Phenotypic and Genotypic Characterization of Cutibacterium acnes Isolated from Shoulder Surgery Reveals Insights into Genetic Diversity.

Specific virulence factors that likely influence C. acnes invasion into deep tissues remain to be elucidated. Herein, we describe the frequency of C. acnes identification in deep tissue specimens of patients undergoing clean shoulder surgery and assess its phenotypic and genetic traits associated with virulence and antibiotic resistance patterns, compared with isolates from the skin of healthy volunteers. Multiple deep tissue specimens from the bone fragments, tendons, and bursa of 84 otherwise healthy patients undergoing primary clean-open and arthroscopic shoulder surgeries were aseptically collected. The overall yield of tissue sample cultures was 21.5% (55/255), with 11.8% (30/255) identified as C. acnes in 27.3% (23/84) of patients. Antibiotic resistance rates were low, with most strains expressing susceptibility to first-line antibiotics, while a few were resistant to penicillin and rifampicin. Phylotypes IB (73.3%) and II (23.3%) were predominant in deep tissue samples. Genomic analysis demonstrated differences in the pangenome of the isolates from the same clade. Even though strains displayed a range of pathogenic markers, such as biofilm formation, patients did not evolve to infection during the 1-year follow-up. This suggests that the presence of polyclonal C. acnes in multiple deep tissue samples does not necessarily indicate infection.

New Allele-Specific Oligonucleotide (ASO) amplifications for Toxoplasma gondii rop18 allele typing: Analysis of 86 human congenital infections in Brazil.

This study aimed to detect and differentiate Toxoplasma gondii by the allele typing of its polymorphic rop18 gene. For this purpose, a novel genotyping system using allele-specific oligonucleotides (ASOs) was designed, consisting of three ASO pairs. The first and third pairs specifically amplify rop18 allele I and allele III, while the second pair amplify both allele I and II. Genomic DNA from 86 congenital infections was analyzed by ASO-PCRs, successfully typing 82 (95.35%) samples. The remaining 4 samples (4.65%) required sequencing and single nucleotide polymorphism (SNP) analysis of the amplification products. The distribution of samples according to rop18 alleles was: 39.5% of allele III, 38.4% of allele II, 19.8% of mixed rop18 alleles (I/III or II/III), and 2.3% of allele I. The six severely compromised infants exhibited the highest parasite load levels and were infected during the first and early second trimesters of pregnancy. Among these cases, two were associated with rop18 allele I parasites, two with mixed rop18 alleles (I/III), one with allele II, and one with allele III parasites. In conclusion, all severe cases of congenital toxoplasmosis were infected during early pregnancy, but they were not exclusively associated with rop18 allele I parasites, as observed in murine toxoplasmosis. Furthermore, nearly one-fifth of parasites were non-archetypal, exhibiting more than one rop18 allele, indicating a higher genetic diversity of Toxoplasma gondii in this South American sample. Overall, a robust T. gondii rop18 allele typing was developed and suggested that congenital toxoplasmosis in humans involves complex mechanisms beyond the parasite genotype.

High Virulence and Multidrug Resistance of Escherichia coli Isolated in Periodontal Disease.

Periodontal disease is caused by different gram-negative anaerobic bacteria; however, Escherichia coli has also been isolated from periodontitis and its role in periodontitis is less known. This study aimed to determine the variability in virulence genotype, antibiotic resistance phenotype, biofilm formation, phylogroups, and serotypes in different emerging periodontal strains of Escherichia coli, isolated from patients with periodontal disease and healthy controls. E. coli, virulence genes, and phylogroups, were identified by PCR, antibiotic susceptibility by the Kirby-Bauer method, biofilm formation was quantified using polystyrene microtiter plates, and serotypes were determined by serotyping. Although E. coli was not detected in the controls (n = 70), it was isolated in 14.7% (100/678) of the patients. Most of the strains (n = 81/100) were multidrug-resistance. The most frequent adhesion genes among the strains were fimH and iha, toxin genes were usp and hlyA, iron-acquisition genes were fyuA and irp2, and protectin genes were ompT, and KpsMT. Phylogroup B2 and serotype O25:H4 were the most predominant among the strains. These findings suggest that E. coli may be involved in periodontal disease due to its high virulence, multidrug-resistance, and a wide distribution of phylogroups and serotypes.

