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Phenotypical characters and ribotyping of pasteurella aerogenes from different sources
Zbl. Bakt. 279, 75-82 (1993) © Gustav Fischer Verlag, Stuttgart· Jena . New York
Phenotypical Characters and Ribotyping of Pasteurella aero genes from Different Sources ANNE LESTER l , PETER GERNER-SMIDT!, BENTE GAHRN-HANSEN 2 , PER S0GAARD 3 , JYTTE SCHMIDT!, and WILHELM FREDERIKSEN l Department of Clinical Microbiology, Statens Seruminstitut, Copenhagen!, Department of Clinical Microbiology, Odense University Hospital, Odense 2 and Department of Clinical Microbiology, Aarhus University Hospital, Aarhus 3 , Denmark
With 2 Figures
Summary On the occasion of five Danish human Pasteurella aerogenes from pig bite lesions, a comparison was made between 6 isolates from man and 15 animal isolates, mainly from pigs. The strains originated from 6 different countries (USA, Canada, Czechoslovakia, France, Belgium and Denmark). The 21 isolates were characterized by conventional biochemical tests, antibiogram and the API 20 NE kit; finally ribotyping was carried out by hybridizing EcoRI-digested chromosomal DNA with a probe derived from E. coli ribosomal RNA. By ribotyping, 19 of the 21 strains clustered at a similarity level of 81 % or more; both phenotypical tests and ribotyping indicated that the remaining two strains did not belong to the species P. aerogenes. In conclusion, despite minor differences our P. aerogenes isolates constituted a well-defined group and they could not be subdivided on basis of animal or geographical origin.
Zusammenfassung Aus Anlag von flinf danischen humanen Pasteurella aerogenes-Fallen nach Schweinebigverletzungen wurde ein Vergleich zwischen 6 Isolaten von Menschen und 15 Isolaten von Tieren, meist Schweinen, durchgeflihrt. Die Stamme kamen aus 6 verschiedenen Landern (USA, Canada, Tschechoslowakei, Frankreich, Belgien und Danemark). Die 21 Isolate wurden durch konventionelle biochemische Reaktionen, das Antibiogramm und den API 20 NE-Kit charakterisiert; weiterhin wurde die Ribotypisierung durch Hybridisierung EcoRIverdauter chromosomaler DNA mit einer Sonde durchgeflihrt, die von ribosomaler E. coliRNA abgeleitet war. Durch die Ribotypisierung wurden 19 der 21 Stamme auf einem Ahnlichkeitsniveau von 81 % oder mehr verbunden; phanotypische Tests und Ribotypisierung zeigten, dag die verbleibenden beiden Stamme nicht der Species P. aerogenes angehorten. Unsere P. aerogenes-Isolate bildeten demzufolge eine wohlabgegrenzte Gruppe und konnten nicht auf der Grundlage der Wirtsspecies oder der geographischen Herkunft unterteilt werden.
76
A. Lester et al. Introduction
Pasteurella aerogenes is a gas producing taxon first described in North America by McAllister and Carter in 1974 (12); a few years later the occurrence of similar strains in Europe was reported (8). P. aerogenes seems to form a rather well defined group at the species level with regard both to genotypical and phenotypical properties (3,4,5,8, 12, 17), but its exact taxonomic position remains unsettled as DNA-DNA hybridization studies have demonstrated that it does not belong to the genus Pasteurella sensu stricto (14). P. aerogenes is associated with swine and is probably a member of the normal intestinal flora of pigs (8, 12). It is seldom isolated from other animal hosts although a few isolates have been reported from rabbits, dogs, cows and man (1, 2, 3, 6). On the occasion of five Danish human P. aerogenes isolates from pig associated lesions, the aim of the present study was to compare isolates from man and from other animal hosts from six different countries by means of ribotyping, conventional biochemical characters, antibiogram and the API 20 NE kit. Materials and Methods
Bacterial strains. 21 strains provisionally identified as P. aerogenes by conventional biochemical tests were studied. They were isolated in the period 1972-1991 and resided in the culture collection of Statens Seruminstitut. Designation and origin of the strains are listed in Table 1. Several of them are mentioned in previous publications (1, 5, 17). Table 1. Designation and origin of the 21 P. aerogenes strains investigated Strain
Original strain designation
Animal origin
Geographical origin
P 591 P 592 P 593 P 594 P 595 P 596 P 626 P 627 P 631 P 632 P 634 T P 673 P 674 P 841 P 1211 P 1286 P 1290 P 1297 P 1323
McAllister, SS-185-72 McAllister, SS-7-73 McAllister, SS-68-73 McAllister, SS-84-73 McAllister, SS-187-74 McAllister, SS-274-74 Weaver, CDC D 7281 Olsen, 34393/79 Dickinson and Mocquot, PD 55 Dickinson and Mocquot, PD 61 ATCC 27883 T , McAllister, P 172-71 Toma, Toronto 770 Toma, Toronto 882 Olsen, 68354/83 Hommez, P.a.9-BA 27/9 Aldovti, Prag 28775 Aldovti, Prag 28926 Damjanov, SSI 5646 Gahrn-Hansen, 14529/90
Pig Pig Pig Pig Pig Pig Human Human Pig Pig Pig Piglet Piglet Human Pig Dog Dog Rabbit Human Human Human
USA USA USA USA USA USA USA Denmark France France USA Canada Canada Denmark Belgium Czechoslovakia Czechoslovakia Denmark Denmark Denmark Denmark
P 1339
