Article Figures & Data
Figures
- FIG. 1.
Restriction length profiles of rfb-encoding locus of E. sakazakii following MboII digestion. Lane 1, ATCC BAA-894; lane 2, E824; lane 3, E825; lane 4, NCTC 11467; lane 5, NCTC 8155; lane 6, 336; lane 7, 344; lane 8, 109; lane 9, CFS06; lane 10, E787; lane 11, 82; lane 12, 102; lane M1, 100-bp DNA ladder (New England Biolabs, Hertfordshire, England); lane M2, DNA molecular weight marker XVI (Roche, Mannheim, Germany). Black and gray boxes indicate profiles of strains corresponding to O:1 and O:2 serotypes, respectively.
- FIG. 2.
Dendrogram generated using BioNumerics software program at a similarity of 80% of MboII restriction digests. Dotted lines indicate uncertain bands. Proposed new species names are included and are marked by asterisks (20). The black and gray boxes in this figure indicate profiles of strains corresponding to serotypes O:1 and O:2, respectively, in the dendrogram (see Fig. 1).
- FIG. 3.
Genetic organization of the NCTC 11467 (O:1) (a) or NCTC 8155 (O:2) (b) O-antigen gene clusters. Arrows labeled with the corresponding gene names represent individual ORFs and the direction of transcription.
- FIG. 4.
A 1.5% agarose gel image of specific-serotype O:1 (top panel) or serotype O:2 (bottom panel) amplicons, generated by PCR. Lane 1, CFS131; lane 2, E824; lane 3, CFS06; lane 4, 44; lane 5, 93; lane 6, E891; lane 7, E784; lane 8, CFS153; lane 9, 88; lane 10, 305N; lane 11, ATCC BAA 893; lane 12, ATCC BAA 894; lane 13, E899; lane 14, 343; lane 15, 80; lane 16, E775; lane 17, 71; lane 18, E846; lane 19, E830; lane 20, 228N; lane 21, CFS136; lane 22, CFS149; lane 23, no-template control; lane M, 100-bp DNA ladder (New England Biolabs, Hertfordshire, England). Dashed-line box indicates serotype O:1 strains, while continuous-line box indicates serotype O:2 strains.
- FIG. 5.
Silver-stained SDS-PAGE of purified LPS samples. The relative positions of the O-antigen side chain and lipid A core are indicated by the brackets. (a) Lane M, rainbow molecular weight marker (high range; RPN 800) (Amersham Life Science, Buckinghamshire, England); lane 1, S. enterica serotype Minnesota LPS (5 μg); lane 2, ATCC BAA-894 (O:1); lane 3, E901 (O:2); lane 4, CFS1001 (O:1); lane 5, CFS10 (O:2); lane 6, 90; lane 7, CFS149 (O:1); lane 8, CFS173 (neither O:1 nor O:2); lane 9, undigested E901. Asterisks indicate O-antigen bands of similar mobilities. (b) Lane M, molecular marker, lane 1, S. enterica serotype Minnesota LPS (5 μg); lane 2, ATCC 12868 (O:2); lane 3, NCTC 8155 (O:2); lane 4, E844 (O:2); lane 5, E892 (O:2); lane 6, 228N (O:2); lane 7, undigested ATCC 12868. Asterisks indicate O-antigen bands of different mobilities in digested NCTC 12868.
Tables
- TABLE 1.
