a Institut für Tierzucht und Tierverhalten der Bundesforschungsanstalt für Landwirtschaft (FAL), Dörnbergstrasse 2527, 29223 Celle, Germany; b Worldwide Animal Health Product Development, Pharmacia Animal Health, Kalamazoo, MI 49001; c Infectious Diseases Biology, Pharmacia Corporation, Kalamazoo, MI 49001, USA
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Abstract |
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Introduction |
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Antimicrobials are still the tools of choice to control Pasteurella and Mannheimia infections in animals. In addition to therapeutic purposes, antimicrobial agents are also used frequently for prophylactic and metaphylactic purposes to prevent either the onset or the further spread of Pasteurella and Mannheimia infections in cattle and pigs.1,2 Among the antimicrobials used in veterinary medicine, tetracyclines account for almost two-thirds of all substances employed, as shown in a survey conducted in the EU member states and Switzerland in 1997 (http://www/fedesa.be/eng/PublicSite/xtra/dossiers/doss9/.). The widespread and multipurpose use of tetracyclines, however, creates a strong selective pressure, under the influence of which resistance genes are exchanged between members of mixed bacterial populations. The association of many tetracycline resistance (tet) genes with plasmids or transposons facilitates the spread of these resistance genes across species and genus borders.6 Despite the extensive knowledge of tet genes and their distribution,6,7 little is known about the tet genes present in P. multocida and Mannheimia spp.812 No data are currently available about antimicrobial resistance in Mannheimia species other than M. haemolytica.
In this study we analysed the types of tet genes present in isolates of P. multocida, M. haemolytica, Mannheimia glucosida and Mannheimia varigena obtained from cases of respiratory diseases in cattle and swine in Germany and the USA. The location of the tet genes, on plasmids or on the chromosome, was investigated with respect to the copy number of tet genes per isolate and the MICs for the isolates. Molecular analyses of the transposon-borne tet genes, tet(B) and tet(H), were performed to identify complete or truncated copies of the respective transposons. Moreover, macrorestriction analysis of isolates carrying the same tet gene on the chromosome or the same tetracycline resistance plasmid was conducted to determine the genomic relationships of these isolates.
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Materials and methods |
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The geographical origins and the animal sources of the 34 isolates included in this study are shown in Table 1. All isolates were obtained from samples from animals suffering from respiratory diseases and were collected between 1996 and 1999. Biochemical confirmation of the species assignment followed standard procedures for the genus Pasteurella,3 as well as the specifications given by Angen et al.13 for the genus Mannheimia. The reference strains P. multocida subsp. multocida DSM5281 and M. haemolytica DSM10531, both obtained from the German national strain collection (DSMZ, Braunschweig, Germany), served as controls. All isolates were cultured overnight at 37°C on blood agar [blood agar base from Oxoid, Wesel, Germany, supplemented with 5% (v/v) sheep blood].
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DNA preparation, PCR, hybridization, transformation and macrorestriction analysis
The preparation of whole-cell DNA was performed as described previously.15 Plasmids were isolated according to a modification of the alkaline lysis procedure described previously that includes purification by affinity chromatography on Qiagen Midi columns (Qiagen, Hilden, Germany).12,15 Plasmid sizes were calculated from logarithmic plots in which the plasmids of E. coli V51716 served as size standards.
The identification of the tet genes was achieved by PCR and by Southern blot hybridization. Primers specific for the detection of tet genes of classes AE and G,9,15 H9,11 and M and O,17 described previously, were used for PCR analyses. Restriction analysis of whole-cell DNA of the Pasteurella and Mannheimia isolates using the enzymes BamHI, HindIII or SfuI, agarose gel electrophoresis of the restriction fragments and Southern blot hybridization were performed as described previously.11,12,15 The specific gene probes of the tet genes of classes B, G and H15 described previously were labelled by the enhanced chemiluminescence system (ECL; Amersham-Pharmacia Biotech, Freiburg, Germany). Hybridization and signal detection followed the manufacturer's recommendations. Plasmid profiles and endonuclease-digested whole-cell DNA served as targets for the tet gene probes. A 1063 bp fragment amplified from Tn570611 using the sequence of the terminal 18 bp inverted repeat as PCR primer served as specific probe for the closely related insertion elements IS1596 and IS1597. Hybridization experiments were performed three times on independent occasions.
