Quantificação de enterobactérias e Clostridium spp. e detecção molecular de Clostridium perfringens, Escherichia coli e Salmonella spp. em pontos de cadeia produtiva da carne de frango

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

PCR-based assay using occult blood detection cards for detection of diarrheagenic Escherichia coli in specimens from U.S. travelers to Mexico with acute diarrhea.

Large field studies of travelers' diarrhea for multiple destinations are limited by the need to perform stool cultures on site in a timely manner. A method for the collection, transport, and storage of fecal specimens that does not require immediate processing and refrigeration and that is stable for months would be advantageous. This study was designed to determine if enterotoxigenic Escherichia coli (ETEC) and enteroaggregative E. coli (EAEC) DNA could be identified from cards that were processed for the evaluation of fecal occult blood. U.S. students traveling to Mexico during 2005 to 2007 were monitored for the occurrence of diarrheal illness. When ill, students provided a stool specimen for culture and occult blood by the standard methods. Cards then were stored at room temperature prior to DNA extraction. Fecal PCR was performed to identify ETEC and EAEC in DNA extracted from stools and from occult blood cards. Significantly more EAEC cases were identified by PCR that was performed on DNA that was extracted from cards (49%) or from frozen feces (40%) than from culture methods that used HEp-2 adherence assays (13%) (P < 0.001). Similarly, more ETEC cases were detected from card DNA (38%) than from fecal DNA (30%) or by culture that was followed by hybridization (10%) (P < 0.001). The sensitivity and specificity of the card test were 75 and 62%, respectively, compared to those for EAEC by culture and were 50 and 63%, respectively, compared to those for ETEC. DNA extracted from fecal cards that was used for the detection of occult blood is of use in identifying diarrheagenic E. coli.

Etiology of childhood diarrhea in the northeast of Brazil: significant emergent diarrheal pathogens

In a study conducted in Joao Pessoa, northeast of Brazil, 2344 Escherichia coli isolated from 290 infants with diarrhea and 290 healthy matched controls were analyzed for virulence traits. Enteroaggregative E. coli (EAEC) was the most prevalent pathogen associated to acute diarrhea. Based on the results of colony blot hybridization, serotyping, and HEp-2 cell adherence assays, strains were separated in categories as typical enteropathogenic E. coli (EPEC) (1.7%), atypical EPEC (a-EPEC) (9.3%), EAEC (25%), enterotoxigenic E. coli (10%), and enteroinvasive E. coli (EIEC) (1.4%). No enterohemorrhagic E. coli strains were isolated. Other enteropathogens were found, including Salmonella (7.9%), Shigella spp. (4.1%), thermophilic Campylobacter spp. (2.4%), Giardia lamblia (9.3%), and Entamoeba histolytica (5.8%). All enteropathogens were associated with diarrhea (P < 0.01). However, the association was lower for EPEC and EIEC (P < 0.03). Different pathogens associated with diarrhea may have been changing in Brazil where EAEC and a-EPEC seem to be the most prevalent pathogens among them. (C) 2010 Elsevier Inc. All rights reserved.

Etiology of diarrheal infections in children of Porto Velho (Rondonia, Western Amazon region, Brazil)

In the present study, 470 children less than 72 months of age and presenting acute diarrhea were examined to identify associated enteropathogenic agents. Viruses were the pathogens most frequently found in stools of infants with diarrhea, including 111 cases of rotavirus (23.6% of the total diarrhea cases) and 30 cases of adenovirus (6.3%). The second group was diarrheogenic Escherichia coli (86 cases, 18.2%), followed by Salmonella sp (44 cases, 9.3%) and Shigella sp (24 cases, 5.1%). Using the PCR technique to differentiate the pathogenic categories of E. coli, it was possible to identify 29 cases (6.1%) of enteropathogenic E. coli (EPEC). Of these, 10 (2.1%) were typical EPEC and 19 (4.0%) atypical EPEC. In addition, there were 26 cases (5.5%) of enteroaggregative E. coli, 21 cases (4.4%) of enterotoxigenic E. coli, 7 cases (1.4%) of enteroinvasive E. coli (EIEC), and 3 cases (0.6%) of enterohemorrhagic E. coli. When comparing the frequencies of diarrheogenic E. coli, EPEC was the only category for which significant differences were found between diarrhea and control groups. A low frequency of EIEC was found, thus EIEC cannot be considered to be a potential etiology agent of diarrhea. Simultaneous infections with two pathogens were found in 39 diarrhea cases but not in controls, suggesting associations among potential enteropathogens in the etiology of diarrhea. The frequent association of diarrheogenic E. coli strains was significantly higher than the probability of their random association, suggesting the presence of facilitating factor(s).

