DOI: 10.1128/AEM.71.8.4919-4924.2005
Article Figures & Data
Figures
- FIG. 1.
Multiplex PCRs targeting the E. coli O45 wzx and wzy (A) and O55 wzx and wzy (B) genes. (A) Lane M, molecular weight markers; lane 1, E. coli O45 (positive control) (multiplex PCR showing the amplified wzx [255 bp] and wzy [451 bp] genes); lane 2, E. coli K-12 (negative control); lane 3, results from a chicken fecal sample spiked with E. coli O45 at 106 CFU/0.2 g; lane 4, result from a chicken fecal sample spiked with E. coli O45 at 108 CFU/0.2 g. (B) Lane M, molecular weight markers; lane 1, E. coli O55 (positive control) (multiplex PCR showing the amplified wzx [683 bp] and wzy [147 bp] genes); lane 2, E. coli K-12, (negative control); lane 3, results from a chicken fecal sample spiked with E. coli O55 at 106 CFU/0.2 g; lane 4, results from a chicken fecal sample spiked with E. coli O55 at 108 CFU/0.2 g.
Tables
- TABLE 1.
Genes in the O-antigen gene cluster of E. coli serogroup O45
ORF no. Proposed gene name Location (nucleotides) No. of amino acids in gene product Putative function Most significant homolog(s) (accession no.) % Identity/% similarity 1 rmlB 59-1198 379 dTDP-d-glucose-4,6-dehydratase RmlB, dTDP-glucose 4,6-dehydratase, Salmonella enterica (AAG09513 ) 83/87 2 wbhP 1246-2196 316 NAD-dependent epimerase/dehydratase ORF_12; similar to NAD-dependent epimerase/dehydratase family, Pseudomonas aeruginosa (AAM27579 ) 42/58 3 wbhQ 2196-3230 344 Glycosyltransferase Probable N-acetylgalatosaminyl transferase, trsF (S51265 ), and WbcO, Yersinia enterocolitica (CAA87703 ) 50/68 4 wbhR 3231-3791 186 Acetyltransferase Acetyltransferase, Oceanobacillus iheyensis (NP_693813 ) (BAC14847 ) 37/58 5 wbhS 3830-5716 628 TrsG protein homolog, role in galactose modification TrsG protein homolog (T44517 ), ORF9P (BAA85014 ), and WbgZ (AAG17416 ), Plesiomonas shigelloides 61/79 6 rmlC 5797-6336 179 dTDP-4-dehydrorhamnose-3,5-epimerase Putative dTDP-4-dehydrorhamnose-3,5-epimerase, Aeromonas hydrophila (AAM22546 ) 66/80 7 wbhT 6333-7151 272 dTDP-glucose-4,6-dehydratase Putative dTDP-glucose-4,6-dehydratase, Aeromonas hydrophila (AAM22547 ) 55/69 8 wzx 7144-8403 419 O-antigen flippase Membrane protein involved in the export of O-antigen and teichoic acid, 12 transmembrane domains, Burkholderia fungorum (ZP_00278649 ) 25/44 9 wbhU 8387-9352 321 Glycosyltransferase Putative glycosyltransferase, Pyrococcus furiosus (NP_579088 ) 29/49 10 wzy 9366-10514 382 O-antigen polymerase Transmembrane protein, 9 transmembrane domains, Ralstonia solanacearum (NP_519421.1 ) 27/45 11 wbhV 10507-11043 178 Serine acetyltransferase Putative acetyltransferase, Clostridium thermocellum (ZP_00313397.1 ) 37/60 12 wbhW 11045-12055 336 Unknown Eps11, Streptococcus thermophilus (AAN63793 ) 30/49 13 rmlA 12102-12974 290 Glucose-1-phosphate thymidylyltransferase RmlA, Raoultella terrigena (AAQ82933.1 ) 78/89 - TABLE 2.