P 1350
SllJgaard 153865190
Gahrn-Hansen, 47288/91
EcoRI Ribotypes of Pasteurella aerogenes
77
For comparison, four additional Pasteurella-like strains derived from pigs (P 1207, P 1208, P 1209 and P 1210) were included in the ribotyping and the API 20 NE testing. Although these 4 strains in some respects resembled P. aerogenes, final identification had not been possible as their phenotypical properties did not fit any known taxon; they were obtained from J. Hommez, Belgium. Conventional biochemical tests were performed as described previously (9, 10). API 20 NE kit. This commercial micromethod combines 20 tests (of which 12 are assimilation tests) for the identification of Gram-negative rods not belonging to the Enterobacteriaceae family. Testing was performed according to the manufacturer's instructions, i. e. incubation at 30°C for up to 48 h. Strains were identified by decoding their numerical profiles in the API 20 NE Analytical Profile Index (4th ed. 1990). Antibiogram. A routine disk pre-diffusion agar method was used for antibiotic susceptibility testing (16). ~-lactamase production was detected by a nitrocefin test. Ribotyping was carried out as described elsewhere (7). In short, chromosomal DNA was digested with EcoRI and fragments separated by agarose gel electrophoresis. Upon southern blotting to a nylon membrane, DNA was hybridized with a non-radioactive digoxigenin labelled eDNA probe derived from E. coli 16S and 23S rRNA. The position of the ribotyping bands was scored visually and the ribotypes analyzed by a numerical approach using UPGMA clustering and the simple matching similarity coefficient.
Results
Conventional biochemical tests The 21 P. aerogenes strains were all facultatively anaerobic and non-motile and gave positive reactions in the following tests: oxydase, urease, ONPG (~-galactosidase) and reduction of nitrate. All produced gas from glucose and acid from glucose, galactose, mannose, sucrose, lactose, and maltose. All strains were indole, VP, gelatinase, arginine and lysine decarboxylase negative and did not produce acid from cellobiose, adonitol, dulcitol, salicin or esculin. Most strains were catalase and ornithine decarboxylase positive and produced acid from arabinose, xylose and inositol but not from trehalose, melibiose, mannitol, sorbitol or rhamnose. Deviations from this main pattern are shown in Table 2. Deviating characters from the P. aerogenes pattern for the 4 unclassified strains and 2 atypical P. aerogenes strains are shown in Table 3. API 20 NE All 21 strains were eventually identified as P. aerogenes despite some difficulties in reading the results of the assimilation tests due to scanty growth. Some strains had to be re-examined - often with a greater inoculum than recommended by the manufacturer - before reliable results could be obtained. Strain P 595 had to be incubated at 35°C to manifest visible growth. None of the strains were positive in the glucose fermentation test (in contrast to the positive results in the conventional test) but this did not result in misidentifications when the numerical profiles were decoded. In accordance with data for P. aerogenes in API's identification table, most strains were positive in 7 of the 12 assimilation tests (glucose, arabinose, mannose, N-acetylglucosamine, maltose, gluconate and malate). Deviations from this pattern are shown in Table 2. With regard to the four unclassified strains, P 1209 was decoded as P. aerogenes, while P 1207, P 1208 and P 1210 were negative in all assimilation tests and
78
A. Lester et al.
Table 2. 21 P. aerogenes strains: Differences in ribotypes, conventional biochemical tests and API 20 NE assimilation tests Deviations in Strain
Ribotype
Biochemical tests
P 591 P 631 P 632 P 592 P 1286 P 1297 P 593 P 1339 P 596 P 674 P 594 P 626 P 627 P 634 P 673 P 841 P 1290 P 1323 P 1211 P 595
1 1 1 2 2 2 3 3 4 4 5 6 7 8 9 10 11 12 13 14
rhamnose
P 1350
15
1
API assimilation tests
+
xylose 0 xylose 0 arabinose 0 arabinose 0 ornithine decarboxylase 0 arabinose 0 arabinose 0, inositol 0 ornithine decarboxylase 0 arabinose 0 inositol 0 inositol 0 arabinose 0, inositol 0, ornithine decarboxylase 0 mannitol +, sorbitol + rhamnose +, inositol 0 trehalose +, melibiose + ornithine decarboxylase 0 catalase 0
arabinose arabinose NAGlO arabinose arabinose
0 0 0 0
arabinose 0
mannitol
+
NAG: N-acetylglucosamine.
Table 3. Differences in biochemical tests for the six strains constituting the three minor clusters by ribotyping. Results of tests not listed in the table were identical to the P. aerogenes main pattern P 595 Catalase Ornithine Indole Arabinose Xylose Rhamnose Trehalose Inositol Melibiose Mannitol Sorbitol
+
P 1208
+
P 1207
+
P 1210
P 1209
P 1350
+
0 0 0
0 0 0
0
0
0 0
0 0
0 0 0
0
0
0 0
0 0
0
0 0
0 0 0
+ +
0
+ +
+ + + + + + +'
+ +
+ + +
0
+ +
0
+ + + +
+ + + +
+
+ + + +
+
EcoRI Ribotypes of Pasteurella aerogenes
79
were decoded as P. pneumotropiea; the latter diagnosis does not correlate to the conventional test results and should be considered as a misidentification.
