Risk-factors for emerging bloodstream infections caused by extended-spectrum beta-lactamase-producing Escherichia coli.

Risk-factors for bloodstream infections caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli were investigated using an exploratory case-double control study in which 43 cases (70% producing CTX-M enzymes) were compared with: (i) 86 patients with bacteraemia caused by non-ESBL-producing E. coli; and (ii) 86 hospitalised patients. Previous follow-up as an outpatient, urinary catheterisation and use of oxyimino-beta-lactams or fluoroquinolones were independent risk-factors for ESBL-producing E. coli among patients with E. coli bacteraemia, and previous use of oxyimino-beta-lactams or fluoroquinolones were also independent risk-factors among hospitalised patients. These findings may help in identifying patients at greater risk for bloodstream infection caused by ESBL-producing E. coli in endemic areas.

Cytomegalovirus mononucleosis as a cause of prolonged fever and prominent weight loss in immunocompetent adults.

Four immunocompetent adults presented with protracted fever lasting > 6 weeks and severe weight loss, associated with primary cytomegalovirus (CMV) infection. Each patient had spleen enlargement, lymphocytosis and hypertriglyceridaemia, but recovered spontaneously. A further 20 immunocompetent patients with primary CMV infection were also reviewed, and all presented the usual clinical picture of CMV mononucleosis. It was concluded that CMV mononucleosis should be considered in the differential diagnosis in patients with prolonged fever and weight loss if lymphocytosis is present.

[In vitro activity of azithromycin against clinical isolates of Acinetobacter baumannii]. / Actividad in vitro de azitromicina frente a aislamientos clínicos de Acinetobacter baumannii.

The activity of azithromycin against 225 clinical strains of Acinetobacter baumannii isolated consecutively from 26 Spanish hospitals in November 2000 was studied. The MICs of azithromycin were determined by microdilution, according to the NCCLS guidelines. The bactericidal activity of azithromycin against 15 clonally unrelated A. baumannii strains with different antimicrobial susceptibility patterns was tested using the subculture method. The killing-curves method was also performed against five strains with different susceptibility to azithromycin. The MIC(50) and MIC(90) of azithromycin were 32 and 64 mg/l, respectively. Moderate bactericidal activity was observed in 14 out of the 15 strains evaluated by the subculture method (MBCs from 1 to 4 dilution steps higher than the MICs) and by the killing-curve method. For three strains the number of CFU/ml was reduced 1 to 1.4 log by concentrations of azithromycin equivalent to 1 and 4 times their MICs. lt is concluded that azithromycin has moderate bactericidal activity against the strains of A. baumannii evaluated.

Evaluation of the WIDER I system for antimicrobial susceptibility testing of clinical isolates of Haemophilus influenzae and Streptococcus pneumoniae.

The aim of this study was to evaluate the WIDER I system for susceptibility testing of Haemophilus influenzae and Streptococcus pneumoniae. MICs of 12 antimicrobials against 42 H. influenzae and 58 S. pneumoniae strains were determined using 1W MIC panels and compared with those obtained by microdilution. Overall essential agreements were >99%. Very major errors were not detected. Major errors occurred with ampicillin (1.7% H. influenzae). Minor errors were 2.3% (amoxicillin-clavulanate, cefuroxime, chloramphenicol), 7.1% (ampicillin) and 16.7% (clarithromycin) for H. influenzae, and 1.7% (chloramphenicol, erythromycin, meropenem), 3.4% (amoxicillin-clavulanate, cefuroxime, tetracycline) and 8.6% (levofloxacin) for S. pneumoniae. The WIDER I system is a reliable method for susceptibility testing of H. influenzae and S. pneumoniae.

Accumulation and activity of cethromycin (ABT-773) within human polymorphonuclear leucocytes.

