Biochemical sequence analyses of GES-1, a novel class A extended-spectrum beta-lactamase, and the class 1 integron In52 from Klebsiella pneumoniae

Klebsiella pneumoniae ORT-1 was isolated in 1998 in France from a rectal sw ab of a 1-month old girl who was previously hospitalized in Cayenne Hospita l, Cayenne, French Guiana. This strain harbored a ca. 140-kb nontransferabl e plasmid, pTK1, that conferred an extended-spectrum cephalosporin resistan ce profile antagonized by the addition of clavulanic acid, tazobactam, or i mipenem. The gene for GES-1 (Guiana extended-spectrum beta-lactamase) was c loned, and its protein was expressed in Escherichia coli DH10B, where this pI-5.8 beta-lactamase of a ca. 31-kDa molecular mass conferred resistance t o oxyimino cephalosporins (mostly to ceftazidime). GES-1 is weakly related to the other plasmid-located Ambler class A extended-spectrum beta-lactamas es (ESBLs). The highest percentage of amino acid identity was obtained with the carbenicillinase GN79 from Proteus mirabilis; with YENT, a chromosome- borne penicillinase from Yersinia enterocolitica; and with L-2, a chromosom e-borne class A cephalosporinase from Stenotrophomonas maltophilia (36% ami no acid identity each). However, a dendrogram analysis showed that GES-1 cl ustered within a class A ESBL subgroup together with ESBLs VEB-1 and PER-1, Sequencing of a 7,098-bp DNA fragment from plasmid pTK1 revealed that the GES-1 gene was located on a novel class 1 integron named In52 that was char acterized by (i) a 5' conserved segment containing an intI1 gene possessing two putative promoters, P-1 and P-2, for coordinated expression of the dow nstream antibiotic resistance genes and an attI1 recombination site; (ii) f ive antibiotic gene cassettes, bla(GES-1), aac(6')Ib' (gentamicin resistanc e and amikacin susceptibility), dfrXVb (trimethoprim resistance), a novel c hloramphenicol resistance gene (cmlA4), and aadA2 (streptomycin-spectinomyc in resistance); and (iii) a 3' conserved segment consisting of qacE Delta 1 and sulI. The bla(GES-1) and aadA2 gene cassettes were peculiar, since the y lacked a typical 59-base element. This work identified the second class A ESBL gene of a non-TEM, non-SHV series which was located in the plasmid an d integron, thus providing it additional means for its spread and its expre ssion.