Experimental evidence for IS1294b-mediated transposition of the blaCMY-2 cephalosporinase gene in Enterobacteriaceae.

Objectives: The objective of this study was to investigate whether the insertion sequence IS1294b (IS91 family) is able to mobilize the blaCMY-2 gene and its adjacent regions from one replicon to another.

Methods: Klebsiella pneumoniae Kp2735 was typed by MLST and its plasmid content was examined by S1-PFGE and PCR-based replicon typing. The genetic blaCMY-2 environment was analysed after cloning experiments and sequencing.

Prevalence and characterization of uropathogenic Escherichia coli harboring plasmid-mediated quinolone resistance in a Tunisian university hospital.

The prevalence of plasmid-mediated quinolone resistance (PMQR) determinants was investigated in a Tunisian collection of 300 uropathogenic Escherichia coli. PMQR genes were detected in 68 isolates (22.7%) as follows: aac(6')-Ib-cr (n=66), qnrB1 (n=3), qnrA6 (n=1), and qnrS1 (n=1).

NDM-1-producing Klebsiella pneumoniae resistant to colistin in a French community patient without history of foreign travel.

A carbapenem-resistant Klebsiella pneumoniae strain, Kp5196, was responsible for an uncomplicated cystitis in a patient living at home and without history of foreign travel. This isolate produced the metallocarbapenemase NDM-1 and was resistant to all antibiotics except tetracyclines and colistin. The K.

Duplication of the chromosomal blaSHV-11 gene in a clinical hypermutable strain of Klebsiella pneumoniae.

In a collection of 110 clinical isolates of Klebsiella pneumoniae, a single strain, Kp593, was found to exhibit a mutator phenotype with a rifampicin mutation frequency 100-fold higher than the modal value for this species. Complementation experiments with the wild-type MutL, one of the main components of the methyl-directed mismatch repair system, allowed the mutator phenotype to be reversed. Sequencing revealed substitution of the conserved residue Lys307 to Arg and site-directed mutagenesis followed by complementation experiments confirmed the critical role of this mutation.

CTX-M-producing Escherichia coli in a maternity ward: a likely community importation and evidence of mother-to-neonate transmission.

Objectives: To investigate the high prevalence of extended-spectrum beta-lactamase (ESBL)-producing strains of Escherichia coli (4%, 10/250 consecutive isolates) recovered during a 5 month period in the maternity ward of the University Hospital of Bordeaux, France.

Methods: beta-Lactam resistance transfer was analysed by conjugation and transformation. ESBLs were characterized by isoelectric focusing, PCR amplification and sequencing.

Activity of linezolid in an in vitro pharmacokinetic-pharmacodynamic model using different dosages and Staphylococcus aureus and Enterococcus faecalis strains with and without a hypermutator phenotype.

The influence of antibiotic dosages and bacterial mutator phenotypes on the emergence of linezolid-resistant mutants was evaluated in an in vitro pharmacokinetic-pharmacodynamic model. A twice-daily 0.5-h infusion of a 200-, 600-, or 800-mg dose for 48 h was simulated against four strains (MIC, 2 microg/ml): Staphylococcus aureus RN4220 and its mutator derivative MutS2, Enterococcus faecalis ATCC 29212, and a mutator clinical strain of E.

A multinational survey of risk factors for infection with extended-spectrum beta-lactamase-producing enterobacteriaceae in nonhospitalized patients.

Background: Infections caused by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae are increasing in frequency and are associated with high mortality rates. Circulation of CTX-M-type ESBLs in the community is of particular concern, because it may confound standard infection-control measures.

Methods: We analyzed the results of epidemiologic studies of infection caused by ESBL-producing Enterobacteriaceae in nonhospitalized patients from 6 centers in Europe, Asia, and North America.

In vitro recombination catalyzed by bacterial class 1 integron integrase IntI1 involves cooperative binding and specific oligomeric intermediates.

Gene transfer via bacterial integrons is a major pathway for facilitating the spread of antibiotic resistance genes across bacteria. Recently the mechanism underlying the recombination catalyzed by class 1 integron recombinase (IntI1) between attC and attI1 was highlighted demonstrating the involvement of a single-stranded intermediary on the attC site. However, the process allowing the generation of this single-stranded substrate has not been determined, nor have the active IntI1*DNA complexes been identified.

