An efficient method for DNA transfer is essential for the genetic manipulation of any organism. Such a capacity will be required for the genetic analysis of Actinobacillus pleuropneumoniae as a swine pathogen, as well as for its manipulation for vaccination purposes. For this reason, the use of electroporation as a means of plasmid DNA introduction into this species was examined. The multiply antibiotic-resistant strain 80-8141 of Actinobacillus pleuropneumoniae harbors 3 plasmids: pYG10, pYG15, and pYG12 of 5.0, 2.7, and 2.5 kb, respectively. Electroporation of A pleuropneumoniae strain 4074 with a plasmid extract of strain 80-8141 showed that pYG10 encodes chloramphenicol resistance and that pYG12 encodes ampicillin resistance. Electrical pulse conditions for efficient electroporation of strain 4074 were examined by use of pYG10 DNA isolated from a 4074 transformant. Efficiency, expressed as transformants per microgram of plasmid DNA, increased directly with pulse amplitude. However, high efficiencies were only observed in a narrow window of pulse duration (gamma = 12 to 22 ms at 6.25 kV/cm). Longer pulse durations resulted in cell death. Electroporation efficiencies increased with cell density. Yield of transformants increased directly with DNA concentration. Results indicate that electroporation can be used to efficiently transform A pleuropneumoniae and that pYG10 and pYG12 are suitable plasmid vectors for use in the genetic manipulation of this organism. Actinobacillus (Haemophilus) pleuropneumoniae is a prominent cause of respiratory infections in swine. Clinical isolates of A pleuropneumoniae have been reported to be resistant to tetracycline, triple sulfonamides, ampicillin, and streptomycin. There has been particular concern over the increasing incidence of resistance to chloramphenicol, which may be related to the extensive use of this antibiotic for treatment of swine pleuropneumonia. In 1980, 95% of the strains of A pleuropneumoniae isolated from the St-Hyacinthe region of Quebec, Canada, were found to be sensitive to chloramphenicol; whereas in 1982, only 57% of surveyed strains were still sensitive to this antibiotic. Resistance to ampicillin, streptomycin, and sulfadiazine in A pl europneumoniae strains has been shown to be plasmid-mediated. The purpose of the study reported here was to use electroporation to analyze plasmids carried by a multiply antibiotic-resistant clinical isolate of A pleuropneumoniae. Electroporation involves the use of brief high-voltage electrical discharges to induce reversible permeability in both prokaryotic and eukaryotic membranes. Using a 5.0-kb A pleuropneumoniae plasmid encoding resistance to chloramphenicol, we have optimized electroporation as a means to transform this species. Conditions permitting an efficiency of over 10(5) transformants (Tfs)/microgram of plasmid DNA are described.

Actinobacillus (Haemophilus) pleuropneumoniae plasmids were characterized and classified. They were isolated from A pleuropneumoniae strains different in serotype, year isolated, or location from which isolated. Six of 8 plasmids encoded streptomycin (Sm) and sulfonamide (Su) resistance (SmSu). One of the other plasmids, pVM105, encoded ampicillin (Ap) resistance and another, pHMO, encoded no drug resistance. All SmSu plasmids were transferred to Escherichia coli strains by transformation. Among them, pABO and pMS260 were 8.1 kb and incompatible with each other; they were stable in E coli. The other SmSu plasmids, pHM1, pVM104, pVM106, and pKD25, were 4.3 kb and did not replicate stably in E coli. The former SmSu plasmids were mobilized in E coli strains by a plasmid RP4, which belonged to incompatibility (Inc) group P, but the latter plasmids were not. Further, each 8.1-kb SmSu plasmid and each 4.3-kb plasmid had the same respective restriction pattern. These results indicated that there were at least 2 types of SmSu plasmids in A pleuropneumoniae. The 2 types were classified in 2 groups: Hl(pMS260 and pABO) and H2(pHM1, pVM104, pVM106, and pKD25). The Hl and H2 plasmids belonged to a different Inc groups, and H2 plasmids belonged to a different Inc group from that of pHMO and pVM105.

Corynebacterium pseudotuberculosis was transformed by electroporation, using pNG2, an erythromycin-resistance plasmid from C diphtheriae. Corynebacterium pseudotuberculosis cultivated in brain-heart infusion broth was washed 3 times with water, and resuspended to a final concentration of about 5 X 10(13) colony-forming units/ml. An electroporator constructed in our laboratory incorporated an electrode with 0.8-mm interelectrode gap, using disposable spectrophotometer cuvettes as containers for electroporation. The pNG2 was prepared in Escherichia coli and 4 to 16 microgram of pNG2 DNA was mixed with 400-microliter amounts of cell suspension in prechilled cuvettes. After incubation on ice for 5 to 10 minutes, the mixture was electroporated at field strengths of up to 18 kV/cm, mixed with 1.5 ml of brain-heart infusion broth, and incubated at 37 C for 2 hours with agitation. Aliquots were then plated on brain-heart infusion blood agar with 15 microgram of erythromycin/ml. Corynebacterium pseudotuberculosis was transformed at a maximal efficiency of approximately 4 X 10(4) transformants/microgram of pNG2 DNA. Most total transformants and most transformants per microgram of pNG2 were generated at a field strength of 18 kV/cm. When the concentration of pNG2 DNA was varied, the average total number of transformants increased through a concentration of 30 microgram/ml, but the efficiency of transformation was highest at the lowest DNA concentration. Transformants contained unmodified pNG2.

The genetic basis of drug-resistant strains of Actinobacillus pleuropneumoniae in Japan was studied. The A pleuropneumoniae strains AV277 and AV281 that belong to serotype 2 were resistant to streptomycin (SM) and sulfonamide (SA). Both strains had an 8.1-kilobase (kb) SM-SA plasmid that was previously classified in the H1 group. The AV177 (serotype 1) strain was resistant to SM, SA, ampicillin, and kanamycin (Km), but did not have any plasmids. The AV319 and AV324 (serotype 1) strains were resistant to Sm, SA, tetracycline (TC), and chloramphenicol (CP). The AV318 (serotype 12) strain was resistant to SM, SA, TC, minocycline, and CP. These 3 strains (AV319, AV324, and AV318) had a 4.3-kb SM-SA plasmid and a 5.2-kb CP plasmid. The 4.3-kb plasmid was classified in the H2 group. The AV263 (serotype 1) strain was resistant to SM, SA, KM, TC, and CP. It had a 5.2-kb CP plasmid and a 6.6-kb SM-SA-KM plasmid. Both plasmids did not replicate stably in Escherichia coli strains. The former 5.2-kb plasmid was mobilized in E coli strains by plasmid RP4, which belonged to incompatibility P with broad host range, but the latter 6.6-kb plasmid was not so mobilized. Three 5.2-kb CP plasmids isolated from strains AV319, AV324, and AV318, had the same restriction endonuclease pattern after digestion with Ava I and EcoRI. They coexisted with H1 group plasmids in the incompatibility test, and coexisted also with H2 group plasmids of the original A pleuropneumoniae strains. Results indicated that the 5.2-kb CP plasmids could be classified in a new incompatibility group, H3. In this study, 4 types of plasmids were isolated, but no plasmids encoded TC and minocycline resistance.