Antibiogram All isolates were resistant to penicillin, vancomycin and clindamycin. All isolates were susceptible to trimethoprim, kanamycin, gentamicin, netilmicin, cefuroxime, cefotaxime, chloramphenicol, ciprofloxacin and polymyxin. Strains P 592, P 593, P 595 and P 634 T were ~-lactamase positive and were also found resistant to ampicillin by agar diffusion testing, while the remaining 17 strains were susceptible to ampicillin. Eleven strains were susceptible to tetracycline.
Ribotyping 15 different ribotypes were found among the 21 P. aerogenes isolates, and the 4 unidentified strains each showed a unique ribotype (Fig. 1 and Table 2). M P 592
P 593 P 1286 PI297
P 1339 P 841
P596 P674 P631 P 632
P 591 P1211
P673
P 1323 PS9S P 1208
PS94 P 1290 P634
Pl209
P626 P627
P 1350 P 1207
P 1210 M
kb
/ I
23.1 19.3
I I It
9.47.76.66.2
1\
4.44.3
( \ 3.5
2.7
2.3
2.0 1.9
1.5
0.9
Fig. 1. EeaRI ribotypes of 21 P. aerogenes and 4 unidentied Pasteurella-like strains. M: molecular size standard (phage lambda DNA digested with HindlII and StyI).
80
A. Lester et al.
At a similarity level of approximately 80% one major and three minor clusters were seen (Fig. 2). All P. aerogenes strains except strains P 595 and P 1350 were found in the major cluster with a similarity of ::::: 81 %. The three minor clusters (a, b and c) contained two strains each; a: consisted of strains P 595 and P 1208; b: of strains P 1207 and 1210, and c: containing strains P 1209 and P 1350. Minor cluster a and b clustered together at a similarity of 76%. The major cluster, cluster c, and cluster a and b showed a similarity of 53-57%. PERCENTAGE SIMILARITY 97
P592, P1286, P1297 P593, P1339 P631, P632, P591 P634
94 92
89
86 84 83 81
P594 P626 P673 P1323 P1290
53
t---
I I
t-t---
P627
P1211
57
l
I
P841
P596, P674
76
r----
r~
~
-
P595 P1208
I
P1207
I
P1210 P1209
I
P1350
Fig. 2. UPGMA clustering dendrogram of the ribotypes shown in Fig. 1. Discussion Eight of the P. aerogenes strains included in the present study have previously been subjected to DNA-DNA hybridization by Ursing (17). He found that seven strains formed a DNA relatedness group together with the type strain with a relative binding ration of 70% or greater, while strain P 595 turned out to be widely separated from the type strain with only 26% binding; obviously, it could not be considered a member of the species (17).
EcoRI Ribotypes of Pasteurella aerogenes
81
This genotypical deviation is confirmed by our ribotyping results. Both P 595 and P 1350 differed from the other 19 P. aerogenes strains and from each other at a similarity level of 53-57%. DNA hybridization has not been carried out with P 1350. As P 1350 and the unidentified strain P 1209 by ribotyping clustered together at a similarity level of the same magnitude as found for the 19 P. aerogenes strains, they may belong to the same species. P 1350 differed considerably from the other P. aerogenes strains from a phenotypical point of view, too (Table 2). When its biochemical characteristics were reviewed after the ribotyping results had become available, it became clear that it was phenotypically identical to strain P 1209 and that it is probably too phenotypically divergent to be classified as P. aerogenes. In fact, both strains seem to fit Bisgaard's taxon 6 (1,4), although this taxon so far has not been observed in pigs. By ribotyping, strains P 595 and P 1208 and strains P 1207 and P 1210 also clustered at a similarity level that might suggest identity to species level. However, phenotypical variations did not corroborate this hypothesis (Table 3). The six strains belonging to the minor clusters all deviated from the main P. aerogenes biochemical pattern in at least five different tests while none of the 19 P. aerogenes in the major cluster deviated in more than two tests (usually one or none) (Table 2 and 3). Thus, divergence in phenotypes was to some extent correlated to divergence in ribotypes. This correlation was not absolute as some strains with identical ribotypes showed different biochemical reactions (Table 2). Antibiotic susceptibility pattern was remarkably uniform except for tetracycline resistance in half of the strains and ampicillin resistance in four strains due to ~-lactamase. These four strains all came from USA. It is known that the ~-lactamase ROB-1 is produced by the type strain P 634 in which it is apparently chromosomally encoded (11), but the type of ~-lactamase present in the other three strains has not been identified. ROB-1 has previously been found in American strains of Haemophilus in{luenzae type b and Actinobacillus (formerly: Haemophilus) pleuropneumoniae (13) and in French strains of P. haemolytica and P. multocida (11,15) as a plasmid-mediated enzyme. Although ROB-1 is probably still rare in H. in{luenzae (13), there seems to be a potential risk for spread of this form of ampicillin resistance between animal bacterial species and from an animal reservoir to human pathogens. In conclusion, our P. aerogenes isolates could not be subdivided on basis of animal or geographical habitat with the methods applied in this study. A partial exception to this was the finding of ~-lactamase production in four strains that were all North American and pig derived. The concept of P. aerogenes as a well-defined taxon was supported, and no conclusive correlation between ribotype and phenotype on one side and geographical or animal origin on the other side was established. Both ribotyping and phenotypical properties indicated that strains P 595 and P 1350 belong to species different from P. aerogenes.
