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Protein Electrophoretic and DNA Homology Analysis of Klebsiella Strains
System. Appl. Microbiol. 11, 121-127 (1989)
Protein Electrophoretic and DNA Homology Analysis of Klebsi-ella Strains C. FERRAGUT,,2, K. KERSTERS" and,]. DE LEY' 1 2
Laboratorium voor Microbiologie en microbiele Genetica, Faculteit Wetenschappen, Rijksuniversiteit, Gent, Belgium, and Institut National de la Sante et de la Recherche medicale, Unite 146, Domaine du Centre d'Enseignement et de Recherches Techniques en Industrie Alimentaire, 59651 Villeneuve-d' Ascq Cedex, France Received August 20, 1988
Summary A total of 175 clinical and non-clinical isolates of Klebsiella were subjected to numerical analysis of electrophoretic patterns of their soluble proteins. Of these strains, 160 clustered into five major groups, ten clustered in groups of two or three strains, and five strains remained ungrouped. Selected strains of the major groups were analyzed by DNA hybridization competition experiments. Almost all strains of the species K. pneumoniae, K. ozaenae and K. rhinoscleromatis occurred in three different electrophoretic groups. The high degree of hybridization obtained between DNA from these strains confirmed their genotypic relatedness and supported the classification proposed by 0rskov. Another group was designated as K. mobilis as its strains shared reassociation values of 88 to 100% with the type strain of K. mobilis. The majority of the remaining strains were clustered in three electrophoretic groups which correlated well with DNA hybridization and phenotypic studies. These were identified as K. oxytoca, K. planticola and K. terrigena.
Key words: Taxonomy - Protein electrophoresis - Numerical analysis - DNA-DNA hybridization - Klebsiella
Introduction
Among the members of the family of the Enterobacteriaceae, the classification of the genus Klebsiella is still controversial. Phenotypical criteria, numerical analysis of phenotypic features and genomic data have lead to different-points of view. Cowan et al. (1960) and Slopek and Durlakowa (1967) subdivided the genus Klebsiella into the following species: K. ozaenae, K. rhinoscleromatis, "K. aerogenes", K. pneumoniae sensu stricto and "K. edwardsii" (var. "edwardsii" and var. "atlantae"), while Johnson et al. (1975) and Sakazaki et al. (1976) recognized three species: K. pneumoniae, K. ozaenae and K. rhinoscleromatis. 0rskov (1984) proposed a different scheme: K. pneumoniae, K. oxytoca, K. terrigena and K. planticola. Because K. pneumoniae, K. rhinoscleromatis and K. ozaenae belong to the same genotypic group (Brenner et aI., 1972), they are now considered as subspecies of K. pneumoniae (0rskov, 1984). K. oxytoca is separated from K. pneumoniae on the basis of indole production, hydrolysis of gelatin and
degradation of polygalacturonate. The present definition of the genus is restricted to non-motile strains, although a close biochemical (Bascomb et aI., 1971) and genotypic (Izard et aI., 1980 b) relationship was observed between Enterobacter aerogenes and the species of Klebsiella. These findings have lead to proposals to transfer E. aerogenes to the genus Klebsiella under the epithet mobilis (Bascomb et aI., 1971; Izard et aI., 1980 b). Both names E. aerogenes and K. mobilis occur on the Approved Lists (Skerman et aI., 1980) with identical type strains. K. terrigena (Izard et aI., 1981) (= group L of Gavini et aI., 1977) and K. planticola (Bagley et aI., 1981) (= Klebsiella sp. 2, Naemura et aI., 1979) are two new species which emerged from the numerical analysis of the phenotypic features of strains isolated from the environment. K. trevisanii, described by Ferragut et al. (1983), belongs to the group K of Gavini et al. (1977) and was later found to be a synonym of K. planticola (Gavini et aI., 1986).
122
C. Ferragut, K. Kersters, and J. de Ley
Considering the different points of view on the taxonomy of the genus Klebsiella, we submitted 175 Klebsiella strains from hospital and environmental origin to a numerical analysis of the electrophoregrams of their soluble proteins. Our aim was to examine the protein electrophoretic groups formed, to study their degree of homogeneity and relationships, and to verify the correspondence with the recognized species. Additional information on the genotypic relationships of the protein electrophoretic groups was obtained through DNA-DNA hybridization experiments on selected strains.
Material and Methods Bacterial strains and media. The 175 strains used in this study are listed in Table 1. For the preparation of DNA, cells were grown in nutrient broth at 30°C during 24 h with shaking. For the electrophoresis of soluble proteins, cells were grown on nutrient agar (Oxoid Ltd., London, England) in Raux flasks at 37°C during 16 h. Polyacrylamide gel electrophoresis of soluble proteins. Cellfree extracts were prepared by the method of Kersters and De Ley (1975) as modified by Izard et al. (1981). Polyacrylamide gel electrophoresis, densitometry, photography of the stained gels
Table. 1. Name of the Klebsiella strains studied. Specl •• (namt H rectlved
or grGIC)
------------_.... -_ .. ------
Strain n· (a)
Electrophorttlc
Klebsiella pne...onlae
ATCC25955 ATCC 27n7 CDC 12t33 NCTC9636 NCTC 9637 Vfron 39 Viron «I Yfron 41 Yiron 42 J31b) J4 J5 J7 J8 J9 JII .lIS JI6 JI7 JI8 J20 J21 J22 J25 .l32 J 34 J38 J41 JS3 J56 J59 J61 J62 J77 030 (bl 037 053 055 063 064 067 072 076
Klebsiella -aerogenes-
E 493 Ib) NCTC 243 NCTCBt46 HCTC 9793
G3B G2 G38
Kiebsl.lIa -edWards!!" var. ·edwardSW
ATCC 13886
G2
Klebsiella ·edWards!!"
E 479 E 50S NCTC 9496 T NeTC 9498
64 GJ8 6JB G38
Klebsiella oxyloca
E 352 E 172/68 .Jain 25871168 Jaln182934/61 Viron 53 V~ron 54 M 101b) MJO M37 M 75 M92 M93 M99 M 104 M 105 M 106 M 114 MilS
64 GSA G4 GSA G4 G4 G4 G3B G4 b G4 G4 G4 G4 G4 G4 G4 G4 G4
ATCC 11297 ATCC 25926 ATCC 29019 coe 276-71 CL.(TN 78-293 E 549 NCTC 5050 T V~ron 47
G3B 62 G38 G2 638 b G2 G38
ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATeC ATCC ATCe ATCC
G2 GlB G2 G3B G2 G38 G2 G3B G3B GlC G36 G38 G3B G3B a
Klebsiella poe .... nla.
4208 4352 4368 4727 65«1 8048 8050 9997 10031 10273 12657 IJBt2 13tBl T 14304 15574 21210
group
~'C
GJC G3C
Klebslell.ozaen.e
ElectraphorttlC
-----_ ..... _- ....... .. ... _----... -- ..... - ....
ATCC 130411 T CI.(1I"178-186 CI.(1I"1 79-235
V~ronSI
Strain n· (a)
or VOI4I ----- ....................... ------- ..
EnterolllCter aerogene. 10 Klebsiella mobilis)
var. -atJantae&
Specl" (namt H rectlved
grGIC)
.,------------ .. -- .. --_ ..... __ .. ---
-
-
"
GJ8 63B
GSA
G2 G3B G3B b GI G38 61 G38 GI 63A 61 G38 G38 GJ8 G38 GI G38 G3A G38 63A G3A G38 G3B G3B G3A G38 G38 G38 G38 GJ8 GSA
c
G38 G38 G3B c 61 G3a
ATCC 13182 T ATCC 13183
G4 G4
KI.bslella rhlnoscleromat 15
ATCC 6908 ATCC 9436 ATCC 13884 T COt B95-68 NCTC 5047 HCTC66« NCTC 1027l Vlron 46
GI G3A GI GJA G3A G3A G3A G3A
Klebsiella SilI>,
• E 522 E 557 E 591 E 685
or VOI4I ....... _-_ .......... - ..... --------- ..
G4 GSA G4 G4
Strain O· la) ElectrophortllC
group
---- ..---- .. --- .. --------------
Klebsiella SlIP.
