Patrice Nordmann

Multiplex PCR for detection of acquired carbapenemase genes

A rapid and reliable PCR-based technique was developed for detection of genes encoding carbapenemases belonging to different classes. Primers were designed to amplify the following 11 genes: bla(IMP), bla(VIM), bla(NDM), bla(SPM), bla(AIM), bla(DIM), bla(GIM), bla(SIM)bla(KPC), bla(BIC), and bla(OXA-48). Three different multiplex reaction mixtures were defined and evaluated for the detection of all these 11 genes. Using optimized conditions, each reaction mixture allowed to identify the respective genes, with PCR giving distinct amplicon sizes corresponding to the different genes for each mixture. We reported here a rapid and reliable technique for screening all clinically relevant carbapenemase genes.

Comparative Analysis of Acinetobacters: Three Genomes for Three Lifestyles

Acinetobacter baumannii is the source of numerous nosocomial infections in humans and therefore deserves close attention as multidrug or even pandrug resistant strains are increasingly being identified worldwide. Here we report the comparison of two newly sequenced genomes of A. baumannii. The human isolate A. baumannii AYE is multidrug resistant whereas strain SDF, which was isolated from body lice, is antibiotic susceptible. As reference for comparison in this analysis, the genome of the soil-living bacterium A. baylyi strain ADP1 was used. The most interesting dissimilarities we observed were that i) whereas strain AYE and A. baylyi genomes harbored very few Insertion Sequence elements which could promote expression of downstream genes, strain SDF sequence contains several hundred of them that have played a crucial role in its genome reduction (gene disruptions and simple DNA loss); ii) strain SDF has low catabolic capacities compared to strain AYE. Interestingly, the latter has even higher catabolic capacities than A. baylyi which has already been reported as a very nutritionally versatile organism. This metabolic performance could explain the persistence of A. baumannii nosocomial strains in environments where nutrients are scarce; iii) several processes known to play a key role during host infection (biofilm formation, iron uptake, quorum sensing, virulence factors) were either different or absent, the best example of which is iron uptake. Indeed, strain AYE and A. baylyi use siderophore-based systems to scavenge iron from the environment whereas strain SDF uses an alternate system similar to the Haem Acquisition System (HAS). Taken together, all these observations suggest that the genome contents of the 3 Acinetobacters compared are partly shaped by life in distinct ecological niches: human (and more largely hospital environment), louse, soil.

Crystal Structure of the OXA-48 β-Lactamase Reveals Mechanistic Diversity among Class D Carbapenemases

Carbapenem-hydrolyzing class D beta-lactamases (CHDLs) are enzymes found in important Gram-negative pathogens (mainly Acinetobacter baumannii and Enterobacteriaceae) that confer resistance to beta-lactam antibiotics, and notably carbapenems. The crystal structure of the OXA-48 carbapenemase was determined at pH 7.5 and at a resolution of 1.9 A. Surprisingly, and by contrast with OXA-24, the only other CHDL of known crystal structure, the structure of OXA-48 was similar to OXA-10, an enzyme devoid of carbapenemase activity, indicating that the hydrolysis of these compounds could depend on subtle changes in the active site region. Moreover, the active site groove of OXA-48 was different from that of OXA-24 in shape, dimensions, and charge distribution. Molecular dynamics pointed to the functional relevance of residues located in or close to the beta5-beta6 loop and allowed us to propose a mechanism for carbapenem hydrolysis by OXA-48.

Performance of chromID ESBL, a chromogenic medium for detection of Enterobacteriaceae producing extended-spectrum beta-lactamases.