Introduction of plasmids into E. coli strains JM107 and JM110 was achieved by heat shock transformation into CaCl2-treated competent E. coli cells. Transformants were selected on LuriaBertani agar supplemented with tetracycline at 20 mg/L.
To determine the genomic relationships between isolates carrying the same tet gene on the chromosome or the same tetracycline resistance plasmid, whole-cell DNA of the corresponding Pasteurella and Mannheimia isolates was digested with SmaI. Subsequent separation of the fragments by pulsed-field gel electrophoresis (SmaI macrorestriction analysis) followed a protocol described previously.12 The pulse times were increased over the first 11 h from 2 to 5 s and for the following 13 h from 20 to 40 s. For a better separation of the multiple fragments in the low molecular weight range, additional macrorestriction analyses were conducted with pulse times increased from 2 to 5 s over 24 h. The SmaI fragments of S. aureus 832518 served as a size standard. The DNA fragments were separated in a CHEF DR III system (Bio-Rad) at 15 V/cm with 0.5x TrisborateEDTA as running buffer.
Cloning and sequence analysis
All tetracycline resistance plasmids were mapped using data obtained from single and double digests with 16 different restriction endonucleases. The tetR-tet(H) gene region and its flanking sequences from plasmid pMHT1 from M. haemolytica M395 were cloned on a c. 3.1 kb BglII fragment into the single BamHI site of pBluescript II SK+ (Stratagene, Amsterdam, The Netherlands). The sequence of a 2980 bp segment of plasmid pMHT1 from M. haemolytica M395, including the entire tetRtet(H) region and its flanking regions, has been deposited with the EMBL database under accession no. Y16103. Of the pMHT1-like plasmids isolated from P. multocida, M. glucosida and M. varigena, only the terminal parts and the respective downstream regions of tetR and tet(H) were sequenced. The internal 1141 bp PCR amplicon of the tet(G) gene of M. haemolytica M166 was cloned into pCR-Blunt II-TOPO (Invitrogen, Groningen, The Netherlands) and sequenced completely. The nucleotide sequence has been deposited with the EMBL database under accession no. AJ276217. Recombinant cloning vectors were propagated in E. coli strain JM107 (Stratagene) or TOP10 (Invitrogen). Sequence analyses with the ALF sequenator (Amersham-Pharmacia Biotech) were performed on both strands using standard M13 universal and reverse primers, as well as another eight primers (Roth, Karlsruhe, Germany) that proved to be suitable for the determination of the tetRtet(H) sequences of plasmids pPMT111 and pPAT1.12 Sequence analysis of the tet(G) amplicon was performed with slightly modified M13 universal and reverse primers (Invitrogen), suitable for sequencing PCR amplicons cloned into pCR-Blunt II-TOPO.
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Results |
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Based on their biochemical properties, the 34 isolates included in this study were assigned to one of four species: P. multocida (eight isolates), M. haemolytica (23 isolates), M. varigena (two isolates) and M. glucosida (one isolate). All 34 isolates exhibited in vitro resistance to tetracycline and streptomycin; in addition 25 of the 34 isolates were also resistant to one to five other antimicrobial agents (Table 1). Among the P. multocida isolates, resistance to ampicillin, kanamycin and/or sulfamethoxazoletrimethoprim, either alone or in combination, was seen in three isolates. Among the M. haemolytica isolates, resistance to ampicillin (16 isolates) and/or chloramphenicol (seven isolates) was detected. The two M. varigena isolates and the single M. glucosida isolate showed unique resistance patterns (Table 1
). The MICTcs varied between 32 and 256 mg/L, with most of the isolates having MICs of 64 or 128 mg/L (Table 2
). An increase in the MIC values by one dilution step was occasionally observed after pre-incubation of the isolates in subinhibitory concentrations of tetracycline.
