Lactobacilli antagonize biological effects of enterohaemorrhagic Escherichia coli in vitro

Aims:  To assess the effect of two lactobacilli on the biological activity of enterohaemorrhagic Escherichia coli (EHEC) in vitro .
Methods and Results:  Strains CIDCA 133 ( Lactobacillus delbrueckii subsp. lactis ) and CIDCA 83114 ( Lactobacillus plantarum ) were studied. Hep-2 cells were used as an in vitro model to assess the biological effect of a clinical isolate of EHEC. Preincubation of cell monolayers with lactobacilli before EHEC prevented detachment of eukaryotic cells and minimizes both F-actin rearrangements and morphological alterations. Interestingly, the protective effect could not be ascribed to pathogen exclusion. In addition, viability of the lactobacilli was not necessary for protection and other species of the genus Lactobacillus failed to protect eukaryotic cells.
Conclusions:  Our results suggest that lactobacilli are antagonizing virulence mechanisms of EHEC either by modification of the microenvironment or by interfering with the signalling cascades triggered by the pathogen.
Significance and Impact of the Study:  Our findings give a rationale basis for the use of specific probiotic strains for the prophylaxis and prevention of intestinal infections due to EHEC.     

A comparison of enteropathogenic and enterohaemorrhagic Escherichia coli pathogenesis

This review covers enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infections, focusing on differences in their virulence factors and regulation. While Shiga-toxin expression from integrated bacteriophages sets EHEC apart from EPEC, EHEC infections often originate from asymptomatic carriage in ruminants whereas human EPEC are considered to be overt pathogens and more host-restricted. In part, these differences reflect variation in adhesin repertoire, type III-secreted effectors and the way in which these factors are regulated.     

Uropathogenic Escherichia coli (UPEC) strains may carry virulence properties of diarrhoeagenic E. coli

To analyze whether Escherichia coli strains that cause urinary tract infections (UPEC) share virulence characteristics with the diarrheagenic E. coli (DEC) pathotypes and to recognize their genetic diversity, 225 UPEC strains were examined for the presence of various properties of DEC and UPEC (type of interaction with HeLa cells, serogroups and presence of 30 virulence genes). No correlation between adherence patterns and serogroups was observed. Forty-five serogroups were found, but 64% of the strains belonged to one of the 12 serogroups (O1, O2, O4, O6, O7, O14, O15, O18, O21, O25, O75, and O175) and carried UPEC virulence genes (pap, hly, aer, sfa, cnf). The DEC genes found were: aap, aatA, aggC, agg3C, aggR, astA, eae, ehly, iha, irp2, lpfA(O113), pet, pic, pilS, and shf. Sixteen strains presented aggregative adherence and/or the aatA sequence, which are characteristics of enteroaggregative E. coli (EAEC), one of the DEC pathotypes. In summary, certain UPEC strains may carry DEC virulence properties, mostly associated to the EAEC pathotype. This finding raises the possibility that at least some faecal EAEC strains might represent potential uropathogens. Alternatively, certain UPEC strains may have acquired EAEC properties, becoming a potential cause of diarrhoea.     

Serotypes and virutypes of enteropathogenic and enterohaemorrhagic Escherichia coli strains from stool samples of children with diarrhoea in Germany

Aims: To investigate the prevalence of traditional and emerging types of enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) strains in stool samples from children with diarrhoea and to characterize their virulence genes involved in the attaching and effacing (A/E) phenotype. Methods and Results: Serological and PCR‐based methods were used for detection and isolation of EPEC and EHEC strains from 861 stool samples from diarrhoeic children. Agglutination with traditional EPEC and EHEC O‐group‐specific antisera resulted in detection of 38 strains; 26 of these carried virulence factors of EPEC or EHEC. PCR screening for the eae gene resulted in isolation of 97 strains, five carried genes encoding Shiga toxins (stx), one carried the bfpA gene and 91 were atypical EPEC. The 97 EPEC and EHEC strains were divided into 36 O‐serogroups and 21 H‐types, only nine strains belonged to the traditional EPEC O‐groups O26, O55, O86 and O128. In contrast, EPEC serotypes O28:H28, O51:H49, O115:H38 and O127:H40 were found in multiple cases. Subtyping the virulence factors intimin, Tir and Tir‐cytoskeleton coupling effector protein (TccP)/TccP2 resulted in further classification of 93·8% of the 97 strains. Conclusions: Our findings show a clear advantage of the eae‐PCR over the serological detection method for identification of EPEC and EHEC strains from human patients. Significance and Impact of the Study: Molecular detection by the eae‐PCR followed by serotyping and virutyping is useful for monitoring trends in EPEC and EHEC infections and to discover their possible reservoirs.     