A PCR based assay for the detection of enteric pathogens from Hemoccult(RTM) cards

Background. Large field studies in travelers' diarrhea (TD) in multiple destinations are limited by the need to perform stool cultures on site in a timely manner. A method for the collection, transport and storage of fecal specimens that does not require immediate processing, refrigeration and is stable for months would be advantageous. ^ Objectives. Determine if enteric pathogen bacterial DNA can be identified in cards routinely used for evaluation of fecal occult blood. ^ Methods. U.S. students traveling to Mexico in 2005-07 were followed for occurrence of diarrheal illness. When ill, students provided a stool specimen for culture and occult blood by the standard method. Cards were then stored at room temperature prior to DNA extraction. A multiplex fecal PCR was performed to identify enterotoxigenic Escherichia coli and enteroaggregative E. coli (EAEC) in DNA extracted from stools and occult blood cards. ^ Results. Significantly more EAEC cases were identified by PCR done in DNA extracted from cards (49%) or from frozen feces (40%) than by culture followed by HEp-2 adherence assays (13%). Similarly more ETEC cases were detected in card DNA (38%) than fecal DNA (30%) or culture followed by hybridization (10%). Sensitivity and specificity of the card test was 75% and 62%, respectively, and 50% and 63%, respectively, when compared to EAEC and ETEC culture, respectively, and 53% and 51%, respectively compared to EAEC multiplex fecal PCR and 56% and 70%, respectively, compared to ETEC multiplex fecal PCR. ^ Conclusions. DNA extracted from fecal cards used for detection of occult blood is of use in detecting enteric pathogens. ^

Susceptibility to travelers' diarrhea and histo-blood group antigens

Histo-blood group antigens (HBGAs) have been associated with susceptibility to enteric pathogens including noroviruses (NoVs), enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni, and Vibrio cholerae. We performed a retrospective cohort study to evaluate the relationship between traveler HBGA phenotypes and susceptibility to travelers' diarrhea (TD) and post-infectious complications. 364 travelers to Guadalajara, Mexico were followed prospectively from June 1 - September 30, 2007 and from June 7–July 28, 2008 for the development of TD and at 6 months for post-infectious irritable bowel syndrome (PIIBS). Noroviruses were detected from illness stool specimens with RT-PCR. Diarrheal stool samples were also assayed for enterotoxigenic and enteroaggregative E. coli, Salmonella species, Shigella species, Vibrio species, Campylobacter jejuni, Yersinia enterocolitica, Aeromonas species, and Plesiomonas species. Diarrheal stools were evaluated for inflammation with fecal leukocytes, mucus, and occult blood. Phenotyping for ABO and Lewis antigens with an ELISA assay and FUT2 gene PCR genotyping for secretor status were performed with saliva. 171 of 364 (47%) subjects developed TD. HBGA typing for the travelers revealed O (62.9%), A (34.6%), B (1.6%), and AB (0.8%) phenotypes. There were 7% nonsecretors and 93% secretors among the travelers. AB phenotypes were more commonly associated with Cryptosporidium species (P=0.04) and ETEC ( P=0.08) as causes of TD. AB and B phenotype individuals were more likely to experience inflammatory diarrhea, particularly mucoid diarrhea ( P=0.02). However, there were relatively few individuals with AB and B phenotypes. GI and GII NoV and Cryptosporidium species infections and PI-IBS were identified only in secretors, but these differences were not statistically significant, (P=1.00), (P=1.00), and (P=0.60), respectively. Additional studies are needed to evaluate whether AB phenotype individuals may be more susceptible to developing TD associated with Cryptosporidium species or ETEC, and whether AB and B phenotype individuals may be more likely to develop inflammatory TD. Further studies are needed to investigate whether nonsecretor travelers may be at less risk for developing infections with NoVs and Cryptosporidium species and PI-IBS.^

