Jean Michel Alonso,w and H. H. Mollaretw. "~'knteric Section, Center for ... raffinose, Simmons" citrate, and o~-methybD~gluco- side, (Mel§ sucrose-negative, ...
CLiRRI NI" Ml{ ROUlOLOGY,Vol. 4 (1980), pp. 195 2011
Current Microbiology An International Journal
Deoxyribonucleic Acid Relatedness in Yersinia enterocolitica and Yersinia enterocolitica-Like Organisms D o n J. Brenner,J-* Jan Ursing,$ Herv6 Bercovier,w A r n o l d G. Steigerwalt,]- G. R i c h a r d F a n n i n g , II Jean Michel Alonso,w and H. H. Mollaretw "~'knteric Section, Center for Disease Control, Public Health Service, U. S. Department of"Health and Human Services. Atlanta. Georgia 3O333, USA :~])epartment et" Clinical Bacteriology, University ~.~tLund. Maim0 General Hospital. Maim6, Sweden w National des Yersinia. Institut Pasteur, Paris, France HDivision of Biochemistry, Walter Reed Army Institute of Research, Washingtom D. C. 20012, USA
Abstract. Yersinia enterocolitica and Y, enterocofitica-[ike strains were characterized by D N A relatedness. These strains l o r m e d four distinct D N A relatedness groups: (it the 5 classical biotypes o f K emerocotitica sensu stricto as designated by Wauters: (ii) strains that are rhamnose positive and also positive in tests for melibiose, ~t~methyl-D-glncoside, raffinose, and S i m m o n s ' citrate; (iii) strains that are r h a m n o s e positive but negative in tests for melibiose, e~-methyl-o-glucoside, and raffinose; (iv) sucrose-negative, Voges-Proskauer-negative, trehalose-positive strains.
Yersmia enterocolitica, isolated by Schleifstein and C o l e m a n [27], was originally called Bacterium enteroco[ilicum. Subsequent isolates were called Pasteuretla pseudotuberculosis rodentium [17] and Pasteuretta X [ 14]. In 1964, Frederiksen [16] examined 55 strains, including isolates from the studies cited above, He concluded: " T h e characteristics of this group o f bacteria are sufficiently distinct to separate them from Yersinia pseudotubercutosLs'. Yet they resemble E pseudotuberculosis sufficiently to justify their classification in the genus Yersinia (within the family Enterobacteriaceae} as a separate species: Yersinia enterocotitica." Yersinia enteroeotitica and Y, enterocolitica-like strains are quite variable biochemically, and m a n y biochemical reactions are temperature dependent. Biotyping schemes were based on different reaction patterns for iipase, lecithinase, indole, xylose, esculin, and salicin [2,18,24,32]. Other groups were included in Y. enterocolitica by some workers and described as Y. emerocotiticalike or separate species by others. These included metabolically inactive strains isolated from hares that are trehalose negative, often sucrose negative, but usually Voges-Proskauer positive [22,24,29,32]; a second sucrose-negative group [4] that is trehalose positive and Voges-Proskauer negative [3,6,13,32]; two *To whom offprint requests should be addressed.
groups o f rhamnose-positive strains [ 1,6,7,8, I 1,19,30], one which is only rhamnose positive (Rh +) and one of which is also positive in reactions for melibiose, raffinose, Simmons" citrate, and o~-methybD~glucoside, (Mel§ sucrose-negative, r h a m n o s e - p o s i t i v e strains (X2) [8]; and sucrose-negative, ornithine dec a r b o x y l a s e - n e g a t i v e , c e l l o b i o s e positive strains (XI). K n a p p and Thal were the first to suggest that the rhamnose-positive and sucrose-negative groups were not part o f Y. enwrocotitica [18]. D N A relatedness studies on various yersiniae, although helpful, do not adequately cover the genus because of a paucity o f strains, biotypes, or species tested [13,15,23,26]. We now present D N A relatedness studies o f Y. enterocolilica and Y. enterocotidcalike organisms. In other papers, D N A relatedness data are correlated with biochemical and other phenotypic characteristics. On the bases of these data we define Yersinia enterocolitica sensu stricto [4], make formal nomenclatural proposals for three new Yersinia species, I"2 intermedia [11], E frederikxenii [30], and Y. kristensenii [5], present evidence that Y. pseudotuberculosis and Y. pestis are genetically the same species [17], and show that "Yersmia'philomiragia does not belong to the genus Yersinia [31]. Materials and Methods Organisms. The bacteria were described previously [28] or in Table I. In ]able I. gersinia enterocohtica is used in the widesl
0343-8651/80/0004-0195 $01.20
196
CURRENT
MICROBIOLOGY.
