Hedi Mammeri

Emergence of Plasmid-Mediated Quinolone Resistance in Escherichia coli in Europe

ABSTRACT Although quinolone resistance results mostly from chromosomal mutations, it may also be mediated by a plasmid-encoded qnr gene in members of the family Enterobacteriaceae. Thus, 297 nalidixic-acid resistant strains of 2,700 Escherichia coli strains that had been isolated at the Bicêtre Hospital (Le Kremlin-Bicêtre, France) in 2003 were screened for qnr by PCR. A single E. coli isolate that carried a ca. 180-kb conjugative plasmid encoding a qnr determinant was identified. It conferred low-level resistance to quinolones and was associated with a chromosomal mutation in subunit A of the topoisomerase II gene. The qnr gene was located on a sul1-type class 1 integron just downstream of a conserved region (CR) element (CR1) comprising the Orf513 recombinase. Promoter sequences for qnr expression overlapped the extremity of CR1, indicating the role of CR1 in the expression of antibiotic resistance genes. This integron was different from other qnr-positive sul1-type integrons identified in American and Chinese enterobacterial isolates. In addition, plasmid pQR1 carried another class 1 integron that was identical to In53 from E. coli. The latter integron possessed a series of gene cassettes, including those coding for the extended-spectrum β-lactamase VEB-1, the rifampin ADP ribosyltransferase ARR-2, and several aminoglycoside resistance markers. This is the first report of plasmid-mediated quinolone resistance in Europe associated with an unknown level of plasmid-mediated multidrug resistance in Enterobacteriaceae.

Origin of Plasmid-Mediated Quinolone Resistance Determinant QnrA

ABSTRACT Plasmid-mediated resistance to quinolones is increasingly reported in studies of Enterobacteriaceae. Using a PCR-based strategy, a series of gram-negative species were screened for qnrA-like genes. Shewanella algae, an environmental species from marine and fresh water, was identified as its reservoir. This is a one of the very few examples of progenitor identification of an acquired antibiotic resistance gene.

Association of Plasmid-Mediated Quinolone Resistance with Extended-Spectrum β-Lactamase VEB-1

ABSTRACT Association of the plasmid-mediated quinolone resistance determinant QnrA and the blaVEB-1 gene was identified in a single Enterobacter cloacae isolate from K.-Bicêtre, France, and in 11 out of 23 blaVEB-1-positive enterobacterial isolates from Bangkok, Thailand. This result may explain in part the association between quinolone and extended-spectrum β-lactam resistance.

Resistance to cefepime and cefpirome due to a 4-amino-acid deletion in the chromosome-encoded ampc β-lactamase of a Serratia marcescens clinical isolate

ABSTRACT A multiresistant Serratia marcescens strain, HD, isolated from a patient with a urinary tract infection, was resistant to amino-, carboxy-, and ureidopenicillins, ceftazidime, and cefepime and was susceptible to cefotaxime and ceftriaxone, according to the guidelines of the NCCLS. No synergy was found between expanded-spectrum cephalosporins and clavulanic acid, according to the double-disk synergy test. The blaAmpC gene of the strain was amplified by PCR and cloned into Escherichia coli DH10B, giving rise to high-level resistance to ceftazidime, cefepime, and cefpirome. Sequencing analysis revealed that the blaAmpC gene from S. marcescens HD had a 12-nucleotide deletion compared to the blaAmpC gene from reference strain S. marcescens S3, leading to a 4-amino-acid deletion located in the H-10 helix of the β-lactamase. Kinetic analysis showed that this enzyme significantly hydrolyzed ceftazidime, cefepime, and cefpirome. This work underlined that resistance to the latest expanded-spectrum cephalosporins may be mediated by structurally modified AmpC-type β-lactamases.

Contribution of extended‐spectrum AmpC (ESAC) β‐lactamases to carbapenem resistance in Escherichia coli

ESAC beta-lactamases have increased catalytic efficiencies toward extended-spectrum cephalosporins and to a lesser extent toward imipenem as compared with the wild-type cephalosporinases. We show here that ESAC expression associated with the loss of both OmpC and OmpF porins conferred in Escherichia coli a high level of resistance to ertapenem and reduced the susceptibility to imipenem. On the contrary, ESAC expressed in the OmpC- or OmpF-deficient E. coli strains or narrow-spectrum cephalosporinase expressed in the OmpC-and OmpF-deficient strain do not confer reduced susceptibility to any of the carbapenems. The production of ESAC beta-lactamase in favorable E. coli background may represent an additional mechanism of resistance to ertapenem.

Phenotypic and Biochemical Comparison of the Carbapenem-Hydrolyzing Activities of Five Plasmid-Borne AmpC β-Lactamases

ABSTRACT The CMY-2, ACT-1, DHA-1, ACC-1, and FOX-1 enzymes are representative of five plasmid-mediated AmpC (pAmpC) β-lactamase clusters. Resistance to imipenem has been reported in Enterobacteriaceae as a result of pAmpC expression combined with decreased outer membrane permeability. The aim of this study was to determine the role of different pAmpCs in carbapenem resistance and to define the structure/activity relationship supporting carbapenemase activity. The ampC genes encoding the five pAmpCs and the chromosomal AmpC of Escherichia coli EC6, which was used as a reference cephalosporinase, were cloned and introduced into wild-type E. coli TOP10 and OmpC/OmpF porin-deficient E. coli HB4 strains. The MICs of β-lactams for the recombinant strains revealed that CMY-2, ACT-1, and DHA-1 β-lactamases conferred a high level of resistance to ceftazidime and cefotaxime once expressed in E. coli TOP10 and reduced significantly the susceptibility to imipenem once expressed in E. coli HB4. In contrast, FOX-1 and ACC-1 enzymes did not confer resistance to imipenem. Biochemical analysis showed that CMY-2 β-lactamase and, to a lesser extent, ACT-1 exhibited the highest catalytic efficiency toward imipenem and showed low Km values. A modeling study revealed that the large R2 binding site of these two enzymes may support the carbapenemase activity. Therefore, CMY-2-type, ACT-1-type, and DHA-1-type β-lactamases may promote the emergence of carbapenem resistance in porin-deficient clinical isolates.