Members of the Enterobacteriaceae used in this study
Species Strain Origin Serotype Enterobacter sakazakii (“Cronobacter” spp.) C. sakazakii CFS06 Acid casein powder 1 C. sakazakii CFS10 Acid casein powder 2 C. sakazakii CFS104 Milk powder 1 C. sakazakii CFS122 Environmental 1 C. malonaticus*a CFS129 Environmental NDb C. sakazakii CFS131 Milk powder 1 C. sakazakii CFS136 Environmental 2 C. sakazakii CFS149 Environmental 2 C. sakazakii CFS153 Environmental 1 C. sakazakii CFS164 Environmental 1 C. sakazakii CFS173 Milk powder ND C. sakazakii CFS175 Environmental 2 C. sakazakii CFS176 Environmental 2 C. dublinensis* CFS237 Milk powder ND C. sakazakii CFS1001 Environmental 1 C. sakazakii 44 Environmental 1 C. sakazakii 71 Environmental 2 C. sakazakii 79 Environmental 2 C. sakazakii 80 Environmental 2 C. sakazakii 82 Environmental ND C. sakazakii 88 Environmental 1 C. sakazakii 90 Environmental 1 C. sakazakii 93 Environmental 1 C. sakazakii 102 Environmental ND C. sakazakii 109 Environmental 2 C. sakazakii 228N Milk powder 2 C. sakazakii 305N Milk powder 1 C. sakazakii 343 Environmental 2 C. sakazakii 344 Environmental ND C. sakazakii 366 Environmental ND C. sakazakii E604 Clinical 2 C. sakazakii E632 Infant food 1 C. sakazakii E770 Milk powder 1 C. sakazakii E775 Milk powder 2 C. sakazakii E784 Neonate 1 C. sakazakii E787 Clinical 2 C. sakazakii E824 (CDC 0996-77) Human spinal (fluid) 1 C. malonaticus* E825 (CDC 1058-77 Human (breast abscess) ND C. sakazakii E826 (CDC 1059-77) Unknown ND C. sakazakii E827 (CDC 0407-77) Human (septum) 1 C. sakazakii E828 (CDC 3128-77) Human (septum) 1 C. malonaticus* E829 (CDC 1716-77) Human (Blood) ND C. sakazakii E830 (CDC 9369-75) Unknown 2 C. sakazakii E837 Clinical 2 C. sakazakii E839 Neonate ND C. sakazakii E844 Neonate 2 C. sakazakii E846 Neonate 2 C. turicensis* E866 Neonate ND C. sakazakii E891 Clinical 1 C. sakazakii E892 Clinical 2 C. sakazakii E899 (CDC 0289-81) Clinical 2 C. sakazakii E900 (CDC 0322-78) Clinical 2 C. sakazakii E901 (CDC 1123-79) Clinical 2 C. sakazakii ATCC 12868 Unknown 2 C. sakazakii NCTC 8155 (CDC 9081-75) Milk powder 2 C. sakazakii NCTC 9238 Human (abdominal puss) 2 C. sakazakii NCTC 29004 Unknown 2 C. genomospecies 1* NCTC 9529 Water ND C. sakazakii NCTC 11467 (ATCC 29544) Human (throat) 1 C. sakazakii ATCC BAA 893 Milk powder 1 C. sakazakii ATCC BAA 894 Neonate 1 C. muytjensii* ATCC 51329 Unknown ND Non-Enterobacter sakazakii (“Cronobacter” spp.) Escherichia coli (EPEC) ND ND Escherichia coli (ETEC) ND ND Escherichia coli K12 NC10538:06 ND ND Salmonella enterica serovar Anatum ND ND Salmonella enterica serovar Agona NCTC11377 ND ND Salmonella enterica serovar Typhimurium LT2 NCTC12416 ND ND Salmonella enterica serovar Enterica PT4 NCTC13349 ND ND Citrobacter youngae ND ND Citrobacter sedlakii ND ND Citrobacter braakii ND ND Citrobacter koseri ND ND Citrobacter freundii ATCC 8090 ND ND Citrobacter koseri NCTC10768 ND ND Klebsiella oxytoca ATCC 43086 ND ND Klebsiella pneumoniae ATCC 13883 ND ND Yersinia enterocolitica ND ND Enterobacter helveticus ND ND Escherichia hermannii ND ND Enterobacter hormaechei ND ND Enterobacter cancerogenes ND ND Escherichia vulneris ND ND Enterobacter cloacae NCTC11933 ND ND Enterobacter aerogenes NCTC10006 ND ND Enterobacter gergoviae NCTC11434 ND ND Enterobacter agglomerans NCTC9381 ND ND - TABLE 2.