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Macrorestriction analysis of the P. multocida isolates revealed unique SmaI fragment patterns for six of the eight isolates. The remaining two pMHT1-bearing P. multocida isolates differed in their SmaI macrorestriction patterns by only four bands (Figure 1a). Among the 23 M. haemolytica isolates nine different genomic groups were identified, the members of which differed in their SmaI fragment patterns by more than six fragments (Figure 1b
). These genomic groups contained one to seven isolates. The American M. haemolytica isolates were assigned to two groups, the German isolates to seven genomic groups. None of these groups contained isolates from both geographical origins. The remaining M. glucosida and M. varigena isolates displayed unique SmaI macrorestriction patterns.
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PCR analysis of the 34 Pasteurella and Mannheimia isolates identified tet genes of classes B, G and H. The respective amplicons were in the size range expected from the corresponding sequences in the databases: 1170 bp [tet(B)], 1141 bp [tet(G)] and 1076 bp [tet(H)]. The identities of the PCR products were confirmed by restriction analysis of the amplicons. EcoRI digestion of the tet(B) amplicon yielded two fragments of 0.56 and 0.61 kb, XmnI digestion of the tet(G) amplicon gave two fragments of 0.28 and 0.86 kb, and BclI digestion of the tet(H) amplicon gave two fragments of c. 0.26 and 0.82 kb. None of the 34 isolates harboured multiple types of tet gene. Genes of class H were the predominant tet genes in the isolates and were found in six of the eight P. multocida isolates, in 17 of the 23 M. haemolytica isolates, in the two M. varigena and in the single M. glucosida isolates. Class B genes were detected in two unrelated P. multocida isolates and class G genes were present in six closely related M. haemolytica isolates.
Plasmid-borne tet(H) genes
Transformation and hybridization experiments identified tet(H) genes exclusively on small plasmids of c. 4.4 kb in three P. multocida isolates, as well as in single isolates of M. haemolytica, M. glucosida and M. varigena. These plasmids have indistinguishable restriction maps. Therefore, a common designation, pMHT1, was chosen. In the tetR-tet(H) gene region plasmid pMHT1 is similar to the tet(H)-carrying plasmids pPMT1 from P. multocida11 and pPAT1 from Pasteurella aerogenes12 described previously, but differs markedly in the remaining parts of the plasmid (Figure 2). Plasmid pMHT1 confers only tetracycline resistance. None of the pMHT1-bearing isolates harbour chromosomal tet genes, as revealed by hybridization experiments.
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Chromosomal tet genes
When plasmids from any one of the 34 isolates did not yield tetracycline-resistant transformants and also did not hybridize with any of the tet gene probes, the isolate from which the plasmids originated was considered to carry the tet gene(s) on the chromosome. Hybridization experiments were then conducted to determine the number of tet gene copies in each of these isolates and to detect any restriction fragment length polymorphisms of tet gene-carrying fragments.
Chromosomally located tet(H) genes were detected in three P. multocida isolates, 16 M. haemolytica isolates and one M. varigena isolate. One to four copies of the tet(H) gene per isolate were observed (Table 2). A total of seven different hybridization patterns, consisting of HindIII fragments of between 2.2 and >23.1 kb, were detected (Figure 3
, Table 4
). Since there are conserved HindIII sites in tetR and in the terminal insertion sequences IS1596/IS1597 of Tn5706, a HindIII fragment of 2.2 kb hybridizing with the tet(H) gene probe might indicate the presence of a complete copy of Tn5706. A hybridizing fragment of that size was observed only in the M. varigena isolate. Larger HindIII fragments of
3.5 kb, as seen for P. multocida and M. haemolytica isolates, suggest the presence of truncated Tn5706 elements. Moreover, none of the P. multocida or M. haemolytica isolates have copies of the Tn5706-associated insertion elements IS1596/IS1597, as indicated by the lack of hybridization to the appropriate probes. The M. varigena isolate, however, carries at least four copies of insertion sequences IS1596/IS1597 on chromosomal EcoRV fragments of c. 2.0, 4.2, 8.0 and 14.5 kb (data not shown).
