Identification of Tn10 insertions in the rfaG, rfaP, and galU genes involved in lipopolysaccharide core biosynthesis that affect Escherichia coli adhesion

Escherichia coli was used as a model to study initial adhesion and early biofilm development to abiotic surface. Tn10 insertion mutants of Escherichia coli K-12 W3110 were selected for altered abilities to adhere to a polystyrene surface. Seven insertion mutants that showed a decrease in adhesion harbored insertions in genes involved in lipopolysaccharide (LPS) core biosynthesis. Two insertions were located in the rfaG gene, two in the rfaP gene, and three in the galU gene. These adhesion mutants were found to exhibit a deep-rough phenotype and to be reduced, at different levels, in type 1 fimbriae production and motility. The loss of adhesion exhibited by these mutants was associated with either the affected type 1 fimbriae production and/or the dysfunctional motility. Apart from the pleiotropic effect of the mutations affecting LPS on type 1 fimbriae and flagella biosynthesis, no evidence for an involvement of the LPS itself in adhesion to polystyrene surface could be observed. Received: 1 December 1998 / Accepted: 3 April 1999     

Chemotaxis and cyclic-di-GMP signalling control surface attachment of Escherichia coli

Attachment to surfaces is an important early step during bacterial infection and during formation of submerged biofilms. Although flagella-mediated motility is known to be important for attachment of Escherichia coli and other bacteria, implications of motility regulation by cellular signalling remain to be understood. Here, we show that motility largely promotes attachment of E. coli, including that mediated by type 1 fimbriae, by allowing cells to reach, get hydrodynamically trapped at and explore the surface. Inactivation or inhibition of the chemotaxis signalling pathway improves attachment by suppressing cell reorientations and thereby increasing surface residence times. The attachment is further enhanced by deletion of genes encoding the cyclic diguanosine monophosphate (c-di-GMP)-dependent flagellar brake YcgR or the diguanylate cyclase DgcE. Such increased attachment in absence of c-di-GMP signalling is in contrast to its commonly accepted function as a positive regulator of the sessile state. It is apparently due to the increased swimming speed of E. coli in absence of YcgR-mediated motor control, which strengthens adhesion mediated by the type 1 fimbriae. Thus, both signalling networks that regulate motility of E. coli also control its engagement with both biotic and abiotic surfaces, which has likely implications for infection and biofilm formation.     

Plasminogen, absorbed by Escherichia coli expressing curli or by Salmonella enteritidis expressing thin aggregative fimbriae, can be activated by simultaneously captured tissue-type plasminogen activator (t-PA)

Curli are fimbrial structures expressed by Escherichia coli that specifically interact with matrix proteins such as fibronectin and laminin. Similar structures are also expressed by Salmonella enteritidis and have been denoted thin aggregative fimbriae. Bacteria expressing curli and thin aggregative fimbriae were found to bind radiolabelled plasminogen as well as the tissue-type plasminogen activator (t-PA). By contrast, E. coli carrying a gene locus with an insertionally inactivated chromosomal curlin subunit were unable to bind the two human proteins. The purified subunit polypeptides of curli and thin aggregative fimbriae bound plasminogen and t-PA with high affinity (1 × 10⁸ to 2 × 10⁸ M^-1). The binding of plasminogen and t-PA to curli-expressing E. coli was only partially inhibited by fibronectin and laminin. Plasminogen absorbed from human plasma by curli-expressing E. coli was readily converted to plasmin by t-PA; both plasmin and t-PA were functionally active when bound to the bacteria. A simultaneous binding of fibrinolytic proteins and matrix proteins to fimbriae of E. coli and S. enteritidis could provide these pathogens with both adhesive and invasive properties.     

Isolation and characterisation of dog uropathogenic Escherichia coli strains and their fimbriae

A number of Escherichia coli strains have been isolated from dogs with urinary tract infections. These strains have been characterised with respect to their O, K, H, and fimbrial antigens, colicin production, antibiotic resistance, plasmid content and their ability to haemagglutinate erythrocytes from various species. Crossed immunoelectrophoresis of fimbrial extracts, as well as the reaction of partly purified fimbriae of a number of these strains with monoclonal antibodies revealed homology or a strong crossereaction with an F12 fimbrial subunit protein of human uropathogenic E. coli strains. Unlike human F12 fimbriae producing strains, the dog isolates did agglutinate dog erythrocytes in the presence of D-mannose but not human erythrocytes, indicating that the adhesin carried by these strains is different from the adhesin on fimbriae of human uropathogenic E. coli. Similar indications were obtained from experiments with latex beads coated with the receptor for P-fimbriae. These beads were agglutinated by Escherichia coli strains from human urinary tract infections, but not by the dog isolates described here. Preliminary adhesion experiments of human and dog Escherichia coli to human bladder epithelial and canine kidney epithelial cells also showed differences in adhesion depending on the origin of the strain tested.     

