OI-57, a Genomic Island of Escherichia coli O157, Is Present in Other Seropathotypes of Shiga Toxin-Producing E. coli Associated with Severe Human Disease

Bacterial strains.All the E. coli strains used in this study were part of the culture collection of the Istituto Superiore di Sanità and have been described in reports from previous studies (17, 18, 24, 37, 38). All the strains had been isolated in Italy between 1988 and 2008. STEC O157 reference strains EDL933 and RIMD0509952/VT2 Sakai, together with E. coli K-12 strain MG1655, were also included in the study. These strains were kindly provided by Roberto La Ragione, Veterinary Laboratories Agency, Weybridge, United Kingdom.

Eighteen STEC strains were used in the microarray experiments. They belonged to (i) serogroups O26 (five strains), O111 (four strains), O103 (two strains), and O145 (two strains), as representatives of seropathotype B; (ii) serogroups O113 (two strains) and O91 (two strains), as representatives of seropathotype C; and (iii) serogroup O45 (one strain), as a representative of seropathotype D.

The PCR analyses for the validation of the microarray results were performed using a panel of 61 additional STEC strains. SPT A included nine O157 strains. SPT B strains belonged to serogroups O26 (nine strains), O111 (ten), O145 (seven), O103 (four), and O121 (two); SPT C strains belonged to serogroups O113 (nine) and O91 (four). SPTs D and E were represented by seven strains of animal origin that were negative for the presence of the intimin-coding eae gene (23) and belonged to serogroups O23, O25, O8, O73, O109, and O nontypeable (ONT).

The presence of the putative virulence gene in OI-57 was also assessed in a panel of Stx-negative E. coli isolates belonging to other pathogroups. These included 42 enteropathogenic E. coli (EPEC) isolates, nine enterotoxigenic E. coli (ETEC) isolates, eight enteroaggregative E. coli (EAEC) isolates, and eight cytotoxic necrotizing factor 1 (CNF1)-producing E. coli (NTEC) isolates. The EPEC strains were characterized as typical or atypical on the basis of the presence or the absence of the EPEC adherence factor (EAF) plasmid as described by Franke et al. (6). The ETEC strains belonged to serogroups O6, O64, and O147; the EAEC strains belonged to serogroups O86, O111, O126, and O128; and the NTEC strains belonged to serogroups O2, O6, O75, and O83.

Twenty-seven nonpathogenic E. coli isolates from the Escherichia coli Reference (ECOR) collection (23) and the K-12 LE 392 strain were also examined.

Microarray hybridizations.The microarray slides used in this study were purchased from Ocimum Biosolutions Ltd. (Hyderabad, India). They were composed of 6,176 oligonucleotides (50-mer) representing almost all the coding genes of the three control strains. In particular, the slides were spotted with oligonucleotides specific for 4,288 open reading frames (ORFs) of E. coli K-12 strain MG1655, 5,358 ORFs of E. coli O157:H7 strain RIMD0509952, and 5,336 ORFs of E. coli O157:H7 strain EDL933.

Total DNA of the test strains was purified from overnight bacterial cultures by using a genomic DNA extraction kit (Gentra Systems) according to the manufacturer's instructions. DNA was labeled with a BioPrime DNA labeling system (InVitrogen Life Technologies, Carlsbad, CA). For a 50-μl reaction mixture, 2.5 μg of DNA was combined with 15 μg of random octamers, heated to 95°C for 5 min, and chilled in ice. The remaining components were added to achieve the final concentration as follows: 0.12 mM dATP-dGTP-dCTP, 0.06 mM dTTP, 0.02 mM Cy3 or Cy5-dUTP (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom), and 40 units of the Klenow fragment of E. coli DNA polymerase. The reaction mixture was incubated at 37°C for 3 h, and the labeled DNA was purified by using a QIAquick PCR purification kit (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. The DNA probe, composed of one of the test DNAs labeled with Cy3 (15 μl) and a mixture of DNA from the three control strains labeled with Cy5 (15 μl), was mixed with 15 μl of hybridization buffer (Ocimum Biosolutions Ltd., Hyderabad, India), heated to 95°C for 5 min, and chilled on ice before hybridization on the slide. The mixture of the three control DNAs was used to check the efficiency of hybridization on the whole slide surface.

