Daniel Aubert

Genetic and Functional Analysis of the Chromosome-Encoded Carbapenem-Hydrolyzing Oxacillinase OXA-40 of Acinetobacter baumannii

ABSTRACT Clinical isolate Acinetobacter baumannii CLA-1 was resistant to a series of antibiotic molecules, including carbapenems. Cloning and expression of the β-lactamase gene content of this isolate in Escherichia coli DH10B identified a chromosome-encoded oxacillinase, OXA-40, that differed by one or two amino acid changes from OXA-24, -25, and -26 and an AmpC-type cephalosporinase. The OXA-40 β-lactamase had a mainly narrow-spectrum hydrolytic profile, but it included ceftazidime and imipenem. Its activity was resistant to inhibition by clavulanic acid, tazobactam, sulbactam, and, like most of the other carbapenem-hydrolyzing oxacillinases, NaCl. OXA-40 had an FGN triad replacing a YGN motif at class D β-lactamase (DBL) positions 144 to 146. Site-directed DNA mutagenesis leading to a Phe-to-Tyr change at DBL position 144 in OXA-40 gave a mutant enzyme with increased hydrolytic activity against most β-lactams, including imipenem. Conversely, with a gene encoding the narrow-spectrum oxacillinase OXA-1 as the template, a nucleotide substitution leading to a Tyr-to-Phe change in the YGN motif of OXA-1 gave a mutant enzyme with decreased hydrolytic activity without an increase in carbapenem-hydrolyzing activity. Thus, the Phe residue in the FGN motif was not associated with carbapenem-hydrolyzing activity by itself but instead was associated with weak overall hydrolytic activity. Finally, this Phe residue in OXA-40 explained resistance to inhibition by NaCl whereas a Tyr residue in motif YGN was related to susceptibility to NaCl.

Molecular and Biochemical Heterogeneity of Class B Carbapenem-Hydrolyzing β-Lactamases in Chryseobacterium meningosepticum

ABSTRACT Although the carbapenem-hydrolyzing β-lactamase (CHβL) BlaB-1 is known to be in Chryseobacterium meningosepticum NCTC 10585, a second CHβL gene, blaGOB-1, was cloned from another C. meningosepticum clinical isolate (PINT). The G+C content of blaGOB-1 (36%) indicated the likely chromosomal origin of this gene. Its expression inEscherichia coli DH10B yields a mature CHβL with a pI of 8.7 and a relative molecular mass of 28.2 kDa. In E. coli, GOB-1 conferred resistance to narrow-spectrum cephalosporins and reduced susceptibility to ureidopenicillins, broad-spectrum cephalosporins, and carbapenems. GOB-1 had a broad-spectrum hydrolysis profile including penicillins and cephalosporins (but not aztreonam). The catalytic efficiency for meropenem was higher than for imipenem. GOB-1 had low amino acid identity with the class B CHβLs, sharing 18% with the closest, L-1 from Stenotrophomonas maltophilia, and only 11% with BlaB-1. Most of the conserved amino acids that may be involved in the active site of CHβLs (His-101, Asp-103, His-162, and His-225) were identified in GOB-1. Sequence heterogeneity was found for GOB-1-like and BlaB-1-like β-lactamases, having 90 to 100% and 86 to 100% amino acid identity, respectively, among 10 unrelated C. meningosepticumisolates. Each isolate had a GOB-1-like and a BlaB-1-like gene. The same combination of GOB-1-like and BlaB-1-like β-lactamases was not found in two different isolates. C. meningosepticum is a bacterial species with two types of unrelated chromosome-borne class B CHβLs that can be expressed in E. coli and, thus, may represent a clinical threat if spread in gram-negative aerobes.