References 1. Aldova, A., W. Frederiksen, V. Pauckova, V. Absolonova, P. Vlad{k, M. Lavickova, O. Hausner, and P. Vokoun: Aerogenic pasteurellas and Pasteurella-like organisms isolated in Czechoslovakia. Zbl. Bakt. 277 (1992) 139-143 2. Barnham, M.: Pig bite injuries and infection: report of seven human cases. Epidem. Infect. 101 (1988) 641-645 6 Zbl. Bakt. 279/1
82
A. Lester et al.
3. Bercovier, H., P. Perreau, F. Escande, J. Brault, M. Kiredjian, and H. H. Mollaret: Characterization of Pasteurella aerogenes in France. In: Haemophilus, Pasteurella and Actinobacillus (M. Kilian, W. Frederiksen, and E. L. Biberstein, eds.), pp. 175-183. Academic Press, London (1981)
4. Bisgaard, M., R. Mutters, and W. Mannheim: Characterization of some previously unreported taxa isolated from guinea pigs (Cavia porcellus) and provisionally classed
with the "HPA-group". Les Colloques de l'INSERM. Les Bacilles a Gram negatif d'interet medical et en Sante Publique: Taxonomie - Identification - Applications. INSERM 114 (1983) 227-244 5. Frederiksen, W.: Gas producing species within Pasteurella and Actinobacillus. In: Haemophilus, Pasteurella and Actinobacillus (M. Kilian, W. Frederiksen, and E. L. Biberstein, eds.), pp. 185-196. Academic Press, London (1981) 6. Frederiksen, W.: Pasteurellosis in man. In: Pasteurella and pasteurellosis (c. Adlam and J. M. Rutter, eds.), pp. 303-320. Academic Press, London (1989) 7. Gerner-Smidt, P.: Ribotyping of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex. J. Clin. Microbiol. 30 (1992) 2680-2685 8. Hommez, J. and L. A. Devriese: Pasteurella aerogenes isolations from swine. Zbl. Vet. Med. B 23 (1976) 265-268 9. Kilian, M. and W. Frederiksen: Identification tables for the Haemophilus-PasteurellaActinobacillus group. In: Haemophilus, Pasteurella and Actinobacillus (M. Kilian, W. Frederiksen, and E. L. Biberstein, eds.), pp. 281-90. Academic Press, London (1981) 10. Lautrop, H.: Gelatin-liquefying Klebsiella strains (Bacterium oxytocum (Fliigge)). Acta path. microbiol. scand. 39 (1956) 375-384 11. Livrelli, V. 0., A. Darfeuille-Richaud, C. D. Rich, B. H. Joly, and J. L. Martel: Genetic determinant of the ROB-1 ~-lactamase in bovine and porcine Pasteurella strains. Antimicrob. Agents Chemother. 32 (1988) 1282-1284 12. McAllister, H. A. and G. R. Carter: An aerogenic Pasteurella-like organism recovered from swine. Am. J. Vet. Res. 35 (1974) 917-922 13. Medeiros, A. A., R. Levesque, and G. A. Jacoby: An animal source for the ROB-1 ~ lactamase of Haemophilus in(luenzae type b. Antimicrob. Agents Chemother. 29 (1986) 212-215 14. Mutters, R., P. Ihm, S. Pohl, W. Frederiksen, and W. Mannheim: Reclassification of the genus Pasteurella Trevisan 1887 on the basis of deoxyribonucleic acid homology, with proposals for the new species Pasteurella dagmatis, Pasteurella canis, Pasteurella stomatis, Pasteurella anatis, and Pasteurella langaa. Int. J. System. Bact. 35 (1985) 309-322 15. Rosenau, A., A. Labigne, F. Escande, P. Courcoux, and A. Philippon: Plasmid-mediated ROB-1 ~-lactamase in Pasteurella multocida from a human specimen. Antimicrob. Agents Chemother. 35 (1991) 2419-2422 16. Thomsen, V. F.: Om teknikken ved resistensbestemmelse med sreriigt henblik pi!. anvendelse af prredifussion. Thesis, Nyt Nordisk Foriag, Arnold Busck, K.0benhavn 1967 (in Danish) 17. Ursing, J.: Deoxyribonucleic acid hybridization studies of gas producing pasteurellae. In: Haemophilus, Pasteurella and Actinobacillus (M. Kilian, W. Frederiksen, and E. L. Biberstein, eds.), pp. 255-263. Academic Press, London (1981)
Anne Lester, Department of Clinical Microbiology, Statens Seruminstitut, Artillerivej 5, DK-2300 Copenhagen S, Denmark
Phenotypical Characters and Ribotyping of Pasteurella aero genes from Different Sources ANNE LESTER l , PETER GERNER-SMIDT!, BENTE GAHRN-HANSEN 2 , PER S0GAARD 3 , JYTTE SCHMIDT!, and WILHELM FREDERIKSEN l Department of Clinical Microbiology, Statens Seruminstitut, Copenhagen!, Department of Clinical Microbiology, Odense University Hospital, Odense 2 and Department of Clinical Microbiology, Aarhus University Hospital, Aarhus 3 , Denmark
With 2 Figures
Summary On the occasion of five Danish human Pasteurella aerogenes from pig bite lesions, a comparison was made between 6 isolates from man and 15 animal isolates, mainly from pigs. The strains originated from 6 different countries (USA, Canada, Czechoslovakia, France, Belgium and Denmark). The 21 isolates were characterized by conventional biochemical tests, antibiogram and the API 20 NE kit; finally ribotyping was carried out by hybridizing EcoRI-digested chromosomal DNA with a probe derived from E. coli ribosomal RNA. By ribotyping, 19 of the 21 strains clustered at a similarity level of 81 % or more; both phenotypical tests and ribotyping indicated that the remaining two strains did not belong to the species P. aerogenes. In conclusion, despite minor differences our P. aerogenes isolates constituted a well-defined group and they could not be subdivided on basis of animal or geographical origin.
