In vitro combinations of five intravenous antibiotics with dalfopristin-quinupristin against Staphylococcus aureus in a 3-dimensional model.

We compared the in vitro activity of dalfopristin and quinupristin combined with five intravenous antibiotics in a 3-dimensional model. We tested six strains of Staphylococcus aureus selected with different patterns of resistance to methicillin and erythromycin. Dalfopristin and quinupristin displayed a very synergistic activity against all the strains with a mean 16- or 32-fold decrease of inhibitory concentrations in combination. That synergy was even better against erythromycin-resistant strains. In combination with tigecycline or fosfomycin, the antibacterial activity could be consistently enhanced with the same decrease of inhibitory concentrations. A synergy was also observed, less regularly and at a lower level, with rifampin, gentamicin or vancomycin. Combinations of dalfopristin and quinupristin with tigecycline or fosfomycin could be very interesting in clinical practice because the inhibitory effect could be achieved with very low concentrations of each component, even when erythromycin-resistant strains are concerned.

Presumed pseudobacteremia outbreak resulting from contamination of proportional disinfectant dispenser.

Reported here are the microbiological and epidemiological details of a presumed outbreak of aerobic gram-negative bacilli infections affecting 19 hematological patients, which was traced to contaminated disinfectant. Over a 5-month period, the following organisms were isolated from the blood cultures of 19 neutropenic patients: Pseudomonas fluorescens (n = 13), Achromobacter xylosoxidans (n = 12), Comamonas testosteroni (n = 2) or Stenotrophomonas maltophilia (n = 1). The affected patients were all treated with an expensive regimen of broad-spectrum antibiotic therapy. The same bacteria were recovered from environmental samples as well as from the water pipes of an apparatus for dispensing disinfectant (didecyldimethylammonium chloride). Genotyping results indicated that many of the clinical strains were identical to strains isolated from the apparatus. It was eventually discovered that the night staff was in the habit of disinfecting the blood-culture bottles before use, thereby contaminating the bottles with bacteria contained in the disinfectant. Contamination of the apparatus resulted from faulty maintenance.

Molecular epidemiology of Enterobacteriaceae producing extended-spectrum beta-lactamase in a French university-affiliated hospital.

We conducted a retrospective study to investigate the epidemiology of Enterobacteriaceae producing extended-spectrum beta-lactamase (ESBLE) in our hospital. We determined the occurrence of extended-spectrum beta-lactamase (ESBL) in Enterobacteriaceae over a 2-year period. We also characterised ESBLs by isoelectric focusing (IEF) and investigated the epidemiological relatedness of EBLSE by pulsed field gel electrophoresis (PFGE). During this period, 70 patients were colonised/infected with one or several strains of EBLSE, giving a crude incidence of 0.095 per 1000 patient-days. We found that ESBL-producing Enterobacter aerogenes were the main source of ESBLE dissemination. Indeed, 59.5% of ESBLE were E. aerogenes and 21.9% of the other ESBLE resulted from a plasmid transfer originating from E. aerogenes. IEF and PFGE analysis demonstrated that the dissemination of ESBL from E. aerogenes in our hospital was due to a single clone that always harbours TEM-24. This emphasises the importance of standard contact isolation precautions and the early detection of ESBLE-colonised patients in high risk departments like intensive care units.

TEM-89 beta-lactamase produced by a Proteus mirabilis clinical isolate: new complex mutant (CMT 3) with mutations in both TEM-59 (IRT-17) and TEM-3.

TEM-89 (CMT-3) is the first complex mutant beta-lactamase produced by a clinical strain of Proteus mirabilis (strain Pm 631). This new enzyme, which has a pI of 6.28, is derived from TEM-3 and has a single amino acid substitution also encountered in TEM-59 (inhibitor-resistant TEM beta-lactamase IRT-17): Ser-130 to Gly. TEM-89 hydrolyzed penicillins to the same extent that TEM-3 did but lost almost all hydrolytic activity for cephalosporins and, like TEM-59, was highly resistant to inhibitors.