Virulence factors of Escherichia coli isolated from urine of diabetic women with asymptomatic bacteriuria: correlation with clinical characteristics

Since Escherichia coli isolated from compromised patients with symptomatic urinary tract infections (UTIs) express fewer virulence factors than those isolated from healthy controls, the question arises whether this is also the case for diabetic patients with asymptomatic bacteriuria (ASB). Polymerase chain reaction (PCR) assays were conducted on 111E. coli strains, isolated from the urine of diabetic women with ASB, using primers for the major subunit A and the G-adhesin (I, II, and III) of P fimbriae, type 1 fimbriae, S fimbriae, afimbrial adhesin, cytotoxic necrotizing factor (CNF), and aerobactin. Phenotypically, hemolysis, mannose-sensitive hemagglutination, mannose-resistant hemagglutination and O:K:H-serotypes were determined. Furthermore, we investigated the associations between virulence factors and patient characteristics (including deterioration of renal function). Type 1 fimbriae were the most prevalent virulence factor (86% by genotyping and 59% phenotypically). Except for a lower prevalence of known uropathogenic O-serotypes, we found the same number of virulence factors in our compromised patient group as listed in the literature in noncompromised patients with ASB. Certain virulence factors (type 1 and S fimbriae and CNF) of the causative E. colicorrelated with the risk of a decline in renal function. In conclusion, the number of virulence factors in E. coli isolated from the urine of diabetic women with ASB are comparable with the results found in other (noncompromised) patients with ASB. Furthermore, certain virulence factors of E. colimight contribute to a decline in renal function.     

Characterization of uropathogenic E. coli O25b-B2-ST131, O15:K52:H1, and CGA: Neutrophils apoptosis,serum bactericidal assay,biofilm formation,and virulence typing

Pathogenic and drug-resistant strains of Escherichia coli (E. coli) O25b-B2-ST131, O15:H1-D-ST393, and CGA (clonal group A) clonal groups have spread worldwide. This study aimed at determining E. coli epidemic clonal groups, their virulence factors, biofilm formation, neutrophils apoptosis, and antimicrobial resistance pattern of uropathogenic E. coli. A total of 95 CTX-M-1-producing E. coli clinical isolates were enrolled. E. coli O25b-B2-ST131, CGA, and O15:K52:H1 were identified by serotyping and phylogrouping and allele-specific polymerase chain reaction-based assay. Antibiotic susceptibility, biofilm formation, hemolysis, and human serum bactericidal assay were performed. Neutrophil apoptosis was assayed by flow cytometry. Nine E. coli clonal groups including six O25b-B2-ST131 strains, two CGA, and one O15:K52:H1-D-ST393 strains were detected. One O25b-B2-ST131 isolate was a strong biofilm-producer. Three ST131 isolates had type I fimbriae. Furthermore, all the CGA and O15:K52:H1 and three of ST131 isolates harbored the P fimbriae. The virulence genes ompT, fimH, and traT were detected among all the clonal groups. The apoptosis was induced by O25b-B2-ST131, CGA, and O15:K52:H1 E. coli. There was no significant difference regarding apoptosis induction among clonal groups. Furthermore, the presence of the cdt, usp, and vat genes was significantly associated with the apoptosis of neutrophils by O25b-B2-ST131, CGA, and O15:K52:H1-D-ST393 clonal groups.     