Vol. 4 (1980)
Table 1+ D N A relatedness among yersiniae. SourCe of labeled DNA
Source ol'uniabeled yersinia DNA Y entcrvcohxh:a
498-70 Strain a
~
498-7(1 (I P 851 We I~1227I 5 2a. 2b. 3 IP 516 f l u 65) P76~ (Th) P766 (Th) p78; (Th) p784 (Th) 184 77 185 77 I 19, @ [p 842 (We B35711 2525-75 3968 76 3974-76 1 ((.'+) 17 1P6155 IKK Yl16) IP 6~66 (KK Yk35) I ( C * ! Ki IP 6168 (KK Y1381 I (Ra ~') 13, 7 IP5919 I 16 3969-76 3970-76 3 3, 2a. 3 IP 39 (131905, Th 3371 Ip 9L IFr P 71, Kn 26/60 6L, 4 I, 2a. 3 Ni 357. Th 357) IP 106 lAb 5819. Fr P3111 I 7. ~ 9 It' 359 (Vp 15/681 2 496 70 (We B2756) 3 IP 1615 (Ra 15} 4 3 6 [p 1646 (~a 1156) ] 5.27 IP 2224 (Zr n:l 9) 2 2 5.27 z IP 3G51 (To 134) 397%76 I P 4052 (Va 3(J/74) [P 4975 (SZ 9435[I) 3 6 IP 5429 (Za 300) PTII Irhl P717 (Th) P719 ITh) P726 (Thl P731 ITh) P733 (Th) P744 (Th) P749 ITh} P750 (Thl FT:', I I'lh) I, 2a IP 6551 I. 2~ IP 6552 1, 24 IP 6554 (LJ)3 IP 4553 {Ze Te 431 (LI)Rough IP 7309 IAi 7803) (U)Rough IP 7308 IAI 153921 IP 7333 (AI 20889) (U) fU )Rough IP 7310 (A145676) P783 ITh) IP 5345 I Fo I ) 4 264%76 265(I 76 IP 64 (Be 5 I/C, Ni 348, 3 I. 24.3 "l'h 348) 1529-75 5, 27 IP97 (Fr P77, Kn 1048/ 2 60-61. Th 359) 9 IP 373 (Ah 246/681 2 1P 161 lAb 33 114, ATCC I 8 9610. F[ P310) 3 IP 175 IWa 14) 4 IP21l (Wa 2[) 2 9 497-70 (We B2336) L 5013-700 P 840. We Bg03 ) 3 1.2a, 3 501-70 (We B282) 3 951 74 2a. 2b, 3 IP4(Lu Lt4. Fr P360) 5 2a, 2b,3 IP 178 IMa 6/53, Lp 2733-53. 5 Ca 255/64, Fr P420, Th 374) 5 24.2b, 3 IP 488 ILu 61) 5 (S-I 2a, 2b, 3 IP 489 (Lu 62) .5 24. 2b, 3 IP517(Lu 661 5 2a. 2b, 3 IP 68~J(Lu 74) IP 2726 [AI Z 49-D 5 24.2b, 3 5 2a, 2b, 3 IP 2727 [AI Z 54-24) 5 24, 2b,3 IP 2879 (La 3274/72) 5 2a, 2b, 3 IF 2880 (La 3522/72) 5 24, 2b, 3 IP 2881 (La 3523/72) 5 (S-) 28, 2b, 3 1P I (Lo 110. Fr P357) 5 ( S ) 24, 2b, 3 IP 2 (Lu 112, Fr P3581 5 ( S ) 2a. 2b. 3 IP 3 ([.u 113. Fr P3591 5(S }2a. 2b, 3 IP 27 fLu 27) 5 (S-) 2a. 2b, 3 IP 37 (Br I. Fr P3711 5 24, 2b, 3 IP 1144 IFi 78/13) S~ 12 IP 103 (Fr P2201 [P 105 IKr MK 134, S [I Fr P226) 9S12 IP4q0 (Lu 63)
~ XI
X,
X,~ X+ X, Xz
[I [[ X,. X IX~ X, X~ Xz
IXa X~
Xl XI XI VIII X,. X~ Xo Xz
60~C 759C 609C RBR" D 't RBR RBR
s ..... ,' Hu I]'a Pi Pi Pi Pi flu Hu Hu Fo Ilu Hu Se S*: Se Ro I-lo Hu Ch Ch 1[~ llu Mo fi Hu ]-[u }tu Ha Hu He Pi Pt Pi Pi Pi Pi Pi s Pi Pi Ro Rt~ Re ~]u
Li
SI SI Th St SI
Sp St St
St Sl St St Ap St S[ St
X SI St St SI
Mi
VIII
Pi Hu
II
Ch Ha Ch
St
X~ X~ X~
Hu Hu
VIII X3
St
Y. enterocolitir ~ (biotype 5)
US FR SW SW SW SW US LIS US NZ LJS US I)E DE DF. FR US US NE DE