Antibiotic susceptibility and resistance among bacterial enteropathogens isolated from international travelers to Mexico, Guatemala, and India, 2006--2008

The incidence rates of travelers' diarrhea (TD) have remained unchanged for the last fifty years. More recently, there have been increasing recommendations for self-initiated therapy and even prophylactic therapy for TD. There is no recent data on the in vitro activities of commonly used antibiotics for TD therapy and whether there have been any changes in susceptibilities over the last ten years. 456 enteropathogens were isolated from adult travelers to Mexico, India, and Guatemala between the years 2006 to 2008. MICs were determined for 10 different antimicrobials by the agar dilution method. Traditional antibiotics such as ampicillin, trimethoprim/sulfamethoxazole, and doxycycline continue to show high levels of resistance. Current first line antibiotic agents including fluoroquinolones and azithromycin had significantly higher MICs when compared to 10 years ago and MIC90 levels were beyond the CSLI cutoffs for resistance. There were significant geographical differences in resistance patterns when comparing Central America with India. Entertoxigenic Escherichia coli (ETEC) isolates were more resistant to ciprofloxacin (p=0.023), and levofloxacin (p=0.0078) in India; whereas, enteroaggregative Escherichia coli (EAEC) isolates from Central America showed more resistance. When compared to MICs of isolates 10 years prior, there was a four to ten-fold increase in MIC90s for ceftriaxone, ciprofloxacin, levofloxacin and azithromycin for both ETEC and EAEC. There were no significant changes in rifaximin MICs over the last ten years, which makes it a promising agent for TD. Rising MICs over time implicate the need for continuous surveillance of susceptibility patterns worldwide and for geography specific recommendations in TD therapy.^

Escherichia coli from Crohn’s disease patient displays virulence features of enteroinvasive (EIEC), enterohemorragic (EHEC), and enteroaggregative (EAEC) pathotypes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Atypical enteropathogenic Escherichia coli genomic background allows the acquisition of non-EPEC virulence factors

Atypical enteropathogenic Escherichia coli (aEPEC) has been associated with infantile diarrhea in many countries. The clonal structure of aEPEC is the object of active investigation but few works have dealt with its genetic relationship with other diarrheagenic E. coli (DEC). This study aimed to evaluate the genetic relationship of aEPEC with other DEC pathotypes. The phylogenetic relationships of DEC strains were evaluated by multilocus sequence typing. Genetic diversity was assessed by pulsed-field gel electrophoresis (PFGE). The phylogram showed that aEPEC strains were distributed in four major phylogenetic groups (A, B1, B2 and D). Cluster I ( group B1) contains the majority of the strains and other pathotypes [enteroaggregative, enterotoxigenic and enterohemorrhagic E. coli ( EHEC)]; cluster II ( group A) also contains enteroaggregative and diffusely adherent E. coli; cluster III ( group B2) has atypical and typical EPEC possessing H6 or H34 antigen; and cluster IV ( group D) contains aEPEC O55:H7 strains and EHEC O157:H7 strains. PFGE analysis confirmed that these strains encompass a great genetic diversity. These results indicate that aEPEC clonal groups have a particular genomic background - especially the strains of phylogenetic group B1 that probably made possible the acquisition and expression of virulence factors derived from non-EPEC pathotypes.

