The plasmid content and molecular distribution of the ROB-1 13-lactamase was investigated in 31 swine isolates of Actinobacillus pleuropneumonuae. Three types of plasmid patterns were observed in ampicillinresistant isolates: a predominant one consisting of 2.6-, 2.9-, and 5.0-kb plasmids and two other patterns consisting of 4.2-and 5.5-kb plasmids and of a 6.8-kb plasmid. Plasmid DNA preparations were hybridized with a blaROB-l intragenic fragment from the Haemophilus influenzae Rr.h plasmid. Positive hybridizations were observed with all ampicillin-resistant isolates. The blaROBl gene was found on 3 plasmids of 2.6, 5.5, and 6.8 kb. One swine isolate of Pasteurella multocida also had a 2.6-kb plasmid bearing blaROB l. ROB-1 was confirmed in typical isolates by isoelectric focusing and blaROB.l sequences were confirmed by polymerase chain reaction amplification with an intragenic set of primers. Plasmids bearing blaROB-i were successfully electrotransformed in a susceptible A. pleuropneumoniae isolate. These results emphasize the importance of ROB-1 in A. pleuropneumoniae and identify a possible reservoir of P-lactam resistance.The antimicrobial susceptibility of Actinobacillus pleuropneumoniae has been studied largely because of the economical importance of porcine pleuropneumonia. The ampicillin resistance of A. pleuropneumoniae is presumably increasing in Quebec (Canada), as demonstrated by susceptibility studies which showed that 53% of isolates were resistant in 1981 and that 74% were resistant in 1986 (24). In Canada (Ontario), the United States (South Dakota and Iowa), Italy, and Japan, more than 80% of isolates were found susceptible to ampicillin (2,7,(17)(18)(19). Plasmid-mediated ampicillin resistance due to a P-lactamase was first described in A. pleuropneumoniae by Hirsh et al. in 1982, and the bla gene was found associated with pVM105 (5.3 kb) (3). Later studies associated ampicillin resistance with 4.3-, 5.3-, and 8.3-kb plasmids (4).The ROB-1 P-lactamase was first described in ampicillinresistant Haemophilus influenzae type b (16). One year later, a plasmid-mediated P-lactamase was found in ampicillinresistant A. pleuropneumoniae but was identified as a TEMtype enzyme (3). Isoelectric focusing and DNA hybridization confirmed that the plasmid-mediated P-lactamases from both H. influenzae and A. pleuropneumoniae were ROB-1 (10). Further studies indicated that the blaROBl gene did not cross-hybridize with other known bla genes (6). Recent analysis at the molecular level clearly showed that ROB-1 is an unusual class A ,-lactamase that could have originated in gram-positive bacteria (5). The P-lactamase was also found in a porcine isolate of Pasteurella multocida and in diverse isolates of Pasteurella species (8). An epidemiological survey of the prevalence of the ROB-1 ,-lactamase among ampicillin-resistant H. influenzae isolates in the United States showed that 92% produced the TEM-1 enzyme, while the remaining isolates produced the ROB-1 enzyme (1).In this study, we report the plasmid content of a...