, diferentes fatores podem controlar a dinâmica populacional em diferentes microhabitats. A hipótese deste estudo é que as variações de microhabitats podem atenuar a influência da sazonalidade climática sobre a dinâmica populacional de espécies herbáceas em uma áreas floresta seca (Caatinga) no Brasil. Então, objetivou-se avaliar a influência de três microhabitats (plano, rochoso e ciliar) sobre a dinâmica das populações de quatro espécies herbáceas (Delilia biflora, Commelina obliqua, Phaseolus peduncularis e Euphorbia heterophylla) em um fragmento de Caatinga da Estação Experimental do Instituto de Pesquisas Agronômicas de Pernambuco, no Brasil, durante um período de três anos. D. biflora, C. obliqua e P. peduncularis foram encontrados em todos os microhabitats, mas elas estavam presentes em baixas densidades no microhabitat ciliar. Não houve registro de E. heterophylla no microhabitat ciliar. O tamanho das populações, as taxas de mortalidade e de natalidade variaram ao longo do tempo em cada microhabitat. Este estudo indica que diferentes as condições de estabelecimento influenciaram o tamanho das populações e a ocorrência das quatro espécies e confirma que microhabitat pode atenuar o efeito do estresse hídrico sobre a mortalidade durante a estação seca, mas a força desse papel atenuador pode variar com o tempo e a espécie considerada.
ABSTRACT
BACKGROUND: Chronic hepatitis B (CHB) and schistosomiasis are prevalent in several countries, but the impact of this association is unknown. We aimed to investigate the prevalence and morbidity of this co-infection in Minas Gerais, an endemic area of schistosomiasis in Brazil. METHODS: In total, 406 adults with CHB (HBsAg positive >6 months) were included in a cross-sectional study. CHB was classified as replicative (HBV DNA ≥ 2.000 IU/ml), and low replicative or inactive hepatitis B carriers (HBV DNA <2.000 IU/ml). Schistosomiasis was confirmed by epidemiological and clinical records. Liver biopsies were scored by METAVIR. The risk of severe fibrosis was estimated by multivariate analysis. RESULTS: Of the 406 patients, 64.8% (263) were male, and the median age was 45 years (IQR 35-54). In total, 57.9% (235) had replicative CHB, and 31.5% (128) had cirrhosis. Schistosoma mansoni was confirmed in 30.5% (124) patients, 81.5% (101) of which were male with a median age of 47 years (IQR 39.5-54). Of the co-infected patients, 61.3% (76) and 38.7% (48) had replicative and inactive CHB, respectively. Schistosomal portal fibrosis (PF) was detected in 69.4% (86/124) patients. Patients with replicative CHB and schistosomal PF had more advanced fibrosis and severe inflammation compared with patients without schistosomal PF (80.8% vs 43.6% for METAVIR F3-F4, p<0.01; 64.0% vs 39.8% for METAVIR A2-A3, p < 0.01). Age >50 years (OR = 1.10; 95% CI 1.06-1.14, p<0.001), male gender (OR = 2.61, 95% CI 1.12-6.09, p = 0.03), schistosomal PF (OR = 4.56, 95% CI 2.10-9.91, p<0.001) and alcoholism (OR = 2.46, 95% CI 1.16-5.19, p = 0.02) were independently associated with cirrhosis. CONCLUSIONS: The association between replicative CHB and schistosomal PF can be a risk factor for more severe liver disease, which can result in deleterious outcomes for patients from endemic areas.
Subject(s)
Hepatitis B, Chronic/epidemiology , Schistosomiasis mansoni/epidemiology , Adult , Age Distribution , Brazil/epidemiology , Comorbidity , Cross-Sectional Studies , Female , Humans , Liver/parasitology , Liver Cirrhosis/epidemiology , Liver Cirrhosis/etiology , Male , Middle Aged , Multivariate Analysis , Prevalence , Sex DistributionABSTRACT
The study was undertaken to determine the clonal relationship and the genetic diversity among Escherichia coli isolates by comparing a non-motile O157 variant with three O157:H7 EHEC isolates and one O55:H7 enteropathogenic E. coli (EPEC) strain. E. coli strains were characterized by sorbitol phenotype, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, random amplification polymorphic DNA, and the presence of specific virulence genes (stx, E-hly and LEE genes). Sorbitol fermentation was observed in O157:H- (strain 116I), O55:H7 and O157:H7 (strain GC148) serotypes. stx1 or stx2 and E-hly genes were only detected among O157:H7 isolates. LEE typing revealed specific allele distribution: eaegamma, tirgamma, espAgamma, espBgamma associated with EPEC O55:H7 and EHEC O157:H7 strains (B1/1 and EDL 933), eaealpha, tiralpha, espAalpha, espBalpha related to the 116I O157:H- strain and the GC148 strain presented non-typable LEE sequences. Multilocus enzyme profiles revealed two main clusters associated with specific LEE pathotypes. E. coli strains were discriminated by random amplification of polymorphic DNA-polymerase chain reaction and pulsed-field gel electrophoresis methodologies. The molecular approaches used in this study allowed the determination of the genetic relatedness among E. coli strains as well as the detection of lineage specific group markers.