Serogroups and virulence genes of Escherichia coli isolated from psittacine birds

Escherichia coli isolates from 24 sick psittacine birds were serogrouped and investigated for the presence of genes encoding the following virulence factors: attaching and effacing (eae), enteropathogenic E. coli EAF plasmid (EAF), pili associated with pyelonephritis (pap), S fimbriae (sfa), afimbrial adhesin (afa), capsule K1 (neu), curli (crl, csgA), temperature-sensitive hemagglutinin (tsh), enteroaggregative heat-stable enterotoxin-1 (astA), heat-stable enterotoxin -1 heat labile (LT) and heat stable (STa and STb) enterotoxins, Shiga-like toxins (stx1 and stx2), cytotoxic necrotizing factor 1 (cnf1), haemolysin (hly), aerobactin production (iuc) and serum resistance (iss). The results showed that the isolates belonged to 12 serogroups: O7; O15; O21; O23; O54; O64; O76; O84; O88; O128; O152 and O166. The virulence genes found were: crl in all isolates, pap in 10 isolates, iss in seven isolates, csgA in five isolates, iuc and tsh in three isolates and eae in two isolates. The combination of virulence genes revealed 11 different genotypic patterns. All strains were negative for genes encoding for EAF, EAEC, K1, sfa, afa, hly, cnf, LT, STa, STb, stx1 and stx2. Our findings showed that some E. coli isolated from psittacine birds present the same virulence factors as avian pathogenic E. coli (APEC), uropathogenic E. coli (UPEC) and Enteropathogenic E. coli (EPEC) pathotypes.

High prevalence of aggregative adherent Escherichia coli strains in the mucosa-associated microbiota of patients with inflammatory bowel diseases

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

P-220-Escherichia coli displaying virulence features of multiple diarrheogenic pathotypes detected in a Crohn's disease patient

Background: The number of Escherichia coli in the gut of Crohn's disease (CD) patients is higher than that of normal subjects, but the virulence potential of these bacteria is not fully known. Previous studies have shown that these E. coli are closely related to extraintestinal pathogenic categories (ExPEC), are able to invade epithelial cells, and usually do not produce exotoxins. We report here the detection, in a CD patient, of an E. coli which belongs to a classical enteropathogenic (EPEC) serotype and displays virulence markers of enteroinvasive (EIEC), enteroaggregative (EAEC) and enterohemorrhagic (EHEC) pathotypes. Methods: The E. coli strain was isolated, in 2009, by classical bacteriological procedures from a 56 year old woman who underwent ileo-terminal resection 1 year before, due to intestinal obstruction. The bacterial characterization was carried out by in vitro adhesion and invasion assays to cultured epithelial cells and macrophages and screening by PCR to identify virulence genetic markers of diarrheogenic E. coli (DEC) and to detect one of the gene combinations which define the phylogroups of the E. coli reference (EcoR) collection. The strain was also tested for the ability to produce biofilm and shiga cytotoxins and had its whole genome sequenced by Ion Torrent Sequencing Technology. Results: The studied strain, which was detected both in ileum biopsies and the stools of the patient, displayed the aggregative adherence (AA) phenotype to Hep-2 cells and an ability to enter Caco-2 cells 3x as high as that of EIEC reference strain and 89% of that of the prototype AIEC LF82 strain. Although it could invade cultured macrophages, the strain was unable to replicate inside these cells. PCR screening revealed the presence of eae, aggR and stx1. Tests with bacterial culture supernatants in Vero cells demonstrating cytotoxicity suggested the production of Stx1. In addition, the strain revealed to be a strong biofilm producer, belonged to the B2 EcoR phylogroup, to the O126:H27 serogroup and to the multilocus sequencing type (MLST) ST3057. The 2 later features were deduced from the whole genome sequence of the strain. Conclusions: The characterization of this E. coli isolate from a CD patient revealed a combination of virulence markers of distinct DEC pathotypes, namely eae and stx1 of EHEC, AA, aggR and biofilm formation of EAEC, and invasiveness of EIEC. These features along with its serotype and phylogroup identity seem to suggest a potential to be involved in CD, an observation which should be tested with additional studies.

Overlapped Sequence Types (sts) And Serogroups Of Avian Pathogenic (apec) And Human Extra-intestinal Pathogenic (expec) Escherichia Coli Isolated In Brazil.