Oligonucleotide primers and probes
Target gene Sequence (5′ to 3′)a Location in cluster (nucleotides) Amplicon size (bp) Accession no. or source Singleplex and multiplex PCR O45 wzx1 (F) CCG GGT TTC GAT TTG TGA AGG TTG 7769-8295 527 This study (R) CAC AAC AGC CAC TAC TAG GCA GAA O45 wzx2 (F) TAT GAC AGG CAC ATG GAT CTG TGG 7347-7601 255 (R) TTG AGA CGA GCC TGG CTT TGA TAC O45 wzy1 (F) GAA ATT ATG CCA TCT TGG CGA GCG 9570-10066 497 This study (R) CAT GTG AAG CCT GAA GGC AAA CTC O45 wzy2 (F) CTG ATG TCA GGC CTC GTG GAA ATA 9858-10308 451 (R) ATG TAA CCA CAA TAA GGG AGC CCG O55 wzx1 (F) AAT GGA ACA TTG CAA CAG CA 10746-10895 150 AF461121 (R) TGT GGA TTC CAG AAA AGC AA O55 wzx2 (F) TCT TGT AAC TAA GTG GCC ACA GGC 10823-11505 683 (R) ATA ACA CCC AAC CTA TAC CTC CCG O55 wzy1 (F) GTG GTT TTG ACG ACT CGC TT 9380-9526 147 AF461121 (R) CCA AAA AGC CCT GCA ACT AA Real-time PCR O45 wzx (F) CGT TGT GCA TGG TGG CAT 7472-7543 72 This study (R) TGG CCA AAC CAA CTA TGA ACT G O45 wzy (F) GGT GCT TTG TGA TAA TTC CTG ATG 9616-9691 76 This study (R) TTA TAG CCG CCC CTA AAT TGC O45 wzx probe 6-FAM d(ATT TTT TGC TGC AAG TGG GCT GTC CA)BHQ-1 7494-7519 This study O45 wzy probe 6-FAM d(TTG CTG CTG GCG GGA TAC CAA TGA T)BHQ-1 9643-9667 This study O55 wzx (F) AAT TAA CGA ACA TAA CAC CCA ACC 11416-11516 101 AF461121 (R) ATA TCT CTT CGT TAC TGT GTG TAT TTC O55 wzy (F) AGC TTT CCT GGC GGG TTT 10118-10204 87 AF461121 (R) GCA CCA CGC TAT CTT TTT TCT TAA T O55 wzx probe 6-FAM d(ACC TCC CGC TAA AAC CCC AAC TCT AGT AG)BHQ-1 11461-11489 AF461121 O55 wzy probe 6-FAM d(CCG CGG CGA TAT TGG GTA CTG C)BHQ-1 10142-10163 ↵ a F, forward; R, reverse.
- TABLE 3.
PCR conditions used for the different assays
PCR gene(s) Primer or primer pair Temp (°C) [(time in s)] Amplicon size(s) (bp) Annealing Extension Singleplex PCR O45 wzx O45wzx1, O45wzx2 59 (0) 72 (30) 527, 255 O45 wzy O45wzy1, O45wzy2 59 (0) 72 (30) 497, 451 O55 wzx O55wzx1, O55wzx2 50 (0) 72 (8) 150, 683 O55 wzy O55wzy1 50 (0) 72 (8) 147 Multiplex PCR O45 (wzx and wzy) O45wzx2, O45wzy2 59 (0) 72 (30) 255, 451 (Fig. 1A) O55 (wzx and wzy) O55wzx2, O55wzy1 55 (0) 72 (32) 683, 147 (Fig. 1B) O45 (wzx) and O55 (wzx) O45wzx2, O55wzx2 55 (0) 72 (32) 255, 683 (not shown) - TABLE 4.
Specificities of 119 putative E. coli O55 isolates and 68 non-O55 E. coli isolates and other bacteria
Serological reaction No. of cultures Sources (no.)b Conclusions from RFLP analyses of O-antigen gene cluster No. of positive isolates O55 wzx PCR O55 wzy PCR O55 102 Humans, animals, and environment Not tested 97 98 O55, O83 4 Cows (3) and unlisted (1) Not O55 0 0 O55, O83, O22 9 Humans (7), mouse (1), and unlisted (1) Not O55 0 0 O55, O23 2 Chicken (1) and environment (1) Not O55 0 0 O55 (weak reaction)a 2 Sea gull (2) Not O55 0 0 Non-O55 47 Human, animals, and environment Not tested 0 0 Non-E. coli 21 ATCC and unknown Not tested 0 0