£739 NCTC91JO NCTC 9170 T 28186 (b) T 37167 T 45/67 T 46/67
Klebsiella group K
K 241b) K26 01 K 33
K42
K 43 K 44 K 47 K 48 K58 K 70 T K 76 K 79 KilO Kill K 117 K 120
a
Klebsiella pneumonloe blolype oxytoca
Klebsiella Nblleeo.,.m
Sptcl" (namt H rtctlvtd
Klebsiella gro"" L
L 27 (bl L 28 L 45 L 49 L 50 L 52 L 63 L66 L 67 L 66 L 69 L72 L 73 L 74 L 76 L 80 L 81 L 82 L 83 L 84 L 8S L 86 L 87 L 89 L 91 L 94
195
L 97 L 98 L 101 L 102 ll08 ll09 lll2 l III l liB L 119
-
GJ8 G3B GSA G3B G3B G3B GSA GSA GSA GSA GSA GSA GSA GSA GSA GSA GSA
-
GSA GSA GSA GSA GSA G5B GSA GSa Gsa G58 G58 GSB G5B GSA GSB GSB GS8 GSB G58 GS8 G5B GS8 G5B G5B GSB G58 GSa G5B GS8 GSA GSB GSB G5B GSB 0
GS8 d G5B G5B G5B GSA G5B
(a) Abbreviations: ATCC, American Type Culture Collection, Rockville, Md., U.S.A.; CDC, Center for Disease Control, Atlanta, Ga., U.S.A.; CUETM, Collection de l'Unite d'Exotoxicologie Microbienne, Villeneuve d'Ascq, France; Jain, K. Jain, Hygiene-Institut der Philipps-Universitiit, MarburgiLahn, Federal Republic of Germany; NCTC, National Collection of Type Cultures, London, England; Veron, M. Veron, Institut Pasteur, Paris, France. (b) Strain numbers beginning with the character D, J, K. Lor M are strain numbers from Gavini et al. (1976 a, b; 1977), with the character E or T are strain numbers from Bascomb et al. (1971).
Relationship among Klebsiella Strains were described previously (Kersters and De Ley, 1975). Each protein extract was examined in at least three different electrophoretic experiments. The normalized densitometric tracings were converted into a sequence of 120 numbers, representing the optical density (expressed as height in millimeters) of each position of the scan. The Pearson product moment correlation coefficient (r), between any pair of densitometric tracings of protein patterns was calculated (Sneath and Sokal, 1973), and clustering was performed by the unweighted average pair group method (UPGMA) (Sneath and Sokal, 1973) using a Siemens model 7755 (BS 2000) computer of the Centraal Digitaal Rekencentrum, RUG, Gent. DNA-DNA hybridization. The following strains were chosen for the preparation of DNA labeled with eH)-thymidine: the type strains of K. mobilis (= Enterobacter aerogenes) ATCC 13048, K. oxytoca ATCC 13182 and K. terrigena (= Klebsiella group L) L 84 (ATCC 33257); the phenotypic centrotypes D 72 and J 22 of K. pneumoniae (Gavini et ai., 1976 a, 1977) and the protein electrophoretic centrostrain K 70 of K. planticola (= K. trevisanii = Klebsiella group K) (Ferragut et aI., 1983). Labeled DNA was prepared according to the method of Marmur (1961) as modified by Ferragut and Leclerc (1978) . Hybridizations were carried out by the competition method used by De Ley and Tijtgat (1970).
,
1
40 j
123
o
I
I< 70 T K 58
(I< 42 Fig. 1. Examples of Klebsiella strains of electrophoretic group G5A (Klebsiella group K) displaying visual similarities in their protein patterns, except for a heavy protein band located at 17 mm from the top of the gel.
Results Comparison of electrophoregrams of the soluble proteins
The reproducibility of the technique was studied by comparing 55 electrophoregrams of ten strains belonging to representative species of the genus Klebsiella. The reproducibility limits were always above r = 0.95 for patterns of the same extract. The most typical protein pattern for each strain was selected, and used in the final numerical analyses. In a preliminary computer analysis, s<;>me isolates belonging to different electrophoretic clusters displayed visually very similar protein patterns, except that a heavy protein band occurred at 17 mm of the top of the gel in some of these strains. Some typical examples of such patterns are shown in Fig. 1. In order to prevent the formation of artificial groupings, the optical densities corresponding to the position of this heavy protein band (i. e. positions 35 to 37) were eliminated from the final numerical analysis of 175 Klebsiella strains. The results are represented in a simplified dendrogram (fig. 2). Above a clustering level of r = 0.87, 160 strains formed five major groups (G1 to G5), ten strains clustered in groups of two or three (groups a, b, c and d) and five strains remained ungrouped. Below this r value, the linkage levels are taxonomically not significant. Group G 1 contains the type strains of K. rhinoscleroma tis ATCC 13884 besides one strain of the same species and six K. pneumoniae strains. Group G2 contains four strains of K. ozaenae including the type strain NCTC 5050, five strains of K. pneumoniae, one strain of "K. aerogenes" and the type strain of "K. edwardsii" var. "edwarsii'!· ATCC 13886. Group G3 consists of 64 strains amongst which 39 belong to K. pneumoniae, including the type strain ATCC
.J
.•
o. I OUt
At~·.' • I
I • I
I
I
All
:'I,,:AI,_. IIHII '
IIUS I S
UUUIUA U
ILIIIIIUA U
02
I
• I
, l
0 •
o.
Fig. 2. Simplified dendrogram showing the relationships among protein electrophoretic groups based on the UPGMA analysis.
124
C. Ferragut, K. Kersters, and J. de Ley
scleromatis and K. ozaenae whereas subgroup G3C contains the three K. mobilis strains. Most K. oxytoca strains, amongst them type strain ATCC 13182, are integrated into group G4. Moreover the latter group includes three strains received as Klebsiella spp. and one "K. edwardsii" var. "atlantae" strains.
13883. Group G3 includes also seven K. rhinoscleromatis, three "K. edwardsii" var. "atlantae", five Klebsiella spp. four K. ozaenae, two "K. aerogenes", one K. oxytoca and three K. mobilis strains. G3 is subdivided into three subgroups A, Band C. Subgroups G3A and G3B are ess~n tially represented by strains of K. pneumoniae, K. rhino-
PAGE Source of unlabeled DNA a ~roup b
" relative DNA binding to labeled DNA from Kpn. E.a. Ko 072 J22
--------------- -------- ----
--- --- ---
K L ----
---
Kpneumonlae 072 J7 J9 J21 J22 J8 J56 JJ2 Veron 40 ATCC 13883 T ATCC 13882 J61 J25 J77 030 JI6 JI5 J3 J20 JII J59 076 064 063 J3B 055 053 067 037 NCTC 9636
GI GI GI a G3A G3A G3A G3A G3B G38 G3B G3B G3B G3B G3B G3B G3B G3B G36 G36 G3B G3B G3B G3B G3B G3B c c G5A G5A
Kozaenae CDC 276-71
G2
82 85
K.rnlnoscleromatl CDC 895-68
G3A
83
E.aerogenes (-K.mobills) ATCC 13048 T CUETM 78- 186 CUETM 79-235
G3C G3C G3C
43 53 100 54 42 59 55 95 51 45 63 44 53 54 88
K.oxytoca ATCC 13182 T V~ron 51 M75 M99 MI04 M92 MilS M93
G4 G4 G4 G4 G4 G4 G4 G4
57 61 52 49 52 57 52 55 56 59
100 81 88 87 66 89 88
86 86 '.
85 81 86
69 66 82 100 89 65 61 92 90 90 90 89 87 87 86 86 86 86 84 83 82 82 79 78
82 58 42 33 61 52 57
65 63
54 62 51
61 57 53 58 53 49 53 52
62
78 I,
55 100 62 58 57 96 60 57 50 87 50 57 85 57 60 83 59 49 50 82 55 57 81 65 62 54
PAGE Source of unlabeled DNA a group b -----
----------
--------
" relative DNA biMlOg 10 labeled DNA from Kpn. Ea Ko 072 J22
----
---
K L
----
---
51
64
K.oxyloca MI14 MIO M37 EI72/68
G4 G38 b GSA
Klebsiella group I K70 T K47 KilO K24 K42 Kill K31 K26 K48 K33 KI20 K58 K43 K79 KI17 K44 K76
GSA GSA GSA GSA GSA GSA GSA GSA GSA GSA GSA GSA GSA GSA GSA GSA
56 48 65 100 86 56 59 56 84 84 84 65 84 83 80 80 79 78 75 49 75 74 73 57
GSB GSB GSB G5B G5B GSB G5B G5B GS6 GSB G5B G5B G5B G5B G5B G5B G5B GSB GSB GSB GSA GSA GSA GSA d d
48 60 57 61
Klebsiella group L L84 T L80 L66 L72 L98 L63 L81 L82 L95 L68 L102 L83 L94 L78 L69 L50 LI 13 L119 L27 L87 L67 L91 L28 L118 LI08 L101
-
54 54
95 82 63
50 82 65 65 63 69 68 68 65 63 70 68 74 61 60
62 64 67 77 62
100 99 99 97 96 96 96 95 95 95 Q5 94 94 93 93 89 88 88 87 87 97 87 69 77 63 67 94 40 18 41 46
58 71 57 50 68 54 54 59 56 54 51 69 63 59 43 56 59 58 58 56 71 51 61 71 86 59 58
Species name as received or group. Polyacrylamide gel electrophoresis group. K.pn. D72 = Klebsiella pneumoniae (Ferragut and Leclerc, 1978), gmup Gl. K.pn. J22 = Klebsiella pneunomiae (Izard et aI., 1980 a, b), group G3A. E.a. = Enterobacter aerogenes (K. m obilis) ATCC 13048 T, group G3C. K.o. = Klebsiella o;ytoca ATCC 13182T, group G4. K = Group K, K70 (Ferragut et aI., 1983), group GSA. L = Group L, L84 T (Izard et aI., 1981), group GSB.