Avian pathogenic Escherichia coli (APEC) strains belong to a category that is associated with colibacillosis, a serious illness in the poultry industry worldwide. Additionally, some APEC groups have recently been described as potential zoonotic agents. In this work, we compared APEC strains with extraintestinal pathogenic E. coli (ExPEC) strains isolated from clinical cases of humans with extra-intestinal diseases such as urinary tract infections (UTI) and bacteremia. PCR results showed that genes usually found in the ColV plasmid (tsh, iucA, iss, and hlyF) were associated with APEC strains while fyuA, irp-2, fepC sitDchrom, fimH, crl, csgA, afa, iha, sat, hlyA, hra, cnf1, kpsMTII, clpVSakai and malX were associated with human ExPEC. Both categories shared nine serogroups (O2, O6, O7, O8, O11, O19, O25, O73 and O153) and seven sequence types (ST10, ST88, ST93, ST117, ST131, ST155, ST359, ST648 and ST1011). Interestingly, ST95, which is associated with the zoonotic potential of APEC and is spread in avian E. coli of North America and Europe, was not detected among 76 APEC strains. When the strains were clustered based on the presence of virulence genes, most ExPEC strains (71.7%) were contained in one cluster while most APEC strains (63.2%) segregated to another. In general, the strains showed distinct genetic and fingerprint patterns, but avian and human strains of ST359, or ST23 clonal complex (CC), presented more than 70% of similarity by PFGE. The results demonstrate that some zoonotic-related STs (ST117, ST131, ST10CC, ST23CC) are present in Brazil. Also, the presence of moderate fingerprint similarities between ST359 E. coli of avian and human origin indicates that strains of this ST are candidates for having zoonotic potential.

Clustering Of Water Bodies In Unpolluted And Polluted Environments Based On Escherichia Coli Phylogroup Abundance Using A Simple Interaction Database.

Different types of water bodies, including lakes, streams, and coastal marine waters, are often susceptible to fecal contamination from a range of point and nonpoint sources, and have been evaluated using fecal indicator microorganisms. The most commonly used fecal indicator is Escherichia coli, but traditional cultivation methods do not allow discrimination of the source of pollution. The use of triplex PCR offers an approach that is fast and inexpensive, and here enabled the identification of phylogroups. The phylogenetic distribution of E. coli subgroups isolated from water samples revealed higher frequencies of subgroups A1 and B23 in rivers impacted by human pollution sources, while subgroups D1 and D2 were associated with pristine sites, and subgroup B1 with domesticated animal sources, suggesting their use as a first screening for pollution source identification. A simple classification is also proposed based on phylogenetic subgroup distribution using the w-clique metric, enabling differentiation of polluted and unpolluted sites.

Influence Of The Major Nitrite Transporter Nirc On The Virulence Of A Swollen Head Syndrome Avian Pathogenic E. Coli (apec) Strain.

Avian Pathogenic Escherichia coli (APEC) strains are extra-intestinal E. coli that infect poultry and cause diseases. Nitrite is a central branch-point in bacterial nitrogen metabolism and is used as a cytotoxin by macrophages. Unlike nitric oxide (NO), nitrite cannot diffuse across bacterial membrane cells. The NirC protein acts as a specific channel to facilitate the transport of nitrite into Salmonella and E. coli cells for nitrogen metabolism and cytoplasmic detoxification. NirC is also required for the pathogenicity of Salmonella by downregulating the production of NO by the host macrophages. Based on an in vitro microarray that revealed the overexpression of the nirC gene in APEC strain SCI-07, we constructed a nirC-deficient SCI-07 strain (ΔnirC) and evaluated its virulence potential using in vivo and in vitro assays. The final cumulative mortalities caused by mutant and wild-type (WT) were similar; while the ΔnirC caused a gradual increase in the mortality rate during the seven days recorded, the WT caused mortality up to 24h post-infection (hpi). Counts of the ΔnirC cells in the spleen, lung and liver were higher than those of the WT after 48 hpi but similar at 24 hpi. Although similar number of ΔnirC and WT cells was observed in macrophages at 3 hpi, there was higher number of ΔnirC cells at 16 hpi. The cell adhesion ability of the ΔnirC strain was about half the WT level in the presence and absence of alpha-D-mannopyranoside. These results indicate that the nirC gene influences the pathogenicity of SCI-07 strain.