I(~ 83 8t 88 9l 8q ~8 S7 .~4 89 86 84 81 86 89 ;36 95 9(~ 90 80
US BE US SA CZ JA r US
87 90 9I 86 89 90 86 I []/,J
HU PO SW S.W SW SW SW SW SW SW SW SW FR FR FR JA CZ CZ CZ CZ SW SP US US SZ
89 88 79 80 76 83 86 87 73 81 81 91 92 87 94 89 85 89 85 86 89 85 81
(J I
D
Y. enrerocol#ica 6048 ( S ' ]
75~C 60~C RBR RBR
D
75~C 6O~C RBR RBR D
1130 77 78
72 77
7 9
55 54
78
74
9
53
59
12
Y. enterocolmca 2581-77 (Rh §
75~C 60~ RBR RBR 24
)0
D 13
Y. enlerocolitico 3953 ([~oLIone 48) (Mel § )
75'~C 6g~ RBR RBR
D
78 4
2
73 87 88 ~2
2
94 92
[
97
2
94 82 87
89 87 7 ~. 72
3
3 4
92 9[
74 92 74 68 80 79 74 79 78 85
9
25
63
13
23
61
12
26
64 fi9 76
I~
34
13
3~
53
76
8
55
75
IO
52
73
II
51
66
12
36
IO
4~ 55 58
74 78 82
75~C RBR
12
60
60
13
25
52
59
25
55 58
24
62
28
71 75 8q 89
62
78
i
84
74
9
54
US DE
87 78
1 I
83 88
74
9
41
St Sk
FI US
83 79
I 2
89 80
75
9
51
BE BE US US US US FR UK
84 86 77
! 2 2
88 86 83
74 74 74
IG 9 I~
51 54 52
II [I
Hu St l'l~ St Mo Hu Mo Hu Ha St Ha Sp
84 87
4 2
80 81
75 72 77
113 9 8
50 55 53
XI I XI XI [[ XI Xl XI XI I I I II Xl XI X~ Xz
Ha Ha Ha Ha go Ro G~ Go Go Ha Ha I3a Ha Ha Ha gh Hu
Sp Li LI Lu St St St St St St St St St Sp
99 76 75 78 77 81 73 74 82 74 80 75 82 84 84 70 75
2 L 2 3 3 2 3 3 2 1 3 2 3 2 3 8 9
74 72 73 71 75 78 75 69 78 67 72 67 80 75 83 45 42
73 75
9 9
51 51
72
~
51
8
Sk Ur
HU FR FR FR CZ CZ NO NO NO FR FR FR FR BE BE FE DE
Xt
Ha
Li
FR
71
8
44
Xl
Y. enrerocolit&~ 6175 (Rh +}
90
0
98
IC~ 98 lO0 97
0 t t 1
I[81 I00 96 I0(I
64 6'~ 73 72
8 9
48 46 49 53
97 61
I 8
98 46
75 g3
9 I
51 83
62
[0
93
0
99
II
34
5~
59
13
27
64 53
12 II
32 20
54
II
27
38
59
12
29
49
13
17
55
14
la
63
13
36
63 55 52
13
30
48 42
13 t5
17 17
63
197
D. J B r e n n e r et al.: D N A R e l a t e d n e s s in Yersinia
IP 1474 ~La 542 DI IP 1475 ( t a 547 C} IP 4764 IAI 18999) IP 5517 IP 55L9 i p 5520 Ip 5553 (A1 t9548) IF 5852 [P 5873 [ P 5874 [ P 5878 (P 5879 IP 5894 [P 5920 [P 5932 1P5933 IP 5943 {KK Y25) 1P 6048 ( M D B 4 5 1 2 0 / 1 7 6 ) IP 6072 IP 7701 IP 7703 IP 7704 IP 7705 968-74 [P 3605 (Ts ~36-3) [p 3606(Ts 140-2) IP 5588 [P 5591 [P 588 I [P 5890 IP 596(! ( K K Y64) [P 5981 ( K K Y 104) [P 5982 ( K K Y 105) )P 6334 (Pc 7%49 i6) IP 6 1 5 0 ( K K YI07) [P bl75 ( K K YI461 0 2 7 0 5 (We) IP 5966 ( K K Y77) IP 5976 [ K K Y98) IP 5975 ( K K Y96) 871-77 8.72-77 2581 77 IP 5630 1p619~ ( K K YI76) IP 6237 ~KK Y258) ZP 6249 (K/~ Y295) lP 6250 ( K K Y279) ]p 6194 ( K K Y183) 17 (Bo, Wi 171 56 (Bo. IP 39,60) 57 (Be, IP 3861) 870-77 1874-77 2580 77 2647-77 2651-77 IP 4679 [P 5797 {Oa 384.24} IP 5983 ( K K u lP 6630 (Da 71902) IP 6L31 ~KK YI08) IP 