a
b
-- ----
Table 2. Relative binding of DNA from 90 Klebsiella strains to 3H-DNA of the representative strains of Klebsiella species
Relationship among Klebsiella Strains Gr~up G5 contains 34 Klebsiella group L strains, 18 Klebsiella. group K, two Klebsiella spp., two K. pneumontae and two K. oxytoca strains. It is subdivided Ir:to two subgroups G5A and G5B represented by Klebsiella group. K a?d ~le~siel~a group L strains, respectively. The speCIes dIstnbutIOFI mto the minor groups is as follows: group a, two "K. rubiacearum" strains and one K. pneumoniae strain; group b, one K. pneumoniae, one K. oxytoca ~nd on~ K. ozaenae strain; group c, two K. pnel!'montae strams; group d, two Klebsiella group L strams.
DNA-DNA Hybridization
The DNA-DNA hybridization values measured between representative strains of the different Klebiella species and the strains of the protein electrophoretic groups are summarized in Table 2. ' . K. pneumoniae strains D 72 and] 22 have hybridizatIOns levels of over 78% with strains of the species K. pneumoniae, K. ozaenae and K. rhinoscleromatis except for strain~ D5~, D67, D37 and NCTC 9636 (33 to 61 %), 49 to 61 Yo wIth K. oxytoca, 49 to 57% with Klebsiella group K, 48 to 60% with Klebsiella group Land 43 to 55% with E. aerogenes (= K. mobilis). The three E. aerogenes (= K. mobilis) strains analyzed reveal a genetic homogeneity of 88 to 100%, reassociation percentages vary from 51 to 65% with K. pneumoniae, K. oxytoca shows 50 to 57% hybridization with strain ATCC 13048\ Klebsiella group K 48 to 56% and Klebsiella group L 40 to 57%. The reassociation percentages between K. oxytoca AT~C 13182T and eight strains belonging to the same speCIes vary from 81 to 96, strain E 172/68 received as K. ox~toca exhibits a lower percentage (63%). The hybridizatIOn levels of K. oxytoca ATCC 13182T with the other species are as follows: K. pneumoniae 62 to 63 % Klebsiella group K 59 to 65%; Klebsiella group L 56 t~ 61 % except for strain L 101 (18%), E. aerogenes (= K. mobilis) 51 to 54%. The DNA homology values within Klebsiella group K amount to at least 73 %. Hybridizations between strain K 70T and Klebsiella group L yield a degree of reassociation of 50 to 71 %, except for strains L 28, L 118 and L 101 (86%,77% and 41 %, respectively), K. pneumoniae shows 53% homology exept for strain NCTC 9636 (78%), K. oxytoca 50 to 65% except for strain E 172/68 (82%) and E. aerogenes (= K. mobilis) 42 to 45%. The relatedness levels within Klebsiella group L reach values, from 80 to 100% except for strains L 28, L 118 and L 101 (69%,63% and 46%, respectively). Klebsiella gr.oup L s~ows hybridization percentages of 60 to 74% wIth Klebsiella group K, 53 to 63% with E. aerogenes (= K: mobilis), 49 to 64% with K. oxytoca and 49 to 58% wIth K. pneumoniae. Discussion
The n~merical analysis of the protein electrophoregrams (FIg. 2) reveals that the clustering is correlated with
125
the origin of the Klebsiella strains. Indeed, strains of groups Gl to G4 originated mostly from the clinical environment, while strains of group G5 were isolated from water and unpolluted soils. This is in agreement with the phenotypic investigations of Gavini et al. (1976 a, 1976 b, 1977) and of Naemura et al. (1979) who mentioned the emergence of new groups in the genus Klebsiella when strains originating from different sources are examined. Strains of the species K. pneumoniae, K. ozaenae and K. r~inoscleromatis occur mainly in the protein electrophoretIc groups Gl, G2 G3A and G3B (Fig. 2). Electrophoresis of soluble proteins does not differentiate between these three species, as strains of the three species occur in almost any of the groups mentioned. We observe also in Table 2 a high degree of DNA hybridization (> 78 %) between DNA of strains D 72 (of group Gl) and] 22 (of group G3A) and strains belonging to groups G1, G2 and G3 (subgroup G3C excepted). This confirms the genomic relatedness of these three groups and agrees with the high DNA-DNA hybridization levels (> 80%) observed by Brenner et al. (1972) between the species K. pneumoniae, K. ozaenae a.nd K. rhinoscleromatis. Our study supports the classificatIOn proposed by 0rskov (1984). The majority of "K. edwardsii" and" K. aerogenes" strains also clustered into groups G2 a.n~ G3.B. Electrophoresis of soluble proteins does not dIstmgmsh them from K. pneumoniae, K. ozaenae or K. rhinoscleromatis. Moreover, Brenner et al. (1972) have shown hybridization levels of 80 and 81 % between pneumoniae and strains ATCC 13886T ("K. edwardsll" var. "edwardsii") and ATCC 13887T ("K. edwardsi~" ~ar. "~tlan~ae"), r~spectively. The phenotypic charactenstIcs whIch dlfferentlate between the species" K. edwardsii" and" K. aerogenes" do not seem to be significant. All these data indicate that the epithets "edwardsii" and "aerogenes" are obsolete. The protein electrophoretic group G.3 (except subgroup G3C) is thus representative of the speCIes K. pneumoniae. The type strain ATCC 13883 is a member of subgroup G3B. Subgroups G3A and G3B can be considered as electrotypes of K. pneumoniae. K. ozaenae and K. rhinoscleromatis should remain subspecies of K. pneumoniae as proposed by 0rskov (1984). Similarly, electrophoretic groups a, G1 and G2 are closely relat~d. to. K. pneumoniae as shown by the DNA-DNA hybndlzatIOns (Table 2). All the strains of the groups G1, G2, G3A, G3B and a form a true continuum. Subgroup G3C consists of three K mobilis strains and K. pneumoniae strain ATCC 10273. On the one hand, one notices the individuality of these four strains within group G3, wher~as on the other hand comparison of the protein patterns lmks them to the species K. pneumoniae (sub~roups G3A and G3B). However, DNA-DNA hybridizatIOns of.T~ble 2 clearly show the individuality of subgroup G3C wlthm the genus Klebsiella. The DNA relatedness v.alues within subgroup G3C are at least 88%. HybridizatIOns between DNA of the type strain of K. mobilis ATCC 13048 ~n~ five strains of K. pneumoniae yield degrees of reaSSOCIatIOn between 51 and 65%. In this case, the taxonomic resolution of the soluble protein electrophoregrams would be reduced by the limited number of K. mobilis strains examined. According to phenotypic and
.!<.
