Community-acquired ESBL-producing Enterobacteriaceae are now prevalent worldwide, necessitating international collaboration. Novel approaches are required to adequately address issues such as empirical treatment for severe community-acquired infection and infection control.

In order to evaluate the impact of an urban effluent on antibiotic resistance of freshwater bacterial populations, water samples were collected from the Arga river (Spain), upstream and downstream from the wastewater discharge of the city of Pamplona. Strains of Enterobacteriaceae (representative of the human and animal commensal flora) (110 isolates) and Aeromonas (typically waterborne bacteria) (118 isolates) were selected for antibiotic susceptibility testing. Most of the Aeromonas strains (72%) and many of the Enterobacteriaceae (20%) were resistant to nalidixic acid. Singly nalidixic acid-resistant strains were frequent regardless of the sampling site for Aeromonas, whereas they were more common upstream from the discharge for enterobacteria. The most common resistances to antibiotics other than quinolones were to tetracycline (24.3%) and beta-lactams (20.5%) for Enterobacteriaceae and to tetracycline (27.5%) and co-trimoxazole (26.6%) for Aeromonas. The rates of these antibiotic resistances increased downstream from the discharge at similar degrees for the two bacterial groups; it remained at high levels for enterobacteria but decreased along the 30-km study zone for Aeromonas. Genetic analysis of representative strains demonstrated that these resistances were mostly (enterobacteria) or exclusively (Aeromonas) chromosomally mediated. Moreover, a reference strain of Aeromonas caviae (CIP 7616) could not be transformed with conjugative R plasmids of enterobacteria. Thus, the urban effluent resulted in an increase of the rates of resistance to antibiotics other than quinolones in the riverine bacterial populations, despite limited genetic exchanges between enterobacteria and Aeromonas. Quinolone resistance probably was selected by heavy antibiotic discharges of unknown origin upstream from the urban effluent.

In 1999, 39 of 2,599 isolates of the family Enterobacteriaceae (1.5%) collected by eight private laboratories in the Aquitaine region in France produced an extended-spectrum ␤-lactamase (ESBL). Among these were 19 Enterobacter aerogenes isolates; 8 Klebsiella pneumoniae isolates; 6 Escherichia coli isolates; 3 Proteus mirabilis isolates; and 1 isolate each of Serratia marcescens, Morganella morganii, and Providencia stuartii. ESBL producers were isolated from 38 patients, including 33 residents of 11 clinics or nursing homes and 5 ambulatory patients. Seven different ESBLs were characterized. These mainly consisted of TEM-24 (25 isolates) and TEM-21 (9 isolates), but TEM-15 (2 isolates) and TEM-3, TEM-19, SHV-4, and CTX-M-1 (1 isolate each) were also characterized. Seven strains showed the coexistence of different TEM-and/or SHV-encoding genes, including a new SHV-1 variant, SHV-44, defined by the substitution R205L previously reported for SHV-3 in association with S238G. The epidemiology of the ESBL producers was investigated by random amplification of polymorphic DNA, typing by enterobacterial repetitive intergenic consensus PCR, analysis of resistance cotransferred with the ESBL, and analysis of the restriction profiles of the ESBL-encoding plasmids. Of the TEM-24-expressing strains, 18 were E. aerogenes isolates, including 9 from the same clinic, that were representatives of the epidemic clone disseminating in France. Of the TEM-21-producing strains that belonged to different species of the family Enterobacteriaceae (E. coli, K. pneumoniae, and P. mirabilis), 8 were isolated in the same nursing home. Outbreaks due to strain and/or plasmid dissemination in these clinic and nursing home were demonstrated. The presence of ESBL producers in five ambulatory patients probably resulted from nosocomial acquisition. Our data highlight the serious need to monitor patients for ESBL-producing Enterobacteriaceae in general practice.

As seen by the disk diffusion method, the clinical strain of Pseudomonas aeruginosa Pa695, resistant to all extended-spectrum cephalosporins and aminoglycosides, exhibited an unusual synergistic effect between ceftazidime and imipenem. This isolate produced an extended-spectrum ␤-lactamase (ESBL) with a pI of 5.8 that appeared to be chromosomally encoded. Cloning experiments revealed that this ESBL was encoded by bla GES-1 , previously described in an integron from Klebsiella pneumoniae. In P. aeruginosa Pa695, a higher level of resistance to ceftazidime than to ticarcillin was observed, and no synergy between the ␤-lactamase inhibitors and extended-spectrum cephalosporins was detected, in contrast to the resistance pattern observed in K. pneumoniae. Further sequence analysis demonstrated that the bla GES-1 gene cassette was located in a class 1 integron, which contained another sequence corresponding to the fused aac(3)-Ib and aac(6)-Ib gene cassettes. The fusion product was functional, as was the product of each gene cloned separately: AAC(3)-I, despite the deletion of the four last amino acids, and AAC(6), which carried three amino acid changes compared with the most homologous sequence. The AAC(3)-I protein conferred an expected gentamicin and fortimicin resistance, and the AAC(6), despite the Leu-1193Ser substitution, yielded resistance to kanamycin, tobramycin, and dibekacin, but slightly affected netilmicin and amikacin, and had no apparent effect on gentamicin. The fusion product conveyed a large profile of resistance, combining the AAC(6) activity with a higher level of gentamicin resistance without accompanying fortimicin resistance.