OBJECTIVES: To evaluate the penetration of ketolide cethromycin (ABT-773) into human polymorphonuclear leucocytes (PMNs) and its intracellular activity. METHODS: The uptake of radiolabelled cethromycin by PMNs was determined by a velocity gradient centrifugation technique. The activity of cethromycin against intracellular Staphylococcus aureus ATCC 25923 in PMNs was also evaluated. RESULTS: The cellular to extracellular concentration (C/E) ratio for cethromycin was >200 at an extracellular concentration of 2 mg/L. The uptake of cethromycin into PMNs was rapid and saturable. Cethromycin was slowly released from the loaded PMNs (cell associated drug>50% after 2 h of incubation). Intracellular penetration was significantly affected by the environmental temperature (C/E ratio at 4 degrees C and 37 degrees C: 13 +/- 6 and 226 +/- 31, respectively; P < 0.05), by cell viability (C/E ratio for dead and viable cells: 100 +/- 38 and 226 +/- 31, respectively; P < 0.05), by pH (C/E ratio was significantly increased at basic pH) and by the metabolic inhibitors 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. The intracellular accumulation of cethromycin also decreased significantly when cells were activated with phorbol myristate acetate or opsonized zymosan. These data indicate that a potentially active mechanism could be involved in the uptake of cethromycin by PMNs. At high extracellular concentrations of 5 and 10 mg/L, cethromycin showed significant intracellular activity against S. aureus. CONCLUSIONS: Cethromycin achieves high intracellular concentrations within human PMNs, remaining active intracellularly.

[The activity of four fluoroquinolones against strains of Pseudomonas aeruginosa with a different sensitivity pattern to ceftazidime and imipenem]. / Actividad de cuatro fluoroquinolonas frente a cepas de Pseudomonas aeruginosa con diferente patrón de sensibilidad a ceftazidima e imipenem.

OBJECTIVES: To evaluate the activity of four fluorquinolones (ciprofloxacin, clinafloxacin, norfloxacin and perfloxacin) against clinical strains of Pseudomonas aeruginosa with different sensitivity patterns to ceftazidime and imipenem. MATERIAL AND METHODS: 156 strains of isolated P. aeruginosa were studied at the Virgin Macarena University Hospital in Seville during 1998 and 1999. The in vitro activity of four fluorquinolones was determined by microdilution in Mueller Hinton bouillon, supplemented with cations, following the NCCLS guidelines. RESULTS: For all the strains evaluated, the minimum inhibitory concentration values (MIC90) of the clinafloxacin (4 mg/l) were significantly less than those for ciprofloxacin (64 mg/l). In the 76 strains resistant to ciprofloxacin, the clinafloxacin and ciprofloxacin MCI90 were 16 and >128 mg/l respectively. Clinafloxacin was more active than ciprofloxacin, norfloxacin and pefloxacin, independent to the sensitivity pattern or the resistance to ceptazidime and imipenem. CONCLUSION: Clinafloxacin was more active in vitro than ciprofloxacin against P. aeruginosa.

Evaluation of the VITEK 2 system for the identification and susceptibility testing of three species of nonfermenting gram-negative rods frequently isolated from clinical samples.