126
C. Ferragut, K. Kersters, and]. de Ley
genotypic data it is logical to consider K. pneumoniae and K. mobilis (synonym of E. aerogenes) as two distinct specIes. Protein electrophoretic group G4 includes 90% of the K. oxytoea strains analyzed, together with the type strain ATCC 13182. This group corresponds to group M of Gavini et al. (1977). DNA-DNA hybridization levels amongst the strains of this group amount to at least 81 % (Table 2) except for strain E 172/68 (63%); they are lower between K. oxytoea ATCC 13182T and the strains of groups G1 (57%), G3A (63%), G3B (62%), G3C (51 to 54%), G5A (59 to 65%) and G5B (18 to 61 %). The high degrees of reassociation (Table 2) observed between K. oxytoea ATCC 13182T and strains M 37 and M 10 (members of electrophoretic groups band G3B, respectively) imply that these two strains should be classified in the species K. oxytoea (Table 2). The delineation of electrophoretic group G4 allows the integration of the following four Klebsiella strains in K. oxytoea : Klebsiella spp. E 522, E 685 and E 591 and "K. edwardsii" E 479. Our DNA-DNA hybridization results confirm almost completely the results of the numerical analysis of the electrophoretic protein patterns and strenghten the independance of K. oxytoea versus K. pneumoniae. Klebsiella groups K and L described by Gavini et al. (1977) correspond in our study to electrophoretic groups G5A and G5B, respectively. Ferragut et al. (1983) and Izard et al. (1981) have previously presented monographs on these two groups and proposed the creation of two new species K. trevisanii and K. terrigena. The DNA reassociation of K. trevisanii K 70 and K. planticola ClP 100.751 with DNAs of all the other known Klebsiella species indicate that these two names are synonyms ;md priority belongs to K. plantieola (Gavini et al., 1986). Strains L 28 and L 118, which are previously classified in group L of Gavini et al. (1977), K. oxytoea E 172/68 and K. pneumoniae NCTC 9636 show high hybridization levels with K. planticola K 70 (86%, 77%, 82% and 78%,
respectively), (Table 2) indicating that these strains belong genotypically to K. planticola. According to the electrophoretic analysis the following strains grouping in G5A should also be included in K. plantieola : Klebsiella spp. E. 557 and T 28/66, K. pneumoniae D 37 and K. oxytoea 182934/68. It should be noted that a high number of K. planticola strains are indole positive, a characteristic which was previously exclusively confined to K. oxytoea. On the other hand, visual inspection of the normalized photographs (Izard et al., 1981) and DNA-DNA hybridization data (Table 2) justifies the classification of strains L 67, L 91 (members of group G5A) and L 108 (member of group d) into the species K. terrigena (group G5B). In summary, our protein electrophoretic and DNADNA hybridization data confirm that the genus Klebsiella is composed of the following five species: K. pneumoniae, K. oxytoea, K. mobilis, (= E. aerogenes) K. terrigena and K. plantieola and three subspecies K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis. The phenotypic characteristics allowing the differentiation of these species are summarized in Table 3. Growth at 4°C differentiates the species K. mobilis, K. terrigena and K. plantieola. K. terrigena does not grow at 41°C. K. mobilis, K. oxytoca and K. terrigena utilize m-hydroxybenzoate as carbon source. K. pneumoniae subsp. rhinoscleromatis and K. mobilis yield negative reactions in the urease test. K. oxytoca and a number of K. planticola strains produce indole. Acknowledgements. The authors thank E. Dewailly, D. Dewettinck, and U. Torck for their technical assistance.
References Bagley, S. T., Seidler,R. j., Brener, D. j.: Klebsiella planticola sp. nov.: a new species of Enterobacteriaceae found primarily in
Table 3. Phenotypic characteristics useful for the differentiation between Klebsiella species (0rskov, 1984, Izard et al. 1981, Ferragut et al., 1983) Test
K. pneumoniae subsp.
pneumoniae Growth at 4 °C Growth at 41°C Presence of: Urease j3-Galactosidase Indole production Voges-Proskauer reaction m-Hydroxybenzoate as carbon source Histamine as carbon source a
subsp.
ozaenae
+
rhinoscleromatis
d +
K. terrigena
K.planticola
+
+ or (+) +
+
+ +
+ + +
+ + +
+ + - or + + - or + +
K.oxytoca K.mobilis
+
+ + +
subsp.
+ + + + +
(+) +
symbols: +, Positive re;!ction for at least 90% of the strains within 24 to 48 h; -, negative reaction for at least 90% of the strains within 24 to 48 h; (+), slow positive reaction; + or (+), - or +, the first sign corresponds to the most frequent result; d, different biochemical types (+, (+), -).
Relationship among Klebsiella Strains non clinical environments. Curr. Microbiol. 6, 105-109 (1981) Bascomb, S., Lapage, S. P., Willcox, W. R., Curtis, M. A.: Numerical classification of the tribe Klebsielleae. J. Gen. MicrobioI. 66, 279-295 Brenner, D. ].,Steigerwalt, A. G., Fanning, G. R.: Differentiation of Enterobacter aerogenes from Klebsiella by deoxyribonucleic acid reassociation. Int. J. System. Bact. 22, 193-200 (1972) Cowan, S. T., Steel, K. ]., Shaw, C. A.: Classification of the Klebsiella group. J. Gen. Microbiol. 23, 601-612 (1960) De Ley, ]., Tijtgat, R.: Evaluation of membrane filter methods for DNA-DNA hybridization. Antonie v. Leeuwenhoek 36, 461-474 (1970) Ferragut, c., Leclerc, H.: Characterization of motile and acetoinnegative Klebsiella pneumoniae strains by DNA:DNA hybrydization. Antonie v. Leeuwenhoek 44, 407-424 (1978) Ferragut, c., Izard, D., Gavini, G., Kersters, K., De Ley, ]., Leclerc, H.: Klebsiella trevisanii: a new species from water and soil. Int. J. System. Bact. 33, 133-142 (1983) Gavini, F., Lefebvre, B., Leclerc, H.: Positions taxonomiques d'enterobacteries HzS- par rapport au genre Citrobacter. Ann. lnst. Pasteur (Paris) 127 A, 275-295 (1976 a) Gavini, F., Ferragut, c., Lefebvre, B., Leclerc, H.: Etude taxonomique d'enterobacteries appartenant ou apparentees au genre Enterobacter. An. lnst. Pasteur (Paris) 127 B, 317-335 (1976b) Gavini, F., Leclerc, H., Lefebvre, B., Ferragut, c., Izard, D.: Etude taxonomique d'enterobacteries appartenant ou apparentees au genre Klebsiella. Ann. lnst. Pasteur (Paris) 128 B, 45-59 (1977) Gavini, F., Izard, D., Grimont, P. A. D., Beji, A., Ageron, E., Leclerc, H.: Priority of K1ebsiella planticola Bagley, Seidler and Brenner 1982 over Klebsiella trevisanii Ferragut,' Izard, Gavini, Kersters, De Ley and Leclerc. 1983. Int. J. System. Bact. 36, 486-488 (1986) Izard, D., Gavini, F., Leclerc, H.: Polynucleotide sequence
127
relatedness and genome size among Enterobacter intermedium sp. nov. and the species Enterobacter cloacae and Klebsiella pneumoniae. Zbl. Bakt., I. Abt. Orig. C 1, 51-60 (1980 a) Izard, D., Gavini, F., Trinel, P. A., Krubwa, F., Leclerc, H.: Contribution of DNA-DNA hybridization to the transfer of Enterobacter aerogenes to the genus Klebsiella as K. mobilis. Zbl. Bakt. Hyg., 1. Abt. Orig. C 1, 257-263 (1980 b) Izard, D., Ferragut, c., Gavini, F., Kersters, K., De Ley, J., Leclerc, H.: Klebsiella terrigena, a new species from soil and water. Int. J. System. Bact. 31, 116-127 (1981) Johnson, R., Colwell, R. R., Sakazaki, R., Tamura, K.: Numerical taxonomy study of the Enterobacteriaceae. Int. J. System. Bact. 25, 13-37 (1975) Kersters, K., De Ley, ].: Identification and grouping of bacteria by numerical analysis of their electrophoretic protein patterns. J. Gen. Microbiol. 87, 333-342 (1975) Marmur, ].: A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Molec. BioI. 3, 208-218 (1961) Naemura, L. G., Bagley, S. T., Seidler, R. ]., Kaper, ]. B., Colwell, R. R .: Numerical taxonomy of Klebsiella pneumoniae strains isolated from clinical and non clinical sources. Curro Microbiol. 2, 175-180 (1979) 0rskov, I.: Genus V. Klebsiella Trevisan 1885, pp.461-465. In: Bergey's Manual of Systematic Bacteriology (N. R. Krieg and J. G. Holt, eds.) Baltimore-London, Williams and Wilkins Co. 1984 Sakazaki, R., Tamura, K., Johnson, R., Colwell, R. R.: Taxonomy of some recently described species in the family Enterobacteriaceae. Int. J. System. Bact. 26, 158-179 (1976) Skerman, V. B. D., Mac Gowan, V., Sneath, P. H. A.: Approved lists of bacterial names. Int. J. System. Bact. 30, 225-420 (1980) Slopek, S., Durlakowa, I.: Studies on the taxonomy of Klebsiella bacilli. Arch. Immunol. Ther. Exp. 15,481-487 (1967) Sneath, P. H. A., Sokal, R. R.: Numerical Taxonomy. San Francisco, Freeman and Co. 1973
Carmen Ferragut, Institut National de la Sante et de la Recherche medicale, Unite 146, Domaine du Centre d'Enseignement et de Recherches Techniques en Industrie Alimentaire, 369, rue Jules Guesde, B.P. 39,59651 Villeneuve-d'Ascq Cedex, France
Protein Electrophoretic and DNA Homology Analysis of Klebsi-ella Strains C. FERRAGUT,,2, K. KERSTERS" and,]. DE LEY' 1 2
Laboratorium voor Microbiologie en microbiele Genetica, Faculteit Wetenschappen, Rijksuniversiteit, Gent, Belgium, and Institut National de la Sante et de la Recherche medicale, Unite 146, Domaine du Centre d'Enseignement et de Recherches Techniques en Industrie Alimentaire, 59651 Villeneuve-d' Ascq Cedex, France Received August 20, 1988
Summary A total of 175 clinical and non-clinical isolates of Klebsiella were subjected to numerical analysis of electrophoretic patterns of their soluble proteins. Of these strains, 160 clustered into five major groups, ten clustered in groups of two or three strains, and five strains remained ungrouped. Selected strains of the major groups were analyzed by DNA hybridization competition experiments. Almost all strains of the species K. pneumoniae, K. ozaenae and K. rhinoscleromatis occurred in three different electrophoretic groups. The high degree of hybridization obtained between DNA from these strains confirmed their genotypic relatedness and supported the classification proposed by 0rskov. Another group was designated as K. mobilis as its strains shared reassociation values of 88 to 100% with the type strain of K. mobilis. The majority of the remaining strains were clustered in three electrophoretic groups which correlated well with DNA hybridization and phenotypic studies. These were identified as K. oxytoca, K. planticola and K. terrigena.