In Vitro Analysis of ISEcp1B-Mediated Mobilization of Naturally Occurring β-Lactamase Gene blaCTX-M of Kluyvera ascorbata

ABSTRACT ISEcp1B has been reported to be associated with and to mobilize the emerging expanded-spectrum β-lactamase blaCTX-M genes in Enterobacteriaceae. Thus, the ability of this insertion sequence to mobilize the blaCTX-M-2 gene was tested from its progenitor, Kluyvera ascorbata. Insertions of ISEcp1B upstream of the blaCTX-M-2 gene in K. ascorbata reference strain CIP7953 were first selected with cefotaxime (0.5 and 2 μg/ml). In those cases, ISEcp1B brought promoter sequences enhancing blaCTX-M-2 expression in K. ascorbata. Then, ISEcp1B-mediated mobilization of the blaCTX-M-2 gene from K. ascorbata to Escherichia coli J53 was attempted. The transposition frequency of ISEcp1B-blaCTX-M-2 occurred at (6.4 ± 0.5) × 10−7 in E. coli. Cefotaxime, ceftazidime, and piperacillin enhanced transposition, whereas amoxicillin, cefuroxime, and nalidixic acid did not. Transposition was also enhanced when studied at 40°C.

Oxacillinase-Mediated Resistance to Cefepime and Susceptibility to Ceftazidime in Pseudomonas aeruginosa

ABSTRACT Pseudomonas aeruginosa clinical isolate SOF-1 was resistant to cefepime and susceptible to ceftazidime. This resistance phenotype was explained by the expression of OXA-31, which shared 98% amino acid identity with a class D β-lactamase, OXA-1. Theoxa-31 gene was located on a ca. 300-kb nonconjugative plasmid and on a class 1 integron. No additional efflux mechanism for cefepime was detected in P. aeruginosa SOF-1. Resistance to cefepime and susceptibility to ceftazidime in P. aeruginosawere conferred by OXA-1 as well.

Functional and Structural Characterization of the Genetic Environment of an Extended-Spectrum β-Lactamase blaVEB Gene from a Pseudomonas aeruginosa Isolate Obtained in India

ABSTRACT A Pseudomonas aeruginosa clinical strain isolated from a patient hospitalized in a New Delhi, India, hospital was resistant to expanded-spectrum cephalosporins, imipenem, and aztreonam. A blaVEB-1-like gene named blaVEB-1a, which codes for the extended-spectrum β-lactamase VEB-1a, was identified. The genetic environment of blaVEB-1a was peculiar: (i) no 5′ conserved sequence (5′-CS) region was present upstream of the β-lactamase gene, whereas blaVEB-1-like genes are usually associated with class 1 integrons; (ii) blaVEB-1a was inserted between two truncated 3′-CS regions in a direct repeat; and (iii) four 135-bp repeated DNA sequences (repeated elements) were located on each side of the blaVEB-1a gene. Expression of the blaVEB-1a gene was driven by a strong promoter located in one of these repeated sequences. In addition, cloning of the β-lactamase content of this P. aeruginosa isolate followed by expression in Escherichia coli identified the naturally occurring AmpC β-lactamase and a gene encoding an OXA-2-like β-lactamase located in a class 1 integron, In78, in which an insertion sequence, ISpa7, was inserted within its 5′-CS region.

Novel Genetic Structure Associated with an Extended-Spectrum β-Lactamase blaVEB Gene in a Providencia stuartii Clinical Isolate from Algeria

ABSTRACT A ceftazidime-resistant Providencia stuartii isolate from Algeria harbored a ca. 160-kb conjugative plasmid that contained a truncated blaVEB-1b gene flanked by three 135-bp repeated elements. This work gives further evidence of the worldwide spread of blaVEB genes that are associated with genetic structures other than class 1 integrons.

Complex Genetic Structures with Repeated Elements, a sul-Type Class 1 Integron, and the blaVEB Extended-Spectrum β-Lactamase Gene

ABSTRACT Two clinical isolates of Pseudomonas aeruginosa, TL-1 and TL-2, were isolated from a patient transferred from Bangladesh and hospitalized for osteomyelitis in Paris, France. P. aeruginosa TL-1 expressed the extended-spectrum β-lactamase VEB-1a and was susceptible only to imipenem and colistin, while P. aeruginosa TL-2 expressed only the naturally occurring blaAmpC gene at a basal level and exhibited a wild-type β-lactam resistance phenotype. In TL-1, the typical 5′-end conserved sequence (5′-CS) region of class 1 integrons usually present upstream of the blaVEB-1a gene was replaced by a truncated 3′-CS and a 135-bp repeated element (Re). Downstream of the blaVEB-1a gene, an insertion sequence, ISPa31 disrupted by ISPa30, and an orf513 sequence, belonging to a common region (conserved region 1 [CR1]) immediately upstream of the aphA-6 gene, were present. Further downstream, a second truncated 3′-CS region in direct repeat belonged to In51, an integron containing two gene cassettes (aadA6 and the OrfD cassette). Thus, the overall structure corresponded to a sul-type class 1 integron termed In121. Genetic analyses revealed that both isolates were clonally related and differed by a ca. 100-kb fragment that contained In121. Both isolates contained another integron, In122, that carried three gene cassettes: aadB, dfrA1, and the OrfX cassette. This work identifies for the first time the spread of Re-associated blaVEB genes located on a sul-type integron. It also reports for the first time a CR1 element in P. aeruginosa that is associated with an aminoglycoside resistance aphA-6 gene that is expressed from a composite promoter.