This first nationwide study reports an ESBLE prevalence of 1.1% in the French community setting in 2006, mainly related to the presence of CTX-M-producing E. coli strains; furthermore, unusual co-resistances rarely found in the community setting were occasionally observed, which may threaten future emergence.

Listeria spp. were found in most treated waters (84.4%) and raw sludge (89.2%) of six French urban wastewater treatment plants and one composting facility, examined monthly over a 1-year period. Most strains belonged to Listeria monocytogenes, serotypes 4b/4e being predominant. Sludge composting and liming reduced or prevented Listeria contamination.

Over a 3-year period, 67 patients of the Hospital of Pau (Pau, France), including 64 patients hospitalized in the adult intensive care unit (ICU), were colonized and/or infected by strains of Pseudomonas aeruginosa P12, resistant to all potentially active antibiotics except colistin. Most patients were mechanically ventilated and presented respiratory tract infections. Since cefepime and amikacin were the least inactive antibiotics by MIC determination, all ICU patients were treated with this combination, and most of them benefited. Cefepimeamikacin was found highly synergistic in vitro. Ribotyping and arbitrary primer-PCR analysis confirmed the presence of a single clonal isolate. Isoelectrofocusing revealed that the epidemic strain produced large amounts of the chromosomal cephalosporinase and an additional enzyme with a pI of 5.7, corresponding to PSE-1, as demonstrated by PCR and sequencing. Outer membrane protein profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the absence of a ca. 46-kDa protein, likely to be OprD, and increased production of two ca. 49-and 50-kDa proteins, consistent with the outer membrane components of the efflux systems, MexAB-OprM and MexEF-OprN. Thus, we report here a nosocomial outbreak due to multiresistant P. aeruginosa P12 exhibiting at least four mechanisms of ␤-lactam resistance, i.e., production of the penicillinase PSE-1, overproduction of the chromosomal cephalosporinase, loss of OprD, and overexpression of efflux systems, associated with a better activity of cefepime than ceftazidime.Pseudomonas aeruginosa, like many other nonfermenting gram-negative rods, is a saprophytic organism widespread in nature, particularly in moist environments (water, soil, plants, and sewage), and endowed with only weak pathogenic potential. However, because of its ability to survive on inert materials and its resistance to most antiseptics and antibiotics, P. aeruginosa has become an important and frequent nosocomial pathogen. Indeed, in hospitals, sinks, respiratory therapy equipment, and antiseptic or detergent solutions can act as reservoirs of P. aeruginosa. This organism is responsible for a wide range of hospital-acquired infections such as pneumonia, urinary tract infections, or bacteremia. Patients with impaired specific or nonspecific defense systems particularly tend to suffer from severe and even fatal infections caused by P. aeruginosa (9,11,23,24). Cross-transmission from patient to patient may occur via the hands of the health care staff or through contaminated materials or reagents (2, 18, 21, 24). Thus, a number of outbreaks of nosocomial infections due to P. aeruginosa have been reported, especially in intensive care units (ICUs) (2, 11, 18, 24), burn wound units (21), and cancer centers (23).One feature of P. aeruginosa is its high level of intrinsic resistance to a number of structurally unrelated antimicrobial agents. Indeed, the broad-spectrum resistance of this organism is largely due to a low outer-membrane permeability (32,33) and to multidrug...

Liquid chromatography with a column-switching technique was developed for simultaneous direct quantification of levofloxacin, gatifloxacin and moxifloxacin in human serum. Serum samples were injected on a LiChroCART 4-4 pre-column (PC) filled with a LiChrospher 100 RP-18, 5 microm where fluoroquinolones (FQs) were purified and concentrated. The FQs were back-flushed from the PC and then separated on a Supelcosil ABZ+ Plus (150 mm x 4.6 mm i.d.) analytical column with a mobile phase containing 10 mM phosphate buffer (pH 2.5), acetonitrile (88:12, v/v) and 2mM tetrabutyl ammonium bromide. The effects of ion-pair reagents, buffer type, pH and acetonitrile concentrations in the mobile phase on the separation of the three FQs were investigated. Fluorescence detection provided sufficient sensitivity to achieve a quantification limit of 125 ng/ml for levofloxacin and moxifloxacin; 162.5 ng/ml for gatifloxacin with a 5 microl sample size. The on-line process of extraction avoids time-consuming treatment of the samples before injection and run time is shortened. The recovery, selectivity, linearity, precision and accuracy of the method are convenient for pharmacokinetic studies or routine assays.