Molecular and biochemical characterization of SHV-56, a novel inhibitor-resistant beta-lactamase from Klebsiella pneumoniae.

A clinical strain of Klebsiella pneumoniae was found to possess the chromosomal gene bla(SHV-56), encoding a new inhibitor-resistant beta-lactamase with a pI of 7.6. SHV-56 is derived from SHV-11 by the single substitution K234R.

Contrasting effects of human THP-1 cell differentiation on levofloxacin and moxifloxacin intracellular accumulation and activity against Staphylococcus aureus and Listeria monocytogenes.

Background And Aims: Listeria monocytogenes and Staphylococcus aureus invade and multiply in THP-1 monocytes. Fluoroquinolones accumulate in these cells, but are less active against intracellular than extracellular forms of L. monocytogenes and S.

Beta-lactam and aminoglycoside resistance rates and mechanisms among Pseudomonas aeruginosa in French general practice (community and private healthcare centres).

Objectives: The aim of this study was to assess antibiotic resistance rates and mechanisms of beta-lactam and aminoglycoside resistance among isolates of Pseudomonas aeruginosa isolated in the extra-hospital setting (community and private healthcare centres).

Patients And Methods: During a 4 month period, 226 non-repetitive strains of P. aeruginosa were collected from patients residing in private healthcare centres (73.

Role of the AheABC efflux pump in Aeromonas hydrophila intrinsic multidrug resistance.

Gene inactivation and complementation experiments showed that the tripartite AheABC efflux pump of Aeromonas hydrophila extruded at least 13 substrates, including nine antibiotics. The use of phenylalanine-arginine-beta-naphthylamide (PAbetaN) revealed an additional system(s) contributing to intrinsic resistance. This is the first analysis of the role of multidrug efflux systems in Aeromonas spp.

A new in vitro strand transfer assay for monitoring bacterial class 1 integron recombinase IntI1 activity.

IntI1 integrase is a tyrosine recombinase involved in the mobility of antibiotic resistance gene cassettes within bacterial class 1 integrons. Recent data have shown that its recombination specifically involves the bottom strand of the attC site, but the exact mechanism of the reaction is still unclear. An efficient in vitro assay is still required to better characterize the biochemical properties of the enzyme.

Synthesis of new 4-[2-(alkylamino) ethylthio]pyrrolo[1,2-a]quinoxaline and 5-[2-(alkylamino) ethylthio]pyrrolo[1,2-a]thieno[3,2-e]pyrazine derivatives, as potential bacterial multidrug resistance pump inhibitors.

The synthesis of new 4-[2-(alkylamino)ethylthio]pyrrolo[1,2-a]quinoxaline derivatives la-1 is described in five or six steps starting from various substituted nitroanilines 2a-e. The bioisostere 5-[2-(alkylamino)ethylthio]pyrrolo[1,2- a]thieno[3,2-e]pyrazine 1m was also prepared. The new derivatives were evaluated as efflux pump inhibitors (EPIs) in a model targeting the NorA system of Staphylococcus aureus.

Extended-spectrum-beta-lactamase-producing Enterobacteriaceae strains in various types of private health care centers.

During a 2004 survey, 49 extended-spectrum-beta-lactamase-producing enterobacteria were collected in 20 French private health care centers and one local hospital. They included 12 CTX-M-producing Escherichia coli strains (1.8% versus 0.

Determination of linezolid in growth media by high-performance liquid chromatography with on-line extraction.

An isocratic high-performance liquid chromatography (HPLC) method with on-line extraction has been developed to determine linezolid in Mueller-Hinton broth. The loading mobile phase consisting of water-acetonitrile 99:1 (v/v) allowed retention of the analyte on a LiChrocart 4-4 pre-column filled with a LiChrospher 100 RP-8, 5 microm. The transfer of the analyte by a backflush mode to a 150 mm x 4.

Factors compromising the activity of moxifloxacin against intracellular Staphylococcus aureus.

Objectives: The aim of this study was to determine the intracellular activity of moxifloxacin against a reference strain and a clinical strain and to study the factors compromising the intracellular activity of moxifloxacin.

Methods: The bactericidal activity of moxifloxacin at therapeutic concentrations was studied against extracellular (broth) and intracellular (infected THP-1 monocytes) forms of Staphylococcus aureus and compared with that of levofloxacin. The activity of moxifloxacin was also evaluated in the presence of alkalinizing agents, in intracellular salt medium mimicking the phagolysosomal environment and in cell lysate.