Zusammenfassung Aus Anlag von flinf danischen humanen Pasteurella aerogenes-Fallen nach Schweinebigverletzungen wurde ein Vergleich zwischen 6 Isolaten von Menschen und 15 Isolaten von Tieren, meist Schweinen, durchgeflihrt. Die Stamme kamen aus 6 verschiedenen Landern (USA, Canada, Tschechoslowakei, Frankreich, Belgien und Danemark). Die 21 Isolate wurden durch konventionelle biochemische Reaktionen, das Antibiogramm und den API 20 NE-Kit charakterisiert; weiterhin wurde die Ribotypisierung durch Hybridisierung EcoRIverdauter chromosomaler DNA mit einer Sonde durchgeflihrt, die von ribosomaler E. coliRNA abgeleitet war. Durch die Ribotypisierung wurden 19 der 21 Stamme auf einem Ahnlichkeitsniveau von 81 % oder mehr verbunden; phanotypische Tests und Ribotypisierung zeigten, dag die verbleibenden beiden Stamme nicht der Species P. aerogenes angehorten. Unsere P. aerogenes-Isolate bildeten demzufolge eine wohlabgegrenzte Gruppe und konnten nicht auf der Grundlage der Wirtsspecies oder der geographischen Herkunft unterteilt werden.
76
A. Lester et al. Introduction
Pasteurella aerogenes is a gas producing taxon first described in North America by McAllister and Carter in 1974 (12); a few years later the occurrence of similar strains in Europe was reported (8). P. aerogenes seems to form a rather well defined group at the species level with regard both to genotypical and phenotypical properties (3,4,5,8, 12, 17), but its exact taxonomic position remains unsettled as DNA-DNA hybridization studies have demonstrated that it does not belong to the genus Pasteurella sensu stricto (14). P. aerogenes is associated with swine and is probably a member of the normal intestinal flora of pigs (8, 12). It is seldom isolated from other animal hosts although a few isolates have been reported from rabbits, dogs, cows and man (1, 2, 3, 6). On the occasion of five Danish human P. aerogenes isolates from pig associated lesions, the aim of the present study was to compare isolates from man and from other animal hosts from six different countries by means of ribotyping, conventional biochemical characters, antibiogram and the API 20 NE kit. Materials and Methods
Bacterial strains. 21 strains provisionally identified as P. aerogenes by conventional biochemical tests were studied. They were isolated in the period 1972-1991 and resided in the culture collection of Statens Seruminstitut. Designation and origin of the strains are listed in Table 1. Several of them are mentioned in previous publications (1, 5, 17). Table 1. Designation and origin of the 21 P. aerogenes strains investigated Strain
Original strain designation
Animal origin
Geographical origin
P 591 P 592 P 593 P 594 P 595 P 596 P 626 P 627 P 631 P 632 P 634 T P 673 P 674 P 841 P 1211 P 1286 P 1290 P 1297 P 1323
McAllister, SS-185-72 McAllister, SS-7-73 McAllister, SS-68-73 McAllister, SS-84-73 McAllister, SS-187-74 McAllister, SS-274-74 Weaver, CDC D 7281 Olsen, 34393/79 Dickinson and Mocquot, PD 55 Dickinson and Mocquot, PD 61 ATCC 27883 T , McAllister, P 172-71 Toma, Toronto 770 Toma, Toronto 882 Olsen, 68354/83 Hommez, P.a.9-BA 27/9 Aldovti, Prag 28775 Aldovti, Prag 28926 Damjanov, SSI 5646 Gahrn-Hansen, 14529/90
Pig Pig Pig Pig Pig Pig Human Human Pig Pig Pig Piglet Piglet Human Pig Dog Dog Rabbit Human Human Human
USA USA USA USA USA USA USA Denmark France France USA Canada Canada Denmark Belgium Czechoslovakia Czechoslovakia Denmark Denmark Denmark Denmark
P 1339
P 1350
SllJgaard 153865190
Gahrn-Hansen, 47288/91
EcoRI Ribotypes of Pasteurella aerogenes
77
For comparison, four additional Pasteurella-like strains derived from pigs (P 1207, P 1208, P 1209 and P 1210) were included in the ribotyping and the API 20 NE testing. Although these 4 strains in some respects resembled P. aerogenes, final identification had not been possible as their phenotypical properties did not fit any known taxon; they were obtained from J. Hommez, Belgium. Conventional biochemical tests were performed as described previously (9, 10). API 20 NE kit. This commercial micromethod combines 20 tests (of which 12 are assimilation tests) for the identification of Gram-negative rods not belonging to the Enterobacteriaceae family. Testing was performed according to the manufacturer's