Subject(s)
Enteropathogenic Escherichia coli/classification , Enteropathogenic Escherichia coli/genetics , Escherichia coli O157/classification , Escherichia coli O157/genetics , Animals , Bacterial Typing Techniques , Cattle , Cell Line , DNA Fingerprinting , DNA, Bacterial/genetics , Electrophoresis, Gel, Pulsed-Field , Enteropathogenic Escherichia coli/enzymology , Escherichia coli Infections/microbiology , Escherichia coli Infections/veterinary , Escherichia coli O157/enzymology , Fermentation , Humans , Random Amplified Polymorphic DNA Technique , Sorbitol/metabolism , Virulence Factors/geneticsABSTRACT
Fifty-six Escherichia coli strains, serogrouped as EPEC, isolated from three different brands of pasteurised milk commercialised in Rio de Janeiro, Brazil, were tested for enteropathogenicity markers. Most of the strains (71.4%) were adherent to HEp-2 cells. The adherent strains were distributed among 7 EPEC serogroups (O26, O55, O111, O114, O125, O127, O128, O158). Although almost half of these strains (33.9%) presented unrecognisable adherence phenotypes, classical adherence patterns (localised-like, aggregative and diffuse adherence) described for E. coli and epidemiologically associated with diarrheagenic strains were observed. None of the strains showed typical localised adherence, usually associated with EPEC strains, but 4 of them displayed a localised-like adherence (LAL) phenotype, characterised by fewer and less compact microcolonies but that is still associated with diarrheagenic strains as well as strains of non-human origin. Indeed, 3 of these 4 strains were able to elicit the attaching-effacing lesion (FAS-positive), the central feature of EPEC pathogenesis, and hybridised with bfpA and eae DNA probes. The other LAL-positive strain hybridised with the bfpA probe but gave negative results for the eae probe and FAS assays. Interestingly, all LAL-positive strains produced amplicons of 200 bp in the PCR for bfpA, instead of the expected 326 bp fragment. PCR reactions for stx1 and stx2, two shiga-toxin-encoding genes, gave negative results. Typing of LEE-associated genes by PCR showed the profile eae (beta), tir (beta), espA (alpha) and espB (alpha) for one of the LAL-positive strain. The most prevalent adherence phenotype was the aggregative pattern which is observed in strains epidemiologically associated with persistent diarrhea. Additionally, one strain promoted complete detachment of the Hep-2 cell monolayer after 3 h of infection which might be related to the production of citotoxins, a feature that has been increasingly observed in clinical strains. The possession of EPEC-related O and H antigens is no longer deemed an essential characteristic of true pathogenic EPEC strains, emphasising the importance of routinely screen for virulence markers in E. coli strains isolated from foods. Our results are in accordance with data from the literature that demonstrate that environmental strains display atypical features but yet are capable of eliciting the classical A/E lesion and thus must be considered as potentially pathogenic. Further, our results demonstrate the potential of pasteurised milk as a vehicle for transmission of diarrheagenic E. coli in Brazil.