Frequency and Diversity of Hybrid Escherichia coli Strains Isolated from Urinary Tract Infections.

(1) Background: Hybrid uropathogenic Escherichia coli (UPEC) strains carry virulence markers of the diarrheagenic E. coli (DEC) pathotypes, which may increase their virulence potential. This study analyzed the frequency and virulence potential of hybrid strains among 452 UPEC strains. (2) Methods: Strains were tested for the DEC virulence diagnostic genes' presence by polymerase chain reaction (PCR). Those carrying at least one gene were classified as hybrid and further tested for 10 UPEC and extraintestinal pathogenic E. coli (ExPEC) virulence genes and phylogenetic classification. Also, their ability to produce hemolysis, adhere to HeLa and renal HEK 293T cells, form a biofilm, and antimicrobial susceptibility were evaluated. (3) Results: Nine (2%) hybrid strains were detected; seven of them carried aggR and two, eae, and were classified as UPEC/EAEC (enteroaggregative E. coli) and UPEC/aEPEC (atypical enteropathogenic E. coli), respectively. They belonged to phylogroups A (five strains), B1 (three), and D (one), and adhered to both cell lineages tested. Only the UPEC/EAEC strains were hemolytic (five strains) and produced biofilm. One UPEC/aEPEC strain was resistant to third-generation cephalosporins and carried blaCTX-M-15. (4) Conclusions: Our findings contribute to understanding the occurrence and pathogenicity of hybrid UPEC strains, which may cause more severe infections.

Genotypic Diversity and Pathogenic Potential of Clinical and Environmental Vibrio parahaemolyticus Isolates From Brazil.

Vibrio parahaemolyticus strains recovered from human diarrheal stools (one in 1975 and two in 2001) and environmental sources (four, between 2008 and 2010) were investigated for the presence of virulence genes (trh, tdh, and vpadF), pandemic markers (orf8, toxRS new), and with respect to their pathogenic potential in two systemic infection models. Based only on the presence or absence of these genetic markers, they were classified as follows: the environmental strains were non-pathogenic, whereas among the clinical strains, the one isolated in 1975 was pathogenic (non-pandemic), and the other two were pathogenic (pandemic). The pathogenic potential of the strains was evaluated in mice and Galleria mellonella larvae infection models, and except for the clinical (pathogenic, non-pandemic) isolate, the others produced lethal infection in both organisms, regardless of their source, serotype, and genotype (tdh, orf8, toxRS new, and vpadF). Based on mice and larval mortality rates, the strains were then grouped according to virulence (high, intermediate, and avirulent), and remarkably similar results were obtained by using these models: The clinical strain (pathogenic and non-pandemic) was classified as avirulent, and other strains (four non-pathogenic and two pandemic) were considered of high or intermediate virulence. In summary, these findings demonstrate that G. mellonella larvae can indeed be used as an alternative model to study the pathogenicity of V. parahaemolyticus. Moreover, they raise doubts about the use of traditional virulence markers to predict pathogenesis of the species and show that reliable models are indispensable to determine the pathogenic potential of environmental isolates considered non-pathogenic, based on the absence of the long-standing virulence indicators.

Virulence Profiling and Molecular Typing of Shiga Toxin-Producing E. coli (STEC) from Human Sources in Brazil.

Since no recent data characterizing Shiga toxin-producing E. coli (STEC) from human infections in Brazil are available, the present study aimed to investigate serotypes, stx genotypes, and accessory virulence genes, and also to perform pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) of 43 STEC strains recovered from 2007 to 2017. Twenty-one distinct serotypes were found, with serotype O111:H8 being the most common. However, serotypes less frequently reported in human diseases were also found and included a hybrid STEC/ETEC O100:H25 clone. The majority of the strains carried stx1a as the sole stx genotype and were positive for the eae gene. Regarding the occurrence of 28 additional virulence genes associated with plasmids and pathogenicity islands, a diversity of profiles was found especially among the eae-harboring strains, which had combinations of markers composed of up to 12 distinct genes. Although PFGE analysis demonstrated genetic diversity between serotypes such as O157:H7, O111:H8, O26:H11, O118:H16, and O123:H2, high genetic relatedness was found for strains of serotypes O24:H4 and O145:H34. MLST allowed the identification of 17 distinct sequence types (STs) with ST 16 and 21 being the most common ones. Thirty-five percent of the strains studied were not typeable by the currently used MLST approach, suggesting new STs. Although STEC O111:H8 remains the leading serotype in Brazil, a diversity of other serotypes, some carrying virulence genes and belonging to STs incriminated as causing severe disease, were found in this study. Further studies are needed to determine whether they have any epidemiological relevance.