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The gene tet(G) was detected in the chromosomal DNA of six closely related M. haemolytica isolates. One isolate harboured copies of tet(G) on HindIII fragments of 8.2 and 11.6 kb, while the remaining five isolates carried tet(G) on a common HindIII fragment of 12.5 kb (data not shown). Since tet(G) has not previously been reported in bacteria of the family Pasteurellaceae, a PCR amplicon that comprised almost the entire tet(G) structural gene was cloned and sequenced. This revealed a single incomplete reading frame. Comparative analysis of the nucleotide sequence showed 94% identity to tet(G) from Pseudomonas spp. (AF133140), Salmonella enterica serovar Typhimurium DT104 (AF071555) and Vibrio anguillarum (S52437) and 93% identity to tet(G) from Pseudomonas spp. (AF133139). The deduced amino acid sequence has 96% identity to the first three Tet(G) proteins and 94% to the last.
Correlation between tet gene type, number of tet gene copies and MICTc values
No correlation was observed between the type of tet gene and the MICTc value when determined by the two-fold broth dilution method. The P. multocida isolates that harboured one or two copies of the tet(B) gene showed the same MICTc of 128 mg/L, as did most of the tet(H)- and tet(G)-carrying M. haemolytica isolates (Table 2). There was also no obvious correlation between the copy number of the tet genes and the MICTc value for the respective isolates. P. multocida and M. haemolytica isolates carrying four copies of tet(H) displayed the same MICTc value as isolates of the same species harbouring a single copy of tet(H). It is noteworthy, however, that plasmid pMHT1 confers different levels of tetracycline resistance in the four different host bacteria. Relatively low levels of tetracycline resistance (MICTc 32 mg/L) were observed for P. multocida and M. varigena, whereas a moderate level of resistance (MICTc 64 mg/L) was displayed by M. haemolytica and a high level (MICTc 256 mg/L) by M. glucosida. Examples of plasmid pMHT1 from all six original Pasteurella and Mannheimia isolates were transformed into the same E. coli recipient strain, JM107. MIC determinations for the six E. coli JM107:pMHT1 transformants revealed the same MICTc of 128 mg/L, which increased to 256 mg/L after induction of the tet(H) system. Repetition of these experiments with E. coli JM110 again gave the same, but slightly lower, MICTc of 64 mg/L for all transformants, which was increased by one dilution step when the transformants were pre-incubated in medium containing tetracycline.
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Discussion |
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Tetracycline resistance in isolates of P. multocida from animal sources has been reported to be due mainly to the presence of the gene tet(H).9 The tet(H) gene was originally detected on plasmid pVM111 of which, however, no restriction map is available. The subsequent detection of tet(H) on other plasmids but also on the chromosome led to the assumption that tet(H) might be part of a mobile genetic element.9 This supposition was confirmed by the identification of the transposon Tn5706 on the 6.8 kb plasmid pPMT1 from bovine P. multocida.11 Tn5706 is a small 4.3 kb composite transposon in which the tetRtet(H) gene cluster is bracketed by the closely related insertion elements IS1596 and IS1597 (Figure 1).11 A truncated version of Tn5706, from which IS1596 has been lost and IS1597 has been deleted by recombinational events, has recently been identified on the 5.5 kb plasmid pPAT1 found in porcine isolates of P. multocida and P. aerogenes (Figure 2
).12
All plasmid-borne tet genes identified in this study were of class H and were associated with a new plasmid, pMHT1. Plasmid pMHT1 is the smallest tet(H)-carrying plasmid reported to date. It differs from pPMT1 and pPAT1 in that it lacks the two insertion elements, IS1596 and IS1597, and their adjacent sequences. The Tn5706-specific spacer sequence between IS1596 and tetR is completely absent and of that between tet(H) and IS1597 only 119 bp is left in pMHT1. These observations indicate that pMHT1 carries a largely truncated Tn5706 relic. Since the sequences adjacent to the Tn5706 relic in pMHT1 do not show identity to known sequences, it is impossible at this juncture to say what led to the truncation of the Tn5706 copy on pMHT1. The finding that plasmids indistinguishable from pMHT1, as judged from their restriction maps and the sequences downstream of tetR and tet(H), were isolated from P. multocida, M. haemolytica, M. glucosida and M. varigena indicates that the plasmid has spread between bacteria of different species and genera that share the same habitat.