Subject(s)
Escherichia coli Proteins , Escherichia coli/classification , Escherichia coli/pathogenicity , Food Microbiology , Milk/microbiology , Shiga Toxins/biosynthesis , Animals , Bacterial Adhesion , Base Sequence , Cell Line , Consumer Product Safety , DNA Probes , Escherichia coli/physiology , Genotype , Humans , Phenotype , Phylogeny , Serotyping , VirulenceABSTRACT
No período de março 2001 a julho 2002 foram estudadas 2823 fêmeas bovinas, em idade de reprodução, mestiças das raças Holandesa e Gir, pertencentes a 34 propriedades da bacia leiteira de Goiânia. A taxa de prenhez foi 47,8%. Entre os animais não gestantes (n= 1473), 17% apresentaram alterações inflamatórias do útero. Outras anormalidades encontradas foram: aplasia parcial do sistema genital (0,04%), feto macerado (0,01%), ovário aderido (0,04%). Casos de natimortalidade (0,04%), retenção placentária (0,01 %) e cervicite (0,6%) e taxas de abortos (0,88%) também foram registrados. Cocos Gram positivos (41,3%) e bastonetes Gram negativos (52,6%) foram os microrganismos mais isolados sendo Staphylococcus aureus e Escherichia colli os principais patógenos encontrados nas infeccções uterinas. Testes in vitro mostraram que esses microrganismos apresentaram maior susceptibilidade frente ao c1oranfenicol, à gentamicina e à neomicina.
Subject(s)
Cattle , Endometritis/epidemiology , Endometritis/etiology , Escherichia coli/isolation & purification , Staphylococcus aureus/isolation & purification , Microbial Sensitivity Tests/methodsABSTRACT
No período de março 2001 a julho 2002 foram estudadas 2823 fêmeas bovinas, em idade de reprodução, mestiças das raças Holandesa e Gir, pertencentes a 34 propriedades da bacia leiteira de Goiânia. A taxa de prenhez foi 47,8%. Entre os animais não gestantes (n= 1473), 17% apresentaram alterações inflamatórias do útero. Outras anormalidades encontradas foram: aplasia parcial do sistema genital (0,04%), feto macerado (0,01%), ovário aderido (0,04%). Casos de natimortalidade (0,04%), retenção placentária (0,01%) e cervicite (0,6%) e taxas de abortos (0,88%) também foram registrados. Cocos Gram positivos (41,3%) e bastonetes Gram negativos (52,6%) foram os microrganismos mais isolados, sendo Staphylococcus aureus e Escherichia coli os principais patógenos encontrados nas infeccções uterinas. Testes in vitro mostraram que esses microrganismos apresentaram maior susceptibilidade frente ao cloranfenicol, à gentamicina e à neomicina.(AU)
An epidemiological study was carried out on 2823 cows from 34 dairy herds from Goiania in the State of Goias-Brazil during 2001 to 2002. The pregnancy rate was 47.8%. In 1473 non-pregnant cows, causes of reproductive failure problems were sought. The most prevalent uterine infection was endometritis (17.0%). Uterine disorders such as partial hypoplasia of the genital system (0.04%), macerate fetus (0.01%), adhesion of ovaries (0.04%), stillbirth (0.04%), retained placenta (0.01%), cervix inflammation (0.6%) and abortion (0.88%) also were found. Uterine swabs were collected aseptically for microbiological culture. Gram positives cocci (41.3%) and Gram negatives rods (52.6%) were found, and Staphylococcus aureus and Escherichia coli were the most prevalent pathogens. Susceptibility patterns of microorganisms suggested the use of chloramphenicol, gentamicin and neomycin for antimicrobial therapy.(AU)
Subject(s)
Endometritis/epidemiology , Endometritis/etiology , Staphylococcus aureus/isolation & purification , Escherichia coli/isolation & purification , Microbial Sensitivity Tests/methods , CattleABSTRACT
The relationship between enteropathogens and severe diarrhoea in the Brazilian Amazon is poorly understood. In 1998, outbreaks of acute diarrhoea clinically diagnosed as cholera occurred in two small villages localized far from the main cholera route in the Brazilian rainforest. PCR was performed on some enteropathogens and heat-labile (LT) and/or heat-stable (STh) toxin genes, the virulence determinants of enterotoxigenic Escherichia coli (ETEC), were detected. Further characterization of ETEC isolates revealed the presence of two clones, one from each outbreak. One presenting serotype O167:H5 harboured LT-I and STh toxin genes and expressed the CS5CS6 colonization factor. The other, a non-typeable serotype, was positive for the LT-I gene and expressed the CS7 colonization factor. The current study demonstrates the importance of molecular diagnosis in regions such as the Amazon basin, where the enormous distances and local support conditions make standard laboratory diagnosis difficult. Here we also show that the mis-identified cholera cases were in fact associated with ETEC strains. This is the first report of ETEC, molecularly characterized as the aetiological agent of severe diarrhoea in children and adults in the Brazilian Amazon Rainforest.