O-serogroups of multi-drug resistant cervicovaginal Escherichia coli harboring a battery of virulence genes.

Multi-drug resistant cervicovaginal Escherichia coli (CVEC) infections are a serious health problem. The aim of this study is to determine the patterns of virulence genes, antibiotic resistance and O-serogroups of CVEC isolated in Mexico. Two hundred strains of CVEC were isolated from women attending two Clinics at the Instituto Mexicano del Seguro Social. E. coli O-serogroups and virulence markers were identified by PCR. Antibiotic susceptibility was determined using the Kirby-Bauer disc-diffusion method. Serogroups O25 (50%), O75 (9%) and O15 (7.5%) were the most frequent among the CVEC strains isolated. The frequencies for antibiotic resistance were ampicillin 97%, (n = 194); carbenicillin 93.5%, (n = 187); cefalotin 77%, (n = 154); and nitrofurantoin 71%, (n = 142). The frequency of multiresistant isolates (3-12 drugs) was 197 (98.5%). The most frequent virulence genes found were feoB (91.5%), fimH (89.5%), kpsMT11 (75%), iutA (66%), and iroN (59%). One hundred and four distinct patterns of virulence markers with antibiotic-resistance genes associated with O-serogroups were identified amongst CVEC isolates. In conclusion: most CVEC strains isolated were multiresistant to antibiotics, belonged to three O-serogroups, and possessed a battery of virulence factors. This knowledge may lead to improved guidelines and standards for treating cervicovaginal infections.

Serotypes, virulence markers and cell invasion ability of Shiga toxin-producing Escherichia coli strains isolated from healthy dairy cattle.

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.

Genotypic diversity and virulence markers of Yersinia enterocolitica biotype 1A strains isolated from clinical and non-clinical origins.

Yersinia enterocolitica biotype 1A (B1A) strains are considered as non-pathogenic; however, some reports have identified some strains as the causal agents of infection. In South America, few studies molecularly characterized the strains of this biotype. This work typed 51 B1A strains isolated from clinical and non-clinical sources from Brazil and Chile by Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) to elucidate their genotypic diversity, and verify the distribution of 11 virulence markers by PCR. The strains were divided into two groups, ERIC-A and ERIC-B, clustered independently of their clinical or non-clinical origin. No differences were observed in the frequencies of the virulence markers between clinical and non-clinical strains. However, the genes ystB, hreP and myfA occurred exclusively in the strains of the group ERIC-A. Some clinical and non-clinical strains were clustered in the same genetic group and presented the same number of virulence markers, which might suggest the role of the environment and food as a potential source of infection for humans and animals. The results corroborate with the hypothesis that B1A strains are divided into two main clusters that differ in the frequency of some virulence markers, a fact observed for the first time in South American strains.

Molecular epidemiology and virulence markers of Salmonella Infantis isolated over 25 years in São Paulo State, Brazil.

