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2.  Biofilm formation as a novel phenotypic feature of adherent-invasive Escherichia coli (AIEC) 
BMC Microbiology  2009;9:202.
Crohn's disease (CD) is a high morbidity chronic inflammatory disorder of unknown aetiology. Adherent-invasive Escherichia coli (AIEC) has been recently implicated in the origin and perpetuation of CD. Because bacterial biofilms in the gut mucosa are suspected to play a role in CD and biofilm formation is a feature of certain pathogenic E. coli strains, we compared the biofilm formation capacity of 27 AIEC and 38 non-AIEC strains isolated from the intestinal mucosa. Biofilm formation capacity was then contrasted with the AIEC phenotype, the serotype, the phylotype, and the presence of virulence genes.
Specific biofilm formation (SBF) indices were higher amongst AIEC than non-AIEC strains (P = 0.012). In addition, 65.4% of moderate to strong biofilms producers were AIEC, whereas 74.4% of weak biofilm producers were non-AIEC (P = 0.002). These data indicate that AIEC strains were more efficient biofilm producers than non-AIEC strains. Moreover, adhesion (P = 0.009) and invasion (P = 0.003) indices correlated positively with higher SBF indices. Additionally, motility (100%, P < 0.001), H1 type flagellin (53.8%, P < 0.001), serogroups O83 (19.2%, P = 0.008) and O22 (26.9%, P = 0.001), the presence of virulence genes such as sfa/focDE (38.5%, P = 0.003) and ibeA (26.9%, P = 0.017), and B2 phylotype (80.8%, P < 0.001) were frequent characteristics amongst biofilm producers.
The principal contribution of the present work is the finding that biofilm formation capacity is a novel, complementary pathogenic feature of the recently described AIEC pathovar. Characterization of AIEC specific genetic determinants, and the regulatory pathways, involved in biofilm formation will likely bring new insights into AIEC pathogenesis.
PMCID: PMC2759958  PMID: 19772580
3.  Invasiveness as a putative additional virulence mechanism of some atypical Enteropathogenic Escherichia coli strains with different uncommon intimin types 
BMC Microbiology  2009;9:146.
Enteropathogenic Escherichia coli (EPEC) produce attaching/effacing (A/E) lesions on eukaryotic cells mediated by the outer membrane adhesin intimin. EPEC are sub-grouped into typical (tEPEC) and atypical (aEPEC). We have recently demonstrated that aEPEC strain 1551-2 (serotype O non-typable, non-motile) invades HeLa cells by a process dependent on the expression of intimin sub-type omicron. In this study, we evaluated whether aEPEC strains expressing other intimin sub-types are also invasive using the quantitative gentamicin protection assay. We also evaluated whether aEPEC invade differentiated intestinal T84 cells.
Five of six strains invaded HeLa and T84 cells in a range of 13.3%–20.9% and 5.8%–17.8%, respectively, of the total cell-associated bacteria. The strains studied were significantly more invasive than prototype tEPEC strain E2348/69 (1.4% and 0.5% in HeLa and T84 cells, respectively). Invasiveness was confirmed by transmission electron microscopy. We also showed that invasion of HeLa cells by aEPEC 1551-2 depended on actin filaments, but not on microtubules. In addition, disruption of tight junctions enhanced its invasion efficiency in T84 cells, suggesting preferential invasion via a non-differentiated surface.
Some aEPEC strains may invade intestinal cells in vitro with varying efficiencies and independently of the intimin sub-type.
PMCID: PMC2724384  PMID: 19622141
4.  Extraintestinal pathogenic Escherichia coli O1:K1:H7/NM from human and avian origin: detection of clonal groups B2 ST95 and D ST59 with different host distribution 
BMC Microbiology  2009;9:132.
Extraintestinal pathogenic Escherichia coli (ExPEC) strains of serotype O1:K1:H7/NM are frequently implicated in neonatal meningitis, urinary tract infections and septicemia in humans. They are also commonly isolated from colibacillosis in poultry. Studies to determine the similarities of ExPEC from different origins have indicated that avian strains potentially have zoonotic properties.