Hybridizations were carried out overnight under glass coverslips in a sealed wet box at 42°C. Following hybridization, slides were washed for 2 min in 1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.05% sodium dodecyl sulfate (SDS) at 42°C and two times for 2 min in 0.06× SSC at room temperature. Following washes, the slides were dried by centrifugation in conical 50-ml tubes.

Hybridized slides were analyzed by using a ProScanArray microarray scanner (Perkin Elmer, Downers Grove, IL). Computer analysis of images was conducted by using ScanArray software (Perkin Elmer, Downers Grove, IL) and MeV software (27). An ORF was considered present in the test DNA when the corresponding spot in the hybridized slide showed a mean fluorescence value higher than that of the background plus 2 standard deviations.

PCR validation of the microarray results.The major differences identified by the microarray analyses were further characterized by developing PCR assays aimed at assessing the presence of the ORFs that had been detected in SPTs A and B but not in SPTs C and D. PCR primers were deployed by using the primer-BLAST tool available at NCBI website (http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome ) and are listed in Table 1 .

All the PCRs were performed using 50 ng of total DNA as the template, 1.5 mM MgCl₂, 200 μM deoxynucleoside triphosphate, 1 μM each primer, and 1 U of Taq polymerase.

PCR detection and analysis of the OI-57 genomic island.The presence of the OI-57 GI was investigated using primer pairs (Table 1) corresponding to the ORFs whose products were annotated with a putative function, namely, the putative virulence factor adfO and ORF Z2054, encoding a putative bacterial cell killing factor, according to the EDL933 strain sequence (GenBank accession no. AE005174). Six additional primer pairs, corresponding to as many ORFs (Z2066, Z2096, Z2104, Z2105, Z2148, and Z2150), were used to assess the integrity of the OI-57 GI (Table 1 and Fig. 1).

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FIG. 1.

Map of OI-57. The ORFs investigated in this study (Table 3) are indicated. The intO gene indicates the beginning of the genomic island. The island architecture and the nomenclature of the ORFs refer to the STEC O157:H7 strain EDL933 sequence (GenBank accession no. AE005174).

Whole-genome comparison between STEC O157 and STEC strains belonging to SPTs B, C, and D.Microarrays were used to compare the whole genomes of STEC strains belonging to SPTs B (13 strains), C (four strains), and D (one strain) with that of STEC O157 (SPT A). Total DNA from each of the selected 18 strains was hybridized onto commercial microarray slides containing the complete gene set of the E. coli O157:H7 strains EDL933 and RIMD0509952/VT2 Sakai and E. coli K-12 strain MG1655.

The analysis of the microarray profiles identified 87 ORFs absent in the strains belonging to SPTs C and D but giving strong hybridization signals with the SPT B strains (not shown). These ORFs represented the most evident difference between the groups studied and were selected for further investigation.

Sequence and functional analyses conducted in silico showed that some (61 out of 87) of the ORFs identified were part of E. coli metabolic pathways, part of the Stx1- or Stx2-converting phages, or part of the K-12-related Rac phage; other ORFs were part of GIs that have also been found in strains belonging to SPTs C and D. None of these ORFs were further investigated, since they were not considered potential markers of the presence of GIs specifically associated with the most pathogenic SPTs, types A and B. The remaining 26 ORFs (Table 2), located in 12 different GIs according to the sequence of the STEC O157 strain EDL933 (25), were considered genes apparently specific for SPTs A and B. To verify this possible association, their presence was further investigated in a larger panel of STEC strains belonging to different SPTs by using specific PCR assays.

PCR validation of the microarray results.The panel included STEC strains belonging to SPTs from A to D and E and also E. coli strains from healthy human subjects included in the ECOR collection (23). The PCR primers specific for the 26 ORFs apparently associated with SPTs A and B are shown in Table 1. The results of the PCR analyses (Table 2) showed that eight ORFs, located in five different GIs according to the EDL933 strain sequence (25), were present only in strains belonging to SPTs A and B.