Infection of humans and animals caused by Salmonella is a major public health problem worldwide. Among the more than 2500 serovars, S. Infantis has been one of the 15 most isolated serovars in the world. Despite its clinical importance, little is known about the molecular characteristics of S. Infantis strains from Brazil. The aims of this study were to type S. Infantis isolates of this country and to assess their pathogenic potential. The molecular epidemiology of 35 S. Infantis strains, isolated from human sources (25) and food items (10) between 1984 and 2009 in São Paulo State, Brazil, were investigated using ERIC-PCR, PFGE and MLST. Furthermore, the presence of some virulence markers from Salmonella pathogenicity islands (SPIs) SPI-1 and SPI-2 and from the virulence plasmid was assessed by PCR. Using ERIC-PCR, 34 S. Infantis strains exhibited a high genetic similarity (≥ 93.7%) and using PFGE, 32 strains exhibited a similarity ≥ 80.6%. Additionally, MLST showed a high clonal similarity among strains that all presented the same ST32. Thirty-two isolates under investigation contained the virulence markers invA, sopB, sopD, sipA, sipD, ssaR, sifA, flgK, fljB and flgL. In conclusion, the S. Infantis strains studied were genetically similar, suggesting that a prevalent subtype has been causing disease and food contamination during a 25year period in São Paulo State, an important metropolitan region in Brazil. Furthermore, the contamination between strains from food items and sick humans indicates that better control measures for S. Infantis may be needed in this country.

Pesquisa dos patotipos enteropatogênico, êntero-hemorrágico e produtor de toxina shiga de Escherichia coli em crianças com diarreia aguda e sem diarreia em Belo Horizonte / Investigation of enteropathogenic, enterohemorrhagic and shiga toxin-producing pathotypes of Escherichia coli in children with and without diarrhea in Belo Horizonte

Diarreia infecciosa aguda é uma das causas mais importantes de morbidade e mortalidade em todo o mundo, principalmente em países em desenvolvimento. Diversos microrganismos são reconhecidos como agentes etiológicos da doença, entre eles Escherichia coli diarreiogênica (DEC). DEC inclui diversos patotipos como E. coli enteropatogênica (EPEC), E. coli enteremorrágica (EHEC) e E. coli produtora de toxina shiga (STEC). EPEC induz lesão A/E (aderência e achatamento de microvilosidades) associada à intimina, proteína codificada pelo gene eae. Com base na expressão de BFP, codificada pelo gene bfp, EPEC é classificada em EPEC típica, bfpA-positiva, e EPEC atípica. EHEC é caracterizada pela expressão de intimina, à semelhança de EPEC, e de toxinas shiga, codificadas pelos genes stx. De forma semelhante a EHEC, STEC expressa toxinas shiga, associadas a inibição da síntese protéica em células eucariotas. O conhecimento relativo à diarreia associada a estes dois patotipos de DEC ainda é escasso e estudos relativos ao tema são desenvolvidos, mais comumente, em países industrializados. Os objetivos deste estudo foram investigar, em crianças com e sem diarreia com idade até 5 anos, a prevalência e o perfil de suscetibilidade a antimicrobianos de EPEC, EHEC e STEC, bem como verificar a existência de relação entre infecção pelos patotipos e parâmetros clínicos e epidemiológicos. Setecentos e oitenta e nove crianças (392 com diarreia aguda e 397 sem diarreia) que procuraram assistência médica entre março/2004 e julho/2007 em um hospital público de Belo Horizonte, referência em pediatria na região, foram estudadas. Amostras de fezes eliminadas espontaneamente foram obtidas e transportadas para o laboratório em banho de gelo em até uma hora e cultivadas em ágar MacConkey e ágar Shigella Salmonella. Após 24 horas de incubação, 5 colônias lactose positivas e 5 lactose negativas foram selecionadas e transferidas para ágar Tríplice Açúcar-Ferro, EPM, meio MILi e ágar Citrato. Todas as colônias identificadas como E. coli foram utilizadas no restante do estudo. EPEC, EHEC e STEC foram identificadas por PCR. O teste de suscetibilidade a antimicrobianos foi feito pela técnica de difusão em ágar. Diarréia aguda foi mais comum nos meses secos e em meninos. EPEC foi detectada em 118 crianças, 12 (2,4%) com EPEC típica e 116 (13.4%) com EPEC atípica. EHEC e STEC foram identificadas em 8 (1,0%) e em 3 (0,4%), respectivamente. Nove crianças apresentavam infecção mista por mais de um patotipo de DEC. Febre e vômito foram relatadas para mais de metade das crianças com diarreia estudadas. Feces com sangue/pus foram raramente reportadas. As maiores taxas de resistência a antimicrobianos de EPEC, EHEC e STEC foram observadas para cefazolina (mais de 50%) and cefalotina (mais de 30%). As menores taxas de resistência foram observadas para ciprofloxacina (1.5%) e ácido nalidíxico (abaixo de 10%). Resistência a ampicilina e sulfametoxazol-trimethoprim foi identificada em mais de um quarto das amostras testadas. Amostras produtoras de ESBL não foram detectadas. EPEC, especialmente EPEC atípica é muito comum tanto em crianças com diarreia como em pacientes sem diarreia. As taxas elevadas de resistência a antimicrobianos são, provavelmente devidas ao uso indiscriminado dos mesmos. Nossos dados reforçam a necessidade de vigilância constante da etiologia da diarreia no nosso meio e do padrão de suscetibilidade de amostras de E. coli associadas à gênese da diarreia aguda. Estes resultados serão úteis para melhorar nosso conhecimento relativo à etiologia da diarreia no nosso meio e contribuirão para que se possam traçar estratégias visando à prevenção e controle da doença.