A total of 59 ExPEC O1:K1:H7/NM isolates (21 from avian colibacillosis, 15 from human meningitis, and 23 from human urinary tract infection and septicemia) originated from four countries were characterized by phylogenetic PCR grouping, Multilocus Sequence Typing (MLST), Pulsed Field Gel Electrophoresis (PFGE) and genotyping based on several genes known for their association with ExPEC or avian pathogenic Escherichia coli (APEC) virulence.
APEC and human ExPEC isolates differed significantly in their assignments to phylogenetic groups, being phylogroup B2 more prevalent among APEC than among human ExPEC (95% vs. 53%, P = 0.001), whereas phylogroup D was almost exclusively associated with human ExPEC (47% vs. 5%, P = 0.0000). Seven virulence genes showed significant differences, being fimAvMT78 and sat genes linked to human isolates, while papGII, tsh, iron, cvaC and iss were significantly associated to APEC. By MLST, 39 of 40 ExPEC belonging to phylogroup B2, and 17 of 19 belonging to phylogroup D exhibited the Sequence Types (STs) ST95 and ST59, respectively. Additionally, two novel STs (ST1013 and ST1006) were established. Considering strains sharing the same ST, phylogenetic group, virulence genotype and PFGE cluster to belong to the same subclone, five subclones were detected; one of those grouped six strains of human and animal origin from two countries.
Present results reveal that the clonal group B2 O1:K1:H7/NM ST95, detected in strains of animal and human origin, recovered from different dates and geographic sources, provides evidence that some APEC isolates may act as potential pathogens for humans and, consequently, poultry as a foodborne source, suggesting no host specificity for this type of isolates. A novel and important finding has been the detection of the clonal group D O1:K1:H7/NM ST59 almost exclusively in humans, carrying pathogenic genes linked to the phylogenetic group D. This finding would suggest D O1:K1:H7/NM ST59 as a host specific pathotype for humans.
PMCID: PMC2713252  PMID: 19583828
5.  Serotypes, virulence genes and intimin types of Shiga toxin (verocytotoxin)-producing Escherichia coli isolates from minced beef in Lugo (Spain) from 1995 through 2003 
BMC Microbiology  2007;7:13.
Shiga toxin-producing Escherichia coli (STEC) have emerged as pathogens that can cause food-borne infections and severe and potentially fatal illnesses in humans, such as haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). In Spain, like in many other countries, STEC strains have been frequently isolated from ruminants, and represent a significant cause of sporadic cases of human infection. In view of the lack of data on STEC isolated from food in Spain, the objectives of this study were to determine the level of microbiological contamination and the prevalence of STEC O157:H7 and non-O157 in a large sampling of minced beef collected from 30 local stores in Lugo city between 1995 and 2003. Also to establish if those STEC isolated from food possessed the same virulence profiles as STEC strains causing human infections.
STEC were detected in 95 (12%) of the 785 minced beef samples tested. STEC O157:H7 was isolated from eight (1.0%) samples and non-O157 STEC from 90 (11%) samples. Ninety-six STEC isolates were further characterized by PCR and serotyping. PCR showed that 28 (29%) isolates carried stx1 genes, 49 (51%) possessed stx2 genes, and 19 (20%) both stx1 and stx2. Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 43 (45%) and in 25 (26%) of the isolates, respectively. Typing of the eae variants detected four types: γ1 (nine isolates), β1 (eight isolates), ε1 (three isolates), and θ (two isolates). The majority (68%) of STEC isolates belonged to serotypes previously detected in human STEC and 38% to serotypes associated with STEC isolated from patients with HUS. Ten new serotypes not previously described in raw beef products were also detected. The highly virulent seropathotypes O26:H11 stx1 eae-β1, O157:H7 stx1stx2 eae-γ1 and O157:H7 stx2eae-γ1, which are the most frequently observed among STEC causing human infections in Spain, were detected in 10 of the 96 STEC isolates. Furthermore, phage typing of STEC O157:H7 isolates showed that the majority (seven of eight isolates) belonged to the main phage types previously detected in STEC O157:H7 strains associated with severe human illnesses.
The results of this study do not differ greatly from those reported in other countries with regard to prevalence of O157 and non-O157 STEC in minced beef. As we suspected, serotypes different from O157:H7 also play an important role in food contamination in Spain, including the highly virulent seropathotype O26:H11 stx1 eae-β1. Thus, our data confirm minced beef in the city of Lugo as vehicles of highly pathogenic STEC. This requires that control measures to be introduced and implemented to increase the safety of minced beef.
