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J Med Microbiol 56 (2007), 177-184; DOI: 10.1099/jmm.0.46775-0
© 2007 Society for General Microbiology
ISSN 1473-5644

Relationship between O-antigen subtypes, bacterial surface structures and O-antigen gene clusters in Escherichia coli O123 strains carrying genes for Shiga toxins and intimin

Lothar Beutin1, Quan Wang2,3, Dieter Naumann4, Weiqing Han2,3, Gladys Krause1, Luciana Leomil5, Lei Wang2,3 and Lu Feng2,3

1 National Reference Laboratory for Escherichia coli, Centre for Infectiology and Pathogen Characterization, Federal Institute for Risk Assessment (BfR), Diedersdorfer Weg 1, D-12277 Berlin, Germany

2 TEDA School of Biological Sciences and Biotechnology, Nankai University, 23 HongDa Street, TEDA, Tianjin 300457, P. R. China

3 Tianjin Key Laboratory for Microbial Functional Genomics, TEDA College, Nankai University, 23 HongDa Street, TEDA, Tianjin 300457, P. R. China

4 Robert Koch Institute, P13, Nordufer 20, D-13353 Berlin, Germany

5 Departamento de Microbiologia, Instituto de Ciências Biomédicas II, Universidade de São Paulo, 05508-900, São Paulo, SP, Brazil

Correspondence
Lu Feng
fenglu63{at}nankai.edu.cn

Received 12 June 2006
Accepted 14 September 2006

Escherichia coli O123 strains express a broad spectrum of phenotypes, H serotypes and virulence markers and are able to colonize and to cause disease in different hosts including humans. In this study, two subtypes of E. coli O123 antigen (group I and group II) have been identified based on their cross-reactions with other E. coli O antigens. Investigation of the relationship between O123 group I and group II strains by O serotyping and Fourier transform infrared spectroscopy of whole bacteria revealed surface structural differences between these two groups of E. coli O123 strains. Nucleotide sequence analysis of the O-antigen gene clusters of two E. coli O123 strains representing O123 group I and group II revealed no change at the amino acid level. These findings indicate that the differences in the surface structures of group I and group II strains are not related to genetic heterogeneity in their O-antigen gene clusters. A PCR assay based on O123 antigen-specific wzx and wzy genes was developed and found to be suitable for reliable detection of all subtypes of E. coli O123 strains, which bears an advantage over traditional serological detection.

Abbreviations: AEEC, attaching and effacing Escherichia coli; FT-IR, Fourier transform infrared; STEC, Shiga toxin-producing Escherichia coli.

The GenBank/EMBL/DDBJ accession numbers for the sequences of the E. coli O123 strains 43w and CB9827 O-antigen gene clusters are DQ676933 and DQ676934, respectively.


   INTRODUCTION
TOP
 INTRODUCTION
METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The O antigen, which consists of many repeats of an oligosaccharide unit (O unit), is the outer component of LPS in the surface of Gram-negative bacteria (Reeves & Wang, 2002). Due to the presence of different sugars and sugar linkages, it is one of the most variable cell constituents which plays an important role in bacterial evasion of host defence systems (Reeves, 1995).

Genes involved in O-antigen synthesis are normally clustered between two housekeeping genes, galF and gnd, in Escherichia coli, and are commonly classified into three main classes: sugar biosynthetic pathway genes, sugar transferase genes and O-antigen processing genes including the flippase (Wzx) and polymerase (Wzy) genes (Reeves & Wang, 2002). Genes encoding Wzx and Wzy are normally specific to different O antigens, and can be used for the development of a PCR assay for the identification and detection of individual strains. Most O-antigen gene clusters have a low G+C content, and it has been proposed that the O-antigen gene cluster was acquired by transfer from other low G+C content species (Wang & Reeves, 2000).