The diversity of Escherichia coli serotypes and biotypes in cattle faeces

AIM: To study the diversity of commensal Escherichia coli populations shed in faeces of cattle fed on different diets. METHODS AND RESULTS: Thirty Brahman-cross steers were initially fed a high grain (80%) diet and then randomly allocated into three dietary treatment groups, fed 80% grain, roughage, or roughage + 50% molasses. Up to eight different E. coli isolates were selected from primary isolation plates of faecal samples from each animal. Fifty-two distinct serotypes, including nine different VTEC strains, were identified from a total of 474 E. coli isolates. Cattle fed a roughage + molasses diet had greater serotype diversity (30 serotypes identified) than cattle fed roughage or grain (21 and 17 serotypes identified respectively). Cluster analysis showed that serotypes isolated from cattle fed roughage and roughage + molasses diets were more closely associated than serotypes isolated from cattle fed grain. Resistance to one or more of 11 antimicrobial agents was detected among isolates from 20 different serotypes. Whilst only 2.3% of E. coli isolates produced enterohaemolysin, 25% were found to produce alpha-haemolysin. CONCLUSIONS: Diverse non-VTEC populations of E. coli serotypes are shed in cattle faeces and diet may affect population diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information on the serotype diversity and phenotypic traits of predominant E. coli populations in cattle faeces, which could be sources of environmental contamination.     

Isolation and characterization of lytic bacteriophages against enterohaemorrhagic Escherichia coli

Aims: The objective of this study was to isolate, identify and characterize a collection of lytic bacteriophages capable of infecting enterohaemorrhagic Escherichia coli (EHEC) serotypes. Methods and Results: Phages were isolated from dairy and cattle feedlot manure using E. coli O157, O26 and O111 strains as hosts. Phages were enriched from faecal slurries by culture in 10× trypticase soy broth at 37°C overnight. Phage plaques were obtained by mixing the filtered culture supernatant with molten tryptone agar containing the phage E. coli host strain, pouring the inoculated agar on top of cooled TS agar and incubating the culture overnight. Phages were purified from plaques and screened against additional E. coli and EHEC strains by the efficiency of plating method (EOP). Phage CEV2, and five other phages previously isolated, were able to lyse all of the 15 O157 strains tested with EOP values consistently above 0·001. Two phages were found to be highly effective against strains of E. coli O157 through EOP tests and against O26 strains through spot tests, but not against the O serogroup 111 strains. A cocktail of eight phage that lyse E. coli O157 strains resulted in >5 log CFU ml^?1 reductions at 37°C. Multiplex‐PCR revealed that none of these eight phages carried stx1, stx2, hlyA or eaeA genes. Conclusions: A cocktail of bacteriophages was capable of lysing most strains of two EHEC serotypes. Significance and Impact of the Study: This collection of phages can be combined and potentially used as an antimicrobial cocktail to inactivate E. coli strains from O serogroups 157 and 26 and reduce their incidence in the food chain.     

Evaluation of the 'GeneDisc' real-time PCR system for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains according to their virulence markers and their O- and H-antigen-associated genes

Aims:  To evaluate the GeneDisc multiplex real-time PCR assay for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains.
Methods and Results:  GeneDiscs for detection of genes encoding Shiga toxins ( stx ), intimins ( eae ), E. coli O157 ( rfbE _O157) and H7 ( fliC _H7) antigens as well as genes specific for EHEC O26 ( wzx _O26), O103 ( wzx _O103), O111 ( wbd1 _O111), O145 ( ihp1 _O145) and O157 ( ihp1 _O157) were evaluated. The assay was run with native bacteria in 1 h in a GeneDisc Cycler. All genotypes of stx and eae , except stx _2f and eae -rho, were identified. Escherichia coli strains belonging to O-groups O26, O103, O111, O157 as well as EHEC O145:[H28] strains were specifically detected with this assay. The ihp1 _O157 gene was not found specific for EHEC O157. O-rough mutants of EHEC and non-motile EHEC O157 strains were reliably identified with the GeneDisc assay. Two to three colonies of EHEC strains were still detectable in a lawn of 50 000 apathogenic E. coli from agar plates.
Conclusions:  The GeneDisc assay is a specific and reliable assay for detection of major EHEC strains. It is robust enough to detect few EHEC colonies in mixed cultures of bacteria.
Significance and Impact of the Study:  The assay is promising for its use in EHEC diagnostics and for EHEC monitoring with different kinds of samples.     