instructions, i. e. incubation at 30°C for up to 48 h. Strains were identified by decoding their numerical profiles in the API 20 NE Analytical Profile Index (4th ed. 1990). Antibiogram. A routine disk pre-diffusion agar method was used for antibiotic susceptibility testing (16). ~-lactamase production was detected by a nitrocefin test. Ribotyping was carried out as described elsewhere (7). In short, chromosomal DNA was digested with EcoRI and fragments separated by agarose gel electrophoresis. Upon southern blotting to a nylon membrane, DNA was hybridized with a non-radioactive digoxigenin labelled eDNA probe derived from E. coli 16S and 23S rRNA. The position of the ribotyping bands was scored visually and the ribotypes analyzed by a numerical approach using UPGMA clustering and the simple matching similarity coefficient.
Results
Conventional biochemical tests The 21 P. aerogenes strains were all facultatively anaerobic and non-motile and gave positive reactions in the following tests: oxydase, urease, ONPG (~-galactosidase) and reduction of nitrate. All produced gas from glucose and acid from glucose, galactose, mannose, sucrose, lactose, and maltose. All strains were indole, VP, gelatinase, arginine and lysine decarboxylase negative and did not produce acid from cellobiose, adonitol, dulcitol, salicin or esculin. Most strains were catalase and ornithine decarboxylase positive and produced acid from arabinose, xylose and inositol but not from trehalose, melibiose, mannitol, sorbitol or rhamnose. Deviations from this main pattern are shown in Table 2. Deviating characters from the P. aerogenes pattern for the 4 unclassified strains and 2 atypical P. aerogenes strains are shown in Table 3. API 20 NE All 21 strains were eventually identified as P. aerogenes despite some difficulties in reading the results of the assimilation tests due to scanty growth. Some strains had to be re-examined - often with a greater inoculum than recommended by the manufacturer - before reliable results could be obtained. Strain P 595 had to be incubated at 35°C to manifest visible growth. None of the strains were positive in the glucose fermentation test (in contrast to the positive results in the conventional test) but this did not result in misidentifications when the numerical profiles were decoded. In accordance with data for P. aerogenes in API's identification table, most strains were positive in 7 of the 12 assimilation tests (glucose, arabinose, mannose, N-acetylglucosamine, maltose, gluconate and malate). Deviations from this pattern are shown in Table 2. With regard to the four unclassified strains, P 1209 was decoded as P. aerogenes, while P 1207, P 1208 and P 1210 were negative in all assimilation tests and
78
A. Lester et al.
Table 2. 21 P. aerogenes strains: Differences in ribotypes, conventional biochemical tests and API 20 NE assimilation tests Deviations in Strain
Ribotype
Biochemical tests
P 591 P 631 P 632 P 592 P 1286 P 1297 P 593 P 1339 P 596 P 674 P 594 P 626 P 627 P 634 P 673 P 841 P 1290 P 1323 P 1211 P 595
1 1 1 2 2 2 3 3 4 4 5 6 7 8 9 10 11 12 13 14
rhamnose
P 1350
15
1
API assimilation tests
+
xylose 0 xylose 0 arabinose 0 arabinose 0 ornithine decarboxylase 0 arabinose 0 arabinose 0, inositol 0 ornithine decarboxylase 0 arabinose 0 inositol 0 inositol 0 arabinose 0, inositol 0, ornithine decarboxylase 0 mannitol +, sorbitol + rhamnose +, inositol 0 trehalose +, melibiose + ornithine decarboxylase 0 catalase 0
arabinose arabinose NAGlO arabinose arabinose
0 0 0 0
arabinose 0
mannitol
+
NAG: N-acetylglucosamine.
Table 3. Differences in biochemical tests for the six strains constituting the three minor clusters by ribotyping. Results of tests not listed in the table were identical to the P. aerogenes main pattern P 595 Catalase Ornithine Indole Arabinose Xylose Rhamnose Trehalose Inositol Melibiose Mannitol Sorbitol
+
P 1208
+
P 1207
+
P 1210
P 1209
P 1350
+
0 0 0
0 0 0
0
0
0 0
0 0
0 0 0
0
0
0 0
0 0
0
0 0
0 0 0
+ +
0
+ +
+ + + + + + +'
+ +
+ + +
0
+ +
0
+ + + +
+ + + +
+
+ + + +
+
EcoRI Ribotypes of Pasteurella aerogenes
79
were decoded as P. pneumotropiea; the latter diagnosis does not correlate to the conventional test results and should be considered as a misidentification.