Subject(s)
Cholera/microbiology , Diarrhea/microbiology , Disease Outbreaks , Escherichia coli Infections , Adolescent , Adult , Brazil/epidemiology , Child , Child, Preschool , Cholera/epidemiology , Diarrhea/epidemiology , Enterotoxins/analysis , Escherichia coli Infections/epidemiology , Escherichia coli Infections/genetics , Female , Humans , Male , Polymerase Chain ReactionABSTRACT
The frequency of diarrhoeagenic Escherichia coli (DEC) strains was investigated in 253 children up to 3 years old, with (patient group, PG, 199 children) and without (control group, CG, 54 children) diarrhoea, living in Rio de Janeiro, Brazil. DEC strains were detected in 70 (27.6%) children, including 54 (27.1%) with diarrhoea and 16 (29.6%) without diarrhoea. Enteroaggregative E. coli (EAEC) was the most frequent DEC category, accounting for 14.6% of the isolates in the PG and for 11.1% in the CG. E. coli strains carrying enteropathogenic E. coli (EPEC) virulence markers showed higher incidence in the CG (12.9%) than in the PG (8.0%). E. coli strains belonging to non-classical EPEC groups that carried eae only or eae and bfpA, designated as attaching-effacing E. coli (AEEC) were the most frequent (79.1%). Simultaneous presence of multiple EPEC virulence factors (EAF/eae/bfpA) were only detected among strains isolated from the PG. Enterotoxigenic E. coli (ETEC) strains were isolated from 5.5% of the children in the CG and from 3.5% of those in the PG. Most of the ETEC isolates were LT-probe positive (70%) and none carried both LT-I and ST-I probe sequences. One enteroinvasive E. coli (EIEC) strain was recovered from a child with diarrhoea. No stx-probe positive E. coli strains were detected. Overall, DEC strains were not found to be significantly associated with diarrhoea (p>0.05). However, the higher incidence of EAEC, the most frequent DEC category, among children with diarrhoea, suggests a potential role of EAEC as an important enteric pathogen in the community investigated.
Subject(s)
Diarrhea/microbiology , Escherichia coli Infections/microbiology , Escherichia coli/classification , Agglutination Tests , Bacterial Toxins/genetics , Bacterial Toxins/isolation & purification , Brazil/epidemiology , Child, Preschool , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Diarrhea/epidemiology , Enterotoxins/genetics , Enterotoxins/isolation & purification , Escherichia coli/genetics , Escherichia coli/isolation & purification , Escherichia coli/pathogenicity , Escherichia coli Infections/epidemiology , Feces/microbiology , Female , Humans , Incidence , Infant , Male , Nucleic Acid Hybridization , VirulenceABSTRACT
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.
Subject(s)
Escherichia coli Infections/veterinary , Escherichia coli/classification , Genes, Bacterial , Rabbits/microbiology , Animals , Bacterial Adhesion/genetics , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , Bacterial Typing Techniques , Brazil , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Diarrhea/microbiology , Diarrhea/veterinary , Escherichia coli/genetics , Escherichia coli/pathogenicity , Escherichia coli Infections/microbiology , Female , HeLa Cells , Humans , O Antigens/genetics , Polymerase Chain Reaction/veterinary , Virulence/geneticsABSTRACT
The ability of ten Bacteroides fragilis strains isolated from intestinal and non-intestinal infections, normal flora and environment to adhere to human colon carcinoma cells, Caco-2, was examined. The adherence capacity varied among the strains tested from strongly adherent (76-100%) to non- or weakly adherent (0-25%). Negative staining with Indian ink showed that all the strains were capsulated, although strain 1032 (strongly adherent and originated from bacteremia) had the highest rate of capsulated cells in the culture. All strains studied presented an electron-dense layer and no fimbrial structures in their surface after PTA negative staining. The analysis of the strains with ruthenium red showed the presence of an acidic polysaccharide and also surface vesicles in all of them. The strain 1032 presented an aggregative adherence pattern toward Caco-2 cells monolayers. It could be seen trapped by elongated microvilli and surrounded by extracellular material in the scanning electron microscope. Treatment with sodium periodate (100 mM/1 h) reduced significantly its adherence capacity and also the expression of an electron-dense layer and of the capsule, detected with PTA and Indian ink staining, respectively. We suggest that the capsular polysaccharide might mediate the adherence of the B. fragilis to Caco-2 cells.