Key words: Taxonomy - Protein electrophoresis - Numerical analysis - DNA-DNA hybridization - Klebsiella
Introduction
Among the members of the family of the Enterobacteriaceae, the classification of the genus Klebsiella is still controversial. Phenotypical criteria, numerical analysis of phenotypic features and genomic data have lead to different-points of view. Cowan et al. (1960) and Slopek and Durlakowa (1967) subdivided the genus Klebsiella into the following species: K. ozaenae, K. rhinoscleromatis, "K. aerogenes", K. pneumoniae sensu stricto and "K. edwardsii" (var. "edwardsii" and var. "atlantae"), while Johnson et al. (1975) and Sakazaki et al. (1976) recognized three species: K. pneumoniae, K. ozaenae and K. rhinoscleromatis. 0rskov (1984) proposed a different scheme: K. pneumoniae, K. oxytoca, K. terrigena and K. planticola. Because K. pneumoniae, K. rhinoscleromatis and K. ozaenae belong to the same genotypic group (Brenner et aI., 1972), they are now considered as subspecies of K. pneumoniae (0rskov, 1984). K. oxytoca is separated from K. pneumoniae on the basis of indole production, hydrolysis of gelatin and
degradation of polygalacturonate. The present definition of the genus is restricted to non-motile strains, although a close biochemical (Bascomb et aI., 1971) and genotypic (Izard et aI., 1980 b) relationship was observed between Enterobacter aerogenes and the species of Klebsiella. These findings have lead to proposals to transfer E. aerogenes to the genus Klebsiella under the epithet mobilis (Bascomb et aI., 1971; Izard et aI., 1980 b). Both names E. aerogenes and K. mobilis occur on the Approved Lists (Skerman et aI., 1980) with identical type strains. K. terrigena (Izard et aI., 1981) (= group L of Gavini et aI., 1977) and K. planticola (Bagley et aI., 1981) (= Klebsiella sp. 2, Naemura et aI., 1979) are two new species which emerged from the numerical analysis of the phenotypic features of strains isolated from the environment. K. trevisanii, described by Ferragut et al. (1983), belongs to the group K of Gavini et al. (1977) and was later found to be a synonym of K. planticola (Gavini et aI., 1986).
122
C. Ferragut, K. Kersters, and J. de Ley
Considering the different points of view on the taxonomy of the genus Klebsiella, we submitted 175 Klebsiella strains from hospital and environmental origin to a numerical analysis of the electrophoregrams of their soluble proteins. Our aim was to examine the protein electrophoretic groups formed, to study their degree of homogeneity and relationships, and to verify the correspondence with the recognized species. Additional information on the genotypic relationships of the protein electrophoretic groups was obtained through DNA-DNA hybridization experiments on selected strains.
Material and Methods Bacterial strains and media. The 175 strains used in this study are listed in Table 1. For the preparation of DNA, cells were grown in nutrient broth at 30°C during 24 h with shaking. For the electrophoresis of soluble proteins, cells were grown on nutrient agar (Oxoid Ltd., London, England) in Raux flasks at 37°C during 16 h. Polyacrylamide gel electrophoresis of soluble proteins. Cellfree extracts were prepared by the method of Kersters and De Ley (1975) as modified by Izard et al. (1981). Polyacrylamide gel electrophoresis, densitometry, photography of the stained gels
Table. 1. Name of the Klebsiella strains studied. Specl •• (namt H rectlved
or grGIC)
------------_.... -_ .. ------
Strain n· (a)
Electrophorttlc
Klebsiella pne...onlae
ATCC25955 ATCC 27n7 CDC 12t33 NCTC9636 NCTC 9637 Vfron 39 Viron «I Yfron 41 Yiron 42 J31b) J4 J5 J7 J8 J9 JII .lIS JI6 JI7 JI8 J20 J21 J22 J25 .l32 J 34 J38 J41 JS3 J56 J59 J61 J62 J77 030 (bl 037 053 055 063 064 067 072 076
Klebsiella -aerogenes-
E 493 Ib) NCTC 243 NCTCBt46 HCTC 9793
G3B G2 G38
Kiebsl.lIa -edWards!!" var. ·edwardSW
ATCC 13886
G2
Klebsiella ·edWards!!"
E 479 E 50S NCTC 9496 T NeTC 9498
64 GJ8 6JB G38
Klebsiella oxyloca
E 352 E 172/68 .Jain 25871168 Jaln182934/61 Viron 53 V~ron 54 M 101b) MJO M37 M 75 M92 M93 M99 M 104 M 105 M 106 M 114 MilS
64 GSA G4 GSA G4 G4 G4 G3B G4 b G4 G4 G4 G4 G4 G4 G4 G4 G4
ATCC 11297 ATCC 25926 ATCC 29019 coe 276-71 CL.(TN 78-293 E 549 NCTC 5050 T V~ron 47
G3B 62 G38 G2 638 b G2 G38
ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATeC ATCC ATCe ATCC
G2 GlB G2 G3B G2 G38 G2 G3B G3B GlC G36 G38 G3B G3B a
Klebsiella poe .... nla.
4208 4352 4368 4727 65«1 8048 8050 9997 10031 10273 12657 IJBt2 13tBl T 14304 15574 21210
group
~'C
GJC G3C
Klebslell.ozaen.e
ElectraphorttlC
-----_ ..... _- ....... .. ... _----... -- ..... - ....
ATCC 130411 T CI.(1I"178-186 CI.(1I"1 79-235
V~ronSI
Strain n· (a)
or VOI4I ----- ....................... ------- ..
EnterolllCter aerogene. 10 Klebsiella mobilis)
var. -atJantae&
Specl" (namt H rectlved
grGIC)
.,------------ .. -- .. --_ ..... __ .. ---
-
-
"
GJ8 63B
GSA
G2 G3B G3B b GI G38 61 G38 GI 63A 61 G38 G38 GJ8 G38 GI G38 G3A G38 63A G3A G38 G3B G3B G3A G38 G38 G38 G38 GJ8 GSA
c
G38 G38 G3B c 61 G3a
ATCC 13182 T ATCC 13183
G4 G4
KI.bslella rhlnoscleromat 15
ATCC 6908 ATCC 9436 ATCC 13884 T COt B95-68 NCTC 5047 HCTC66« NCTC 1027l Vlron 46
GI G3A GI GJA G3A G3A G3A G3A
Klebsiella SilI>,
• E 522 E 557 E 591 E 685
or VOI4I ....... _-_ .......... - ..... --------- ..
G4 GSA G4 G4
Strain O· la) ElectrophortllC
group
---- ..---- .. --- .. --------------
Klebsiella SlIP.