Chromosomal Integration of a Cephalosporinase Gene from Acinetobacter baumannii into Oligella urethralis as a Source of Acquired Resistance to β-Lactams

ABSTRACT Clinical Oligella urethralis isolate COH-1, which was uncommonly resistant to penicillins and narrow-spectrum cephalosporins, was recovered from a 55-year-old patient with a urinary tract infection. Shotgun cloning into Escherichia coli and expression experiments gave recombinant clones expressing either an AmpC β-lactamase-type phenotype of resistance or a carbenicillin-hydrolyzing β-lactamase-type phenotype of resistance. The AmpC β-lactamase identified (ABA-1), which had a pI value of 8.2, had 98% amino acid identity with a chromosomally encoded cephalosporinase of Acinetobacter baumannii. A 820-bp insertion sequence element, ISOur1, belonging to the IS6 family of insertion sequence elements, was identified immediately upstream of blaABA-1, providing a −35 promoter sequence and likely giving rise to a hybrid promoter region. The carbenicillin-hydrolyzing β-lactamase identified (CARB-8), which had a pI value of 6.4, differed from CARB-5 by two amino acid substitutions. Hybridization of CeuI fragment I-restricted DNA fragments of O. urethralis COH-1 with blaABA-1-, blaCARB-8-, and 16S rRNA-specific probes indicated the chromosomal integration of the β-lactamase genes. PCR and hybridization experiments failed to detect blaCARB-8- and blaABA-1-like genes in three O. urethralis reference strains, indicating that the β-lactamase genes identified were the source of acquired resistance in O. urethralis COH-1. This is one of the few examples of the interspecies transfer and the chromosomal integration of a gene encoding a naturally occurring β-lactamase.

Chromosome-Encoded β-Lactamases TUS-1 and MUS-1 from Myroides odoratus and Myroides odoratimimus (Formerly Flavobacterium odoratum), New Members of the Lineage of Molecular Subclass B1 Metalloenzymes

ABSTRACT Myroides odoratus and Myroides odoratimimus (formerly designated in a single species as Flavobacterium odoratum) are gram-negative aerobes and sources of nosocomial infections in humans. They have variable susceptibility to β-lactams and a decreased susceptibility to carbapenems. Using genomic DNAs of M. odoratus CIP 103105 and M. odoratimimus CIP 103073 reference strains, shotgun cloning of β-lactamase genes was performed, followed by protein expression in Escherichia coli. The deduced amino acid sequences of these β-lactamase genes revealed that TUS-1 and MUS-1 from M. odoratus CIP 103105 and M. odoratimimus CIP 103073, respectively, shared 73% amino acid identity. Mature proteins TUS-1 and MUS-1, with pI values of 7.8 and 5.2, respectively, had relative molecular masses of ca. 26 kDa. These β-lactamases are members of the subclass B1 of metallo-β-lactamases and are distantly related to other metalloenzymes, being most closely related to IND-1 from Chryseobacterium indologenes (42% amino acid identity). However, phylogenic analysis showed that TUS-1 and MUS-1 belong to the same phylogenic lineage of subclass B1 enzymes that groups the subclass B1 β-lactamases of Flavobacterium species. Kinetic parameters of purified β-lactamases TUS-1 and MUS-1 detailed their hydrolysis spectra, which encompass most β-lactams except aztreonam. β-Lactamases TUS-1 and MUS-1 were classified in functional subgroup 3a of metalloenzymes. This work further characterizes chromosome-encoded metalloenzymes from Flavobacteriaceae species that explain at least part of their intrinsic resistance to β-lactams.

Extended-spectrum cephalosporinases: structure, detection and epidemiology

Extended-spectrum AmpC beta-lactamases of Enterobacteriaceae, which are chromosomally or plasmid-encoded, possess structural modifications in the vicinity of the active site compared with their progenitors. They display an increased catalytic efficiency against extended-spectrum beta-lactams, such as ceftazidime, cefotaxime, cefepime, cefpirome and, in some cases, also against imipenem. An overview of the molecular and biochemical characterization of this recently identified mechanism of resistance to beta-lactams is provided as well as its prevalence and possible clinical significance.

Coexistence of SHV-4- and TEM-24-Producing Enterobacter aerogenes Strains before a Large Outbreak of TEM-24-Producing Strains in a French Hospital

ABSTRACT In 1996, a monitoring program was initiated at the teaching hospital of Amiens, France, and carried out for 3 years. All extended-spectrum β-lactamase (ESBL)-producing Enterobacter aerogenes isolates recovered from clinical specimens were collected for investigation of their epidemiological relatedness by pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and determination of the type of ESBL harbored by isoelectric focusing and DNA sequencing. Molecular typing revealed the endemic coexistence, during the first 2 years, of two clones expressing, respectively, SHV-4 and TEM-24 ESBLs, while an outbreak of the TEM-24-producing strain raged in the hospital during the third year, causing the infection or colonization of 165 patients. Furthermore, this strain was identified as the prevalent clone responsible for outbreaks in many French hospitals since 1996. This study shows that TEM-24-producing E. aerogenes is an epidemic clone that is well established in the hospitals ecology and able to spread throughout wards. The management of the outbreak at the teaching hospital of Amiens, which included the reinforcement of infection control measures, failed to obtain complete eradication of the clone, which has become an endemic pathogen.