Evidence of in vivo transfer of a plasmid encoding the extended-spectrum beta-lactamase TEM-24 and other resistance factors among different members of the family Enterobacteriaceae.

The epidemiological study of several multidrug-resistant Enterobacteriaceae isolated from five patients demonstrated in vivo dissemination of a 100-kb plasmid encoding the extended-spectrum beta-lactamase TEM-24 from a clonal strain of Enterobacter aerogenes to different strains of Klebsiella pneumoniae, Escherichia coli, Proteus vulgaris, Proteus mirabilis, and Serratia marcescens.

Characterization of TEM-56, a novel beta-lactamase produced by a Klebsiella pneumoniae clinical isolate.

TEM-56 produced by a Klebsiella pneumoniae clinical isolate is a novel beta-lactamase of isoelectric point 6.4 that confers a moderate resistance level to expanded-spectrum cephalosporins. The amino acid sequence deduced from the corresponding bla gene showed two amino acid replacements with respect to the TEM-2 sequence: Glu-104 to Lys and His-153 to Arg. This enzyme showed catalytic properties close to those of TEM-18. Thus, TEM-56 appears as a new TEM mutant, an intermediary between TEM-18 and the extended-spectrum beta-lactamase TEM-21.

[A three-dimensional model for in vitro study of the association of three antibiotics. Application to the activity of piperacillin, tazobactam and amikacin against five strains of enterobacteria as a function of their phenotype of beta-lactam resistance]. / Modèle tridimensionnel pour l'étude in vitro de l'association de trois antibiotiques. Application à l'activité de pipéracilline, tazobactam et amikacine sur cinq souches d'entérobactéries en fonction de leur phénotype de résistance aux bêta-lactamines.

We described an in vitro 3-dimensional model to study the bactericidal activity of piperacillin (P), tazobactam (T) and amikacin (A) in combination against 5 strains of enterobacteria with different resistance patterns of beta-lactam antibiotics. A synergy was defined by calculation of sigma FBCP,T,A = BCp/MBCp + BCT/MBCT + BCA/MBCA and classic sigma FBCs for each double combination. The therapeutic value of each antibiotic was estimated by comparison of its bactericidal concentrations alone and in double or triple combination.

Outbreak of TEM-24-producing Enterobacter aerogenes in an intensive care unit and dissemination of the extended-spectrum beta-lactamase to other members of the family enterobacteriaceae.

We report an outbreak of Enterobacter aerogenes in an intensive care unit (ICU) and two medicine departments that produced the extended-spectrum beta-lactamase TEM-24, which was difficult to detect by disk agar diffusion. The strains were compared by DNA restriction fragment length polymorphism after pulsed-field gel electrophoresis following cleavage with XbaI. This typing method indicated that a single strain, first isolated in the ICU, spread throughout the other medical departments as a result of patient transfer. We also observed the transfer in vivo of the plasmid encoding TEM-24 from the strain of Enterobacter aerogenes to different strains of Escherichia coli and Citrobacter freundii in the ICU. It therefore appears that the epidemic involved results from two events: dissemination of one strain of Enterobacter aerogenes and dissemination of the plasmid encoding TEM-24 among various members of the family Enterobacteriaceae.

Imipenem resistance in clinical isolates of Proteus mirabilis associated with alterations in penicillin-binding proteins.

Two strains of Proteus mirabilis, IpR1 and IpR2, resistant to both imipenem and mecillinam, but susceptible to other beta-lactams were isolated from blood cultures of patients who had been treated with imipenem. Strain IpR1 was isolated in the same sample as a P. mirabilis IpS1 which was susceptible to imipenem and mecillinam. Strains IpR1 and IpR2 did not produce a beta-lactamase and their outer membrane protein profiles were similar to those of IpS1 and P. mirabilis ATCC 29906. Electrophoretic profiles of penicillin-binding proteins (PBPs) showed a decrease in PBP 1A of strains IpR1 and IpR2 compared with IpS1 and ATCC 29906. Competition experiments revealed a decrease in affinity of PBP 2 for imipenem from strain IpR1. These findings suggest that imipenem resistance in P. mirabilis might result from altered PBPs, as reported for Acinetobacter baumanii and Pseudomonas aeruginosa.