The E. coli serogroup O123 was defined by Orskov in 1952 with prototype strain 43w isolated from blood of a calf with septicaemia (Orskov, 1952; Orskov et al., 1977). Interest in E. coli O123 strains has recently been regained because of their roles as Shiga toxin-producing E. coli (STEC) and as attaching and effacing E. coli (AEEC) from animals and humans. STEC O123 strains have been isolated from healthy sheep (Beutin et al., 1993; Djordjevic et al., 2004) and diarrhoeic cattle and humans (Beutin et al., 2004; Leomil et al., 2003; Mercado et al., 2004). Non-motile STEC O123 and O123 : H10 strains from sheep were associated with production of stx1c- and stx2d-variant toxins and most of these strains were negative for intimin (Beutin et al., 2004; Brett et al., 2003; Ramachandran et al., 2001). Stx1- and Stx2-producing O123 : H2 and O123 : H11 strains positive for intimins ß1 and {varepsilon} have been isolated from diarrhoeic cattle and human patients (Beutin et al., 2004; Leomil et al., 2003; Mercado et al., 2004). Stx-negative E. coli O123 strains producing intimin (AEEC) have been isolated as causative agents of diarrhoea in calves and piglets (Malik et al., 2006; Orden et al., 1998). Current data indicate that E. coli O123 strains express a broad spectrum of phenotypes, H serotypes and virulence markers and are able to colonize and to cause disease in different hosts including humans.

We previously reported that E. coli O123 strains from pigs were different from the prototype O123 strains by showing an unusual cross-reaction with E. coli O4 specific antiserum that had not been described before (Malik et al., 2006). We became interested in characterizing the differences between two O-antigen types of E. coli O123; therefore, in this report, we analysed the representative E. coli O123 strains of two O-antigen types for their serological cross-reactions by O serotyping, for alterations in their surface structures by using Fourier transform-infrared (FT-IR) spectroscopy, and for their O-antigen-encoding genes by nucleotide sequencing. A PCR assay specific for all types of E. coli O123 strains was developed on the basis of E. coli O123 wzx and wzy genes in the O-antigen gene cluster and found to be useful for the detection of E. coli O123 strains.


   METHODS
TOP
 INTRODUCTION
 METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Bacterial strains. E. coli O123 reference strain 43w (laboratory stock no. G1169) was obtained from the Institute of Medical and Veterinary Science, Adelaide, Australia (IMVS). Other E. coli and Shigella reference strains used were as previously described (Feng et al., 2004c). The clinical isolates of E. coli O123, O4 and O12 listed in Table 1Go were from the strain collection of the Federal Institute for Risk Assessment (BfR), Berlin. Serotyping of O and H antigens was performed in titration assays as described by Orskov & Orskov (1984). The strength of the agglutination reaction was indicated as the reciprocal value of the highest dilution of antiserum causing visible agglutination of bacteria (Ewing, 1986; Orskov, 1952; Orskov et al., 1977). Rabbit antisera against E. coli O and H antigens were produced at the BfR, according to standard methods (Orskov & Orskov, 1984). Subtyping of flagellar (fliC) genes by PCR was performed as described previously (Beutin et al., 2005).


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  		Table 1. Properties of E. coli strains used in this work

 
Phenotypical and genotypical characterization of virulence markers. The strains were investigated for production of cytotoxins with the Vero cell toxicity test and for cytolysins (haemolysins) on washed blood agar plates (Beutin et al., 2004). Subtyping of Shiga toxin, haemolysin and intimin genes was performed as described previously (Beutin et al., 2005).

FT-IR spectroscopy. For FT-IR spectroscopy the E. coli strains were subcultured on Caso agar (Merck) for 24 h as overnight cultures applying a four quadrant streak pattern. Cells were harvested and prepared for FT-IR measurements as already described by Helm et al. (1991a, b). Briefly, 35 µl of a bacterial cell suspension in distilled water was transferred to a ZnSe optical plate and dried to a transparent film under mild vaccum (2.5–7.5 kPa). All spectra between 500 and 4000 cm–1 were recorded on an IFS 28/B Fourier transform infrared spectrometer (Bruker Optik) equipped with a deuterated triglycerine sulphate detector. Nominal physical resolution was set to 6 cm–1, a Blackman/Harris apodization was used for Fourier transformation and a zerofilling factor of 4 was applied to yield an encoding interval of approximately one data point per wavenumber. Spectral data were collected and evaluated with OPUS 3.0 software (Opus Software). Data processing included calculation of second derivatives to minimize baseline problems and to enhance apparent resolution. For hierarchical cluster analysis, an unsupervised classification technique, the Ward's algorithm (Helm et al., 1991a, b), was used to construct the dendrograms. As a distance measure, Pearson's product moment correlation coefficient was used as defined previously (Helm et al., 1991a, b).