6172 ( K K YI42) IP 0187 i K K Y 166) 1P 6262 ( K K Y296) [P 6300 { K K Y353) IP 63 [ I ( K K Y3871 [P 6521 IP 66[8 A0235 (Fe. Le~ 8 [ I ) AI251 {Fr Hi D3) A 1262 [Fr Va 3 ) AI263 (Fe. Va h75) AL2M (Fe. Va IIJ0~l AI265 (Fe, Va 1049) A 1266 (Fe. Va 10591 Al267 (Fe, Va 1178) C365 L (We} C3741 (We) I P 3953 { Bo 48, C h 48) 1P 955 ( L a 333 B) 1093 74 IP 7702 [P 7706 I P 5587 (P 6553 [P 867 ( O r 19) (P 6005 (AI I9955} [P 5850
S 28 S16 S Ii S I, 2a S'I~ SI~ S 5 12 SI, 2a Sl, 2a S I S [.2a S" I, 2a S12 S 12 S ~2 SSI2 S N S 12 S~2 S16 S 12 S S'" 12 S 12 s 12 S 12 SI. 2a S I[ Rh q N R~ ' N Rh ~ N Rh' N Rh ~ N Rh + N Rh § Rh ~ N Rh* N Rh*" N Rh* Rh* gh ~ Mel* 4 Mel ~ N Mel + N Mel + N Mel ~ 15,7 Mel* 6 Mel + L7 Mel * Mel + 16 Mei* MeI* Mel + Mel ~ MeI* MoI § 4 Mcl ~ t4 Mel ~ ~3, 7 M e I ' 14 Mel ~ N Mel ~ 12 Mel + 6 Mel ' t4 Mel ' N Mel + 4 M~I + N Mel + 4 Mel + Mel* Me]* MeI* MoI" Mel + MeI* Mel + Mql ~ Mcl + M,.:I~ 17 MeI* 17 Mr ~ XI 16 XI 16 S 12 I I. 2a Rh + 16 X2 N X2 1(I
Xz X~ Xo Xz Xz X~ Xo X~ X[ XI Xz XI X~ Xt YW. Xt X~ Xz Xz Xz X, X~ Xz Xz X,. Xz X~ X] Xz Xz Xz X~ Xz X~ Xr Xz Xz X~
X~ Xz X~, X~ X,, X.z X~ Y~ X•
Xz X~ X~ Xz Xz X~ X~ Xz Xz Xz Xz Xz
X~ Xz X~ Xz X~ Xl X~ Xo Xz
Wa Wa Fo IRo Ro Ro Wa So Ro Ro go Ro Ro Ro Ro Re Se 13u Ro So So So Era Flu Mo Mc Ro Ro Ro Ro Se Se ~ Hu S~ ~ Hu Se Se Se Hu Hu Hu He Se Sr Pi Pi S~ 1"1~ Hu H~ Hu He Itu H~ Hu Wa Hu Se Hu Sr Se Se Fi Fi Fi Wa BI Fo Wa Fo Fo FO Fo Fo Fo Hu Hu Hu Wa Hu So Ro Ro Ro f']tl Wa So
Sp St St
Sp St St St St St Sp St $1 St
St St St Sp Sp St
BI
St St St
Th Ey $I St S~
B] BI
Li
Sp St St
NO NO CZ FR FR FR CZ FK FR FR FR FR FR FR FR FR DE AO FR FR FR FR FR US IA IA FR FR FR FR DE DE D I" ErR DE DE US. DE DE DE U~; US US FR DE DE DE DE DE US US US US US US US FR FR D~h FR DE DE DE DE DE DE FR FR US US US US US US US US US US US NO US FR FR FR FR BE CZ FR
72 69 77 73
9 9 8 8
39
50 48 44 5t
24
69 65
L0 ~0
38 20
66
10
3A
68
I~
33
71 69 66 65 69 69 69 73 73 74 76 80 72 72
10 9 I1 II II
31 45 34 36 34 31 27 43 39 35 37 58 4g 47
8 ;~ 10 9 9 9
65
12
39
66 ~ 65 7[ 71 69
I1 13 12 11 10 II
3g 31 30 38 36 30
55 58 60 63 56 67 67 50 59 58 58
68 66 69 64 62 58 62 62 69 67 63 67 62 65 64 65 66
48 445 49 8 49 45 9 9 48 7 l0 8 9 9 9 9
46 44
86 84 &5 90 92 84 86 91 82 81 83 77
5
91 93 95 8.7 10D 84 85 94 85 94
I
86 92 91 81
62
7
4
76 74 72 79 78 70 78 89 79
12
21
~
L3
9
57
12
20
52
13
l0
79 79
5 0
I
1
92 97 99 77 I(~l. 89 75
61 6~ 60 63 62
75 99
2/
93 92 94 gO
H
3l 13 I1
54
23 26 35
20 25 71 60 68 72
45 59 77 79 82 77 80 100 79 79 94
12 5 6 6 5 6 0 5 5 i
100 69 66 ~.