Acute infectious diarrhea is a leader cause of morbidity and mortality worldwide, mainly in developing countries. Several organisms have been implicated in the ethiopathogenesis of diarrheal disease among them diarrheagenic Escherichia coli (DEC). DEC includes several pathotypes such as enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC) and shiga toxin producing E. coli (STEC). EPEC induces eae encoded intimin-dependent A/E (attaching and effacing) lesion. On the basis of BFP expression, encoded by bfpA, EPEC strains are named typical or atypical. EHEC is characterized by expression of intimin, such as EPEC, and shiga toxins, encoded by stx. Similarly to EHEC, STEC express shiga toxins, associated to protein synthesis impairment in eukaryotic cells. Knowledge on disease associated to EHEC and STEC is still scarce and studies on the organisms have been conducted more frequently in industrialized countries. We aimed to investigate the prevalence and antimicrobial susceptibility profile of EPEC, EHEC and STEC as well as clinical and epidemiological parameters associated to infection with these pathotypes in children younger than five years with and without diarrhea. A total of 789 children (392 with acute diarrhea and 397 without diarrhea) who searched for medical assistance from March 2004 to July 2007 at a public hospital in Belo Horizonte which is reference in paediatrics the region were consecutively studied. Passed fecal samples were obtained and transported to the laboratory in an ice bath whithin an hour. Stools were plated onto MacConkey Agar and Shigella-Salmonella Agar. After 24-hours incubation, 5 lactose-positive and 5 lactose-negative colonies were picked from each medium and transferred to Triple Sugar Iron Agar, EPM medium, MILi medium, and Citrate Agar. All colonies identified as E. coli were further studied. EPEC, EHEC, and STEC were identified by PCR and antimicrobial susceptibility testing was performed by an agar diffusion technique. Acute diarrhea was more common in dry months and in males. EPEC was found in 118 children, 12 of them colonized with typical EPEC (2.4%) and 106 with atypical EPEC (13.4%). EHEC and STEC were identified in eight (1.0%) and three (0.4%) patients, respectively. Nine children presented mixed infection by more them one DEC pathotype. Fever and vomits were reported for more than half of the diarrhea patients studied. Bloody/purulent feces were rarely reported. The highest levels of antimicrobial resistance among EPEC, EHEC, and STEC were detected for Cefazolin (higher than 50%) and Cephalotin (higher than 30%). The lowest antimicrobial resistance levels were found for Ciprofloxacin (1.5%) and Nalidixic Acid (under 10%). Resistance to Ampicillin and to Sulphametoxazole-Thrimethoprim was identified in more than 25% of the examined strains. ESBL testing was negative for all EPEC, EHEC, and STEC strains. These results demonstrates thac EPEC, specially atypical EPEC, are common in children with and without diarrhea in our region. The high levels of resistance are probably due to the indiscriminate use of antimicrobials. Our data reinforce the need for constant epidemiological surveillance to improve our knowledge on several aspects of infectious diarrhea in order to give clues for designing strategies for preventing, diagnosing and treating patients presenting with the disease.