Construction of DNaseI shotgun bank, and sequencing and analysis of the O-antigen gene cluster. Chromosomal DNA was prepared as previously described (Bastin & Reeves, 1995). The primer pair 5'-ATTGGTAGCTGTAAGCCAAGGGCGGTAGCGT-3' and 5'-CACTGCCATACCGACGACGCCGATCTGTTGCTTGG-3', based on JUMPstart sequence and gnd, respectively (Wang & Reeves, 1998), was used to amplify the O-antigen gene clusters of E. coli O123 type I strain 43w and E. coli O123 type II strain CB9827. The PCR cycles used were as follows: denaturation at 94 °C for 10 s, annealing at 60 °C for 30 s, and extension at 68 °C for 15 min. Shotgun banks for each strain were constructed as described previously (Wang & Reeves, 1998). Sequencing was carried out using an ABI 3730 automated DNA sequencer and sequence data were analysed using computer programs as described previously (Feng et al., 2004a).

Specificity and sensitivity tests of O-serogroup specific PCR assay. Chromosomal DNA was prepared from each of 186 reference strains to represent the broadest range of O antigens of E. coli (including Shigella) and used to make DNA pools as described by Feng et al. (2004c). A total of 13 pools were made, each containing DNA from 12–19 strains (Feng et al., 2004c). Primer pairs based on wzx and wzy genes of E. coli O123 (Table 3Go) were used to screen the DNA pools. The PCR cycles used were as follows: denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s, extension at 72 °C for 1 min, 30 cycles. Template DNA from 48 clinical E. coli isolates including 22 E. coli O123 and 26 other O-serogroup strains (data not shown) was prepared as described by Guo et al. (2004) and screened using the same primers in a double-blind test.


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  		Table 3. Primers used for genetic detection of all types of E. coli O123 strains

 
To test the sensitivity of the PCR assay, 10-fold serial dilutions of chromosomal DNA from E. coli O123 strain 43w were amplified using all of the primer pairs designed. Primer pairs wl-2400/wl-2401 and wl-2406/wl-2407 from E. coli O123 were also used to screen E. coli O123 strain 43w in pork and water samples. Raw pork was purchased from three local butchers, weighed into 20 g portions, and stored at –40 °C before use. Serial 10-fold dilutions (10–3–10–9) from the full-grown culture of E. coli O123 strain 43w were added to each portion of pork or 20 ml Milli-Q water. The concentration of the O123 cells in pork or water samples was determined by checking the c.f.u. on agar plates. Samples spiked with different concentrations of E. coli O123 cells were homogenized in 200 ml Luria broth culture. The homogenized samples were cultured at 200 r.p.m. at 37 °C for 12 h and chilled at 4 °C. The culture was passed through a six-chamber filter bag, and 3 ml filtrate was collected for use as the template DNA in the PCR as described above (Guo et al., 2004).


   RESULTS AND DISCUSSION
TOP
 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Properties of E. coli O123 strains

We have investigated 22 E. coli O123 strains that were isolated from diarrhoeic humans, calves, pigs and healthy sheep. The strains, except E. coli O123 reference strain 43w, were from Europe, Latin America and Australia and had been isolated between 1989 and 2003 (Table 1Go). All these E. coli O123 strains except 43w carried one or more virulence markers associated with AEEC (eae gene), with STEC [stx gene(s)] or with enterohaemorrhagic E. coli (EHEC) [stx, eae and E-hlyA (EHEC haemolysin) genes]. E. coli O123 strains could be associated with multiple H types such as H2, H10, H11, H16, H19, H25, H40 and H45. Taken together, E. coli O123 strains presented as a group of human and animal pathogenic strains with a broad host spectrum and a high diversity in their virulence attributes.