ABSTRACT
Escherichia coli strains of non-EPEC serotypes that carry eae and lack the EAF and the Shiga toxin (stx) gene sequences have been found in acute diarrhea. Both the cell association and the cell entry of these strains in human intestinal epithelial cells were studied as a function of cell differentiation and polarization. The eae+/EAF-/stx- non-EPEC E. coli strains invaded undifferentiated Caco-2 cells more efficiently than differentiated cells. In contrast, prototype EPEC strain E2348/69 did not show significative differences from invasion rates of undifferentiated and differentiated cells. The uptake of these strains was greatly enhanced by pretreatment of differentiated Caco-2 cells with EGTA. These results suggest that the eae+/EAF-/stx- non-EPEC E. coli invasion of intestinal cells may be dependent on receptors expressed on the surface of undifferentiated cells and the basolateral pole of differentiated cells.
Subject(s)
Adhesins, Bacterial , Carrier Proteins , Enterocytes/microbiology , Escherichia coli Proteins , Escherichia coli/pathogenicity , Genes, Bacterial , Intestines/microbiology , Bacterial Adhesion , Bacterial Outer Membrane Proteins/genetics , Caco-2 Cells , Cell Differentiation , Cell Polarity , Enterocytes/cytology , Enterocytes/ultrastructure , Escherichia coli/genetics , Escherichia coli/ultrastructure , Humans , Intestines/cytology , Intestines/ultrastructure , Plasmids/genetics , Serotyping , Shiga Toxin/geneticsABSTRACT
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.
Subject(s)
Adhesins, Bacterial , Bacterial Outer Membrane Proteins/metabolism , Carrier Proteins , Diarrhea/veterinary , Escherichia coli Proteins , Escherichia coli/classification , Escherichia coli/pathogenicity , Swine Diseases/microbiology , Animals , Bacterial Adhesion , Bacterial Outer Membrane Proteins/genetics , Blotting, Western , Chlorocebus aethiops , Diarrhea/microbiology , Escherichia coli/genetics , Escherichia coli/isolation & purification , Escherichia coli Infections/microbiology , Escherichia coli Infections/veterinary , Genotype , HeLa Cells , Humans , Microscopy, Electron , Phenotype , Polymerase Chain Reaction , Shiga Toxins/metabolism , Shiga Toxins/toxicity , Swine , Vero CellsABSTRACT
A Vibrio cholerae cytotoxin, designated VcVac, was found to cause vacuolation in Vero cells. It was originally detected in the pathogenic O1 Amazonia variant of V. cholerae and later shown to be produced in environmental strains and some El Tor strains. Comparison of VcVac production in various strains suggested that hemolysin was responsible for the vacuolating phenotype. Genetic experiments established a firm correlation between vacuolation and hemolysin production. The mammalian cell vacuolating activity of the V. cholerae hemolysin is a new property of this protein and points to a previously unknown type of interaction between V. cholerae and its host.
Subject(s)
Cytotoxins/toxicity , Escherichia coli Proteins , Hemolysin Proteins/toxicity , Macrolides , Vacuoles/drug effects , Vibrio cholerae/pathogenicity , Animals , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/toxicity , Base Sequence , Chlorocebus aethiops , Hemolysin Proteins/genetics , Hemolysis , Molecular Sequence Data , Temperature , Vero CellsABSTRACT
In order to evaluate the prevalence of Shiga toxin-producing Escherichia coli (STEC) strains, 197 fecal samples of healthy cattle from 10 dairy farms, four beef farms and one slaughterhouse at Rio de Janeiro State, Brazil, were examined for Shiga toxin (Stx) gene sequences by polymerase chain reaction (PCR). For presumptive isolation of O157:H7 E. coli, the Cefixime-potassium tellurite-sorbitol MacConkey Agar (CT-SMAC) was used. A high occurrence (71%) of Stx was detected, and was more frequently found among dairy cattle (82% vs. 53% in beef cattle), in which no differences were observed regarding the age of the animals. Dot blot hybridization with stx1 and stx2 probes revealed that the predominant STEC type was one that had the genes for both stx1 and stx2 in dairy cattle and one that had only the stx1 gene for beef cattle. Three (1.5%) O157:H7 E. coli strains were isolated from one beef and two dairy animals by the use of CT-SMAC. To our knowledge, this is the first report of O157:H7 isolation in Brazil. A PCR-based STEC detection protocol led to the isolation of STEC in 12 of 16 randomly selected PCR-positive stool samples. A total of 15 STEC strains belonging to 11 serotypes were isolated, and most of them (60%) had both stx1 and stx2 gene sequences. Cytotoxicity assays with HeLa and Vero cells revealed that all strains except two of serotype O157:H7 expressed Stx. The data point to the high prevalence of STEC in our environment and suggest the need for good control strategies for the prevention of contamination of animal products.