[In vitro antibacterial activity of piperacillin-tazobactam in combination with netilmicin or amikacin against Enterobacteriaceae resistant to amoxicillin]. / Activité antibactérienne in vitro de pipéracilline-tazobactam en association avec la nétilmicine ou l'amikacine sur des entérobactéries résistantes à l'amoxicilline.

The antibacterial in vitro activity of piperacillin and tazobactam (in a concentration ratio of 8/1) was studied in combination with netilmicin or amikacin by a microtiter checkerboard assay against 162 strains of Enterobacteriaceae. These strains were selected for their resistance pattern to beta-lactam antibiotics and their beta-lactamases were characterized by the mean of isoelectric focusing in comparison with reference strains. A comparison of the MICs of piperacillin, alone and in combination, assessed the efficacy of tazobactam as beta-lactamase inhibitor, particularly when a TEM-1 beta-lactamase was produced. When the strains were sensitive to the aminoglycosides (111 netilmicin-sensitive ones and 131 amikacin-sensitive ones), we observed 55% of synergistic effects and 45% of additions with the combinations piperacillin-tazobactam-netilmicin or amikacin. A synergistic effect was usually encountered with P. mirabilis, P. vulgaris, M. morganii and with the strains of E. coli, E. cloacae and S. marcescens which produced a cephalosporinase only. Among the 51 strains that were intermediate or resistant to netilmicin, 8 ones were inhibited by piperacillin-tazobactam-netilmicin at therapeutic levels (3 synergisms, 5 additions). Among the 31 strains that were intermediate or resistant to amikacin, 24 ones (18 synergisms, 6 additions) were inhibited by piperacillin-tazobactam-amikacin at therapeutic concentrations. In most of the cases, the combination of piperacillin-tazobactam with an aminoglycoside enhanced the antibacterial activity of these agents by decreasing the concentrations necessary to inhibit the strains.

[Uptake of ofloxacin by Escherichia coli]. / Pénétration de l'ofloxacine chez Escherichia coli.

The uptake of ofloxacin by Escherichia coli NIHJ-JC2 was determined by a sensitive and convenient method using high-performance liquid chromatography (HPLC) with a fluorometric detection (sensitivity level: 1 ng/ml). Concentrations of ofloxacin were measured in bacteria after contact with 5 micrograms/ml of antibiotic for 1, 2, 3, 5, 10, 20 and 30 min. Ofloxacin uptake was rapid, 70% of broth concentration occurring within the first min and 96% after 5 min; then it reached a plateau which was 1.16 times as high as the broth concentration.

[Diffusion of imipenem in synovial fluid]. / Diffusion de l'imipénème dans le liquide synovial.

The diffusion of imipenem (IMP) in the knee joint was studied after a 1 g i.v. administration of Tienam over one hour. The synovial fluid was collected under anesthesia during arthroscopy carried out for mechanical lesions of the knee (meniscal lesions after ligamental injuries or sequelae after meniscectomy), in 3 groups of six patients at one, two, or three hours after the end of injection of IMP. The concentrations of IMP determined by high performance liquid chromatography (HPLC) were: 42.5, 20.1, 9.3 and 5.7 mg/l in the blood at T0, T1, T2 and T3 hr, respectively; 20.4, 13.0 and 7.9 mg/l in the synovial fluid at T1, T2 and T3, respectively. The decrease of IMP concentrations in the synovial fluid was 1,5 times as low as in serum. On account of its broad-spectrum antibacterial activity and our data, IMP could be used in perioperative prophylaxis of the knee joint surgery.

[In vitro synergistic activity of piperacillin-tazobactam combination against beta-lactam antibiotics resistant strains of Enterobacteriaceae]. / Activité synergique in vitro de l'association pipéracilline-tazobactam vis-à-vis d'entérobactéries résistantes aux bêtalactamines.