The incidence of Shiga toxin-producing Escherichia coli in cattle with mastitis in Brazil

AIMS: To determine the prevalence and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) isolates from bovine mastitic milk in Brazil. METHODS AND RESULTS: A total of 2144 milk samples from dairy cattle showing mastitis were screened for the presence of E. coli. A total of 182 E. coli isolates were selected and examined. All were subjected to dot blot analysis using the CVD419 probe for the detection of the enterohaemolysin (hly) gene, and to a multiplex PCR for the detection of stx1, stx2 and eaeA genes. STEC were isolated from 22 (12.08%) milk samples. All the STEC isolates were tested for sensibility to 10 antimicrobials; the resistances most commonly observed were to cephalothin (86.3%), tetracycline (63.6%) and doxycycline (63.6%). CONCLUSION: STEC isolates were found in bovine mastitic milk in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: STEC isolates from mastitic milk were potentially pathogenic for human in that they belonged to serogroups associated with diarrhoea and haemolytic-uraemic syndrome, some of them were stx2, eaeA and hly positive.     

禽致病性大肠杆菌IMT5155 疑似毒力基因在人源及动物源大肠杆菌中的分布

[目的]检测禽致病性大肠杆菌IMT5155自分泌黏附素基因等具有代表性的疑似毒力基因在不同来源大肠杆菌中的分布,为进一步研究其致病机理提供依据.[方法]采用PCR和Dot blot,检测疑似毒力基因在不同地区(101株大肠杆菌中国分离株和121株大肠杆菌德国分离株)、不同来源(人源、禽源及猪源)大肠杆菌中的分布,并分析其和大肠杆菌系统进化分群的关系.[结果]自分泌黏附素基因B11等11个疑似毒力基因在禽致病性大肠杆菌中分布率较高,阳性率分别为:A1 36.4%(32/88)、A8 53.4%(47/88)、A1063.6%(56/88)、B1137.5%(33/88)、F3 59.1%(52/88)等,且疑似毒力基因主要存在于大肠杆菌B2进化群中.值得注意的是,D1、E9和F11基因片段在新生儿脑膜炎大肠杆菌中有较高的分布率,分别为60%(6/10)、80%(8/10)和90%(9/10),而在新生儿脑膜炎大肠杆菌中未检测到B11基因.[结论]自分泌黏附素B11等疑似毒力基因与禽致病性大肠杆菌关系密切,但疑似毒力基因D1、E9和F11与新生儿脑膜炎大肠杆菌密切相关,提示禽致病性大肠杆菌可能是新生儿脑膜炎大肠杆菌的毒力基因储库.     

PCR detection of seven virulence and toxin genes of Campylobacter jejuni and Campylobacter coli isolates from Danish pigs and cattle and cytolethal distending toxin production of the isolates

AIMS: To study the prevalence of seven virulence and toxin genes, and cytolethal distending toxin (CDT) production of Campylobacter jejuni and C. coli isolates from Danish pigs and cattle. METHODS AND RESULTS: The presence of the cadF, ceuE, virB11, flaA, cdtA, cdtB, cdtC and the cdt gene cluster among 40 C. jejuni and C. coli isolates was detected by polymerase chain reaction. The CDT production of the isolates was determined on Vero, colon 205 and chicken embryo cells. The cadF, flaA, ceuE and cdtB genes were detected from 100% of the isolates. The cdtA and cdtC genes were found in 95.0 and 90.0% of the isolates, respectively. The cdt gene cluster was detected in 82.5% isolates. Only 7.5% of the isolates were positive for virB11. Ninety-five per cent of the isolates produced CDT in Vero and colon 205 cell assays, and 90% of the isolates produced CDT in chicken embryo cell assays. CONCLUSIONS: High prevalence of the cadF, ceuE, flaA and cdtB genes was found. Data of the prevalence of cdt genes was consistent with the CDT titres produced by the isolates. Campylobacter coli from pigs produced high CDT titres. SIGNIFICANCE AND IMPACT OF THE STUDY: The high prevalence of seven virulence and toxin genes demonstrated that these putative pathogenic determinants are widespread among Campylobacter isolates from pigs and cattle. Campylobacter coli isolates from pigs produced much higher CDT titres compared with C. coli isolates from other sources suggesting that C. coli may be particularly adapted to or associated with this species.     