VITEK 2 is a new automatic system for the identification and susceptibility testing of the most clinically important bacteria. In the present study 198 clinical isolates, including Pseudomonas aeruginosa (n = 146), Acinetobacter baumannii (n = 25), and Stenotrophomonas maltophilia (n = 27) were evaluated. Reference susceptibility testing of cefepime, cefotaxime, ceftazidime, ciprofloxacin, gentamicin, imipenem, meropenem, piperacillin, tobramycin, levofloxacin (only for P. aeruginosa), co-trimoxazole (only for S. maltophilia), and ampicillin-sulbactam and tetracycline (only for A. baumannii) was performed by microdilution (NCCLS guidelines). The VITEK 2 system correctly identified 91.6, 100, and 76% of P. aeruginosa, S. maltophilia, and A. baumannii isolates, respectively, within 3 h. The respective percentages of essential agreement (to within 1 twofold dilution) for P. aeruginosa and A. baumannii were 89.0 and 88.0% (cefepime), 91.1 and 100% (cefotaxime), 95.2 and 96.0% (ceftazidime), 98.6 and 100% (ciprofloxacin), 88.4 and 100% (gentamicin), 87.0 and 92.0% (imipenem), 85.0 and 88.0% (meropenem), 84.2 and 96.0% (piperacillin), and 97.3 and 80% (tobramycin). The essential agreement for levofloxacin against P. aeruginosa was 86.3%. The percentages of essential agreement for ampicillin-sulbactam and tetracycline against A. baumannii were 88.0 and 100%, respectively. Very major errors for P. aeruginosa (resistant by the reference method, susceptible with the VITEK 2 system [resistant to susceptible]) were noted for cefepime (0.7%), cefotaxime (0.7%), gentamicin (0.7%), imipenem (1.4%), levofloxacin (2.7%), and piperacillin (2.7%) and, for one strain of A. baumannii, for imipenem. Major errors (susceptible to resistant) were noted only for P. aeruginosa and cefepime (2.0%), ceftazidime (0.7%), and piperacillin (3.4%). Minor errors ranged from 0.0% for piperacillin to 22.6% for cefotaxime against P. aeruginosa and from 0.0% for piperacillin and ciprofloxacin to 20.0% for cefepime against A. baumannii. The VITEK 2 system provided co-trimoxazole MICs only for S. maltophilia; no very major or major errors were obtained for co-trimoxazole against this species. It is concluded that the VITEK 2 system allows the rapid identification of S. maltophilia and most P. aeruginosa and A. baumannii isolates. The VITEK 2 system can perform reliable susceptibility testing of many of the antimicrobial agents used against P. aeruginosa and A. baumannii. It would be desirable if new versions of the VITEK 2 software were able to determine MICs and the corresponding clinical categories of agents active against S. maltophilia.

Activities of gemifloxacin and five other antimicrobial agents against Listeria monocytogenes and coryneform bacteria isolated from clinical samples.

The in vitro activities of gemifloxacin, ciprofloxacin, ampicillin, doxycycline, gentamicin, and vancomycin were evaluated against 15 Listeria monocytogenes strains and 205 coryneform bacteria isolated from clinical samples. The percentages of strains inhibited by gemifloxacin at 0.5 microg/ml were 100% (L. monocytogenes), 93.3% (Brevibacterium spp.), 90% (Corynebacterium minutissimum), 42.5% (Corynebacterium amycolatum), 20% (Corynebacterium striatum), 12.5% (Corynebacterium jeikeium), and 10% (Corynebacterium urealyticum). One hundred percent of the L. monocytogenes strains were inhibited by 0.25 microg of gemifloxacin per ml, whereas 0% of the strains were inhibited by 0.25 microg of ciprofloxacin per ml. Vancomycin at 2 microg/ml inhibited all strains. Doxycycline and gentamicin at 4 microg/ml inhibited 94 and 49% of the strains, respectively, while ampicillin at 0.5, 2, and 8 microg/ml inhibited 24, 61, and 66% of the strains, respectively. It is concluded that gemifloxacin shows good in vitro activity against L. monocytogenes and coryneform bacteria except C. jeikeium and C. urealyticum.

Uptake and intracellular activity of ketolide HMR 3647 in human phagocytic and non-phagocytic cells.

OBJECTIVE: To evaluate the uptake of HMR 3647 into human neutrophils (PMNs), human peritoneal macrophages (PMOs) and tissue-cultured cells (epithelial cells and fibroblasts), and to assess the intracellular activity of this drug. METHOD: Cell uptake of HMR 3647 was measured by radiometric assay, as described by Klemper and Styrt. Intracellular activity was determined by incubation for 3 h of PMNs containing bacteria in the presence of HMR 3647. RESULTS: The intracellular concentrations were 130 and 71 times higher than extracellular concentrations in PMNs and PMOs, respectively (extracellular concentrations: 2-25 mg/L). The cellular-to-extracellular concentration ratios (C/E) for tissue-cultured cells were lower than those obtained in phagocytic cells but still greater than 5. The uptake of HMR 3647 was rapid and non-saturable in all cells. HMR 3647 was released slowly from phagocytic cells. HMR 3647 (extracellular concentration: 0.5-10 mg/L) did not significantly reduce the intracellular survival rate of Staphylococcus aureus ATCC 25923 in PMNs. CONCLUSIONS: HMR 3647 reaches intracellular concentrations several times higher than extracellular concentrations within phagocytic and non-phagocytic cells. The slow efflux of this drug from phagocytic cells suggests that these cells may be a vehicle for it, delivering it to sites of infection.