The chromogenic agar medium chromID ESBL (bioMérieux) was compared with BLSE agar medium (AES) for selective isolation and presumptive identification of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae from clinical samples. A total of 765 samples (468 rectal swabs, 255 urine samples and 42 pulmonary aspirations) obtained from 547 patients was processed. All bacterial strains isolated on either medium were further characterized using biochemical tests, and ESBL producers were confirmed by synergy testing. Genetic characterization of ESBL genes was determined by PCR. A total of 33 ESBL-producing Enterobacteriaceae strains [Escherichia coli (n=16), Klebsiella pneumoniae (n=8), Enterobacter spp. (n=3), Citrobacter spp. (n=5) and Proteus mirabilis (n=1)] was recovered. The sensitivity after 24 h incubation was 88 % for chromID ESBL and 85 % for BLSE agar. At 48 h, the sensitivity of chromID ESBL increased to 94 % and was higher than that obtained with BLSE agar. The positive predictive value at 24 h for chromID ESBL was 38.7 % [95 % confidence interval (95 % CI) 28.3 -50.2 %)], which was significantly higher than that for BLSE agar [15.4 %, 95 % CI 10.1 -21.5 %]. On both media, false-positive results were mostly due to Pseudomonas aeruginosa and to Enterobacteriaceae overproducing chromosomal cephalosporinase (Enterobacter spp.) or a chromosomal penicillinase (Klebsiella oxytoca). This study showed that chromID ESBL, a ready-to-use chromogenic selective medium, is sensitive and specific for rapid, presumptive identification of ESBL-producing Enterobacteriaceae. Its chromogenic properties and its selectivity are particularly useful in specimens containing resident associated flora.

Updated multiplex polymerase chain reaction for detection of 16S rRNA methylases: high prevalence among NDM-1 producers

We develop a rapid (<4 h) and reliable multiplex polymerase chain reaction for screening of 16S rRNA methylase genes conferring resistance to aminoglycosides. This study particularly underlined that 16S rRNA methylases are frequently (75%) identified among Enterobacteriaceae isolates producing the carbapenemase NDM-1.

Complex dynamics of hepatitis B virus resistance to adefovir

In patients with hepatitis B e antigen‐negative chronic hepatitis B, adefovir dipivoxil administration selects variants bearing reverse transcriptase rtN236T and/or rtA181V/T substitutions in 29% of cases after 5 years. The aim of this study was to characterize the dynamics of adefovir‐resistant variant populations during adefovir monotherapy in order to better understand the molecular mechanisms underlying hepatitis B virus resistance to this class of nucleotide analogues. Patients included in a 240‐week clinical trial of adefovir monotherapy who developed adefovir resistance‐associated substitutions were studied. The dynamics of hepatitis B virus populations were analyzed over time, after generating nearly 4,000 full‐length reverse transcriptase sequences, and compared with the replication kinetics of the virus during therapy. Whatever the viral kinetics pattern, adefovir resistance was characterized by exclusive detection of a dominant wild‐type, adefovir‐sensitive variant population at baseline and late and gradual selection by adefovir of several coexisting resistant viral populations, defined by the presence of amino acid substitutions at position rt236, position rt181, or both. The gain in fitness of one or the other of these resistant populations during adefovir administration was never associated with the selection of additional amino acid substitutions in the reverse transcriptase. Conclusion: Our results suggest that adefovir administration selects poorly fit preexisting or emerging viral populations with low‐level adefovir resistance, which subsequently compete to fill the replication space. Viral kinetics depends on the initial virological response to adefovir. Lamivudine add‐on restores some antiviral efficacy, but adefovir‐resistant variants remain predominant. Whether these adefovir resistance–associated substitutions may confer cross‐resistance to tenofovir in vivo will need to be determined. (HEPATOLOGY 2009;49:50‐59.)

Metallo-β-lactamase-producing Pseudomonas aeruginosa isolates in Tunisia

This study was conducted to identify metallo-beta-lactamase (MBL) producers among a collection of 75 nonrepetitive carbapenem-resistant Pseudomonas aeruginosa isolates recovered between November 2003 and May 2007 at the University Hospital Sahloul, Sousse, Tunisia. Five isolates produced the MBL VIM-2. Those bla(VIM-2)-positive isolates were clonally related according to pulsed-field gel electrophoresis analysis. This carbapenemase was very likely chromosomally located and as a form of a gene cassette in a class 1 integron. This is the 1st report of spread of VIM-2 producers in Tunisia.