Distribution of additional virulence factors related to adhesion and toxicity in Shiga toxin‐producing Escherichia coli isolated from raw products in Argentina

A total of 73 Shiga toxin‐producing Escherichia coli (STEC) isolates, belonging to 25 serotypes and isolated from raw products in Argentina, were examined for the occurrence of genes responsible for bacterial adhesions to intestine, ehaA (EHEC autotransporter), lpfAO113 (long polar fimbriae), sab (STEC autotransporter [AT] contributing to biofilm formation), ecpA (E. coli common pilus), hcpA (haemorrhagic coli pilus), elfA (E. coli laminin‐binding fimbriae), sfpA (sorbitol‐fermenting EHEC O157 fimbriae plasmid‐encoded) and of the toxigenic gene cdt‐V (cytolethal distending toxin). Our study showed different adhesin profiles that are not linked to one specific serotype and that all analysed isolates possess, besides stx genes, some adherence genes. Several of the isolates contained also multiple toxin genes. The results of the present work alert the presence of genes coding for additional adhesins and cdt‐V toxin in LEE‐negative STEC strains that occur in foods, and this traits could increase their pathogenic potential.

Significance and Impact of the Study

Meat products are one of the main vehicles of Shiga toxin‐producing E. coli, and the presence of genes coding for additional adhesins and toxins could increase their pathogenic potential. There is a need for a more detailed characterization of the strains in regard to these extra virulence factors.     

Fimbria targeting superparamagnetic iron oxide nanoparticles enhance the antimicrobial and antibiofilm activity of ciprofloxacin against quinolone-resistant E. coli

High quinolone resistance of Escherichia coli limits the therapy options for urinary tract infection (UTI). In response to the urgent need for efficient treatment of multidrug-resistant infections, we designed a fimbriae targeting superparamagnetic iron oxide nanoparticle (SPION) delivering ciprofloxacin to ciprofloxacin-resistant E. coli. Bovine serum albumin (BSA) conjugated poly(acrylic acid) (PAA) coated SPIONs (BSA@PAA@SPION) were developed for encapsulation of ciprofloxacin and the nanoparticles were tagged with 4-aminophenyl-α-D-mannopyrannoside (mannoside, Man) to target E. coli fimbriae. Ciprofloxacin-loaded mannoside tagged nanoparticles (Cip-Man-BSA@PAA@SPION) provided high antibacterial activity (97.1 and 97.5%, respectively) with a dose of 32 μg/mL ciprofloxacin against two ciprofloxacin-resistant E. coli isolates. Furthermore, a strong biofilm inhibition (86.9% and 98.5%, respectively) was achieved in the isolates at a dose 16 and 8 times lower than the minimum biofilm eradication concentration (MBEC) of ciprofloxacin. Weaker growth inhibition was observed with untargeted nanoparticles, Cip-BSA@PAA@SPIONs, confirming that targeting E. coli fimbria with mannoside-tagged nanoparticles increases the ciprofloxacin efficiency to treat ciprofloxacin-resistant E. coli. Enhanced killing activity against ciprofloxacin-resistant E. coli planktonic cells and strong growth inhibition of their biofilms suggest that Cip-Man-BSA@PAA@SPION system might be an alternative and/or complementary therapeutic option for the treatment of quinolone-resistant E. coli infections.     

Biochemical characteristic of biofilm of uropathogenic Escherichia coli Dr+ strains

Urinary tract infections caused by Escherichia coli are very common health problem in the developed countries. The virulence of the uropathogenic E. coli Dr⁺ IH11128 is determined by Dr fimbriae, which are homopolymeric structures composed of DraE subunits with the DraD protein capping the fiber. In this study, we have analyzed the structural and biochemical properties of biofilms developed by E. coli strains expressing Dr fimbriae with or without the DraD tip subunit and the surface-exposed DraD protein. We have also demonstrated that these E. coli strains form biofilms on an abiotic surface in a nutrient-dependent fashion. We present evidence that Dr fimbriae are necessary during the first stage of bacterial interaction with the abiotic surface. In addition, we reveal that the DraD alone is also sufficient for the initial surface attachment at an even higher level than Dr fimbriae and that chloramphenicol is able to reduce the normal attachment of the analyzed E. coli. The action of chloramphenicol also shows that protein synthesis is required for the early events of biofilm formation. Additionally, we have identified reduced exopolysaccharide coverage in E. coli that express only Dr fimbrial polyadhesins at the cell surface with or without the DraD capping subunit.     

