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Methods

Development of PCR Assays Targeting Genes in O-Antigen Gene Clusters for Detection and Identification of Escherichia coli O45 and O55 Serogroups

Chitrita DebRoy, Pina M. Fratamico, Elisabeth Roberts, Michael A. Davis, Yanhong Liu
Chitrita DebRoy
1Gastroenteric Disease Center, Department of Veterinary Science, The Pennsylvania State University, University Park, Pennsylvania 16802
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  • For correspondence: rcd3@psu.edu
Pina M. Fratamico
2United States Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, Pennsylvania 19038
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Elisabeth Roberts
1Gastroenteric Disease Center, Department of Veterinary Science, The Pennsylvania State University, University Park, Pennsylvania 16802
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Michael A. Davis
1Gastroenteric Disease Center, Department of Veterinary Science, The Pennsylvania State University, University Park, Pennsylvania 16802
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Yanhong Liu
2United States Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, Pennsylvania 19038
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DOI: 10.1128/AEM.71.8.4919-4924.2005
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    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.

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  • TABLE 1.

    Genes in the O-antigen gene cluster of E. coli serogroup O45

    ORF no.Proposed gene nameLocation (nucleotides)No. of amino acids in gene productPutative functionMost significant homolog(s) (accession no.)% Identity/% similarity
    1 rmlB 59-1198379dTDP-d-glucose-4,6-dehydrataseRmlB, dTDP-glucose 4,6-dehydratase, Salmonella enterica (AAG09513 )83/87
    2 wbhP 1246-2196316NAD-dependent epimerase/dehydrataseORF_12; similar to NAD-dependent epimerase/dehydratase family, Pseudomonas aeruginosa (AAM27579 )42/58
    3 wbhQ 2196-3230344GlycosyltransferaseProbable N-acetylgalatosaminyl transferase, trsF (S51265 ), and WbcO, Yersinia enterocolitica (CAA87703 )50/68
    4 wbhR 3231-3791186AcetyltransferaseAcetyltransferase, Oceanobacillus iheyensis (NP_693813 ) (BAC14847 )37/58
    5 wbhS 3830-5716628TrsG protein homolog, role in galactose modificationTrsG protein homolog (T44517 ), ORF9P (BAA85014 ), and WbgZ (AAG17416 ), Plesiomonas shigelloides61/79
    6 rmlC 5797-6336179dTDP-4-dehydrorhamnose-3,5-epimerasePutative dTDP-4-dehydrorhamnose-3,5-epimerase, Aeromonas hydrophila (AAM22546 )66/80
    7 wbhT 6333-7151272dTDP-glucose-4,6-dehydratasePutative dTDP-glucose-4,6-dehydratase, Aeromonas hydrophila (AAM22547 )55/69
    8 wzx 7144-8403419O-antigen flippaseMembrane protein involved in the export of O-antigen and teichoic acid, 12 transmembrane domains, Burkholderia fungorum (ZP_00278649 )25/44
    9 wbhU 8387-9352321GlycosyltransferasePutative glycosyltransferase, Pyrococcus furiosus (NP_579088 )29/49
    10 wzy 9366-10514382O-antigen polymeraseTransmembrane protein, 9 transmembrane domains, Ralstonia solanacearum (NP_519421.1 )27/45
    11 wbhV 10507-11043178Serine acetyltransferasePutative acetyltransferase, Clostridium thermocellum (ZP_00313397.1 )37/60
    12 wbhW 11045-12055336UnknownEps11, Streptococcus thermophilus (AAN63793 )30/49
    13 rmlA 12102-12974290Glucose-1-phosphate thymidylyltransferaseRmlA, Raoultella terrigena (AAQ82933.1 )78/89
  • TABLE 2.