In vitro activity of ceftazidime, ceftaroline and aztreonam alone and in combination with avibactam against European Gram-negative and Gram-positive clinical isolates

Recent clinical isolates of key Gram-negative and Gram-positive bacteria were collected in 2012 from hospitalised patients in medical centres in four European countries (France, Germany, Italy and Spain) and were tested using standard broth microdilution methodology to assess the impact of 4 mg/L avibactam on the in vitro activities of ceftazidime, ceftaroline and aztreonam. Against Enterobacteriaceae, addition of avibactam significantly enhanced the level of activity of these antimicrobials. MIC(90) values (minimum inhibitory concentration that inhibits 90% of the isolates) of ceftazidime, ceftaroline and aztreonam for Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii and Morganella morganii were reduced up to 128-fold or greater when combined with avibactam. A two-fold reduction in the MIC(90) of ceftazidime to 8 mg/L was noted in Pseudomonas aeruginosa isolates when combined with avibactam, whereas little effect of avibactam was noted on the MIC values of the test compounds when tested against Acinetobacter baumannii isolates. Avibactam had little effect on the excellent activity of ceftazidime, ceftaroline and aztreonam against Haemophilus influenzae. It had no impact on the in vitro activity of ceftazidime and ceftaroline against staphylococci and streptococci. This study demonstrates that addition of avibactam enhances the activities of ceftazidime, ceftaroline and aztreonam against Enterobacteriaceae and P. aeruginosa but not against A. baumannii.

Role of Ser-237 in the substrate specificity of the carbapenem-hydrolyzing class A β-lactamase Sme-1

The role of the serine residue found at position 237 in the carbapenemase Sme-1 has been investigated by constructing a mutant in which Ser-237 was replaced by an alanine. The S237A mutant showed a catalytic behavior against penicillins and aztreonam very similar to that of Sme-1. By contrast, S237A was characterized by a reduced catalytic efficiency against cephems, such as cephalothin and cephaloridine. In addition, the weak activity of Sme-1 against the cephamycin cefoxitin was hardly detectable with the mutant enzyme. Finally, the Ser-237-->Ala mutation resulted in a marked decrease in catalytic activity against imipenem, showing that Ser-237 contributes to the carbapenemase activity of the class A beta-lactamase Sme-1.

Résistance plasmidique aux quinolones

Resume Objectifs Analyser les mecanismes de resistance aux fluoroquinolones, rechercher la place des determinants plasmidiques. Methodes Elles sont etablies a partir des donnees de la litterature et experience personnelle. Pendant plus de 30 ans, les seuls mecanismes de resistance aux quinolones connus ont ete de support chromosomique. Le premier determinant d’origine plasmidique, QnrA, fut decouvert en 1998, suivi en 2004 par deux autres determinants QnrB et QnrS chez plusieurs especes d’enterobacteries. Ces determinants s’intercaleraient entre les quinolones, les topoisomerases et l’ADN, limitant ainsi la fixation ulterieure des quinolones sur leur cible. En 2005, un second mecanisme de resistance plasmidique contribuant de facon independante a la resistance aux quinolones par modification de la molecule a ete decrit. Ce determinant (AAC (6’)-1b-cr) est un variant de l’acetyl transferase en 6’ dont le spectre d’acetylation, habituellement limite aux aminosides, est elargi a la ciprofloxacine et a la norfloxacine. Epidemiologie de la resistance plasmidique Les mecanismes plasmidiques de resistance aux quinolones sont, d’un point de vue international, dissemines parmi les souches d’enterobacteries. Ils conferent une resistance de haut niveau aux quinolones de premiere generation et une diminution de sensibilite aux fluoroquinolones. Conclusion Il n’existe pas de criteres phenotypiques sur un antibiogramme permettant de distinguer les mecanismes de resistance chromosomiques et plasmidiques aux quinolones. La detection de ces mecanismes de resistance repose donc sur des techniques de biologie moleculaire.