PMCID: PMC1810539  PMID: 17331254
6.  Serotypes, intimin variants and other virulence factors of eae positive Escherichia coli strains isolated from healthy cattle in Switzerland. Identification of a new intimin variant gene (eae-η2) 
BMC Microbiology  2005;5:23.
Enteropathogenic Escherichia coli (EPEC) and Shigatoxin-producing Escherichia coli (STEC) share the ability to introduce attaching-and-effacing (A/E) lesions on intestinal cells. The genetic determinants for the production of A/E lesions are located on the locus of enterocyte effacement (LEE), a pathogenicity island that also contains the genes encoding intimin (eae). This study reports information on the occurrence of eae positive E. coli carried by healthy cattle at the point of slaughter, and on serotypes, intimin variants, and further virulence factors of isolated EPEC and STEC strains.
Of 51 eae positive bovine E. coli strains, 59% were classified as EPEC and 41% as STEC. EPEC strains belonged to 18 O:H serotypes, six strains to typical EPEC serogroups. EPEC strains harbored a variety of intimin variants with eae-β1 being most frequently found. Moreover, nine EPEC strains harbored astA (EAST1), seven bfpA (bundlin), and only one strain was positive for the EAF plasmid. We have identified a new intimin gene (η2) in three bovine bfpA and astA-positive EPEC strains of serotype ONT:H45. STEC strains belonged to seven O:H serotypes with one serotype (O103:H2) accounting for 48% of the strains. The majority of bovine STEC strains (90%) belonged to five serotypes previously reported in association with hemolytic uremic syndrom (HUS), including one O157:H7 STEC strain. STEC strains harbored four intimin variants with eae-ε1 and eae-γ1 being most frequently found. Moreover, the majority of STEC strains carried only stx1 genes (13 strains), and was positive for ehxA (18 strains) encoding for Enterohemolysin. Four STEC strains showed a virulence pattern characteristic of highly virulent human strains (stx2 and eae positive).
Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-harboring E. coli strains. Moreover, cattle have not only to be considered as important asymptomatic carriers of O157 STEC but can also be a reservoir of EPEC and eae positive non-O157 STEC, which are described in association with human diseases.
PMCID: PMC1142320  PMID: 15882459
7.  First isolation of the enterohaemorrhagic Escherichia coli O145:H- from cattle in feedlot in Argentina 
BMC Microbiology  2002;2:6.
Enterohaemorrhagic Escherichia coli (EHEC) is considered to be common cause of haemorrhagic colitis (HC), thrombotic thrombocytopenic purpura and haemolytic uraemic syndrome (HUS) in humans. In a previous paper, we have demonstrated that EHEC are commonly found in the intestines of livestock. Infections in humans are, in part, a consequence of consumption of undercooked meat or raw milk. Argentina has one of the highest records of HUS (300–400 cases/year; 22/100,000 children under 4 years of age). The aim of this work is to communicate the first isolation of O145:H-from cattle in this country and characterize the virulence cassette, providing useful information to evaluate the risk of foodborne transmission of this emergent non-O157:H7 serotype.
EHEC O145:H- was isolated from cattle in an Argentinian feedlot. Pheno- and genotype of nine strains were characterized, corresponding to several virulence cassettes: VT2+eaeA+ Mp+ (n = 5), VT2+eaeA+ (n = 1), VT1+eaeA+ Mp+ (n = 2), and VT1+eaeA+ (n = 1). Strains isolated from the same animal were considered only when they showed a different virulence pattern. The clonal relationship was studied by RAPD. Strains were distributed in two RAPD profiles, which corresponded to the presence of either, VT1+ or VT2+ genotype. No difference was detected by RAPD analysis between Mp+ or Mp- strains.
This was the first isolation of EHEC O145:H- serotype in Argentina enlarging the list of non-O157:H7 serotypes isolated from cattle in this country by us. All O145:H-strains carried several virulence factors which allow us to predict their potential ability to develop haemolytic uraemic syndrome in humans.
PMCID: PMC102760  PMID: 11936956

Results 1-7 (7)