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 ORI-1 was isolated in 1998 in France from a rectal swab of a 1-month-old girl who was previously hospitalized in Cayenne Hospital, Cayenne, French Guiana. This strain harbored a ca. 140-kb nontransferable plasmid, pTK1, that conferred an extended-spectrum cephalosporin resistance profile antagonized by the addition of clavulanic acid, tazobactam, or imipenem. The gene for GES-1 (Guiana extended-spectrum beta-lactamase) was cloned, 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 to oxyimino cephalosporins (mostly to ceftazidime). GES-1 is weakly related to the other plasmid-located Ambler class A extended-spectrum beta-lactamases (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 chromosome-borne class A cephalosporinase from Stenotrophomonas maltophilia (36% amino acid identity each). However, a dendrogram analysis showed that GES-1 clustered 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 characterized by (i) a 5' conserved segment containing an intI1 gene possessing two putative promoters, P(1) and P(2), for coordinated expression of the downstream antibiotic resistance genes and an attI1 recombination site; (ii) five antibiotic gene cassettes, bla(GES-1), aac(6')Ib' (gentamicin resistance and amikacin susceptibility), dfrXVb (trimethoprim resistance), a novel chloramphenicol resistance gene (cmlA4), and aadA2 (streptomycin-spectinomycin resistance); and (iii) a 3' conserved segment consisting of qacEDelta1 and sulI. The bla(GES-1) and aadA2 gene cassettes were peculiar, since they 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 and integron, thus providing it additional means for its spread and its expression.
Poirel
L
L
Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, France.
Le Thomas
I
I
Naas
T
T
Karim
A
A
Nordmann
P
P
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D019426:Integrases / Q000235:genetics*; D007710:Klebsiella Infections / Q000382:microbiology; D007711:Klebsiella pneumoniae / Q000145:classification / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D047090:beta-Lactams
D000900:Anti-Bacterial Agents; D047090:beta-Lactams; D019426:Integrases; C405902:GES-1 beta-lactamase; D001618:beta-Lactamases