Antibiogram All isolates were resistant to penicillin, vancomycin and clindamycin. All isolates were susceptible to trimethoprim, kanamycin, gentamicin, netilmicin, cefuroxime, cefotaxime, chloramphenicol, ciprofloxacin and polymyxin. Strains P 592, P 593, P 595 and P 634 T were ~-lactamase positive and were also found resistant to ampicillin by agar diffusion testing, while the remaining 17 strains were susceptible to ampicillin. Eleven strains were susceptible to tetracycline.
Ribotyping 15 different ribotypes were found among the 21 P. aerogenes isolates, and the 4 unidentified strains each showed a unique ribotype (Fig. 1 and Table 2). M P 592
P 593 P 1286 PI297
P 1339 P 841
P596 P674 P631 P 632
P 591 P1211
P673
P 1323 PS9S P 1208
PS94 P 1290 P634
Pl209
P626 P627
P 1350 P 1207
P 1210 M
kb
/ I
23.1 19.3
I I It
9.47.76.66.2
1\
4.44.3
( \ 3.5
2.7
2.3
2.0 1.9
1.5
0.9
Fig. 1. EeaRI ribotypes of 21 P. aerogenes and 4 unidentied Pasteurella-like strains. M: molecular size standard (phage lambda DNA digested with HindlII and StyI).
80
A. Lester et al.
At a similarity level of approximately 80% one major and three minor clusters were seen (Fig. 2). All P. aerogenes strains except strains P 595 and P 1350 were found in the major cluster with a similarity of ::::: 81 %. The three minor clusters (a, b and c) contained two strains each; a: consisted of strains P 595 and P 1208; b: of strains P 1207 and 1210, and c: containing strains P 1209 and P 1350. Minor cluster a and b clustered together at a similarity of 76%. The major cluster, cluster c, and cluster a and b showed a similarity of 53-57%. PERCENTAGE SIMILARITY 97
P592, P1286, P1297 P593, P1339 P631, P632, P591 P634
94 92
89
86 84 83 81
P594 P626 P673 P1323 P1290
53
t---
I I
t-t---
P627
P1211
57
l
I
P841
P596, P674
76
r----
r~
~
-
P595 P1208
I
P1207
I
P1210 P1209
I
P1350
Fig. 2. UPGMA clustering dendrogram of the ribotypes shown in Fig. 1. Discussion Eight of the P. aerogenes strains included in the present study have previously been subjected to DNA-DNA hybridization by Ursing (17). He found that seven strains formed a DNA relatedness group together with the type strain with a relative binding ration of 70% or greater, while strain P 595 turned out to be widely separated from the type strain with only 26% binding; obviously, it could not be considered a member of the species (17).
EcoRI Ribotypes of Pasteurella aerogenes
81
This genotypical deviation is confirmed by our ribotyping results. Both P 595 and P 1350 differed from the other 19 P. aerogenes strains and from each other at a similarity level of 53-57%. DNA hybridization has not been carried out with P 1350. As P 1350 and the unidentified strain P 1209 by ribotyping clustered together at a similarity level of the same magnitude as found for the 19 P. aerogenes strains, they may belong to the same species. P 1350 differed considerably from the other P. aerogenes strains from a phenotypical point of view, too (Table 2). When its biochemical characteristics were reviewed after the ribotyping results had become available, it became clear that it was phenotypically identical to strain P 1209 and that it is probably too phenotypically divergent to be classified as P. aerogenes. In fact, both strains seem to fit Bisgaard's taxon 6 (1,4), although this taxon so far has not been observed in pigs. By ribotyping, strains P 595 and P 1208 and strains P 1207 and P 1210 also clustered at a similarity level that might suggest identity to species level. However, phenotypical variations did not corroborate this hypothesis (Table 3). The six strains belonging to the minor clusters all deviated from the main P. aerogenes biochemical pattern in at least five different tests while none of the 19 P. aerogenes in the major cluster deviated in more than two tests (usually one or none) (Table 2 and 3). Thus, divergence in phenotypes was to some extent correlated to divergence in ribotypes. This correlation was not absolute as some strains with identical ribotypes showed different biochemical reactions (Table 2). Antibiotic susceptibility pattern was remarkably uniform except for tetracycline resistance in half of the strains and ampicillin resistance in four strains due to ~-lactamase. These four strains all came from USA. It is known that the ~-lactamase ROB-1 is produced by the type strain P 634 in which it is apparently chromosomally encoded (11), but the type of ~-lactamase present in the other three strains has not been identified. ROB-1 has previously been found in American strains of Haemophilus in{luenzae type b and Actinobacillus (formerly: Haemophilus) pleuropneumoniae (13) and in French strains of P. haemolytica and P. multocida (11,15) as a plasmid-mediated enzyme. Although ROB-1 is probably still rare in H. in{luenzae (13), there seems to be a potential risk for spread of this form of ampicillin resistance between animal bacterial species and from an animal reservoir to human pathogens. In conclusion, our P. aerogenes isolates could not be subdivided on basis of animal or geographical habitat with the methods applied in this study. A partial exception to this was the finding of ~-lactamase production in four strains that were all North American and pig derived. The concept of P. aerogenes as a well-defined taxon was supported, and no conclusive correlation between ribotype and phenotype on one side and geographical or animal origin on the other side was established. Both ribotyping and phenotypical properties indicated that strains P 595 and P 1350 belong to species different from P. aerogenes.