Chromosomal tet genes
Although tet(H) was detected previously on chromosomal DNA of five P. multocida isolates9 and the tet(B) gene was found on chromosomal DNA of a single P. haemolytica isolate,10 no information on the sizes of the fragments carrying these tet genes, the number of tet gene copies per isolate or the association of the tet genes with complete or truncated copies of the transposon Tn5706 or Tn10 are available. Therefore, hybridization experiments were conducted to gain information about restriction fragment length polymorphisms of tet gene-carrying fragments (Figure 3), and, from these data, the presence of complete or truncated Tn5706 or Tn10 elements.
The experiments identified truncated Tn5706 copies in the chromosomes of all P. multocida and M. haemolytica isolates carrying tet(H) but not pMHT1, and indicated the presence of a complete copy of Tn5706 on the chromosome of the M. varigena isolate P677. The tet(B) genes detected on the chromosomes of two porcine P. multocida isolates showed hybridizing fragments that suggest that isolate U-P207 carries an incomplete Tn10 element while isolate B624 has two complete copies of Tn10, one of which has an additional BamHI site.
Since no integration site specificity has been reported for Tn5706 or Tn10, integration of these transposons at different chromosomal locations in closely related isolates would be expected to result in different sized hybridizing fragments. A comparison of the tet gene hybridization patterns and macrorestriction patterns of the M. haemolytica and P. multocida isolates was performed to provide insight into the relationships of these isolates. The three P. multocida isolates that carried chromosomal tet(H) genes revealed different tet(H)-specific hybridization patterns and also differed distinctly in their macrorestriction patterns, suggesting that they are unrelated isolates. The two tet(B)-carrying P. multocida also differed distinctly in their hybridization and macrorestriction patterns, again indicating unrelated isolates. Although all M. haemolytica isolates were obtained from different animals, some of the M. haemolytica isolates proved to be either indistinguishable or closely related, on the basis of their hybridization and/or macrorestriction patterns (Table 4), suggesting a wide distribution of members of the same M. haemolytica clone among cattle. M. haemolytica isolates belonging to the same genomic group exhibited not more than two different tet(H)-specific hybridization patterns (Table 4
).
So far, no association of tet(G) with a transposable element has been described. However, tet(G) has been found on plasmids in Vibrio anguillarum21 and Pseudomonas spp.22 Unfortunately, no restriction maps of these plasmids, or the sequences flanking the tetRtet(G) gene regions are available. Moreover, the host range of these plasmids is unknown. If tet(G)-carrying plasmids have been transferred into M. haemolytica hosts and they were unable to replicate in the new host, integration of the plasmids or their tetRtet(G) genes into the chromosome of the new host might explain the occurrence of tet(G) in the chromosome of M. haemolytica. Similar situations can be assumed for the Tn10-borne tet(B) genes commonly found on enterobacterial plasmids that are replication deficient in Pasteurella hosts.23
Tetracycline resistance mediated by tet genes
No significant differences between the levels of tetracycline resistance observed in isolates carrying the tet(H), tet(B) or tet(G) gene were detected. Furthermore, the number of tet gene copies had no obvious influence on the MICTc values for the respective isolates. A single chromosomal copy per isolate of a particular tet gene seems to be as effective as two to four copies of the same gene (Table 2). Thus, more than a single tet gene copy appears not to be necessary to mediate the maximal level of resistance to clinically achievable levels of tetracyclines. However, two or more chromosomal copies of tet or even a tet gene on a multi-copy plasmid, such as pMHT1, may be of advantage to the particular bacterial isolate by ensuring the persistence of the resistance property even if one of the tet gene copies is inactivated.
In summary, the results of this study show that tetracycline resistance in P. multocida and Mannheimia spp. is mediated by at least three different tet genes, most of which are located on the chromosome. A new tet(H)-carrying plasmid was identified and, for the first time to our knowledge, tet(B) was detected in P. multocida and tet(G) in M. haemolytica. Finally, the finding of pMHT1 in M. glucosida and M. varigena is the first report of resistance plasmids in these two species.
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Acknowledgements |
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Notes |
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References |
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Received 1 March 2001; returned 20 July 2001; revised 30 July 2001; accepted 22 August 2001