53 55 55
12 12 12
17 t7 16
53 52 56 51 50 61 54 5~i 51 5~ 52 54 95 ~3 100
13
18 19 16
13
17
14
12
13
16
2 3 O
63 ~5 100
65 71
12 12
58 58 59
63 65 57 58
45 58
36 34 26 21 29
13 13
13
20 3~ 24 25
15 2A~
89 82
9~ 87
36
65 62
90 ~9 94 91 98
26 32
0 i 0
90 ~8 96 87 ~00
100 77 10o
55
99 97 99 98 $4 93 99 99
58
60 62 86 90 53 63 65
2~.
3 II
31 34 79 86 24 36 28
59 56 57 4~ 54 61 55 61 60
12 1L lI
46 65 60
I1
58 53
63
35
1l
24 34
10 I1
68
L2
31
61 65
iI 11
31 33
65 66 94 92 61 66 60
[2 12 I 3
11 1{]
33 37 95 9I 32 33 35
5~ 64 62
11 L0
64 53 56
ltl [3 ~I
29 30 23
34 21
50
13
[6
14 14
i3 ~ 23
48
52 53 43
98 92 85 94 93 g6 94 95 98 94 93 100 95 94 60 60
59 57 5l
I 4 I 0 5 2 o 0 0 0 2 1
t3
94 77 92 97 79 93 93 92 95 94 100 94 89
24 29 31
" b t r a i n r ~ u m l ~ Dsed Jn lEis paper are given first Strains from the Enteric S~cdon at C D C are . o r credited (498-70 a n d 184-77 are such strains). [P: culture collection of the National Ycr~inia Center. [ l ~ t i t u t Pasteur SourOes of other straiz~ and synonyms for $lram numbers ~t~e given in patenthr Abb;ev~atiol~: We, Weaver; Lu, Lucas; Th, ThM; K K , Kro~gaard Kris~ensen; DI, Dtmir Ft. Frederiksen: Kn, Knox: Ni, Nd~hn; Ab, Atbany; Vp, V~ndepitte: Ra. Rafison: Ha, Hausaerova; Ze. Ze n Yogi ~Lnd. M a r u y z m E To, Toma: Sz, Szita: Za. Za~emba; AI. A[dova; Fo. F o u n d a t i o n Jimenez Diar Be, Becht; Ah. Ahvonr A']'CC. A m e r i c a . Type Cu]turr C o l l ~ t i o ~ We,. Wautr Ma. M~r Lp. L~pagr La. L a z ~ ; Br. Beerens: Ft. Fievr Kr. Kri,sw. r~';r MD, McDermotl; Ts. Tsubokara; Pc, P e r i l Bo, BoHone~ Wi. W m b l s d : Da~ Dabe[naL Fe~ Fr162 Hi, Highsmith; Va, Vznde.,zanL Ch, Che.~le~; G L G raux Source is given firSl by httman, animal, ot environmemal source followed by specific site and osunzry of origin. Abbreviations: Hu. h,.tmam; Ha, h~rr Pi. pig: Fo. food; Se, r:,cwage; Ro. wild rodent; Ch, chinchilla; Mo. mo~lkey; He. hea; ML mhk: Go. goat: Sh. s h ~ p ; Wa. water: So. soil: Ez, 0arthworm: Ft. fish: BL bbd: LL liver. St. s~eol: T~-~ Ihro~l; Sp. spleen: Ap, appendlx: Lu, lung: Ur. urine; BI, blnod; Ey, eye: US, United States; FR. France; SW~ Sweden: NZ. New Zealand; DE. Denmar/~; NE, Netherlands; BE. Belgium; SA+ Soalh A fdc,~t; C Z , Czechoslovakm~ JA. l a p a n ; CA, Canada; H U, H~ngary; PO, Pe4an~; SP, Spaia; SZ, Swi~erland; F[, Finland; LIK, U m t e d Ki~,gdom; NO, Norway; FE. Feroe Island; AU, AuslraJia_ c R B R . re|ative b i n d i n g ta;io - % (heterologo~s D N A b o n n d to HA)/(horaologo~s D N A bound to H A ) ~ 100. R B R is a convemenl way to expre~ I~rc~nt rr162 a D. percenl divergonc~ D is calculated on the & ~ m p b o n teat a IoC d,~r--At~ in 1hetm&l stability of It heterologous D H A duplex ex~mpazed to thai of the homult~gous D N A duplex is c~us~d by each I% of~hc base.~ within the duplex thKI are unpaired [ 131=F e r example, c o ~ i d e r organisms A and B which are 50% r=la(ed ( R B R = 50%). T h e thin'real stabilky of a~ A-A duplex i~ 91 ~ and the thermal ~.tability of ~.~. A-B dupiex is 76~C: the i:-erC~nl divergenc~ of the t~late..d D N A is 15.