Ribotyping and virulence markers of Yersinia pseudotuberculosis strains isolated from animals in Brazil

Ribotyping and virulence markers has been used to investigate 68 Yersinia pseudotuberculosis strains of serogroups O:1a and O:3. The strains were isolated from clinical material obtained from healthy and sick animals in the Southern region of Brazil. Ribotypes were identified by double digestion of extracted DNA with the restriction endonucleases SmaI and PstI, separation by electrophoresis and hybridization with a digoxigenin-labeled cDNA probe. The presence of the chromosomal virulence marker genes inv, irp1, irp2, psn, ybtE, ybtP-ybtQ, and ybtX-ybtS, of the IS100 insertion sequence, and of the plasmid gene lcrF was detected by polymerase chain reaction. The strains were grouped into four distinct ribotypes, all of them comprising several strains. Ribotypes 1 and 4 presented distinct profiles, with 57.3 percent genetic similarity, ribotypes 2 and 3 presented 52.5 percent genetic similarity, and genetic similarity was 45 percent between these two groups (1/4 and 2/3). All strains possessed the inv, irp1, and irp2 genes. Additionally, strains of serogroup O:1a carried psn, ybtE, ybtP-ybtQ, ybtX-ybtS, and IS100. As expected lcrF was only detected in strains harboring the virulence plasmid. These data demonstrate the presence of Y. pseudotuberculosis strains harboring genotypic virulence markers in the livestock from Southern Brazil and that the dissemination of these bacteria may occur between herds.

Genetic analysis of Escherichia coli strains carrying enteropathogenic Escherichia coli (EPEC) markers, isolated from children in Rio de Janeiro city, Brazil

In the present study, 47 enteropathogenic Escherichia coli strains identified according to serotyping, presence of eae, bfp and EAF sequences, adherence phenotype and ability to induce attaching-effacing lesions were analyzed by pulsed-field gel electrophoresis (PFGE), multilocus enzyme electrophoresis (MLEE), and the presence of LEE genes (eae, espA, espB, tir) as well as the respective alleles. Amplification of LEE genes subtypes revealed 18 different pathotypes. Typing of the eae gene showed that most strains contained nontypable intimin (42%) followed by beta (35%), gamma and alpha genes (12% each). PFGE analysis revealed a variable degree of polymorphism among isolates and, in general, no clear correlation was observed among PFGE profiles and the virulence markers identified. Otherwise, grouping based on MLEE analysis showed a close association between eae allele and clonal cluster distribution leading us to indicate the eae profile as a promising marker to establish relatedness among such microorganisms.

No presente estudo, 47 amostras enteropatogênicas de Escherichia coli, previamente caracterizadas pelo sorotipo, fenótipo de aderência, habilidade de induzir a formação da lesão histopatológica e presença das seqüências genéticas eae, bfp e EAF, foram analisadas de acordo com o perfil de fragmentação do DNA cromossômico pela técnica de eletroforese em campo pulsado (PFGE), as variantes isoenzimáticas através da eletroforese de isoenzimas (MLEE) e a presença de seqüências específicas da região LEE (eae, espA, espB, tir) e respectivos alelos. A amplificação destas seqüências mostrou a presença de 18 padrões genéticos distintos. A tipagem do gene eae revelou que a maior parte das amostras apresentou intimina não-tipável (42%) seguida dos tipos alélicos beta (35%), gama e alfa (12% cada). A fragmentação do DNA cromossômico detectou um elevado polimorfismo genético entre as amostras estudadas e não foi observada uma correlação com os marcadores de virulência investigados. Por outro lado, a análise das variantes isoenzimáticas sugeriu uma distribuição clonal específica de variantes genéticas do locus eae, o que nos leva a indicar a sua utilização como um marcador promissor para definir as relações genéticas neste grupo de microrganismos.