IS1999 Increases Expression of the Extended-Spectrum β-Lactamase VEB-1 in Pseudomonas aeruginosa

The integron-borne bla(VEB-1) gene encodes an extended-spectrum beta-lactamase. This gene was associated mostly with IS1999 and rarely with an additional IS2000 element in Pseudomonas aeruginosa isolates from Thailand, whereas IS1999 was only very rarely associated with bla(VEB-1) in Enterobacteriaceae. Expression experiments and promoter study identified promoter sequences in IS1999 that increased the expression of VEB-1 in P. aeruginosa.

Chromosome-Encoded Narrow-Spectrum Ambler Class A β-Lactamase GIL-1 from Citrobacter gillenii

ABSTRACT A novel β-lactamase gene was cloned from the whole-cell DNA of an enterobacterial Citrobacter gillenii reference strain that displayed a weak narrow-spectrum β-lactam-resistant phenotype and was expressed in Escherichia coli. It encoded a clavulanic acid-inhibited Ambler class A β-lactamase, GIL-1, with a pI value of 7.5 and a molecular mass of ca. 29 kDa. GIL-1 had the highest percent amino acid sequence identity with TEM-1 and SHV-1, 77%, and 67%, respectively, and only 46%, 31%, and 32% amino acid sequence identity with CKO-1 (C. koseri), CdiA1 (C. diversus), and SED-1 (C. sedlaki), respectively. The substrate profile of the purified GIL-1 was similar to that of β-lactamases TEM-1 and SHV-1. The blaGIL-1 gene was chromosomally located, as revealed by I-CeuI experiments, and was constitutively expressed at a low level in C. gillenii. No gene homologous to the regulatory ampR genes of chromosomal class C β-lactamases was found upstream of the blaGIL-1 gene, which fits the noninducibility of β-lactamase expression in C. gillenii. Rapid amplification of DNA 5′ ends analysis of the promoter region revealed putative promoter sequences that diverge from what has been identified as the consensus sequence in E. coli. The blaGIL-1 gene was part of a 5.5-kb DNA fragment bracketed by a 9-bp duplication and inserted between the d-lactate dehydrogenase gene and the ydbH genes; this DNA fragment was absent in other Citrobacter species. This work further illustrates the heterogeneity of β-lactamases in Citrobacter spp., which may indicate that the variability of Citrobacter species is greater than expected.

Integrase-Mediated Recombination of the veb1 Gene Cassette Encoding an Extended-Spectrum β-Lactamase

The veb1 gene cassette encodes the extended spectrum β-lactamase, VEB-1 that is increasingly isolated from worldwide Gram-negative rods. Veb1 is commonly inserted into the variable region of different class 1 integrons in which it is always associated with a downstream-located aadB gene cassette encoding an aminoglycoside adenylyltransferase. In Pseudomonas aeruginosa, the majority of veb1-containing integrons also carry an insertion sequence, IS1999 that is inserted upstream of the veb1 gene cassette and disrupts the integron specific recombination site, attI1. Investigation of the recombination properties of the sites surrounding veb1 revealed that insertion of IS1999 reduces significantly the recombination frequency of attI1 and that veb1 attC is not efficient for recombination in contrast to aadB attC. Subsequent sequence optimisation of veb1 attC by mutagenesis, into a more consensual attC site resembling aadB attC, successfully improved recombination efficiency. Overall, this work gives some insights into the organisation of veb1-containing integrons. We propose that IS1999 and the nature of veb1 attC stabilize the veb1 gene cassette environment likely by impairing recombination events upstream or downstream of veb1, respectively.