The combination of piperacillin and tazobactam was studied in a microtiter checkerboard assay against 160 strains of Enterobacteriaceae selected for their resistance pattern to amoxicillin, ticarcillin, cephalothin and cefotaxime. Mean FIC indices were respectively 0.02, 0.17 and 0.01 for RRSS strains of E. coli, K. pneumoniae and P. mirabilis. Mean FIC indices were 0.03 for RRRI/R strains of K. pneumoniae producing a broad spectrum beta-lactamase, but the synergistic effect was out of therapeutic range for 16 strains of 20. Mean FIC indices were 0.53, 0.90, 0.55, 0.10 and 0.21 respectively against RSRS strains of E. coli, E. cloacae, S. marcescens, M. morganii and P. vulgaris. Mean FIC indices were 0.02, 0.07, 0.02 and 0.01 against RRRS/R strains of E. coli, E. cloacae, S. marcescens and M. morganii but they were in a therapeutical range (piperacillin less than or equal to 8 mg/l and tazobactam less than or equal to 4 mg/l) respectively for 7, 0, 4 and 10 strains of 10 in each species. Tazobactam restored the antibacterial activity of piperacillin against enterobacterial strains mainly producing a penicillinase or a cephalosporinase for M. morganii and P. vulgaris.

[In vitro activity of beta-lactam antibiotics in combination with sulbactam against enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter. Results of a multicenter study]. / Activité des bêtalactamines en association avec le sulbactam chez les entérobactéries Pseudomonas aeruginosa et Acinetobacter. Résultats d'une étude multicentrique.

The in vitro antimicrobial activity of ticarcillin (TICAR), mezlocillin (MEZLO), piperacillin (PIPER), cefoperazone (CPZ), cefotaxime (CTX) and ceftazidime (CAZ), alone and in combination with 8 micrograms/ml of sulbactam (SULB), was studied by agar dilution against TICAR resistant strains isolated in 8 hospitals over a period of 3 months in 1989 (747 enterobacteria, 110 Ps. aeruginosa and 48 Acinetobacter sp.). SULB did not modify the activity of beta-lactam antibiotics against Ps. aeruginosa. The 6 beta-lactam antibiotics SULB combinations were only active for 27% of Acinetobacter SULB sensitive. SULB restored the activity of: MEZLO, PIPER, CPZ in Enterobacteria producing a penicillinase; PIPER, CTX and CAZ in Enterobacteria producing a broad-spectrum beta-lactamase; MEZLO, PIPER, CTX and CAZ in M. morganii producing a derepressed cephalosporinase.

[Comparative study of the antibacterial activity of cefpodoxime against enterobacteria producing beta lactamases]. / Etude comparative de l'activité antibactérienne du cefpodoxime sur les entérobactéries productrices de bêta lactamases.

Cefpodoxime proxetil (RU 51 807) is the oral prodrug of cefpodoxime (RU 51 763), a third generation cephalosporin. The antibacterial activity of cefpodoxime was compared with the activities of amoxicillin in combination with clavulanic acid (AUG), cefaclor (CCl), cefuroxime (CXM) and cefotaxime (CTX), against species of Enterobacteriaceae showing a resistance pattern against ampicillin (AMP), ticarcillin (TIC), cefalothin (CFT) and cefotaxime (CTX) respectively. For strains AMP and TIC R, CFT and CTX S, MICs 90% of cefpodoxime were 1 mg/l (E. coli), 0.5 (K. pneumoniae), 0.06 (P. mirabilis), 0.5 (Shigella sp.) and 1 (Salmonella sp.); they were 4 to 16 times as high for AUG -CCL -CXM and 4 to 16 times as low for CTX. For K. pneumoniae AMP and TIC R, CFT I/R and CTX S, similar résults were obsereved for the 5 beta-lactam antibiotics, but with an activity 10 times as low. Among the species AMP R, TIC S, CFT R and CTX S, cefpodoxime was active against P. rettgeri, P. stuartii, C. diversus, E. aerogenes and Y. enterocolitica (MICs 90% ranging from 2 to 4 mg/l; from 0.12 to 1 mg/l for CTX) and less active or inactive against P. vulgaris, E. cloacae, S. marcescens, M. morganii and E. coli (MICs 90% ranged from 16 to 32 mg/l; from 1 to 4 mg/l for CTX).(ABSTRACT TRUNCATED AT 250 WORDS)

Differences between clavulanic acid and sulbactam in induction and inhibition of cephalosporinases in enterobacteria.