Genetic analysis of Escherichia coli strains carrying enteropathogenic Escherichia coli (EPEC) markers, isolated from children in Rio de Janeiro city, Brazil

In the present study, 47 enteropathogenic Escherichia coli strains identified according to serotyping, presence of eae, bfp and EAF sequences, adherence phenotype and ability to induce attaching-effacing lesions were analyzed by pulsed-field gel electrophoresis (PFGE), multilocus enzyme electrophoresis (MLEE), and the presence of LEE genes (eae, espA, espB, tir) as well as the respective alleles. Amplification of LEE genes subtypes revealed 18 different pathotypes. Typing of the eae gene showed that most strains contained nontypable intimin (42%) followed by beta (35%), gamma and alpha genes (12% each). PFGE analysis revealed a variable degree of polymorphism among isolates and, in general, no clear correlation was observed among PFGE profiles and the virulence markers identified. Otherwise, grouping based on MLEE analysis showed a close association between eae allele and clonal cluster distribution leading us to indicate the eae profile as a promising marker to establish relatedness among such microorganisms.

No presente estudo, 47 amostras enteropatogênicas de Escherichia coli, previamente caracterizadas pelo sorotipo, fenótipo de aderência, habilidade de induzir a formação da lesão histopatológica e presença das seqüências genéticas eae, bfp e EAF, foram analisadas de acordo com o perfil de fragmentação do DNA cromossômico pela técnica de eletroforese em campo pulsado (PFGE), as variantes isoenzimáticas através da eletroforese de isoenzimas (MLEE) e a presença de seqüências específicas da região LEE (eae, espA, espB, tir) e respectivos alelos. A amplificação destas seqüências mostrou a presença de 18 padrões genéticos distintos. A tipagem do gene eae revelou que a maior parte das amostras apresentou intimina não-tipável (42%) seguida dos tipos alélicos beta (35%), gama e alfa (12% cada). A fragmentação do DNA cromossômico detectou um elevado polimorfismo genético entre as amostras estudadas e não foi observada uma correlação com os marcadores de virulência investigados. Por outro lado, a análise das variantes isoenzimáticas sugeriu uma distribuição clonal específica de variantes genéticas do locus eae, o que nos leva a indicar a sua utilização como um marcador promissor para definir as relações genéticas neste grupo de microrganismos.

Genetic analysis of Escherichia coli strains carrying enteropathogenic Escherichia coli (EPEC) markers, isolated from children in Rio de Janeiro city, Brazil

In the present study, 47 enteropathogenic Escherichia coli strains identified according to serotyping, presence of eae, bfp and EAF sequences, adherence phenotype and ability to induce attaching-effacing lesions were analyzed by pulsed-field gel electrophoresis (PFGE), multilocus enzyme electrophoresis (MLEE), and the presence of LEE genes (eae, espA, espB, tir) as well as the respective alleles. Amplification of LEE genes subtypes revealed 18 different pathotypes. Typing of the eae gene showed that most strains contained nontypable intimin (42%) followed by beta (35%), gamma and alpha genes (12% each). PFGE analysis revealed a variable degree of polymorphism among isolates and, in general, no clear correlation was observed among PFGE profiles and the virulence markers identified. Otherwise, grouping based on MLEE analysis showed a close association between eae allele and clonal cluster distribution leading us to indicate the eae profile as a promising marker to establish relatedness among such microorganisms.

No presente estudo, 47 amostras enteropatogênicas de Escherichia coli, previamente caracterizadas pelo sorotipo, fenótipo de aderência, habilidade de induzir a formação da lesão histopatológica e presença das seqüências genéticas eae, bfp e EAF, foram analisadas de acordo com o perfil de fragmentação do DNA cromossômico pela técnica de eletroforese em campo pulsado (PFGE), as variantes isoenzimáticas através da eletroforese de isoenzimas (MLEE) e a presença de seqüências específicas da região LEE (eae, espA, espB, tir) e respectivos alelos. A amplificação destas seqüências mostrou a presença de 18 padrões genéticos distintos. A tipagem do gene eae revelou que a maior parte das amostras apresentou intimina não-tipável (42%) seguida dos tipos alélicos beta (35%), gama e alfa (12% cada). A fragmentação do DNA cromossômico detectou um elevado polimorfismo genético entre as amostras estudadas e não foi observada uma correlação com os marcadores de virulência investigados. Por outro lado, a análise das variantes isoenzimáticas sugeriu uma distribuição clonal específica de variantes genéticas do locus eae, o que nos leva a indicar a sua utilização como um marcador promissor para definir as relações genéticas neste grupo de microrganismos.