Delphine Girlich

Value of the Modified Hodge Test for Detection of Emerging Carbapenemases in Enterobacteriaceae

ABSTRACT The modified Hodge test has an excellent sensitivity for detecting enterobacterial isolates producing Ambler class A (KPC) and class D (OXA-48) carbapenemases. Its sensitivity is low for NDM-1 producers (50%) but is increased to 85.7% by adding ZnSO4 (100 μg/ml) in the culture medium. However, this test has a low specificity and is time-consuming.

Molecular Epidemiology of the Integron-Located VEB-1 Extended-Spectrum β-Lactamase in Nosocomial Enterobacterial Isolates in Bangkok, Thailand

ABSTRACT Over a 2½-month period in 1999, 37 ceftazidime-resistant nonrepetitive enterobacterial isolates were collected from 37 patients in a Bangkok hospital, Thailand. Eighty-one percent of these strains expressed a clavulanic acid-inhibited extended-cephalosporin resistance profile. An identical extended-spectrum β-lactamase (ESBL), VEB-1, was found in 16 unrelated enterobacterial isolates (Escherichia coli, n = 10; Enterobacter cloacae, n = 2;Enterobacter sakazakii, n= 1; and Klebsiella pneumoniae, n = 3) and in two clonally related E. cloacae isolates. TheblaVEB-1 gene was located on mostly self-conjugative plasmids (ca. 24 to 200 kb) that conferred additional non-β-lactam antibiotic resistance patterns. Additionally, theblaVEB-1 gene cassette was part of class 1 integrons varying in size and structure. TheblaVEB-1-containing integrons were mostly associated with blaOXA-10-like andarr-2-like gene cassettes, the latter conferring resistance to rifampin. These data indicated the spread ofblaVEB-1 in Bangkok due to frequent transfer of different plasmids and class 1 integrons and rarely to clonally related strains. Plasmid- and integron-mediated resistance to rifampin was also found in enterobacterial isolates.

OXA-28, an Extended-Spectrum Variant of OXA-10 β-Lactamase from Pseudomonas aeruginosa and Its Plasmid- and Integron-Located Gene

ABSTRACT Pseudomonas aeruginosa ED-1, isolated from a pulmonary brush of a patient hospitalized in a suburb of Paris, France, was resistant to ceftazidime and of intermediate susceptibility to ureidopenicillins and to cefotaxime. Cloning and expression of the β-lactamase gene content of this isolate in Escherichia coli DH10B identified a novel OXA-10 variant, OXA-28, with a pI value of 8.1 and a molecular mass of 29 kDa. It differed from OXA-10 by 10 amino acid changes and from OXA-13 and OXA-19 by 2 amino acid changes, including a glycine instead of tryptophan at position 164, which is likely involved in its resistance to ceftazidime. Like OXA-11, -14, -16, and -19 and as opposed to OXA-17, OXA-28 predominantly compromised ceftazidime and had only marginal effect on the MICs of aztreonam and cefotaxime in P. aeruginosa. Once expressed in E. coli, OXA-28 raised the MIC of ceftazidime to a much higher level than those of amoxicillin, cephalothin, and cefotaxime (128, 16, 8, and 4 μg/ml, respectively). OXA-28 β-lactamase had a broad spectrum of activity, including ceftazidime. Its activity was partially antagonized by clavulanic acid (50% inhibitory concentration, 10 μM) and NaCl addition. The oxa28 gene cassette was inserted in the variable region of a class 1 integron, In57, immediately downstream of an amino 6′-N-acetyltransferase gene cassette,aac(6′)Ib. The structures of the integrons carrying eitheroxa28, oxa13, or oxa19 gene cassettes were almost identical, suggesting that they may have derived from a common ancestor as a result of the common European origin of theP. aeruginosa isolates. In57 was located on a self-transferable plasmid of ca. 150 kb that was transferred fromP. aeruginosa to P. aeruginosa.

Extended-Spectrum β-Lactamase CTX-M-1 in Escherichia coli Isolates from Healthy Poultry in France

ABSTRACT Genes encoding extended-spectrum β-lactamase CTX-M-1 were detected in 12 Escherichia coli isolates recovered over a 7-month period from the ceca of healthy poultry in seven districts in France in 2005. Eleven of those strains were not clonally related and had a blaCTX-M-1 gene located on transferable plasmids of different sizes and structures.

Nosocomial Spread of the Integron-Located veb-1—Like Cassette Encoding an Extended-Spectrum β-Lactamase in Pseudomonas aeruginosa in Thailand

The beta-lactamase gene content and epidemiology of ceftazidime-resistant Pseudomonas aeruginosa isolates (24% of the total number of P. aeruginosa isolates) were investigated at a University Hospital in Thailand during a 4-month period in 1999. Of 33 nonrepetitive clinical isolates, 31 produced a VEB-1-like clavulanic acid-inhibited extended-spectrum beta-lactamase (ESBL). These isolates belonged to different pulsed-field gel electrophoresis types and subtypes. In 1 case, the bla(VEB-1)-like gene was plasmid located. The bla(VEB-1)-like genes were present as a gene cassette on class 1 integrons that varied in size and structure. In most cases, the veb-1 cassette was associated with an arr-2 cassette (rifampin resistance), aminoglycoside resistance gene cassettes, and an oxa-10-like cassette encoding a narrow-spectrum oxacillinase-type beta-lactamase. The present study indicates that ESBLs may be endemic in P. aeruginosa and illustrates that integrons are efficient means for their spread.