Effect of Escherichia coli Lipopolysaccharide on Bacteroides fragilis Abscess Formation and Mortality in Mice

To study the mechanism of synergism between Bacteroides fragilis and Escherichia coli, the effect of sublethal dose of E. coli lipopolysaccharide (LPS) (25μg/mouse) was checked on B. fragilis abscess formation. LPS was administered prior or after inoculum injection. No significant difference in the abscess size was observed at necropsy on day 6. However, all the groups receiving LPS showed higher incidence of recovery of additional intestinal bacteria (23.5–45.5%) from the abscess pus. When LPS was given 4 hr prior to inoculum administration, 83–100% mortality was observed. Detailed investigation showed autoclaved cecal contents alone could also cause similar mortality. Studies with stimulation of endogenous cytokines by E. coli LPS demonstrated induction of all of them within 3 hr in the blood stream with TNF-α demonstrating peak at 1 hr, IL-1α and IL-6 at 4 hr and IFN-γ between 6–9 hr with moderately high levels at 4 hr. This E. coli LPS-triggered cytokine cascade possibly gets further stimulated by injection of autoclaved cecal contents containing high concentration of endotoxins (1.6 × 10⁵ EU/ml) contributed by dead bacteria and lead to the mortality of animals.     

Role of Type 1 Fimbriae in the Adhesion of Escherichia coli to Salivary Mucin and Secretory Immunoglobulin A

Saliva is known to modulate the adhesion of bacteria in the oral cavity. The present work was performed to assess the effect of salivary components on the adhesion of Escherichia coli to a model oral surface. Several genetically engineered E. coli strains were used to examine the role of type 1 fimbriation in the interaction of these strains with salivary components in solution or adsorbed to hydroxyapatite. High (MG1) and low (MG2) molecular weight salivary mucins, and secretory immunoglobulin A (sIgA), were found to interact with the surface of E. coli, and these interactions were independent of the expression of fimbriae or capsule. In contrast, fimbriated strains of E. coli adhered to a greater extent to saliva-coated synthetic hydroxyapatite (HAP) than did nonfimbriated strains. Testing of salivary components separated by gel filtration chromatography revealed that only high-molecular-weight components promoted adhesion of E. coli to HAP. Additional studies found that purified MG2 and sIgA promoted the adhesion of E. coli to HAP. Expression of type 1 fimbriae enhanced adhesion, while mannose inhibited adhesion of fimbriated strains, to saliva-coated HAP and to HAP coated with MG2 and sIgA. We conclude that salivary MG2 and sIgA may provide receptors for the adhesion of type 1 fimbriated E. coli to oral surfaces. Received: 10 February 1996 / Accepted: 11 March 1996     

The influence of artificial colonization withE. coli strain O83 on the intestinal flora in infants

Dominant bacterial strains present in stool (with particular emphasis onE. coli strains) were examined in 4 groups of healthy infants: breast-fed and bottle-fed, colonized withE. coli O83, and control (non-colonized) breast-fed and bottle-fed newborns. The presence of fimbriae was examined by hemagglutination, the P-fimbriae-bearing strains were tested by the PPA latex test. In addition, adherence to cell line HT-29 and serotyping was performed in selected strains. TheE. coli strain O83 was found to possess type 1 fimbriae. Fewer bacterial strains possessing type 1 fimbriae were found inE. coli O83-colonized infants (except the O83 serotype) than in control infants. TheE. coli O83 strain colonized significantly better the breast-fed than the bottle-fed infants; its higher adherence activity was demonstrated even in cell line HT-29. Finally, colonization withE. coli O83 influenced the character of microbial intestinal flora: the frequency of positiveE. coli isolates was significantly higher in colonized (both breast- and bottle-fed) than noncolonized infants.     

Basement membrane carbohydrate as a target for bacterial adhesion: binding of type I fimbriae of Salmonella enterica and Escherichia coli to laminin