Subject(s)
Bacterial Toxins/biosynthesis , Cattle Diseases/epidemiology , Cytotoxins/biosynthesis , Enterotoxins/biosynthesis , Escherichia coli Infections/veterinary , Escherichia coli/isolation & purification , Animals , Brazil/epidemiology , Cattle , Dairying , Escherichia coli/metabolism , Escherichia coli Infections/epidemiology , Feces/microbiology , Female , Male , Polymerase Chain Reaction/veterinary , Prevalence , Shiga Toxin 1ABSTRACT
The pathogenic O1 Amazonia variant of Vibrio cholerae has been shown previously to have a cytotoxin acting on cultured Vero and Y-1 cells, and to lack important virulence factors such as the cholera toxin (Coelho et al. 1995a). This study extends the molecular analysis of the Amazonia strains, detecting the presence of the toxR gene, with a very similar sequence to that of the E1 Tor and classical biotypes. The outer membrane proteins are analyzed, detecting a variation among the group of Amazonia strains, with three different patterns found. As a by-product of this work a polymerase chain reaction fragment was sequenced, reading part of the sequence of the Lon protease of the Amazonia strains. This gene was not previously described in V. cholerae, but its sequence is present in the TIGR database specific for this species.
Subject(s)
DNA, Bacterial/analysis , DNA, Bacterial/isolation & purification , Genes, Bacterial/genetics , Vibrio cholerae/genetics , Vibrio cholerae/pathogenicity , ATP-Dependent Proteases , Amino Acid Sequence , Base Sequence , Brazil , Cholera Toxin/genetics , Cloning, Molecular , Heat-Shock Proteins/genetics , Molecular Sequence Data , Serine Endopeptidases/genetics , Vibrio cholerae/isolation & purification , Virulence/geneticsABSTRACT
Faeces from urban children < 2 years old with acute diarrhoeal illness and from non-diarrhoeal infants (controls) were examined for Escherichia coli and other enteropathogens. A total of 990 E. coli isolates from 100 patients and 50 controls was tested for enteropathogenic E. coli (EPEC) serotype (O:H), adherence to HEp-2 cells after incubation for 3 and 6 h, fluorescent actin staining (FAS), DNA hybridisation with EAF, eaeA, STh, STp and EAggEC probes and production of heat-labile enterotoxin (LT) and verocytotoxin (VT) with Y1 and Vero cells. EPEC were the most prevalent enteropathogens in patients (32.7%; and 14% in controls). Enteroinvasive E. coli (EIEC) and Vero cytotoxin-producing E. coli (VTEC) were not detected. The rate of isolation of enterotoxigenic E. coli (ETEC) was identical in both groups. Among the EPEC isolates the prevalent serotypes were O111:H2, O55:NM and O119:H6. Localised adherence (LA) was found significantly more frequently in isolates from patients (19.6%) than controls (2.1%). All LA-positive EPEC isolates were FAS+ and eaeA+, but only 75.2% of them hybridised with the EAF probe. Diffusely adhering E. coli (DAEC) and enteroaggregative E. coli (EAggEC) were found with equal frequency in patients and controls. Twenty-seven E. coli isolates were negative for EAF but positive for eaeA and FAS and produced LA in 6-h adherence tests. These EAF-/eaeA+ strains were the only putative enteropathogen identified in seven patients and were not found in controls. The ability of these strains to elicit ultrastructural cell alterations and cell-signalling events was evaluated in Caco-2 cells (human colon carcinoma cell line) by the gentamicin invasion assay and by transmission electron microscopy. The numbers of intracellular bacteria in cell invasion tests varied from 0.4% to 1.6% of the cell-associated bacteria after a 6-h incubation period. Tyrosine phosphorylation of host cell proteins was assessed in HEp-2 cells by immunofluorescence microscopy and all strains gave positive results. EAF-/eaeA+ E. coli strains express most of the virulence properties found among true EPEC strains and can be a relevant cause of infant diarrhoea in developing countries.