High genetic stability of integrons in clinical isolates of Shigella spp. of worldwide origin.

Over a 12-year period, 68 Shigella strains (31 S. sonnei, 30 S. flexneri, 4 S.

Synthesis of omeprazole analogues and evaluation of these as potential inhibitors of the multidrug efflux pump NorA of Staphylococcus aureus.

A series of 11 pyrrolo[1,2-a]quinoxaline derivatives, 1a to 1k, sharing structural analogies with omeprazole, a eukaryotic efflux pump inhibitor (EPI) used as an antiulcer agent, was synthesized. Their inhibitory effect was evaluated using Staphylococcus aureus strain SA-1199B overexpressing NorA. By determinations of the MIC of norfloxacin in the presence of these EPIs devoid of intrinsic antibacterial activity and used at 128 microg/ml, and by the checkerboard method, compound 1e (MIC decrease, 16-fold; fractional inhibitory concentration index [SigmaFIC], 0.

Activity of gatifloxacin in an in vitro pharmacokinetic-pharmacodynamic model against Staphylococcus aureus strains either susceptible to ciprofloxacin or exhibiting various levels and mechanisms of ciprofloxacin resistance.

Gatifloxacin (GAT) is a new 8-methoxy fluoroquinolone with enhanced activity against gram-positive cocci. Its activity was studied in an in vitro pharmacokinetic-pharmacodynamic model against five Staphylococcus aureus strains, either susceptible to ciprofloxacin or exhibiting various levels and mechanisms of ciprofloxacin (CIP) resistance: the ATCC 25923 reference strain (MICs of CIP and GAT: 0.5 and 0.

Factors influencing the intracellular activity of fluoroquinolones: a study using levofloxacin in a Staphylococcus aureus THP-1 monocyte model.

Objectives: Recent studies have raised the question of whether the intracellular activity of quinolones is optimal with respect to their cellular accumulation. The aim of this study was to compare the intracellular and extracellular activities of a commonly used quinolone, levofloxacin, and to examine the causes of the possible inconsistency between intracellular and extracellular effects.

Methods: The bactericidal activity of levofloxacin at therapeutic levels, alone or in combination with various efflux-pump inhibitors or alkalinizing agents, was studied against Staphylococcus aureus ATCC 25923 in Mueller-Hinton (MH) broth and in a THP-1 monocytic cell model, using intracellular salt medium (ISM) mimicking the phagolysosomal environment, and in cell lysate.

Occurrence of Listeria spp. in effluents of French urban wastewater treatment plants.

Listeria spp. were found in most treated waters (84.4%) and raw sludge (89.

Clinical and molecular analysis of extended-spectrum {beta}-lactamase-producing enterobacteria in the community setting.

During a previous survey, five extended-spectrum beta-lactamase (ESBL)-producing enterobacteria (ESBLE) (two Enterobacter aerogenes isolates expressing TEM-24 b, two Escherichia coli isolates expressing TEM-21 or TEM-24 b, and one Klebsiella pneumoniae isolate expressing SHV-4/TEM-15) responsible for urinary tract infections (UTIs) were found among 1,584 strains collected from community patients. The aim of the present study was to elucidate the route of emergence of these typically nosocomial organisms in the community. Thus, the files of the five patients were analyzed over at least a decade, and potentially related ESBLE from hospitals or clinics were examined.

Prolonged outbreak of infection due to TEM-21-producing strains of Pseudomonas aeruginosa and enterobacteria in a nursing home.

Over a 6-year period, 24 extended-spectrum beta-lactamase (ESBL)-producing isolates of Pseudomonas aeruginosa were collected from 18 patients living in a nursing home. These isolates had a delayed development of a red pigment and exhibited a similar antibiotype (resistance to all beta-lactams except for imipenem and to gentamicin, tobramycin, netilmicin, ciprofloxacin, and rifampin) associated with the production of the TEM-21 beta-lactamase and a type II 3'-N-aminoglycoside acetyltransferase [AAC(3)-II] enzyme. Surprisingly, serotyping showed that these isolates belonged to four successive serotypes (P2, P16, P1, and PME), although molecular typing by PCR methods and pulsed-field gel electrophoresis yielded identical or similar profiles.