£739 NCTC91JO NCTC 9170 T 28186 (b) T 37167 T 45/67 T 46/67
Klebsiella group K
K 241b) K26 01 K 33
K42
K 43 K 44 K 47 K 48 K58 K 70 T K 76 K 79 KilO Kill K 117 K 120
a
Klebsiella pneumonloe blolype oxytoca
Klebsiella Nblleeo.,.m
Sptcl" (namt H rtctlvtd
Klebsiella gro"" L
L 27 (bl L 28 L 45 L 49 L 50 L 52 L 63 L66 L 67 L 66 L 69 L72 L 73 L 74 L 76 L 80 L 81 L 82 L 83 L 84 L 8S L 86 L 87 L 89 L 91 L 94
195
L 97 L 98 L 101 L 102 ll08 ll09 lll2 l III l liB L 119
-
GJ8 G3B GSA G3B G3B G3B GSA GSA GSA GSA GSA GSA GSA GSA GSA GSA GSA
-
GSA GSA GSA GSA GSA G5B GSA GSa Gsa G58 G58 GSB G5B GSA GSB GSB GS8 GSB G58 GS8 G5B GS8 G5B G5B GSB G58 GSa G5B GS8 GSA GSB GSB G5B GSB 0
GS8 d G5B G5B G5B GSA G5B
(a) Abbreviations: ATCC, American Type Culture Collection, Rockville, Md., U.S.A.; CDC, Center for Disease Control, Atlanta, Ga., U.S.A.; CUETM, Collection de l'Unite d'Exotoxicologie Microbienne, Villeneuve d'Ascq, France; Jain, K. Jain, Hygiene-Institut der Philipps-Universitiit, MarburgiLahn, Federal Republic of Germany; NCTC, National Collection of Type Cultures, London, England; Veron, M. Veron, Institut Pasteur, Paris, France. (b) Strain numbers beginning with the character D, J, K. Lor M are strain numbers from Gavini et al. (1976 a, b; 1977), with the character E or T are strain numbers from Bascomb et al. (1971).
Relationship among Klebsiella Strains were described previously (Kersters and De Ley, 1975). Each protein extract was examined in at least three different electrophoretic experiments. The normalized densitometric tracings were converted into a sequence of 120 numbers, representing the optical density (expressed as height in millimeters) of each position of the scan. The Pearson product moment correlation coefficient (r), between any pair of densitometric tracings of protein patterns was calculated (Sneath and Sokal, 1973), and clustering was performed by the unweighted average pair group method (UPGMA) (Sneath and Sokal, 1973) using a Siemens model 7755 (BS 2000) computer of the Centraal Digitaal Rekencentrum, RUG, Gent. DNA-DNA hybridization. The following strains were chosen for the preparation of DNA labeled with eH)-thymidine: the type strains of K. mobilis (= Enterobacter aerogenes) ATCC 13048, K. oxytoca ATCC 13182 and K. terrigena (= Klebsiella group L) L 84 (ATCC 33257); the phenotypic centrotypes D 72 and J 22 of K. pneumoniae (Gavini et ai., 1976 a, 1977) and the protein electrophoretic centrostrain K 70 of K. planticola (= K. trevisanii = Klebsiella group K) (Ferragut et aI., 1983). Labeled DNA was prepared according to the method of Marmur (1961) as modified by Ferragut and Leclerc (1978) . Hybridizations were carried out by the competition method used by De Ley and Tijtgat (1970).
,
1
40 j
123
o
I
I< 70 T K 58
(I< 42 Fig. 1. Examples of Klebsiella strains of electrophoretic group G5A (Klebsiella group K) displaying visual similarities in their protein patterns, except for a heavy protein band located at 17 mm from the top of the gel.
Results Comparison of electrophoregrams of the soluble proteins
The reproducibility of the technique was studied by comparing 55 electrophoregrams of ten strains belonging to representative species of the genus Klebsiella. The reproducibility limits were always above r = 0.95 for patterns of the same extract. The most typical protein pattern for each strain was selected, and used in the final numerical analyses. In a preliminary computer analysis, s<;>me isolates belonging to different electrophoretic clusters displayed visually very similar protein patterns, except that a heavy protein band occurred at 17 mm of the top of the gel in some of these strains. Some typical examples of such patterns are shown in Fig. 1. In order to prevent the formation of artificial groupings, the optical densities corresponding to the position of this heavy protein band (i. e. positions 35 to 37) were eliminated from the final numerical analysis of 175 Klebsiella strains. The results are represented in a simplified dendrogram (fig. 2). Above a clustering level of r = 0.87, 160 strains formed five major groups (G1 to G5), ten strains clustered in groups of two or three (groups a, b, c and d) and five strains remained ungrouped. Below this r value, the linkage levels are taxonomically not significant. Group G 1 contains the type strains of K. rhinoscleroma tis ATCC 13884 besides one strain of the same species and six K. pneumoniae strains. Group G2 contains four strains of K. ozaenae including the type strain NCTC 5050, five strains of K. pneumoniae, one strain of "K. aerogenes" and the type strain of "K. edwardsii" var. "edwarsii'!· ATCC 13886. Group G3 consists of 64 strains amongst which 39 belong to K. pneumoniae, including the type strain ATCC
.J
.•
o. I OUt
At~·.' • I
I • I
I
I
All
:'I,,:AI,_. IIHII '
IIUS I S
UUUIUA U
ILIIIIIUA U
02
I
• I
, l
0 •
o.
Fig. 2. Simplified dendrogram showing the relationships among protein electrophoretic groups based on the UPGMA analysis.
124
C. Ferragut, K. Kersters, and J. de Ley
scleromatis and K. ozaenae whereas subgroup G3C contains the three K. mobilis strains. Most K. oxytoca strains, amongst them type strain ATCC 13182, are integrated into group G4. Moreover the latter group includes three strains received as Klebsiella spp. and one "K. edwardsii" var. "atlantae" strains.
13883. Group G3 includes also seven K. rhinoscleromatis, three "K. edwardsii" var. "atlantae", five Klebsiella spp. four K. ozaenae, two "K. aerogenes", one K. oxytoca and three K. mobilis strains. G3 is subdivided into three subgroups A, Band C. Subgroups G3A and G3B are ess~n tially represented by strains of K. pneumoniae, K. rhino-
PAGE Source of unlabeled DNA a ~roup b
" relative DNA binding to labeled DNA from Kpn. E.a. Ko 072 J22
--------------- -------- ----
--- --- ---
K L ----
---
Kpneumonlae 072 J7 J9 J21 J22 J8 J56 JJ2 Veron 40 ATCC 13883 T ATCC 13882 J61 J25 J77 030 JI6 JI5 J3 J20 JII J59 076 064 063 J3B 055 053 067 037 NCTC 9636
GI GI GI a G3A G3A G3A G3A G3B G38 G3B G3B G3B G3B G3B G3B G3B G3B G36 G36 G3B G3B G3B G3B G3B G3B c c G5A G5A
Kozaenae CDC 276-71
G2
82 85
K.rnlnoscleromatl CDC 895-68
G3A
83
E.aerogenes (-K.mobills) ATCC 13048 T CUETM 78- 186 CUETM 79-235
G3C G3C G3C
43 53 100 54 42 59 55 95 51 45 63 44 53 54 88
K.oxytoca ATCC 13182 T V~ron 51 M75 M99 MI04 M92 MilS M93
G4 G4 G4 G4 G4 G4 G4 G4
57 61 52 49 52 57 52 55 56 59
100 81 88 87 66 89 88
86 86 '.
85 81 86
69 66 82 100 89 65 61 92 90 90 90 89 87 87 86 86 86 86 84 83 82 82 79 78
82 58 42 33 61 52 57
65 63
54 62 51
61 57 53 58 53 49 53 52
62
78 I,
55 100 62 58 57 96 60 57 50 87 50 57 85 57 60 83 59 49 50 82 55 57 81 65 62 54
PAGE Source of unlabeled DNA a group b -----
----------
--------
" relative DNA biMlOg 10 labeled DNA from Kpn. Ea Ko 072 J22
----
---
K L
----
---
51
64
K.oxyloca MI14 MIO M37 EI72/68
G4 G38 b GSA
Klebsiella group I K70 T K47 KilO K24 K42 Kill K31 K26 K48 K33 KI20 K58 K43 K79 KI17 K44 K76
GSA GSA GSA GSA GSA GSA GSA GSA GSA GSA GSA GSA GSA GSA GSA GSA
56 48 65 100 86 56 59 56 84 84 84 65 84 83 80 80 79 78 75 49 75 74 73 57
GSB GSB GSB G5B G5B GSB G5B G5B GS6 GSB G5B G5B G5B G5B G5B G5B G5B GSB GSB GSB GSA GSA GSA GSA d d
48 60 57 61
Klebsiella group L L84 T L80 L66 L72 L98 L63 L81 L82 L95 L68 L102 L83 L94 L78 L69 L50 LI 13 L119 L27 L87 L67 L91 L28 L118 LI08 L101
-
54 54
95 82 63
50 82 65 65 63 69 68 68 65 63 70 68 74 61 60
62 64 67 77 62
100 99 99 97 96 96 96 95 95 95 Q5 94 94 93 93 89 88 88 87 87 97 87 69 77 63 67 94 40 18 41 46
58 71 57 50 68 54 54 59 56 54 51 69 63 59 43 56 59 58 58 56 71 51 61 71 86 59 58
Species name as received or group. Polyacrylamide gel electrophoresis group. K.pn. D72 = Klebsiella pneumoniae (Ferragut and Leclerc, 1978), gmup Gl. K.pn. J22 = Klebsiella pneunomiae (Izard et aI., 1980 a, b), group G3A. E.a. = Enterobacter aerogenes (K. m obilis) ATCC 13048 T, group G3C. K.o. = Klebsiella o;ytoca ATCC 13182T, group G4. K = Group K, K70 (Ferragut et aI., 1983), group GSA. L = Group L, L84 T (Izard et aI., 1981), group GSB.