|98
DNA meth~d~. The methods used for DNA hybridization have been described [12[. The guanine-plus-cytosine composition (tool%) was determined by optical thermal denaluration [20,25] and by CsCI buoyant density gradient uhracentrifi,gatmn [21].
Results T h e G + C content in D N A s from 22 strains representing Yersinia enterocolitica biotypes L, 4, and 5, and Mel ~, R h ' , and S groups was 48.5 • 1.5 tool%. Results from b u o y a n t density determinations (kindly done by M. Mandet) were consistently I% higher than those obtained by thermal denaturation. D N A relatedness studies were d o n e on 175, strains chosen from biotypes 1 5 and the Mel +, R h +, S . and X I and X2 groups (Table 1). Eighty-one strains, from biotypes I 5, including strains with atypical reactions for urease, lactose, rallinose, citrate. melibiose, or c~-methyl-o-glucoside were highly related to biotype l strain 498-70. Both sucrose-positive and sucrose-negative biotype 5 strains were tested. In 60~ reactions, the average relatedness o f these 81 strains to 498-70 was 85%. Fifty strains were also tested in 75~ reactions where only closely related D N A sequences can reassociate. Relatedness in these reactions remained high--79%. A further indication of the high degree of relatedness was obtained from thermal elution profiles that assessed the percent divergence (%D) within related D N A sequences (Table 1). D N A from strain 498-70 showed 0.5-3.5% D with D N A from 16 highly related strains. Similar results were obtained with labeled D N A from sucrose-negative biotype 5 strain 1 (Table 1). ~fypical biogroup 1-5 strains were 88% related to strain I in 60~ reactions (1% D, 91% relatedness at 75~ Eight o f these strains had not been tested with strain 498-70. Three additional strains (500-70, 50170, and 74-951) were not tested here, but are included because they were previously shown to be highly related to strain 498-70 [13]. Thus, 8L strains belonged to the biotype I 5 D N A relatedness group. Strains from the S- group were 70% related to strain 498-70 and 64% related to strain 1. In these reactions, D was 9%. Average relatedness of strains 498-70 and 1 to S group strains was 39% and 47%, respectively, at 75~ Average relatedness o f 29 S strains to strain 6048 was 88% in 60~ reactions and 84% in 75~ reactions. T h e r m a l elution profiles d o n e on 10 of these strains showed an average o f 2.5% D. Strains from the rhamnose-positive, raffinosenegative, melibiose-aegative group (Rh +) formed two D N A relatedness groups. Nine R h ~ strains were highly related to R h ~ strain 6175 (81% in 60~ reactions; 5% D; 68% relatedness in 75~ reactions). The second Rh + relatedness g r o u p was c o m p o s e d o f strain 2581-77 and two strains that were 94% related
CURRENT
MICROBIOLOGY.