One of these ORFs (Z4325) was part of the OI-122 PAI, which has already been shown to be associated with SPTs A and B (9, 19). ORF Z1142 was part of the operon encoding the components of the urea metabolic pathway and related to the ureD gene from Klebsiella aerogenes. A similar operon is located on a large GI that is present in two copies (OI-43 and OI-48) in the EDL933 strain (24). OI-43 and OI-48 also contain other 4 of the 26 investigated ORFs (Z1154, Z1174, Z1214, and Z1611), which were also present in strains belonging to SPT C and SPTs D and E and in strains from the ECOR collection (Table 2).

ORF Z6020 was specific to SPTs A and B. However, it was located in OI-71 (25), which also contained the ORFs Z6044 and Z6021, frequently observed among strains belonging to SPT C and SPTs D and E, and Z6044, found in 9 of 27 ECOR collection isolates.

The remaining ORFs exclusively present in SPTs A and B were located in OI-57 (Z2097, Z2098, Z2121, and Z2149) and in OI-52 (Z1914). These GIs appeared to be specific to SPT A (OI-52) and SPTs A and B (OI-57), and in silico sequence analyses showed that both carry putative virulence genes. In particular, OI-52 harbors the genes conferring resistance to the herbicide methylviologen (Z1870) and encoding the RUS endodeoxyribonuclease (Z1873) (25). OI-57 carries adfO, a gene coding for a factor that has been shown to promote the adherence of STEC O157 to in vitro-cultured cells (8). OI-57 also carries a gene, hereby termed ckf, encoding a putative phage-associated killer protein. Paralogues of this protein have been reported to disrupt the bacterial host membranes when produced in excess (25).

Analysis of OI-57 in STEC strains.The apparent association of OI-57 with both SPTs A and B prompted us to investigate its structure in the positive strains. These were analyzed by PCR for the presence of the above-mentioned virulence gene adfO as well as for the presence ckf and of six additional ORFs scattered along the entire GI and selected for their positions (Table 3). The presence of these eight ORFs was also investigated in STEC strains belonging to SPT C and SPTs D and E and in the ECOR strains, which had been negative for the OI-57 ORFs previously investigated. The results of the PCR assays (Table 3) indicated a strong association of the entire OI-57 with SPTs A and B. In particular, the virulence gene adfO was present, always together with ckf, in 92.7% of the strains belonging to these SPTs, while both genes were absent from STEC strains belonging to SPT C and SPTs D and E and from the nonpathogenic strains from the ECOR collection. As for the other OI-57 ORFs investigated (Fig. 1), most of them were present in the majority of the SPT A and B strains, suggesting the presence of an entire OI-57. Conversely, these ORFs were rarely observed among the SPT C strains and in ECOR collection isolates (Table 3).

Presence of OI-57 in other E. coli pathogroups.In order to verify the specificity of the association of OI-57 with STEC SPTs A and B, we investigated its presence in a set of E. coli strains belonging to other pathogroups. The E. coli isolates included 42 EPEC, 9 ETEC, 8 EAEC, and 8 NTEC strains, which were tested by PCR using primers specific for adfO and ckf (Table 1). The results of the PCR experiments (Table 4) showed that both genes were present in 71% of EPEC strains, while they could not be detected in the other E. coli pathogroups, with the exception of one EAEC strain. In particular, OI-57 was present in three of the eight typical EPEC strains and in 27 of the 34 atypical EPEC strains (Table 5).

Analysis of OI-57 in EPEC strains.The structure of OI-57 in the positive EPEC strains was investigated by assessing the presence of the additional six OI-57-associated ORFs (Fig. 1). Variability higher than that observed in STEC was detected, with only 7 of the 30 OI-57-positive EPEC strains harboring the complete set of ORFs. Among the remaining strains, 10 were positive for five of the ORFs (9 for Z2066, Z2104, Z2105, Z2148, and Z2150 and 1 with ORF Z2096 instead of ORF Z2105), 7 were positive for four ORFs (4 strains possessed ORFs Z2066, Z2104, Z2148, and Z2150 and 3 strains had ORF Z2105 instead of ORF Z2150), 4 possessed ORFs Z2066, Z2104, and Z2148, and 2 strains were positive for ORF Z2148 only.