    Oligonucleotide primers and probes

    Target geneSequence (5′ to 3′)aLocation in cluster (nucleotides)Amplicon size (bp)Accession no. or source
    Singleplex and multiplex PCR
        O45 wzx1(F) CCG GGT TTC GAT TTG TGA AGG TTG7769-8295527This study
    (R) CAC AAC AGC CAC TAC TAG GCA GAA
        O45 wzx2(F) TAT GAC AGG CAC ATG GAT CTG TGG7347-7601255
    (R) TTG AGA CGA GCC TGG CTT TGA TAC
        O45 wzy1(F) GAA ATT ATG CCA TCT TGG CGA GCG9570-10066497This study
    (R) CAT GTG AAG CCT GAA GGC AAA CTC
        O45 wzy2(F) CTG ATG TCA GGC CTC GTG GAA ATA9858-10308451
    (R) ATG TAA CCA CAA TAA GGG AGC CCG
        O55 wzx1(F) AAT GGA ACA TTG CAA CAG CA10746-10895150 AF461121
    (R) TGT GGA TTC CAG AAA AGC AA
        O55 wzx2(F) TCT TGT AAC TAA GTG GCC ACA GGC10823-11505683
    (R) ATA ACA CCC AAC CTA TAC CTC CCG
        O55 wzy1(F) GTG GTT TTG ACG ACT CGC TT9380-9526147 AF461121
    (R) CCA AAA AGC CCT GCA ACT AA
    Real-time PCR
        O45 wzx(F) CGT TGT GCA TGG TGG CAT7472-754372This study
    (R) TGG CCA AAC CAA CTA TGA ACT G
        O45 wzy(F) GGT GCT TTG TGA TAA TTC CTG ATG9616-969176This study
    (R) TTA TAG CCG CCC CTA AAT TGC
        O45 wzx probe6-FAM d(ATT TTT TGC TGC AAG TGG GCT GTC CA)BHQ-17494-7519This study
        O45 wzy probe6-FAM d(TTG CTG CTG GCG GGA TAC CAA TGA T)BHQ-19643-9667This study
        O55 wzx(F) AAT TAA CGA ACA TAA CAC CCA ACC11416-11516101 AF461121
    (R) ATA TCT CTT CGT TAC TGT GTG TAT TTC
        O55 wzy(F) AGC TTT CCT GGC GGG TTT10118-1020487 AF461121
    (R) GCA CCA CGC TAT CTT TTT TCT TAA T
        O55 wzx probe6-FAM d(ACC TCC CGC TAA AAC CCC AAC TCT AGT AG)BHQ-111461-11489 AF461121
        O55 wzy probe6-FAM d(CCG CGG CGA TAT TGG GTA CTG C)BHQ-110142-10163
    • ↵ a F, forward; R, reverse.

  • TABLE 3.

    PCR conditions used for the different assays

    PCR gene(s)Primer or primer pairTemp (°C) [(time in s)]Amplicon size(s) (bp)
    AnnealingExtension
    Singleplex PCR
        O45 wzxO45wzx1, O45wzx259 (0)72 (30)527, 255
        O45 wzyO45wzy1, O45wzy259 (0)72 (30)497, 451
        O55 wzxO55wzx1, O55wzx250 (0)72 (8)150, 683
        O55 wzyO55wzy150 (0)72 (8)147
    Multiplex PCR
        O45 (wzx and wzy)O45wzx2, O45wzy259 (0)72 (30)255, 451 (Fig. 1A)
        O55 (wzx and wzy)O55wzx2, O55wzy155 (0)72 (32)683, 147 (Fig. 1B)
        O45 (wzx) and O55 (wzx)O45wzx2, O55wzx255 (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 reactionNo. of culturesSources (no.)bConclusions from RFLP analyses of O-antigen gene clusterNo. of positive isolates
    O55 wzx PCRO55 wzy PCR
    O55102Humans, animals, and environmentNot tested9798
    O55, O834Cows (3) and unlisted (1)Not O5500
    O55, O83, O229Humans (7), mouse (1), and unlisted (1)Not O5500
    O55, O232Chicken (1) and environment (1)Not O5500
    O55 (weak reaction)a2Sea gull (2)Not O5500
    Non-O5547Human, animals, and environmentNot tested00
    Non-E. coli21ATCC and unknownNot tested00
    • ↵ a These strains may not be O55 but may be an unidentified O type and/or related to O55.

    • ↵ b ATCC, American Type Culture Collection.

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Development of PCR Assays Targeting Genes in O-Antigen Gene Clusters for Detection and Identification of Escherichia coli O45 and O55 Serogroups
Chitrita DebRoy, Pina M. Fratamico, Elisabeth Roberts, Michael A. Davis, Yanhong Liu
Applied and Environmental Microbiology Aug 2005, 71 (8) 4919-4924; DOI: 10.1128/AEM.71.8.4919-4924.2005

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Development of PCR Assays Targeting Genes in O-Antigen Gene Clusters for Detection and Identification of Escherichia coli O45 and O55 Serogroups
Chitrita DebRoy, Pina M. Fratamico, Elisabeth Roberts, Michael A. Davis, Yanhong Liu
Applied and Environmental Microbiology Aug 2005, 71 (8) 4919-4924; DOI: 10.1128/AEM.71.8.4919-4924.2005
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KEYWORDS

Escherichia coli
Escherichia coli Proteins
Multigene Family
O Antigens
Polymerase Chain Reaction

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