Vo[. 4
(1980)
Table 2. Percentage of DNA relatedness in 60~ reactions between Yersinia groups. Source of anlabeled D N A ~ SourCe ~!" t,a beled D N A BioLype~ I 5 t498-Tq)) S I~a(Nltl Rh+16175) Rh* (258i 77) Mel § (3953)
8iolype~; ]-~
S-
Rh ~ 161751
Rh ~ (2581-77)
MeI "~
g6(l~JIJ)
70(69)
67(65~"
57(581
59(5,8)
74(721 6(1(591 53151~} fi21 )
N,+',,il~b 55(~7) 53(52) 621 1
4 ) Nl(lll0) 54(54) 60(63)
t I 54(5"~1 g4(l[~)) 52t581
641611 6L(581 4915f~1 95(ll~)j
'~ ['he first number is the average ~-el~tedness iff aLl unlabeled strains with lhe specLbed labe]eci DN A. Tbe number in parentheses repl r the relatednes~ between Ihe slram s thai were labeled for each group Fr~r example, la~eled S slrain 6L,aS D N A sho~ed an average of 74% rel:~tedness to all slrains ~f biagroups I 5 wit~ which it was tested, and shc~wed 72~'4 relatedne~is Io ~train 49b,-70. - . Nat tested.
to it at 60~ and 84% at 75~ Relatedness between these two Rh* groups was approximately 55%. All 37 strains from the melibiose-positive, rhamnose-positive, raflinose-positive ( M e ] * ) g r o u p were in a single D N A relatedness group when tested with D N A from Mel § strain 3953, These strains were 95% related in 60~ reactions (i.5% D) and relatedness remained above 90% in 75~ reactions. Intergroup D N A relatedness is summarized in Table 2. Except lot the approximately 70% relatedhess between biotype 1-5 strains and the S group, intergroup relatedness is 50 64%, D is greater than 10%, and relatedness falls to between 15% and 35% in 75~ reactions. Seven strains (bottom o f Table 1) either did not belong to any o f the D N A relatedness groups or showed high relatedness to two groups (see Discussion). Brenner et al. [13] showed that 1< enterocotitica strain 501-70 (biotype 1) was 15 25% related to species of Enterobacteriaceae other than members o f the genus Yersinia, about 30% related to K ruckeri, and between 45% and 60% related to strains of K pseudotuberculosis. Some o f their data are presented in Table 3 for comparison with relatedness o f Yersinia to other Enterobacteriaceae obtained in the present study. Labeled D N A from strains 27 (biotype 5), 3953 (Melt), and 6048 ( S ) was about 50% related to Y. pseudotuberculosis, 40% related to Y. ruckeri, and between 10% and 25% related to more than 30 other species of Enterobacteriaceae.
Discussion Yersinia ruckeri was not considered in this study because this species was recently characterized [15]. Y. ruckeri was included in Yersinia because, despite significantly different biochemical reactions, its G + C composition and D N A relatedness were closest to those o f Yersinia species. Y. enterocolitiea biotypes 1-4 formed one D N A relatedness group. Also in this group were biotype 5 strains. Biotype 5 contains metabolically inactive
D. J. Brenner eL al.: DNA Relatedness in Yersinia
199
Table 3~ DNA relatedness between yersiniae and other Enterobacteriaceae. R B R at 61):~( using labeled D N A [rOl% Y~rslr?lo sLraic1
Sourceofuniahe~ed
I)NA
Yervb6a pxe~dotreherctdu.~Tx P62 ~lwinia r u , keri 453"~-69 Evr']lerwhla coff g 12 Ed~ardsir r 3592 64 .r typhitm~rium L T 2 (" ~trobtz~.terj~eu*uh~ 461)-61 ('itrrJbacter dire~iT~s !l~fl.7 I Klel,~ella pneum,sniat" 2 Klebsw]ta o~t'soca 13182 Emerobacter ao'ogene~ 1627-6fi L'mertd~acter cloaca,, 134? "/I Fnlerohacter gerga ~'ir1627t~ (I ~ Serratla ~r 88- >7 Serratta hqlle/~Cli'lll 44~1-fi8 ,S'l,rraOa tr~bidaea 93a 72 SerroliafimHcola d'55(1-7 I ttqfi;~a al~'ei 5632..72 O h e ~Ttz'ibacterium proreu ~ ~.3112-74 [,tllerl,bttcter ctgg~on]ergzns 2780 70 t"./er.bacler aggiumerans 1429-7~ Enzer,hrsc~er agglu~ncran~ 6071!-69 Ernq~na ttmvh~v~rez EA 178 12_'r~,inla ~l,~riflut'ns E N li~4 Lru?t~m quercma [ Q 1112 l:rw#na rubr!Ja~iens E R 105 E r w m m sah'cix ES 11/2 ErllTctta rtlalJoti~ora 285 [ P~,t'lahac'lert'tenl {Er~*2nltt~ t arerlorr 495 Pet'hthafls {Era,rata! chrysantherni SR 32 PectobLtcterlum f Erwima) clprlpedii I"C 155 Pectobt~r (~;FMrl'eetr rhaFontlci E R 106 M~rganell~ ~torganii 2 5 8 3 0 Prr162 ,nirahitix PR 1,1 Pro~'Men,'m alcal(laeicn~ ~ ~ 711-67 Providencia ya~arm 2R~t~ 6~
'l'h'r
27 ( b i ~ y p e 3)
3,153 (Me] ~ )
6~8 IS )
5(I 40 25 16 22 20
54 %8 I% 8 I fi 20
~O
17
15
19 ~7 i5 2g