By O serotyping, the E. coli O123 strains fall into two groups by their cross-reactions with antisera directed to E. coli O antigens other than O123. The prototype O123 strain 43w was previously reported to cross-react with E. coli O12 specific antiserum (Ewing, 1986; Orskov, 1952; Orskov et al., 1977). This cross-reaction was confirmed in this study (O titres 1 : 800–1 : 1600) with 43w and also found with four other E. coli O123 strains (CB1654, CB6793, CB6805 and CB9716) (Table 1Go). These E. coli O123 strains were designated ‘O123 group I’ and none of these agglutinated significantly with E. coli O4 specific antiserum (agglutination titres <1 : 50). In contrast, all other E. coli O123 strains from this study did not agglutinate with E. coli O12 antiserum (agglutination titres <1 : 50), but did agglutinate with E. coli O4 specific antiserum (here called ‘O123 group II’ strains). The two groups of O123 strains differed in their agglutination reactions with the O123 specific antiserum made with strain 43w (O123 group I) as immunizing antigen. With this serum O123 group I strains showed higher agglutination titres (1 : 3200–1 : 6400) than O123 group II strains (titres 1 : 400–1 : 1600) (Table 1Go). To further analyse the specificity of cross-reactions obtained with the two groups of O123 strains we produced an O123 specific antiserum with E. coli O123 strain CB9827 as a prototype strain for the O123 group II strains. O antiserum made against CB9827 showed similar agglutination titres with E. coli O123 group I and group II strains but did not cause significant agglutination (titres 1 : 50–1 : 100) with E. coli O4 strains (Table 1Go).

FT-IR spectroscopy

FT-IR spectroscopy of intact micro-organisms provides information on the composition and structure of the whole cell (Naumann et al., 1991; Naumann, 2000). These spectra are complex spectroscopic patterns encoding the signals of thousands of bands that cannot be resolved easily. Thus pattern recognition techniques are generally used to extract the essential information. It has been shown by several groups that FT-IR may provide information useful for discrimination at the genus, species and even strain level (Helm et al., 1991b; Horbach et al., 1988; Kirschner et al., 2001; Maquelin et al., 2003; Tintelnot et al., 2000). Since all cell components depend on the expression of smaller or larger parts of the genome, the FT-IR spectra of micro-organisms display specifically a complete phenotypic and genetic fingerprint of the cells under study.

The purpose of this FT-IR analysis was to group the E. coli isolates according to their O-antigenic properties which reside in the structure of O-specific side chains of LPS. Since it was expected that differences in O antigen would primarily be expressed in the spectral range where the vibrational modes of cellular carbohydrates dominate the experimentally observed spectral contour, data analysis was directed to the spectral range of 900–1200 cm–1 (Helm et al., 1991b). Using the information contained in this spectral range as input for cluster analysis a distinct clustering was observed according to the dendrogram shown in Fig. 1Go. This dendrogram clearly showed the presence of three main clusters according to the grouping scheme suggested by O serotyping. It was particularly notable that the two groups of O123 strains are spectroscopically more related than the O4 group to both, suggesting that the two underlying carbohydrate structures found at the cell surface of the two O123 group strains were indeed more similar to each other than to the carbohydrate structure of the O4 strains.


Figure 1
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  		Fig. 1. FT-IR classification of the E. coli isolates. Cluster analysis was performed using the second derivatives of the spectra and the spectral information contained in the spectral range between 900 and 1200 cm–1. Ward's algorithm was used for calculating the interrelationship between the strains. The numbers at the side of the dendrogram correspond to the different strains measured by FT-IR spectroscopy. All strains have been measured twice from independent cultivations.