The ability of clavulanic acid and sulbactam to induce and inhibit cephalosporinases was evaluated in 16 clinical isolates of enterobacteria. Using the quantitative induction assay, the checkerboard method and the disc approximation test, clavulanic acid was shown to act as inducer for all species, whereas sulbactam only induced strains of Providencia stuartii. Antagonism was achieved using a combination of clavulanic acid and cefotaxime but a combination of sulbactam and cefotaxime was either synergistic or indifferent. This variation in effect was probably due to the fact that sulbactam, but not clavulanic acid could inhibit cephalosporinases. The data revealed a significant difference between sulbactam and clavulanic acid, which may have relevance to their relative usefulness in combination with beta-lactam antibiotics for the treatment of infections due to enterobacteria that produce inducible cephalosporinase.

Sulbactam: secondary mechanisms of action.

Light and scanning electron microscopy showed that 0.25, 0.5 and 1 times the minimum inhibitory concentrations (MICs) of sulbactam (SULB) caused filament formation in different species of Enterobacteriaceae, while 2 and 4 times the MICs caused spheroplast formation and subsequent lysis. By using a competitive assay with 125I-penicillin X, SULB showed a primary affinity for the PBP 1a and PBP3 of Escherichia coli, as well as for the PBP1a of Proteus mirabilis. The bactericidal interaction of human polymorphonuclear leukocytes (PMN) and SULB against E. coli K-1 resistant to the bactericidal activity of human serum was studied in vitro; however, SULB concentrations showed variations in the medium according to human kinetic data. Under these conditions, bacterial growth occurred in Hanks balanced salt solution containing SULB, PMN, or SULB-PMN in combination. In addition, bactericidal activity was observed in serum, with a killing rate of 90% for PMN or SULB, and 95% for SULB-PMN in combination. The postantibiotic enhancement of PMN bactericidal function was assessed against E. coli K1 pretreated with 0.5 the MIC of SULB (32 micrograms/ml) for 0.5 hr. The 90% bacterial killing rate of PMN occurred by 1.5 hr for pretreated bacteria and by 2.5 hr for untreated bacteria. Furthermore, the luminol-enhanced chemiluminescence (CL) assay using an E. coli stimulus showed that SULB does not modify PMN activity.

[Bactericidal activity of daptomycin and vancomycin alone or in combination with tobramycin, netilmicin or ampicillin against enterococcus]. / Activité bactéricide de la daptomycine et de la vancomycine seules ou en association avec la tobramycine, la nétilmicine ou l'ampicilline, sur les entérocoques.

The in vitro bactericidal activity of daptomycin and vancomycin alone or in combination was studied against enterococci in a microtiter checkerboard assay and by kill-kinetic experiments. Daptomycin was more active than vancomycin and was bactericidal. Better Fractional Bactericidal Concentration indices were observed with combinations of vancomycin with aminoglycosides, but in kill-kinetic studies, the combinations of 4 MICs of daptomycin with 4 mg/l of tobramycin or netilmicin produced the more lethal effect; these combinations were even more lethal than ampicillin and aminoglycosides in the same conditions. While vancomycin and ampicillin were antagonistic, synergistic FBC indices were observed with daptomycin and ampicillin in combination, but at 4 MICs of each antibiotic, the combination was less bactericidal than ampicillin alone. The bactericidal effect obtained with daptomycin in combination with aminoglycosides suggest that further evaluation of these combinations in enterococcal endocarditis could have a clinical interest if this bactericidal effect was confirmed by in vivo studies.