Biochemical Characterization of the Naturally Occurring Oxacillinase OXA-50 of Pseudomonas aeruginosa

ABSTRACT The blaOXA-50 gene (formerly known as the PA5514 gene) is an oxacillinase gene identified in silico in the genome of Pseudomonas aeruginosa PAO1. By using a mutant strain of P. aeruginosa PAO1 that had an inactivated blaAmpC cephalosporinase gene, the blaOXA-50 gene was shown to be expressed constitutively in P. aeruginosa. This β-lactamase gene was cloned onto a multicopy plasmid and expressed in P. aeruginosa and Escherichia coli. It conferred decreased susceptibility to ampicillin and ticarcillin and, interestingly, to moxalactam and meropenem in P. aeruginosa but not in E. coli. Overexpression and purification enabled us to determine the molecular mass (25 kDa), the pI value (8.6), and the hydrolysis spectrum of the OXA-50 β-lactamase. It is a narrow-spectrum oxacillinase that uncommonly hydrolyzes imipenem, although at a low level. Very similar oxacillinase genes were identified in all P. aeruginosa isolates from various geographical origins tested. The weak variability of the nucleotide sequence of this gene (0 to 2%) corresponded to that found for the naturally occurring blaAmpC cephalosporinase gene of P. aeruginosa. The study indicated that P. aeruginosa harbors two naturally encoded β-lactamase genes, one of which encodes an inducible cephalosporinase and the other of which encodes a constitutively expressed oxacillinase.

Detection of Carbapenemase Producers in Enterobacteriaceae by Use of a Novel Screening Medium

ABSTRACT A Drigalski agar-based culture medium containing an ertapenem, cloxacillin, and zinc sulfate (Supercarba medium) was tested for screening carbapenemase-producing members of the family Enterobacteriaceae. OXA-48 (n = 44), NDM (n = 25), VIM or IMP (n = 27), and KPC producers (n = 18) were detected with a low detection limit. Its overall sensitivity (95.6%) was higher than those of the currently available ChromID ESBL (bioMérieux) and CHROMagar KPC (CHROMagar) screening media. The Supercarba medium provides a significant improvement for detection of the most common types of carbapenemase producers.

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.

Comparison of the SUPERCARBA, CHROMagar KPC, and Brilliance CRE screening media for detection of Enterobacteriaceae with reduced susceptibility to carbapenems

The recently developed SUPERCARBA medium was evaluated together with 2 commercially available selective culture media containing carbapenems: CHROMagar KPC (CHROMagar) and Brilliance CRE (Oxoid, Thermofisher Scientific). A total of 142 enterobacterial isolates were tested, including 131 isolates with reduced susceptibility to carbapenems. The SUPERCARBA medium has the highest sensitivity (96.5%) (detecting virtually all carbapenemase producers including OXA-48-like producers) as compared to Brilliance CRE (76.3%) and CHROMagar KPC (43%). The specificity of the screening media was similar, ranging from 57% to 68%.

Biochemical-Genetic Characterization and Regulation of Expression of an ACC-1-Like Chromosome-Borne Cephalosporinase from Hafnia alvei

ABSTRACT A naturally occurring AmpC β-lactamase (cephalosporinase) gene was cloned from the Hafnia alvei 1 clinical isolate and expressed in Escherichia coli. The deduced AmpC β-lactamase (ACC-2) had a pI of 8 and a relative molecular mass of 37 kDa and showed 50 and 47% amino acid identity with the chromosome-encoded AmpCs from Serratia marcescens andProvidentia stuartii, respectively. It had 94% amino acid identity with the recently described plasmid-borne cephalosporinase ACC-1 from Klebsiella pneumoniae, suggesting the chromosomal origin of ACC-1. The hydrolysis constants (kcat and Km) showed that ACC-2 was a peculiar cephalosporinase, since it significantly hydrolyzed cefpirome. Once its gene was cloned and expressed inE. coli (pDEL-1), ACC-2 conferred resistance to ceftazidime and cefotaxime but also an uncommon reduced susceptibility to cefpirome. A divergently transcribed ampR gene with an overlapping promoter compared with ampC(blaACC-2) was identified in H. alvei 1, encoding an AmpR protein that shared 64% amino acid identity with the closest AmpR protein from P. stuartii. β-Lactamase induction experiments showed that the ampCgene was repressed in the absence of ampR and was activated when cefoxitin or imipenem was added as an inducer. From H. alvei 1 cultures that expressed an inducible-cephalosporinase phenotype, several ceftazidime- and cefpirome-cross-resistant H. alvei 1 mutants were obtained upon selection on cefpirome- or ceftazidime-containing plates, and H. alvei 1 DER, a ceftazidime-resistant mutant, stably overproduced cephalosporinase. Transformation of H. alvei 1 DER or E. coliJRG582 (ampDE mutant) harboring ampC andampR from H. alvei 1 with a recombinant plasmid containing ampD from E. coli resulted in a decrease in the MIC of β-lactam and recovery of an inducible phenotype for H. alvei 1 DER. Thus, AmpR and AmpD proteins may regulate biosynthesis of the H. alvei cephalosporinase similarly to other enterobacterial cephalosporinases.