 
E. coli O123 group I and group II strains share the same O-antigen gene cluster

A sequence of 15 783 bases between JUMPStart and gnd was obtained from each of E. coli O123 strains 43w (group I) and CB9827 (group II), and each contained 16 genes (excluding gnd) with transcription direction from JUMPStart to gnd (Fig. 2Go).


Figure 2
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  		Fig. 2. O-antigen gene cluster organization of both E. coli O123 group I and group II. All the genes are transcribed in the direction from JUMPStart to gnd.

 
The two sequences shared 99.94 % identity at DNA level and 100 % at protein level, and were regarded as identical. Therefore, their genes were given the same names and the functions of the genes were predicted based on their similarity to those from available databases (Table 2Go).


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  		Table 2. Putative genes in the E. coli O123 O antigen gene cluster

 
Sugar biosynthesis pathway genes. Orfs 1 and 2 shared high level identity (78–92 %) to many other known RmlB and RmlA proteins from other E. coli, Shigella and Salmonella enterica strains, respectively. Orf4 shared 60 % identity to VioA of E. coli O7. RmlB, RmlA and VioA were proposed as dTDP-D-4-amino-4,6-dideoxyglucose biosynthesis pathway enzymes (Marolda et al., 1999). Therefore, orf1, orf2 and orf4 were identified as rmlB, rmlA and vioA, and named accordingly.

Orfs 12, 13 and 14 shared 74, 53 and 62 % identity to WbvB (FnlA), WbvR (QnlA) and WbvD (QnlB) of the Vibrio cholerae O37 O antigen gene cluster, respectively, which were identified as UDP-L-N-acetyl-quinovosamine biosynthesis pathway enzymes experimentally (Kneidinger et al., 2003). Therefore, orf12, orf13 and orf14 were identified as fnlA, qnlA and qnlB, and named accordingly.

Sugar transferase genes. Orf9 and Orf11 belonged to glycosyltransferase family 2 (PF00535, E value=9.0xe–25) and glycosyltransferase family 1 (PF00534, E value=1.1xe–12), and shared 50 and 63 % similarity to glycosyltransferases of Clostridium acetobutylicum and Shigella boydii type 13, respectively. Therefore, orf9 and orf11 were proposed to be glycosyltransferase genes and named wfbE and wfbF, respectively. Orf15 belonged to glycosyltransferase family 1 (PF00534, E value=5.7xe–8), and shared 55 % identity to WbwH of S. boydii type 13, which was a putative L-N-acetyl-quinovosamine transferase (Feng et al., 2004a). Therefore, orf15 was proposed to be a glycosyltransferase gene and named wbwH.

O-antigen processing genes. Both orf3 and orf10 were found to encode predicted membrane proteins. Orf3 had 10 predicted transmembrane segments, which is a typical topological character of Wzx proteins (Liu et al., 1996). Orf3 belonged to the protein family PF01943 (E value=6.3xe–22), and members of this family including RfbX are involved in the export of the O antigen and teichoic acid. It also shared 66 and 65 % similarity to the Wzx proteins of E. coli O7 and E. coli O113, respectively. Therefore, orf3 was proposed to be an O-unit flippase gene (wzx) and named accordingly. Orf10 had eight predicted transmembrane segments and a large periplasmic loop of 113 amino acid residues, which is a typical topological character of Wzy proteins (Daniels et al., 1998). It also shared 48 % similarity to the Wzy protein of E. coli O121. Therefore, orf10 was proposed to be an O-antigen polymerase gene (wzy) and named accordingly.

Other genes. Orf7 belonged to the acetyltransferase (GNAT) family PF00583 (E value=5.6xe–7), and it also shared 56 % similarity to the acetyltransferase of Bacillus cereus ATCC 14579. Therefore, Orf7 was proposed to be an acetyltransferase. Orf5 shared 60 % identity to WbnG of Shigella dysenteriae type 7, which was proposed to be a glycine transferase (Feng et al., 2004b). Orf8 belonged to the protein family PF01575 (E value=5.6xe–7), members of which include monoamine oxidase C dehydratase. It also shared 60–70 % similarity to hydratase or dehydratase with monoamine oxidase C-like domain of many bacteria strains. No function can be assigned to orf6 by searching currently available databases. As the E. coli O123 O-antigen structure has not been identified, only general functions of orfs 5–8 could be proposed, and orfs 5–8 were named wfbA, wfbB, wfbC and wfbD, respectively.