a
b
-- ----
Table 2. Relative binding of DNA from 90 Klebsiella strains to 3H-DNA of the representative strains of Klebsiella species
Relationship among Klebsiella Strains Gr~up G5 contains 34 Klebsiella group L strains, 18 Klebsiella. group K, two Klebsiella spp., two K. pneumontae and two K. oxytoca strains. It is subdivided Ir:to two subgroups G5A and G5B represented by Klebsiella group. K a?d ~le~siel~a group L strains, respectively. The speCIes dIstnbutIOFI mto the minor groups is as follows: group a, two "K. rubiacearum" strains and one K. pneumoniae strain; group b, one K. pneumoniae, one K. oxytoca ~nd on~ K. ozaenae strain; group c, two K. pnel!'montae strams; group d, two Klebsiella group L strams.
DNA-DNA Hybridization
The DNA-DNA hybridization values measured between representative strains of the different Klebiella species and the strains of the protein electrophoretic groups are summarized in Table 2. ' . K. pneumoniae strains D 72 and] 22 have hybridizatIOns levels of over 78% with strains of the species K. pneumoniae, K. ozaenae and K. rhinoscleromatis except for strain~ D5~, D67, D37 and NCTC 9636 (33 to 61 %), 49 to 61 Yo wIth K. oxytoca, 49 to 57% with Klebsiella group K, 48 to 60% with Klebsiella group Land 43 to 55% with E. aerogenes (= K. mobilis). The three E. aerogenes (= K. mobilis) strains analyzed reveal a genetic homogeneity of 88 to 100%, reassociation percentages vary from 51 to 65% with K. pneumoniae, K. oxytoca shows 50 to 57% hybridization with strain ATCC 13048\ Klebsiella group K 48 to 56% and Klebsiella group L 40 to 57%. The reassociation percentages between K. oxytoca AT~C 13182T and eight strains belonging to the same speCIes vary from 81 to 96, strain E 172/68 received as K. ox~toca exhibits a lower percentage (63%). The hybridizatIOn levels of K. oxytoca ATCC 13182T with the other species are as follows: K. pneumoniae 62 to 63 % Klebsiella group K 59 to 65%; Klebsiella group L 56 t~ 61 % except for strain L 101 (18%), E. aerogenes (= K. mobilis) 51 to 54%. The DNA homology values within Klebsiella group K amount to at least 73 %. Hybridizations between strain K 70T and Klebsiella group L yield a degree of reassociation of 50 to 71 %, except for strains L 28, L 118 and L 101 (86%,77% and 41 %, respectively), K. pneumoniae shows 53% homology exept for strain NCTC 9636 (78%), K. oxytoca 50 to 65% except for strain E 172/68 (82%) and E. aerogenes (= K. mobilis) 42 to 45%. The relatedness levels within Klebsiella group L reach values, from 80 to 100% except for strains L 28, L 118 and L 101 (69%,63% and 46%, respectively). Klebsiella gr.oup L s~ows hybridization percentages of 60 to 74% wIth Klebsiella group K, 53 to 63% with E. aerogenes (= K: mobilis), 49 to 64% with K. oxytoca and 49 to 58% wIth K. pneumoniae. Discussion
The n~merical analysis of the protein electrophoregrams (FIg. 2) reveals that the clustering is correlated with
125
the origin of the Klebsiella strains. Indeed, strains of groups Gl to G4 originated mostly from the clinical environment, while strains of group G5 were isolated from water and unpolluted soils. This is in agreement with the phenotypic investigations of Gavini et al. (1976 a, 1976 b, 1977) and of Naemura et al. (1979) who mentioned the emergence of new groups in the genus Klebsiella when strains originating from different sources are examined. Strains of the species K. pneumoniae, K. ozaenae and K. r~inoscleromatis occur mainly in the protein electrophoretIc groups Gl, G2 G3A and G3B (Fig. 2). Electrophoresis of soluble proteins does not differentiate between these three species, as strains of the three species occur in almost any of the groups mentioned. We observe also in Table 2 a high degree of DNA hybridization (> 78 %) between DNA of strains D 72 (of group Gl) and] 22 (of group G3A) and strains belonging to groups G1, G2 and G3 (subgroup G3C excepted). This confirms the genomic relatedness of these three groups and agrees with the high DNA-DNA hybridization levels (> 80%) observed by Brenner et al. (1972) between the species K. pneumoniae, K. ozaenae a.nd K. rhinoscleromatis. Our study supports the classificatIOn proposed by 0rskov (1984). The majority of "K. edwardsii" and" K. aerogenes" strains also clustered into groups G2 a.n~ G3.B. Electrophoresis of soluble proteins does not dIstmgmsh them from K. pneumoniae, K. ozaenae or K. rhinoscleromatis. Moreover, Brenner et al. (1972) have shown hybridization levels of 80 and 81 % between pneumoniae and strains ATCC 13886T ("K. edwardsll" var. "edwardsii") and ATCC 13887T ("K. edwardsi~" ~ar. "~tlan~ae"), r~spectively. The phenotypic charactenstIcs whIch dlfferentlate between the species" K. edwardsii" and" K. aerogenes" do not seem to be significant. All these data indicate that the epithets "edwardsii" and "aerogenes" are obsolete. The protein electrophoretic group G.3 (except subgroup G3C) is thus representative of the speCIes K. pneumoniae. The type strain ATCC 13883 is a member of subgroup G3B. Subgroups G3A and G3B can be considered as electrotypes of K. pneumoniae. K. ozaenae and K. rhinoscleromatis should remain subspecies of K. pneumoniae as proposed by 0rskov (1984). Similarly, electrophoretic groups a, G1 and G2 are closely relat~d. to. K. pneumoniae as shown by the DNA-DNA hybndlzatIOns (Table 2). All the strains of the groups G1, G2, G3A, G3B and a form a true continuum. Subgroup G3C consists of three K mobilis strains and K. pneumoniae strain ATCC 10273. On the one hand, one notices the individuality of these four strains within group G3, wher~as on the other hand comparison of the protein patterns lmks them to the species K. pneumoniae (sub~roups G3A and G3B). However, DNA-DNA hybridizatIOns of.T~ble 2 clearly show the individuality of subgroup G3C wlthm the genus Klebsiella. The DNA relatedness v.alues within subgroup G3C are at least 88%. HybridizatIOns between DNA of the type strain of K. mobilis ATCC 13048 ~n~ five strains of K. pneumoniae yield degrees of reaSSOCIatIOn between 51 and 65%. In this case, the taxonomic resolution of the soluble protein electrophoregrams would be reduced by the limited number of K. mobilis strains examined. According to phenotypic and
.!<.