Adherence of type-1-fimbriate Salmonella enterica and Escherichia coli to immobilized proteins of the extracellular matrix and reconstituted basement membranes was studied. The type-1-fimbriate strain SH401 of S. enterica serovar Enteritidis showed good adherence to laminin, whereas the adherence to fibronectin, type I, type III, type IV or type V collagens was poor. Only minimal adherence to the matrix proteins was seen with a non-fimbriate strain of S. enterica serovar Typhimurium. A specific and mannoside-inhibitable adhesion to laminin was exhibited by the recombinant E. coli strain HB101(plSF101) possessing fim genes of Typhimurium. Adherence to laminin of strain SH401 was inhibited by Fab fragments against purified SH401 fimbriae, and a specific binding to laminin, of the purified fimbriae, was demonstrated using fimbriae-coated fluorescent microparticles. Periodate treatment of laminin abolished the bacterial adhesion as well as the fimbrial binding. Specific adhesion to immobilized laminin was also shown by the type-1 -fimbriate E. coli strain 2131 and the recombinant strain E. coli HB101(pPKL4) expressing the cloned type-1-fimbriae genes of E. coli. Adhesion to laminin of strain HB101(pPKL4) was inhibited by mannoside, and no adherence was seen with the fimH mutant E. coli HB101(pPKL5/pPKL53) lacking the fimbrial lectin subunit. The type-1 fimbriate strains also adhered to reconstituted basement membranes from mouse sarcoma cells and human placenta. Adhesion of strains HB101(plSF101) and HB101(pPKL4) to both basement membrane preparations was inhibited by mannoside. We conclude that type-1 fimbriae of S. enterica and E. coli bind to oMgomannoside chains of the lamjnjn network in basement membranes.     

Escherichia coli K‐12 possesses multiple cryptic but functional chaperone–usher fimbriae with distinct surface specificities

Commensal and pathogenic Escherichia coli adherence to host and environmental surfaces is mediated by a variety of adhesins. Although extensively studied as a model bacterium, 34% of the genes in the E. coli K‐12 genome have no known function. We hypothesized that some of them may correspond to functional adhesins. We characterized E. coli K‐12 ycb, ybg, yfc, yad, yra, sfm and yeh operons, which display sequence and organizational homologies to type 1 fimbriae exported by the chaperone/usher pathway. We showed that, although these operons are poorly expressed under laboratory conditions, six of them are nevertheless functional when expressed, and promote adhesion to abiotic and/or epithelial cell surfaces. While the studied fimbriae display different binding specificities, we obtained evidence of synergy/interference with other adhesins such as Ag43 or type 1 fimbriae. We showed that their expression is under the negative control of H‐NS and, except for yad, subjected to cAMP receptor protein‐mediated activation and carbon catabolite repression. These results therefore demonstrate that ycb, yfc, yad, yra, sfm and yeh operons encode cryptic but functional fimbriae adhesins whose expression following environmental modifications could contribute to E. coli's ability to adhere to and colonize a wide diversity of surfaces in its various ecological niches.     

Type 1 Fimbriae Contribute to Catheter-Associated Urinary Tract Infections Caused by Escherichia coli

Biofilm formation on catheters is thought to contribute to persistence of catheter-associated urinary tract infections (CAUTI), which represent the most frequent nosocomial infections. Knowledge of genetic factors for catheter colonization is limited, since their role has not been assessed using physicochemical conditions prevailing in a catheterized human bladder. The current study aimed to combine data from a dynamic catheterized bladder model in vitro with in vivo expression analysis for understanding molecular factors relevant for CAUTI caused by Escherichia coli. By application of the in vitro model that mirrors the physicochemical environment during human infection, we found that an E. coli K-12 mutant defective in type 1 fimbriae, but not isogenic mutants lacking flagella or antigen 43, was outcompeted by the wild-type strain during prolonged catheter colonization. The importance of type 1 fimbriae for catheter colonization was verified using a fimA mutant of uropathogenic E. coli strain CFT073 with human and artificial urine. Orientation of the invertible element (IE) controlling type 1 fimbrial expression in bacterial populations harvested from the colonized catheterized bladder in vitro suggested that the vast majority of catheter-colonizing cells (up to 88%) express type 1 fimbriae. Analysis of IE orientation in E. coli populations harvested from patient catheters revealed that a median level of ∼73% of cells from nine samples have switched on type 1 fimbrial expression. This study supports the utility of the dynamic catheterized bladder model for analyzing catheter colonization factors and highlights a role for type 1 fimbriae during CAUTI.     