Putative genes in E. sakazakii O:1 or O:2 O-antigen gene cluster
ORF Location in sequence (bp) G+C content (%) Size (aaa) Similar protein Source (accession no.) % Identical/% similar (no. of aa) Putative function O:1 cluster orf1 1-891 53.4 296 GalF Salmonella enterica (AE017220) 87/95 (295) UDP-glucose pyrophosphorylase orf2 1282-2361 50.3 282 RfbB Shigella flexnerii (NP_707936) 87/93 (273) dTDP-d-glucose 4,6 dehydratase orf3 2358-3230 40.9 290 RmlA Escherichia coli (AAT77170) 81/90 (287) dTDP-glucose pyrophosphorylase orf4 3232-3633 35.8 133 WbtA Escherichia coli (ABK27315) 55/73 (131) Putative isomerase orf5 3626-4078 31.8 150 FdtC Escherichia coli (AAT77171) 49/58 (147) Acetyltransferase orf6 4079-5014 34.4 311 Xanthomonas axonopodis pv. Citri (NP_642021) 45/62 (306) Hypothetical protein orf7 5011-6114 36.7 367 FdtB Escherichia coli (AAT77174) 58/73 (365) Aminotransferase orf8 6111-7373 37.3 420 Wzx Escherichia coli (AAT77175) 46/67 (413) O-antigen flippase orf9 7373-8317 32.5 314 WbtE Escherichia coli (AAS73170) 28/50 (254) Putative glycosyltransferase orf10 8356-9636 31.9 426 Wzy Escherichia coli (AAZ85713) 19/43 (272) O-antigen polymerase orf11 9623-10501 36.7 292 WbgQ Shigella boydii (AAL27350) 32/51 (281) Putative glycosyltransferase orf12 10491-11255 34.0 254 Shewanella baltica (ZP_01435692) 55/68 (243) Glycosyltransferase orf13 11469-12875 54.3 468 Gnd Escherichia coli (ZP_00926655) 93/97 (469) 6-Phosphogluconate dehydrogenase O:2 cluster orf1 1-594 55.1 197 GalF Escherichia coli (AAS99158) 83/90 (197) UDP-glucose pyrophosphorylase orf2 986-2068 50.3 360 RmlB Escherichia coli (ABI98981) 84/91 (360) dTDP-glucose 4,6-dehydratase orf3 2065-2967 49.5 300 RmlD Shigella boydii (AAY23334) 80/88 (298) dTDP-4-dehydrorhamnose reductase orf4 3017-3895 46.6 292 RmlA Salmonella enterica (P55254) 91/96 (292) dTDP-glucose synthase orf5 3899-4441 35.9 180 RfbC Shigella boydii (YP_407363) 81/90 (180) dTDP-4-dehydrorhamnose 3,5-epimerase orf6 4488-5744 30.2 418 RfbX Escherichia coli (NP_416541) 27/52 (406) Putative O-antigen transporter orf7 5741-6718 33.2 325 Escherichia coli (ZP_01699967) 22/42 (280) Glycosyltransferase orf8 6730-7485 29.4 251 WbsV Shigella boydii (AAS98030) 30/48 (241) Glycosyltransferase orf9 7475-8473 31.0 332 Wzy Shigella boydii (AAS98031) 27/43 (317) O-antigen polymerase orf10 8493-9332 28.9 279 WbsW Shigella boydii (AAS98032) 30/47 (268) Glycosyltransferase orf11 9334-10341 31.9 335 WbgM Escherichia coli (YP_541302) 30/46 (110) Putative galactosyltransferase orf12 10331-11095 29.9 254 WbbL Escherichia coli (P36667) 40/58 (257) Putative lipopolysaccharide biosynthesis glycosyltransferase orf13 11462-12868 54.4 468 Gnd Escherichia coli (ZP_00926655) 93/97 (469) 6-Phosphogluconate dehydrogenase ↵a aa, amino acids.