猪源大肠杆菌(ETEC、STEC、AEEC)毒力基因及其与O抗原型的关系

[目的]揭示从我国部分地区仔猪腹泻或水肿病病猪体内分离到的300个大肠杆菌分离株所属病原型(pathotype)、毒力基因及其与O血清型的关系.[方法]O血清型采用常规的凝集试验进行测定,毒力基因采用PCR方法检测.[结果]通过对这300个分离株的O血清型及其毒素、紧密素和黏附素基因进行鉴定,结果显示除50株未定型、17株自凝外,测定出233个分离株的血清型,这些分离株覆盖了45个血清型,其中以0149、0107、0139、093和091为主,共133株,占定型菌株的57.1%;拥有est Ⅰ、estⅡ、elt、stx2e和eae A基因的菌株分别为102(34.0%)、190(63.3%)、81(27.0%)、57(19.0%)和54(18.0%)株;分离株中有51株K88基因阳性(其中菌毛表达率为100%),75株F18基因阳性(其中菌毛表达率为50.7%),在K88菌株中,0149血清型与est Ⅰ或estⅡ elt密切相关,在F18菌株中,0107血清型与est Ⅰ或estⅡ、0139血清型与stx2e紧密相关.依其毒力特征可将这些分离株分为以下6种类型:ETEC、STEC、AEEC、ETEC/STEC、AEEC/ETEC和AEEC/ETEC/STEC,分别拥有190、24、36、32、17和1个菌株,占分离株的63.3%、8.0%、12.0%、10.7%、5.7%和0.3%.通过分析这些分离株的O血清型、毒素类型和黏附素型之间的相关性:猪源ETEC以0149、0107、093和098等血清型为主,0149:K88菌株主要与estⅡ或estⅡ elt肠毒素相关,0107:F18菌株主要与estⅡ相关,093和098血清型菌株主要与estⅡ肠毒素相关;STEC菌株以0139:F18血清型为主,拥有stx2e;AEEC菌株拥有紧密素,无明显优势血清型;ETEC/STEC菌株以0107:F18和0116:F18血清型为主,主要与est Ⅰ stx2e或estⅡ stx2e密切相关,ETEC/AEEC菌株以091和0107血清型为主,全部拥有肠毒素est Ⅰ和紧密素基因.[结论]我国至少存在6种病原型的猪肠道致病性大肠杆菌,其中ETEC为我国部分地区猪大肠杆菌病的主要病原,同时其病原型日益复杂.     

Prevalence of virulence genes in Escherichia coli strains isolated from Romanian adult urinary tract infection cases

A total of 78 E. coli strains isolated from adults with different types of urinary tract infections were screened by polymerase chain reaction for prevalence of genetic regions coding for virulence factors. The targeted genetic determinants were those coding for type 1 fimbriae ( fimH ), pili associated with pyelonephritis ( pap ), S and F1C fimbriae ( sfa and foc ), afimbrial adhesins ( afa ), hemolysin ( hly ), cytotoxic necrotizing factor ( cnf ), aerobactin ( aer ). Among the studied strains, the prevalence of genes coding for fimbrial adhesive systems was 86 %, 36%, and 23% for fimH, pap , and sfa/foc , respectively. The operons coding for Afa afimbrial adhesins were identified in 14% of strains. The hly and cnf genes coding for toxins were amplified in 23% and 13% of strains, respectively. A prevalence of 54% was found for the aer gene. The various combinations of detected genes were designated as virulence patterns. The strains isolated from the hospitalized patients displayed a greater number of virulence genes and a diversity of gene associations compared to the strains isolated from the ambulatory subjects. A rapid assessment of the bacterial pathogenicity characteristics may contribute to a better medical approach of the patients with urinary tract infections.     

Virulence markers and serotypes of Shiga toxin-producing Escherichia coli, isolated from cattle in Rio Grande do Sul, Brazil

AIMS: To determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) and serotypes and virulence markers of the STEC isolates from beef and dairy cattle in Rio Grande do Sul, Brazil. METHODS AND RESULTS: Faecal samples from beef cattle were collected at slaughterhouses. The isolates were submitted to colony hybridization assay with specific DNA probes for stx1, stx2 and eae genes, and serotyped for the identification of O and H antigens. Thirty-nine per cent of beef cattle surveyed harboured at least one STEC strain. Among the distinct serotypes identified, 10 were shared by both beef and dairy cattle. Most of the strains isolated harboured stx2. Genotypic and phenotypic profiles allowed the identification of 34 and 31 STEC strains, isolated from beef and dairy cattle, respectively. Serotypes O10:H14, O15:H21, O96:H21, O119:H4, O124:H11, O128:H21, O137:H-, O141:H19, O159:H42, O160:H2 and O177:H11, identified in this study, have not been previously reported as STEC isolated from cattle. CONCLUSIONS: Cattle are an important reservoir of STEC strains associated with human diseases in South America. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the prevalence, genotypic profile and serotypes of STEC strains isolated from cattle enables the prediction of possible risk for public health.     