16 Iq 12 2~
20 I5
211 1%
22 12 21 17 22 17 25 17 17 1% ~9 29 19 29 2k i5
17
12
Iq
20
13
I
2fJ
IS
13
21 26 I1 21 IF; [9 2(I
J~;' 23 ik 13 14 I~ 15
19 I~: 18 2~1 II 28 ~9
7
27
17
I ~l
18
25
15
28
17 13 13 II
[I 6 ", ~
2 ~, II I 19
501-70 (bi~qype I f ~ 3I 20 15 21
19 2.0 ]8 ~
15
lq
17 [0
dat;i itle frlml relerencc 2b
strains isolated mainly from hares [22,32]. These strains are trehalose negative, negative in reactions for indole and nitrate reductase, and often negative in reactions for sucrose, ornithine decarboxylase, flgalactosidase, and sorbose. Strains from biotypes 1 5 constitute the species Y, enterocolitica. Biochemical characterization and designation of a type strain for K enterocolitica are presented elsewhere [4]. A second D N A relatedness group was formed from sucrose-negative, Voges-Proskauer-negative strains ( S ) . The S- group is separable from those sucrose-negative strains in biotype 5 because S strains are always trehalose positive and positive in reactions for xylose, ornithine decarboxylase, and fi-galactosidase. S strains are 60~75% related to Y. enterocotitica compared to 88% intragroup relatedness in 60~ reactions. There was almost no divergence among Sstrains, and average relatedness was above 90% in 75~ reactions. Average divergence between Sstrains and Y. enterocolitica was 9%, and relatedness fell to about 45% in 75~ reactions.
With essentially no exceptions, species of Enterobacteriaceae are at least 70% related under optimal (60~ conditions for D N A reassociation, relatedness is 60% or higher in 75~ reactions, and %D is 5 or iess [9]. S- strains and K enterocoftica are at the border of species level relatedness at 60~ but %D is 9 and relatedness in 75~C reactions falls well below 60%. We conch]de that the S strains do not belong to Y, enterocotitica. These strains are described as a new species, Yersinia kristensenii, elsewhere [5]. Strains positive in reactions for melibiose, rhamnose, raffinose, c~-methyl-D-glucoside, and Simmons" citrate were all in one lightly clustered D N A relatedness group (MeP). They were 50-60% related to other Yersinia groups. An occasional strain negative in one of the above reactions was still in this D N A relatedness group, Brenner et al. described this group with only a few strains [13], and informally called them Y. intermedia [10]. Y. mtermedia has been for~ really proposed and the type strain designated [11]. Four rhamnose-positive strains, negative in reactions for rneIibiose, raffinose, and or glucoside (Rh" ), were previously shown to be genetically distinct from Y. enteroeotitica, Y, kristenseniL and Y. intermedia, although they were not tested for intragroup relatedness [13]. This group was tentatively designated as Y,/?ederiksenii [10]. We have now studied 14 Rh ' strains. Ten, all from Denmark, were in one D N A relatedness group, and three others, isolated in the United States, were in a second relatedness group. A single strain, 867, was not in either of these relatedness groups, nor was it in any of the previously described relatedness groups corresponding to Y. enterocolitica, Y. kristensenti, or Y. intermedia, Only minimal biochemical differences were detected between the two Rh § relatedness groups [30]. Because of this and because the "U.S?' group contains only three strains, the name Y. frederiksenii was proposed for all Rh § strains [30]. In addition to strain 867, six other Yersinia strains either did not belong to any D N A relatedness group or were highly related to two relatedness groups, One of these is a biotype I (strain 6553) that shows high relatedness to both E enterocotitica and Y, kristensenii. Strain 5587, biochemically }I. kristenseniL is very highly related to Y. kristensenii, but is almost as highly related to E enterocolitica. The rem a i n i n g u n g r o u p e d strains showed genus level relatedness to Yersinia. but were not more than 65% related to any species. Two of these strains (6005, 5850) are from group X2 (rhamnose positive, sucrose negative), and the other two (7702, 7706) are from group X I (sucrose negative, ornithine decarboxylase negative, Voges-Proskauer negative).
200
CurrENT
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