Adhesion of canine and human uropathogenicEscherichia coli andProteus mirabilis strains to canine and human epithelial cells

Escherichia coli strains causing urinary tract infections in dogs produce fimbriae composed of fimbrial subunits closely related to the F12 and F13 fimbriae of human uropathogenic strains [4]. The adhesins carried by the fimbriae of human and canine isolates differ, however, as concluded from a different hemagglutination pattern and from the fact that the dog strains do not agglutinate latex beads coated with P-fimbriae receptor. This possible difference in adhesive specificity was confirmed by experiments in which the adhesion of human and dog isolates to dog kidney epithelial cells (MDCK cells) and human bladder epithelial cells (T24 cells) was compared. Dog uropathogenic strains, in contrast to human uropathogenicE. coli strains, adhere to MDCK cells but hardly to T24 cells. Adhesion to MDCK cells correlates with the presence of F12 or F13 fimbriae on the dog strains. These results suggest that homologous fimbrial subunits can carry different adhesin molecules and that these adhesin molecules can be responsible for species-specific adherence. On the contrary, adhesion of a number of dog uropathogenicProteus mirabilis strains to MDCK and T24 cells was not species specific; it depended on the mere presence of fimbriae.     

Antibacterial and membrane‐damaging activities of β‐bungarotoxin B chain

This study investigates whether the B chain of β‐bungarotoxin exerted antibacterial activity against Escherichia coli (Gram‐negative bacteria) and Staphylococcus aureus (Gram‐positive bacteria) via its membrane‐damaging activity. The B chain exhibited a growth inhibition effect on E. coli but did not show a bactericidal effect on S. aureus. The B‐chain bactericidal action on E. coli positively correlated with an increase in membrane permeability in the bacterial cells. Lipopolysaccharide (LPS) layer destabilization and lipoteichoic acid (LTA) biosynthesis inhibition in the cell wall increased the B‐chain bactericidal effect on E. coli and S. aureus. The B chain induced leakage and fusion in E. coli and S. aureus membrane‐mimicking liposomes. Compared with LPS, LTA notably suppressed the membrane‐damaging activity and fusogenicity of the B chain. The B chain showed similar binding affinity with LPS and LTA, whereas LPS and LTA binding differently induced B‐chain conformational change as evidenced by the circular dichroism spectra. Taken together, our data indicate that the antibacterial action of the B chain is related to its ability to induce membrane permeability and suggest that the LPS‐induced and LTA‐induced B‐chain conformational change differently affects the bactericidal action of the B chain. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

The DraC usher in Dr fimbriae biogenesis of uropathogenic E. coli Dr+ strains

Biogenesis of Dr fimbriae encoded by the dra gene cluster of uropathogenic Escherichia coli strains requires the chaperone-usher pathway. This secretion system is based on two non-structural assembly components, the DraB periplasmic chaperone and DraC outer-membrane usher. The DraB controls the folding of DraE subunits, and DraC forms the assembly and secretion platform for polymerization of subunits in linear fibers. In this study, mutagenesis of the DraC N-terminus was undertaken to select residues critical for Dr fimbriae bioassembly. The DraC-F4A, DraC-C64, DraC-C100A and DraC-W142A significantly reduced the adhesive ability of E. coli strains. The biological activity of the DraC mutants as a assembly platform for Dr fimbriae polymerization was verified by agglutination of human erythrocytes and adhesion to DAF localized at the surface of CHO-DAF⁺ and HeLa cells. The residue F4 of the DraC usher conserved among FGL and FGS chaperone-assembled adhesive organelles can be used to design pillicides blocking the biogenesis of Dr fimbriae. Because the draC and afaC-III genes share 100% identity the range of the virulence determinant inhibitors could also be extended to E. coli strains encoding afa-3 gene cluster. The investigations performed showed that the usher N-terminus plays an important role in biogenesis of complete fiber.     

Production of Monoclonal Antibody Discriminating Serological Difference in Escherichia coli O9 and O9a Polysaccharides

A monoclonal antibody (mAb) with a unique antigenic specificity against Escherichia coli O9 was produced. The O9a mAb was reactive with a part of the strains in E. coli O9. The O9a mAb did not react with LPS from the E. coli O9 test strain Bi316-42. The distribution of the antigen defined by the O9a mAb in E. coli O9 was consistent with that of E. coli O9a present in E. coli O9 strains. The chemical structure of the repeating unit of the O-specific polysaccharide detected by the mAb was demonstrated to be a mannotetraose by two-dimensional nuclear magnetic resonance spectroscopy. It was confirmed that the mAb recognized E. coli O9a serotype in E. coli O9 serotype strains, suggesting that E. coli O9a serotype might be a dominant strain in E. coli O9.