126
C. Ferragut, K. Kersters, and]. de Ley
genotypic data it is logical to consider K. pneumoniae and K. mobilis (synonym of E. aerogenes) as two distinct specIes. Protein electrophoretic group G4 includes 90% of the K. oxytoea strains analyzed, together with the type strain ATCC 13182. This group corresponds to group M of Gavini et al. (1977). DNA-DNA hybridization levels amongst the strains of this group amount to at least 81 % (Table 2) except for strain E 172/68 (63%); they are lower between K. oxytoea ATCC 13182T and the strains of groups G1 (57%), G3A (63%), G3B (62%), G3C (51 to 54%), G5A (59 to 65%) and G5B (18 to 61 %). The high degrees of reassociation (Table 2) observed between K. oxytoea ATCC 13182T and strains M 37 and M 10 (members of electrophoretic groups band G3B, respectively) imply that these two strains should be classified in the species K. oxytoea (Table 2). The delineation of electrophoretic group G4 allows the integration of the following four Klebsiella strains in K. oxytoea : Klebsiella spp. E 522, E 685 and E 591 and "K. edwardsii" E 479. Our DNA-DNA hybridization results confirm almost completely the results of the numerical analysis of the electrophoretic protein patterns and strenghten the independance of K. oxytoea versus K. pneumoniae. Klebsiella groups K and L described by Gavini et al. (1977) correspond in our study to electrophoretic groups G5A and G5B, respectively. Ferragut et al. (1983) and Izard et al. (1981) have previously presented monographs on these two groups and proposed the creation of two new species K. trevisanii and K. terrigena. The DNA reassociation of K. trevisanii K 70 and K. planticola ClP 100.751 with DNAs of all the other known Klebsiella species indicate that these two names are synonyms ;md priority belongs to K. plantieola (Gavini et al., 1986). Strains L 28 and L 118, which are previously classified in group L of Gavini et al. (1977), K. oxytoea E 172/68 and K. pneumoniae NCTC 9636 show high hybridization levels with K. planticola K 70 (86%, 77%, 82% and 78%,
respectively), (Table 2) indicating that these strains belong genotypically to K. planticola. According to the electrophoretic analysis the following strains grouping in G5A should also be included in K. plantieola : Klebsiella spp. E. 557 and T 28/66, K. pneumoniae D 37 and K. oxytoea 182934/68. It should be noted that a high number of K. planticola strains are indole positive, a characteristic which was previously exclusively confined to K. oxytoea. On the other hand, visual inspection of the normalized photographs (Izard et al., 1981) and DNA-DNA hybridization data (Table 2) justifies the classification of strains L 67, L 91 (members of group G5A) and L 108 (member of group d) into the species K. terrigena (group G5B). In summary, our protein electrophoretic and DNADNA hybridization data confirm that the genus Klebsiella is composed of the following five species: K. pneumoniae, K. oxytoea, K. mobilis, (= E. aerogenes) K. terrigena and K. plantieola and three subspecies K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis. The phenotypic characteristics allowing the differentiation of these species are summarized in Table 3. Growth at 4°C differentiates the species K. mobilis, K. terrigena and K. plantieola. K. terrigena does not grow at 41°C. K. mobilis, K. oxytoca and K. terrigena utilize m-hydroxybenzoate as carbon source. K. pneumoniae subsp. rhinoscleromatis and K. mobilis yield negative reactions in the urease test. K. oxytoca and a number of K. planticola strains produce indole. Acknowledgements. The authors thank E. Dewailly, D. Dewettinck, and U. Torck for their technical assistance.
References Bagley, S. T., Seidler,R. j., Brener, D. j.: Klebsiella planticola sp. nov.: a new species of Enterobacteriaceae found primarily in
Table 3. Phenotypic characteristics useful for the differentiation between Klebsiella species (0rskov, 1984, Izard et al. 1981, Ferragut et al., 1983) Test
K. pneumoniae subsp.
pneumoniae Growth at 4 °C Growth at 41°C Presence of: Urease j3-Galactosidase Indole production Voges-Proskauer reaction m-Hydroxybenzoate as carbon source Histamine as carbon source a
subsp.
ozaenae
+
rhinoscleromatis
d +
K. terrigena
K.planticola
+
+ or (+) +
+
+ +
+ + +
+ + +
+ + - or + + - or + +
K.oxytoca K.mobilis
+
+ + +
subsp.
+ + + + +
(+) +
symbols: +, Positive re;!ction for at least 90% of the strains within 24 to 48 h; -, negative reaction for at least 90% of the strains within 24 to 48 h; (+), slow positive reaction; + or (+), - or +, the first sign corresponds to the most frequent result; d, different biochemical types (+, (+), -).
Relationship among Klebsiella Strains non clinical environments. Curr. Microbiol. 6, 105-109 (1981) Bascomb, S., Lapage, S. P., Willcox, W. R., Curtis, M. A.: Numerical classification of the tribe Klebsielleae. J. Gen. MicrobioI. 66, 279-295 Brenner, D. ].,Steigerwalt, A. G., Fanning, G. R.: Differentiation of Enterobacter aerogenes from Klebsiella by deoxyribonucleic acid reassociation. Int. J. System. Bact. 22, 193-200 (1972) Cowan, S. T., Steel, K. ]., Shaw, C. A.: Classification of the Klebsiella group. J. Gen. Microbiol. 23, 601-612 (1960) De Ley, ]., Tijtgat, R.: Evaluation of membrane filter methods for DNA-DNA hybridization. Antonie v. Leeuwenhoek 36, 461-474 (1970) Ferragut, c., Leclerc, H.: Characterization of motile and acetoinnegative Klebsiella pneumoniae strains by DNA:DNA hybrydization. Antonie v. Leeuwenhoek 44, 407-424 (1978) Ferragut, c., Izard, D., Gavini, G., Kersters, K., De Ley, ]., Leclerc, H.: Klebsiella trevisanii: a new species from water and soil. Int. J. System. Bact. 33, 133-142 (1983) Gavini, F., Lefebvre, B., Leclerc, H.: Positions taxonomiques d'enterobacteries HzS- par rapport au genre Citrobacter. Ann. lnst. Pasteur (Paris) 127 A, 275-295 (1976 a) Gavini, F., Ferragut, c., Lefebvre, B., Leclerc, H.: Etude taxonomique d'enterobacteries appartenant ou apparentees au genre Enterobacter. An. lnst. Pasteur (Paris) 127 B, 317-335 (1976b) Gavini, F., Leclerc, H., Lefebvre, B., Ferragut, c., Izard, D.: Etude taxonomique d'enterobacteries appartenant ou apparentees au genre Klebsiella. Ann. lnst. Pasteur (Paris) 128 B, 45-59 (1977) Gavini, F., Izard, D., Grimont, P. A. D., Beji, A., Ageron, E., Leclerc, H.: Priority of K1ebsiella planticola Bagley, Seidler and Brenner 1982 over Klebsiella trevisanii Ferragut,' Izard, Gavini, Kersters, De Ley and Leclerc. 1983. Int. J. System. Bact. 36, 486-488 (1986) Izard, D., Gavini, F., Leclerc, H.: Polynucleotide sequence
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relatedness and genome size among Enterobacter intermedium sp. nov. and the species Enterobacter cloacae and Klebsiella pneumoniae. Zbl. Bakt., I. Abt. Orig. C 1, 51-60 (1980 a) Izard, D., Gavini, F., Trinel, P. A., Krubwa, F., Leclerc, H.: Contribution of DNA-DNA hybridization to the transfer of Enterobacter aerogenes to the genus Klebsiella as K. mobilis. Zbl. Bakt. Hyg., 1. Abt. Orig. C 1, 257-263 (1980 b) Izard, D., Ferragut, c., Gavini, F., Kersters, K., De Ley, J., Leclerc, H.: Klebsiella terrigena, a new species from soil and water. Int. J. System. Bact. 31, 116-127 (1981) Johnson, R., Colwell, R. R., Sakazaki, R., Tamura, K.: Numerical taxonomy study of the Enterobacteriaceae. Int. J. System. Bact. 25, 13-37 (1975) Kersters, K., De Ley, ].: Identification and grouping of bacteria by numerical analysis of their electrophoretic protein patterns. J. Gen. Microbiol. 87, 333-342 (1975) Marmur, ].: A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Molec. BioI. 3, 208-218 (1961) Naemura, L. G., Bagley, S. T., Seidler, R. ]., Kaper, ]. B., Colwell, R. R .: Numerical taxonomy of Klebsiella pneumoniae strains isolated from clinical and non clinical sources. Curro Microbiol. 2, 175-180 (1979) 0rskov, I.: Genus V. Klebsiella Trevisan 1885, pp.461-465. In: Bergey's Manual of Systematic Bacteriology (N. R. Krieg and J. G. Holt, eds.) Baltimore-London, Williams and Wilkins Co. 1984 Sakazaki, R., Tamura, K., Johnson, R., Colwell, R. R.: Taxonomy of some recently described species in the family Enterobacteriaceae. Int. J. System. Bact. 26, 158-179 (1976) Skerman, V. B. D., Mac Gowan, V., Sneath, P. H. A.: Approved lists of bacterial names. Int. J. System. Bact. 30, 225-420 (1980) Slopek, S., Durlakowa, I.: Studies on the taxonomy of Klebsiella bacilli. Arch. Immunol. Ther. Exp. 15,481-487 (1967) Sneath, P. H. A., Sokal, R. R.: Numerical Taxonomy. San Francisco, Freeman and Co. 1973
Carmen Ferragut, Institut National de la Sante et de la Recherche medicale, Unite 146, Domaine du Centre d'Enseignement et de Recherches Techniques en Industrie Alimentaire, 369, rue Jules Guesde, B.P. 39,59651 Villeneuve-d'Ascq Cedex, France