[Comparison of the activity of different macrolides on strains of Haemophilus influenzae]. / Actividad comparada de diversos macrólidossobre cepas de Haemophilus influenzae. Grupo Español de Estudio de la Actividad de Macrólidos sobre H. influenzae.

In this study we evaluated the activity of macrolides and b-lactam antimicrobials on Haemophilus influenzae isolated in 1998 in eight Spanish cities. A total of 174 clinical isolates were examined. Overall, 29% of the isolates were found to produce b-lactamase. Azithromycin was the most active of the macrolides tested in this study (MIC90 of 4 mg/l); no azithromycin-resistant strains were found. Ampicillin resistance was 29%. We found one strain intrinsically resistant to beta-lactam agents (0.65% overall); and two beta-lactamase-positive strains that were resistant to amoxicillin-clavulanic acid (1.2%). The presence of these strains, while uncommon at present, makes it necessary to test the activity of antimicrobial drugs on H. influenzae.

Intracellular penetration and activity of gemifloxacin in human polymorphonuclear leukocytes.

The intracellular penetration and activity of gemifloxacin in human polymorphonuclear leukocytes (PMN) were evaluated. Gemifloxacin reached intracellular concentrations eight times higher than extracellular concentrations. The uptake was rapid, reversible, and nonsaturable and was affected by environmental temperature, cell viability, and membrane stimuli. At therapeutic extracellular concentrations, gemifloxacin showed intracellular activity against Staphylococcus aureus.

Uptake and intracellular activity of ofloxacin isomers in human phagocytic and non-phagocytic cells.

The penetration and intracellular activity of ofloxacin and its isomers (levofloxacin and D-ofloxacin) into human polymorphonuclear leucocytes (PMN), human peritoneal macrophages (PMphi) and tissue cultured epithelial cells (McCoy) were evaluated. The cellular to extracellular concentration (C/E) values of the three fluoroquinolones were higher than 3.6 and 2.6 in PMN and PMphi, respectively. The C/E ratios in McCoy cells were lower than those in PMN, but still higher than 2.0. The uptake of ofloxacin and its isomers was rapid, non-saturable and reversible. All quinolones (extracellular concentrations: 2, 5 and 10 mg/l) produced a significant reduction of viable intraphagocytic Staphylococcus aureus in phagocytic cells. We concluded that ofloxacin and its isomers reach high intracellular concentrations in phagocytic and non phagocytic cells while remaining active in the former.

Activities of imipenem and cephalosporins against clonally related strains of Escherichia coli hyperproducing chromosomal beta-lactamase and showing altered porin profiles.

Forty clonally related clinical isolates of Escherichia coli from hospitalized patients were resistant to cefoxitin (MICs, >256 microg/ml) and ceftazidime (MICs, 32 to 256 microg/ml) and were intermediate or resistant to cefotaxime (MICs, 16 to 128 microg/ml) but susceptible to both cefepime (MICs, 0.5 to 2 microg/ml) and imipenem (MICs, 0.125 to 0.25 microg/ml). Resistance to beta-lactams was related to high-level production of AmpC beta-lactamase and loss of OmpF porin.

In vitro adherence of Enterococcus faecalis and Enterococcus faecium to urinary catheters.

The in vitro adherence of ten strains of Enterococcus faecalis and ten strains of Enterococcus faecium to siliconized latex urinary catheters and to silicone elastomer was evaluated. Bacterial suspensions (2.5x10(5) cfu/ml) in tryptic soy broth containing 0.5 cm segments from each type of catheter were incubated at 37 degrees C. At specified intervals, the segments were washed to remove nonadherent bacteria and sonicated for 1 min, and colony-forming units were quantified. Bacterial adherence occurred rapidly, reaching maximal peaks after 24 h of incubation. Enterococcus faecium adherence to both biomaterials was significantly lower than that of Enterococcus faecalis. No differences were observed between the two elastomers. Bacterial adherence was not related to bacterial surface hydrophobicity, hemolysin or gelatinase production.