Genetic diversity of Escherichia coli recovered from the oral cavity of beef cattle and their relatedness to faecal E. coli

AIMS: To determine the genetic diversity of generic Escherichia coli recovered from the oral cavities of beef cattle and their relatedness to E. coli isolated from the faeces of cattle during pasture grazing and feedlot finishing. METHODS AND RESULTS: A total of 484 E. coli (248 oral and 236 faecal isolates) were obtained from eight beef cattle after 1 and 5 months of grazing on pasture and after 1 and 5 months in a feedlot. The random amplification of polymorphic DNA (RAPD) method was used to genetically characterize these isolates. The RAPD patterns showed that ca 60% of E. coli recovered from the oral cavities and faeces during pasture and feedlot shared a close genetic relatedness. A number of E. coli with unique RAPD types were also found either in the oral cavities or faeces. Most of the E. coli RAPD types recovered from the oral cavities were shared among animals, but there were also RAPD types which were unique to individual animals. The E. coli populations of the oral cavities were genetically diverse and changed over time. CONCLUSIONS: This study indicates that there are large numbers of E. coli carried in the oral cavities of beef cattle and those E. coli are closely related to strains found in the faeces. The oral cavities of cattle harbour a genetically diverse E. coli population. SIGNIFICANCE AND IMPACT OF THE STUDY: The oral cavity may be an important reservoir of enteric pathogens which may transfer to meat during carcass dressing. A better understanding of the molecular ecology of E. coli in cattle would assist the design of approaches to control pathogenic strains during beef production and processing.     

Fate of Escherichia coli O157 and detection of stx phage during fermentation of maize, an animal feedstuff

AIMS: The fate of inoculated Escherichia coli O157, stx phages and the physico-chemical properties of maize were studied during laboratory-scale fermentation by naturally occurring lactic acid bacteria. METHODS AND RESULTS: Before fermentation, chopped maize was inoculated with 6.2 log(10) CFU g(-1) of a five-isolate mixture of E. coli O157. After fermentation, the silage contained 70.6 g kg(-1) dry matter (DM) lactic acid and 12.8 g kg(-1) DM acetic acid and was pH 4.0. Levels of E. coli O157 fell rapidly, and none of the five isolates could be recovered from the fermenting maize after 8 days. Using a resuscitation step did not consistently enhance recovery of E. coli O157. Stx phages were not isolated from the fermenting maize at any time. Pulsed-field gel electrophoresis (PFGE) genotyping showed that E. coli O157 2975 and 64a/01 survived better than the other three isolates studied. Escherichia coli O157 isolate 1474/00 was particularly sensitive to the laboratory procedures used to harvest the inocula and contaminate the maize. Some colonies recovered during the fermentation had one to three band alterations compared with the initial PFGE genotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: None of the five different E. coli O157 genotypes survived maize fermentation. Fermentation of maize produces an animal feedstuff that is unlikely to contain E. coli O157 or stx phages.     

Detection of virulent Escherichia coli O157 strains using multiplex PCR and single base sequencing for SNP characterization

Aims: To compare 167 Norwegian human and nonhuman Escherichia coli O157:H7/NM (nonmotile) isolates with respect to an A/T single nucleotide polymorphism (SNP) in the tir gene and to detect specific SNPs that differentiate STEC O157 into distinct virulence clades (1–3 and 8). Methods and Results: We developed a multiplex PCR followed by single base sequencing for detection of the SNPs, and examined the association among SNP genotype, virulence profile (stx and eae status), multilocus variable number of tandem repeats analysis (MLVA) profile and clinical outcome. We found an over‐representation of the T allele among human strains compared to nonhuman strains, including 5/6 haemolytic‐uraemic syndrome cases. Fourteen strains belonged to clade 8, followed by two clade 2 strains. No clade 1 nor 3 isolates were observed. stx1 in combination with either stx2_EDL933 or stx2c were frequently observed among human strains, whereas stx2c was dominating in nonhuman strains. MLVA indicated that only single cases or small outbreaks with E. coli O157 have been observed in Norway through the years 1993–2008. Conclusion: We observed that the tir‐255 A/T SNP and the stx status were different between human and nonhuman O157 strains. No major outbreaks were observed, and only a few strains were differentiated into the virulence clades 2 and 8. Significance and Impact of the Study: The detection of virulence clade‐specific SNPs enables the rapid designation of virulent E. coli O157 strains, especially in outbreak situations.     

