Sequence and molecular characterization of the ROB-1 beta-lactamase gene from Pasteurella haemolytica

Abstract

The ROB-1 ,I-lactamase-encoding plasmids from eight Pasteurella and two Haemophilus strains were compared by restriction endonuclease and hybridization analyses. Two types of ROB-i-encoding plasmids, which differed in size, were detected. One (4.1 kb) was found only in Pasteurella strains. The other (4.4 kb) was found in both Haemophilus influenzae and in one of the eight Pasteurella strains examined. These two plasmids shared multiple homologous fragments, suggesting that one was derived from the other. The ROB-i-encoding gene from Pasteurella P-Lactamases are the major determinants of resistance to P-lactam antibiotics among microorganisms and are widely distributed in both gram-positive and gram-negative bacteria (11). These enzymes were first classified according to their substrate profiles, isoelectric points, and other properties such as molecular weight. A classification scheme based on amino acid composition and sequence homologies was later proposed by Ambler (1), who defined classes A and B.3-Lactamases are now divided into four classes. Class A includes serine penicillinases such as chromosomally encoded 13-lactamases from Bacillus spp. (37, 42), Streptomyces spp. (17, 18), and Klebsiella spp. (3, 4); plasmid-mediated enzymes from Staphylococcus aureus PC1 (46); and TEM-and SHV-type enzymes from members of the family Enterobacteriaceae (2,6,43). All these class A P-lactamases show amino acid sequence homology, especially around the active serine site. Class B includes metalloenzymes (1), class C includes chromosomally encoded cephalosporinases (24), and class D includes oxacillinases (23). Most ,-lactamases have not yet been classified according to this scheme because their amino acid sequences are not available.The ROB-1 ,B-lactamase was first described in a human Haemophilus influenzae strain. The new enzyme had a TEM-like substrate profile, and its isoelectric point was estimated to be 8.1. In this strain ROB-1 was encoded by a plasmid of 4.4 kb, RROb (40). ROB-1 was later found in several H. influenzae strains and in porcine Actinobacillus pleuropneumoniae strains throughout the United States (34). More recently, in France we found (31) bovine and porcine Pasteurella multocida, Pasteurella haemolytica, and Pasteurella aerogenes strains that produce ROB-1. Furthermore, study of a ROB-1-encoding plasmid from an H. influenzae strain isolated in France revealed that it is identical to the 4.4-kb plasmid RROb (30).Recently, the nucleotide sequence of the ROB-1 13-lacta-* Corresponding author.mase gene from H. influenzae plasmid RROb was determined (26). We report here the molecular characterization of the ROB-1-encoding plasmids from Pasteurella and Haemophilus strains. The nucleotide sequence of the ,-lactamase gene from P. haemolytica LNPB 51 was determined, and the deduced amino acid sequence was compared with those of other enzymes. Our data confirmed that ROB-1 is a class A 13-lactamase which shares several features with gram-positive bacterial 1-lactamases. MATERIALS AND METHODSBacterial strain...

Antimicrobial resistance in Pasteurella and Mannheimia: epidemiology and genetic basis

“…The sequence of ROB1 protein was compared with the sequences of other class A β-lactamases and showed about 40% homology with all the known class A enzymes. The ROB1 enzyme exhibited the highest similarity to the β-lactamases from gram-positive bacteria and appeared as a possible link between the β-lactamases of gram-positive and gram-negative bacteria [45].…”

“…Since the Mob proteins originating from one plasmid may be used for the mobilisation of all plasmids of the same bacterial cellprovided these plasmids carry an "origin of transfer" (oriT) -, plasmids which do not harbour mob genes can also be mobilized. Mobilisation is also the most reasonable explanation for the occurrence of indistinguishable type pPAT1 tet(H)-carrying plasmids in porcine P. multocida and P. aerogenes isolates [36], but also for the presence of identical plasmid-borne bla ROB1 genes in members of the genera Pasteurella [44,45], Actinobacillus [34], and Haemophilus [18,48]. Conjugative plasmids not associated with antimicrobial resistance, but promoting mobilisation of co-resident small resistance plasmids have been described in P. multocida from turkeys [29].…”

“…pleuropneumoniae (AB034202; S51028 or M97481), and one from Haemophilus (H.) influenzae (AF022114). All ROB1-β-lactamase protein sequences of 305 amino acids (aa) are identical [34,44,45,48]. The sequence of ROB1 protein was compared with the sequences of other class A β-lactamases and showed about 40% homology with all the known class A enzymes.…”

“…The corresponding bla ROB1 gene was on a plasmid of 4.4 kb. Later on, the ROB1 enzyme was detected in porcine Actinobacillus pleuropneumoniae isolates throughout the USA, and 81 87 82 52 86 -36 -1991 310 87 76 81 77 42 77 74 27 69 1992 471 90 89 70 77 39 58 71 25 85 1993 428 90 87 59 73 27 45 51 16 79 1994 488 90 81 74 80 35 64 45 26 84 1995 685 91 71 77 81 42 64 38 23 77 1996 560 90 66 70 72 38 56 51 in bovine and porcine P. multocida, P. haemolytica and P. aerogenes in France [44,45,48,51]. In all the bovine strains of Pasteurella studied at that time, the bla ROB1 genes were present on plasmids of 4.1 kb except in one strain of P. haemolytica harbouring a plasmid of 4.4 kb.…”

Identification of a ROB-1 beta-lactamase in Haemophilus ducreyi

“…Three other ROB-1 P-lactamase plasmids have been described previously. A 4.1-kb ROB-1 plasmid was isolated from Pasteurella strains (8), while a 4.4-kb plasmid, Rrob, was found in Haemophilus strains and at least one isolate of Pasteurella haemolytica (7). A third plasmid, of 5.4 kb, having the bla ROB-1 gene has been described only with A. pleuropneumoniae (10).…”