[False resistance to imipenem in gram negative bacilli with and automatized system]. / Falsa resistencia a imipenema en bacilos gramnegativos mediante un sistema automatizado.

BACKGROUND: The aim of this study was to evaluate the reliability of MIC values of imipenem against gramnegative rods obtained with the automated system WalkAway-98 (MicroScan, Dade, USA). MATERIAL AND METHODS: One-hundred and seventy three consecutive clinical isolates of Gram-negative rods for which the MIC of imipenem were > or = 4 mg/l (Urine-Combo 6I panels, U6I) or > or = 8 mg/l (Neg-Combo 6I panels, N6I) were evaluated, including 104 non-fermenting gram-negative rods (NFGNR) and 69 enterobacteria. Panels were inoculated and read according to manufacturer's instructions. Microdilution, according to NCCLS guidelines, was used as the method of reference. MIC of imipenem determined by WalllAway-96 and microdilution differing > or = 2 dilution steps from those obtained with mirodilution were considered as discrepant results. Discrepancies in clinical categories were also evaluated by calculating three types of errors: very major (false susceptibility), major (false resistance) and minor (either susceptible or resistant by one method and intermediate by the other one). RESULTS: The percentages of discrepancies in the MIC of imipenem determined with U6I panels were 74% and 84% for NFGNR and enterobacteria, respectively. No very major errors were detected. Major errors were observed for 6% and 12% of the strains with U6I panels in NFGNR and enterobacteria, respectively, and in 12% (NFGNR) and 50% (enterobacteria) with N61 panels. With U61 panels minor errors were observed in 11% and 25% of NFGNR and enterobacteria, respectively, while with N61 panels minor error were observed in 39% and 45% of both groups, respectively. CONCLUSIONS: MIC of imipenem > or = 4 mg/l obtained with the WalkAway-96 system against gramnegative rods, particularly in the case of enterobacteria, should be confirmed with a reference susceptibility method.

In-vitro activity of levofloxacin, ofloxacin and D-ofloxacin against coryneform bacteria and Listeria monocytogenes.

The objective of this study was to evaluate the in-vitro activity of levofloxacin, ofloxacin and D-ofloxacin compared with ciprofloxacin, norfloxacin and sparfloxacin against coryneform bacteria and Listeria monocytogenes isolated from clinical samples. The following organisms (and number of strains) were studied: Corynebacterium jeikeium (20), Corynebacterium urealyticum (20), Corynebacterium minutissimum (20), Corynebacterium striatum (20), Corynebacterium amycolatum (30), Brevibacterium spp. (15) and Listeria monocytogenes (15). Antimicrobial activity was determined by microdilution using cation-adjusted Mueller-Hinton broth supplemented with 0.5% Tween 80 when testing C. jeikeium or C. urealyticum. Fluoroquinolones were used in the range 0.015-16 mg/L. Plates were incubated in air at 35 degrees C for 18-20 h (24 h when testing C. jeikeium or C. urealyticum). The following MIC50 values were obtained for all 140 organisms tested: levofloxacin, 1 mg/L; ofloxacin, 2 mg/L; D-ofloxacin, > 16 mg/L; ciprofloxacin, 1 mg/L; norfloxacin, 16 mg/L; sparfloxacin, 1 mg/L. MIC90 values were > 16 mg/L for all test antibiotics with the exception of levofloxacin, which had an MIC90 value of 16 mg/L. At a concentration of 2 mg/L, levofloxacin inhibited all L. monocytogenes strains and 35-93% of the remaining species. MIC90 values of ofloxacin were one dilution step higher than those of levofloxacin against C. minutissimum, C. striatum, Brevibacterium spp. and L. monocytogenes. Levofloxacin showed similar (C. jeikeium, C. urealyticum, C. amycolatum and Brevibacterium spp.) or greater (C. minutissimum and C. striatum) activity than ciprofloxacin and sparfloxacin, and higher than D-ofloxacin or norfloxacin against all species studied. In conclusion, levofloxacin was the most active of the six fluoroquinolones evaluated against coryneform bacteria isolated from clinical samples and could therefore be a promising treatment option in this setting.