Serogroup distribution and virulence characteristics of sorbitol‐negative Escherichia coli from food and cattle stool

Aims: To (i) study the serogroup distribution and virulence characteristics of non‐sorbitol‐fermenting Escherichia coli isolates from foods of animal origin and cattle faeces and (ii) re‐examine the true sorbitol and β‐d ‐glucuronidase (GUD) reactions of sorbitol‐negative (Sor^?) strains from MacConkey sorbitol agar (SMAC) to assess their phenotypic similarity with E. coli O157. Methods and Results: One hundred and thirty Sor^?E. coli were isolated from 556 food samples and 177 cattle stool samples using cefixime tellurite–supplemented SMAC (CT‐SMAC) and chromogenic HiCrome MS.O157 agar respectively. Based on typing of somatic antigen, the isolates were classified into 38 serogroups. PCR results identified about 40% strains, belonging to O5, O8, O20, O28, O48, O60, O78, O82, O84, O101, O110, O123, O132, O156, O157, O‐rough and OUT as Shiga toxigenic. Majority of O5, O84, O101, O105, O123, O157, O‐rough and OUT strains were enterohaemolytic. Further, 39·2% and 63·1% of Sor^? isolates from CT‐SMAC fermented sorbitol in phenol red broth and hydrolysed 4‐methylumbelliferyl‐β‐d ‐glucuronide (MUG) respectively. Members of serogroups O5, O28, O32, O81, O82, O84, O101, O‐rough lacked both the sorbitol fermentation (broth test) and GUD activity and might create confusion in phenotypic identification of E. coli O157. Conclusions: Sor^?E. coli isolates from raw meat, milk, shrimp and cattle stool belonged to 38 serogroups, with E. coli O157 constituting only 14·6% of the isolates. Many of these nonclinical Sor^? strains were potentially pathogenic. Nearly 39% of these Sor^?E. coli from CT‐SMAC fermented sorbitol in broth, indicating the need for confirmation of sorbitol reaction in broth. Significance and Impacts of the Study: Classical sorbitol utilization and GUD tests are not likely definitive tests for E. coli O157. Further improvement of differential media based on these phenotypic properties is necessary for detection of pathogenic serotypes from foods and environmental samples.     

A specific genetic background is required for acquisition and expression of virulence factors in Escherichia coli

In bacteria, the evolution of pathogenicity seems to be the result of the constant arrival of virulence factors (VFs) into the bacterial genome. However, the integration, retention, and/or expression of these factors may be the result of the interaction between the new arriving genes and the bacterial genomic background. To test this hypothesis, a phylogenetic analysis was done on a collection of 98 Escherichia coli/Shigella strains representing the pathogenic and commensal diversity of the species. The distribution of 17 VFs associated to the different E. coli pathovars was superimposed on the phylogenetic tree. Three major types of VFs can be recognized: (1) VFs that arrive and are expressed in different genetic backgrounds (such as VFs associated with the pathovars of mild chronic diarrhea: enteroaggregative, enteropathogenic, and diffusely-adhering E. coli), (2) VFs that arrive in different genetic backgrounds but are preferentially found, associated with a specific pathology, in only one particular background (such as VFs associated with extraintestinal diseases), and (3) VFs that require a particular genetic background for the arrival and expression of their virulence potential (such as VFs associated with pathovars typical of severe acute diarrhea: enterohemorragic, enterotoxigenic, and enteroinvasive E. coli strains). The possibility of a single arrival of VFs by chance, followed by a vertical transmission, was ruled out by comparing the evolutionary histories of some of these VFs to the strain phylogeny. These evidences suggest that important changes in the genome of E. coli have occurred during the diversification of the species, allowing the virulence factors associated with severe acute diarrhea to arrive in the population. Thus, the E. coli genome seems to be formed by an "ancestral" and a "derived" background, each one responsible for the acquisition and expression of different virulence factors.