Orf16 shared 67 and 62 % similarity to a proposed remnant gene product, WbuC, of S. boydii type 13 (Feng et al., 2004a) and E. coli O26 (D'Souza et al., 2002). orf16 was proposed to be non-functional and named wbuC.

Possible factors responsible for the differences between the O antigens of the two E. coli O123 groups

The data above indicated that the differences observed between the E. coli O123 group I and group II strains were not attributable to the genes encoded by the O-antigen gene cluster but to other functions responsible for the structures of the O antigen in E. coli O123 strains. These must be encoded by genes located outside of the O-antigen gene clusters between galF and gnd in E. coli O123 strains and their functions need to be explored. It was reported that the O antigen of E. coli O4 has a side branch UDP-D-Glu residue, which was proposed to be added after synthesis and translocation of the O unit, and the genes responsible for the side chain transferase were located outside of the O-antigen gene cluster (D'Souza et al., 2005). Therefore, we proposed that the cross-reaction between E. coli O123 group II strains and E. coli O4 specific antiserum may be due to the side chain of the O antigen in both strains, although the O-antigen structures of E. coli O123 group I and group II strains were not characterized. The differences found between the E. coli O123 groups by serotyping could be related to differences in their surface structures (presumably the addition of side branch residues), which was also indicated by the results from FT-IR spectroscopy.

Identification of E. coli O123 O-antigen specific genes

Four primer pairs based on the proposed O unit processing genes wzx and wzy (two pairs for each) were designed (Table 3Go). All the primers were used to screen DNA pools containing representatives of the 186 known O antigens of typical E. coli and Shigella strains, and no PCR products were detected from other pools, except the pools containing E. coli O123, which gave the expected PCR products. All of the primer pairs were further used for a double-blind test on E. coli clinical isolates including 22 E. coli O123 group I and group II strains (Table 1Go) and 26 E. coli strains of other O serogroups (data not shown). All of the E. coli O123 strains gave the expected PCR products corresponding to the primer pairs used, and no PCR products were obtained from other O-serotype strains. Therefore, all of these primer pairs are highly specific and can be used for the identification and detection of both serological groups of E. coli O123 strains.

Sensitivity of the serogroup specific PCR assay

All the four primer pairs of E. coli O123 were used to amplify a dilution series of chromosomal DNA from E. coli O123 type I strain 43w, and obtained a sensitivity of 1 pg µl–1. The primer pairs of wl-2400/wl-2401 and wl-2406/wl-2407 of E. coli O123 were used to screen E. coli O123 strain 43w in pork and water samples. By this, as few as 103 c.f.u. g–1 were detected in samples which were examined directly and 0.1 c.f.u. g–1 could be detected in samples which were further incubated at 37 °C for 12 h. In contrast to traditional serological testing, which is slow, labour-intensive and subject to interference by cross-reactions, the specific PCR assay is specific, sensitive and could be easily performed to detect E. coli O123 group I and group II strains belonging to different clonal groups, and sero- and pathotypes, and changes of the bacterial surface which may cause difficulties in O serotyping are avoided.


   ACKNOWLEDGEMENTS
 
The authors are grateful to Béla Nagy, Budapest, Hungary, for supplying porcine pathogen Escherichia coli O123 strains. This work was supported by the NSFC General Program (30370023, 30370339) and the NSFC Key Program (30530010). L. L. was supported from Brazil by ‘Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)’, number BEX 1686/03-8 and by ‘Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)’ grant no. 03/01823-9. The authors are also grateful to Maren Stämmler for carefully helping in the FT-IR measurements.


   REFERENCES
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 INTRODUCTION
 METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
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