Molecular characterization of chromosomal class C beta-lactamase and its regulatory gene in Ochrobactrum anthropi

Abstract

Ochrobactrum anthropi, formerly known as CDC group Vd, is an oxidase-producing, gram-negative, obligately aerobic, non-lactose-fermenting bacillus of low virulence that occasionally causes human infections. It is highly resistant to all beta-lactams except imipenem. A clinical isolate, SLO74, and six reference strains were tested. MICs of penicillins, aztreonam, and most cephalosporins tested, including cefotaxime and ceftazidime, were >128 microg/ml and of cefepime were 64 to >128 microg/ml. Clavulanic acid was ineffective and tazobactam had a weak effect in association with piperacillin. Two genes, ampR and ampC, were cloned by inserting restriction fragments of genomic DNA from the clinical strain O. anthropi SLO74 into pBK-CMV to give the recombinant plasmid pBK-OA1. The pattern of resistance to beta-lactams of this clone was similar to that of the parental strain, except for its resistance to cefepime (MIC, 0.5 ,micro/ml). The deduced amino acid sequence of the AmpC beta-lactamase (pI, 8.9) was only 41 to 52% identical to the sequence of other chromosomally encoded and plasmid-encoded class C beta-lactamases. The kinetic properties of this beta-lactamase were typical for this class of beta-lactamases. Upstream from the ampC gene, the ampR gene encodes a protein with a sequence that is 46 to 62% identical to those of other AmpR proteins and with an amino-terminal DNA-binding domain typical of transcriptional activators of the Lys-R family. The deduced amino acid sequences of the ampC genes of the six reference strains were 96 to 99% identical to the sequence of the clinical strain. The beta-lactamase characterized from strain SLO74 was named OCH-1 (gene, bla(OCH-I)).

FIG. 1

Schematic restriction endonuclease map of the recombinant plasmids pBK-OA1 and pSK⁺-OA2. The genes ampR and ampC and the other ORFs are indicated.

FIG. 2

Nucleotide sequence of the 2,244-bp fragment of pBK-OA1 containing the ampC and ampR coding regions. The putative promoter sequences are indicated as −35 and −10 regions (boxed). The start codons and the Lys-R motif are indicated by arrows, and the stop codons are underlined. The PCR primers upper ochro and lower ochro are in italics.

FIG. 3

Multiple alignment of AmpC amino acid sequences deduced from the sequence of the ampC gene present in O. anthropi SLO74 and in the six reference strains of O. anthropi. The specific SVSK and KTG boxes of the serine-active β-lactamases and the KTG box specific for class C β-lactamases are boxed. Identical amino acids are indicated by dashes.

FIG. 4

Schematic dendrogram obtained for 16 representative chromosomally encoded and plasmid-encoded class C β-lactamases. Percentages in brackets are percent identities between the indicated amino acid sequence and that of OCH-1.

FIG. 5

Multiple alignment of deduced amino acid sequences of AmpR, which regulates cephalosporinase genes. The origins of the AmpR sequences are as follows: 1, E. cloacae MHN-1; 2, Citrobacter freundii OS 60; 3, Yersinia enterocolitica IP97; 4, Morganella morganii GUI-1; 5, O. anthropi SLO74; 6, P. aeruginosa PAO01; 7, Providencia stuartii VDG 96. Identical amino acids are indicated by an asterisk. The predicted helix-turn-helix motif (HTH) of the Lys-R family is indicated by arrows.

FIG. 6

Alignment of the ampC-ampR intercistronic region from the β-lactamase of the following strains: 1, O. anthropi SLO74; 2, P. aeruginosa PAO01; 3, P. stuartii VDG 96; 4, M. morganii SLM 01; 5, Y. enterocolitica IP 97; 6, C. freundii OS 60. The Lys-R motif is boxed.