References 1. Aldova, A., W. Frederiksen, V. Pauckova, V. Absolonova, P. Vlad{k, M. Lavickova, O. Hausner, and P. Vokoun: Aerogenic pasteurellas and Pasteurella-like organisms isolated in Czechoslovakia. Zbl. Bakt. 277 (1992) 139-143 2. Barnham, M.: Pig bite injuries and infection: report of seven human cases. Epidem. Infect. 101 (1988) 641-645 6 Zbl. Bakt. 279/1
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3. Bercovier, H., P. Perreau, F. Escande, J. Brault, M. Kiredjian, and H. H. Mollaret: Characterization of Pasteurella aerogenes in France. In: Haemophilus, Pasteurella and Actinobacillus (M. Kilian, W. Frederiksen, and E. L. Biberstein, eds.), pp. 175-183. Academic Press, London (1981)
4. Bisgaard, M., R. Mutters, and W. Mannheim: Characterization of some previously unreported taxa isolated from guinea pigs (Cavia porcellus) and provisionally classed
with the "HPA-group". Les Colloques de l'INSERM. Les Bacilles a Gram negatif d'interet medical et en Sante Publique: Taxonomie - Identification - Applications. INSERM 114 (1983) 227-244 5. Frederiksen, W.: Gas producing species within Pasteurella and Actinobacillus. In: Haemophilus, Pasteurella and Actinobacillus (M. Kilian, W. Frederiksen, and E. L. Biberstein, eds.), pp. 185-196. Academic Press, London (1981) 6. Frederiksen, W.: Pasteurellosis in man. In: Pasteurella and pasteurellosis (c. Adlam and J. M. Rutter, eds.), pp. 303-320. Academic Press, London (1989) 7. Gerner-Smidt, P.: Ribotyping of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex. J. Clin. Microbiol. 30 (1992) 2680-2685 8. Hommez, J. and L. A. Devriese: Pasteurella aerogenes isolations from swine. Zbl. Vet. Med. B 23 (1976) 265-268 9. Kilian, M. and W. Frederiksen: Identification tables for the Haemophilus-PasteurellaActinobacillus group. In: Haemophilus, Pasteurella and Actinobacillus (M. Kilian, W. Frederiksen, and E. L. Biberstein, eds.), pp. 281-90. Academic Press, London (1981) 10. Lautrop, H.: Gelatin-liquefying Klebsiella strains (Bacterium oxytocum (Fliigge)). Acta path. microbiol. scand. 39 (1956) 375-384 11. Livrelli, V. 0., A. Darfeuille-Richaud, C. D. Rich, B. H. Joly, and J. L. Martel: Genetic determinant of the ROB-1 ~-lactamase in bovine and porcine Pasteurella strains. Antimicrob. Agents Chemother. 32 (1988) 1282-1284 12. McAllister, H. A. and G. R. Carter: An aerogenic Pasteurella-like organism recovered from swine. Am. J. Vet. Res. 35 (1974) 917-922 13. Medeiros, A. A., R. Levesque, and G. A. Jacoby: An animal source for the ROB-1 ~ lactamase of Haemophilus in(luenzae type b. Antimicrob. Agents Chemother. 29 (1986) 212-215 14. Mutters, R., P. Ihm, S. Pohl, W. Frederiksen, and W. Mannheim: Reclassification of the genus Pasteurella Trevisan 1887 on the basis of deoxyribonucleic acid homology, with proposals for the new species Pasteurella dagmatis, Pasteurella canis, Pasteurella stomatis, Pasteurella anatis, and Pasteurella langaa. Int. J. System. Bact. 35 (1985) 309-322 15. Rosenau, A., A. Labigne, F. Escande, P. Courcoux, and A. Philippon: Plasmid-mediated ROB-1 ~-lactamase in Pasteurella multocida from a human specimen. Antimicrob. Agents Chemother. 35 (1991) 2419-2422 16. Thomsen, V. F.: Om teknikken ved resistensbestemmelse med sreriigt henblik pi!. anvendelse af prredifussion. Thesis, Nyt Nordisk Foriag, Arnold Busck, K.0benhavn 1967 (in Danish) 17. Ursing, J.: Deoxyribonucleic acid hybridization studies of gas producing pasteurellae. In: Haemophilus, Pasteurella and Actinobacillus (M. Kilian, W. Frederiksen, and E. L. Biberstein, eds.), pp. 255-263. Academic Press, London (1981)
Anne Lester, Department of Clinical Microbiology, Statens Seruminstitut, Artillerivej 5, DK-2300 Copenhagen S, Denmark