Susceptibility to beta-lactams was determined in 203 recent Spanish E. coli isolates from urinary tract infections exhibiting different resistance phenotypes: a) susceptible (n = 60); b) quinolone-resistant (n = 45); c) penicillinase (n=64); d) hyperproduction of penicillinase (n=8); e) inhibitor resistant TEM (IRT) (n=18), and f) extended spectrum betalactamase (ESBL) (n=8).Minimum inhibitory concentration (MIC) determination by agar dilution and susceptibility tests for ESBL detection by macrodilution were performed following CLSI recommendations. All the beta-lactams tested showed high activity against susceptible and penicillinase phenotypes, with close to 100 % susceptibility. Hyperproduction of penicillinase increased MIC90 values for all antibiotics except for meropenem, with 100% resistance to cefuroxime and amoxicillin/clavulanic acid, and 100% susceptibility to cefotaxime, piperacillin/tazobactam and meropenem. All the antibiotics, except for amoxicillin/clavulanic acid, exhibited high activity against IRT. Meropenem, cefminox and piperacillin/tazobactam exhibited the highest activity against ESBL, followed by amoxicillin/clavulanic acid. The most active compound among the parenteral antibiotics was meropenem, regardless of the resistance phenotype. Among the oral antibiotics, the most active compound was cefditoren with the exception of ESBL where amoxicillin/clavulanic acid where the MIC90 value was one dilution lower.

The monomer of 2-butanone peroxide is a novel peroxygen derivative with potential use as biocide in the hospital environment. The aim of this study was to test the biocidal activity of different concentrations of the compound against American Tissue Culture Collection strains from 11 different micro-organisms, including bacteria, mycobacteria, spores, fungi and virus, following the European Standard guidelines. Toxicity tests were also carried out following United States Environmental Protection Agency Standards. 2-Butanone peroxide exhibited biocidal activity at 0.12% against Legionella pneumophila, at 0.5% against Escherichia coli, Pseudomonas aeruginosa and Enterococcus hirae, and at 1% against Staphylococcus aureus after 5min contact at room temperature. Mycobactericidal activity was obtained at 0.5% after 60min contact at 20°C, and sporicidal activity was obtained at 4% after 60min at 40°C. Good fungicidal (against yeasts and moulds) and virucidal (adenovirus and poliovirus) activities were obtained at 0.5% after 60min contact. Toxicity assessment showed negative results in the acute dermal irritation test, acute eye irritation test and acute oral toxicity test. The skin sensitisation test was negative. The safety profile in the toxicity tests and the basic cidal activity against the strains tested suggest that 2-butanone peroxide in the control of hospital infections.

Nineteen β-lactamase–negative ampicillin-resistant (BLNAR) and 2 β-lactamase–positive amoxicillin-clavulanic acid–resistant Canadian Haemophilus influenzae strains were characterized. All 21 isolates were found to have the N526K mutations in their ftsI genes, and their ampicillin MIC50 values were 4–8 times that of β-lactamase–negative ampicillin-susceptible strains. The difficulty in detection of BLNAR strains was discussed.

This study explores the influence on the intrinsic activity of different oral beta-lactams of beta-lactamase production in Haemophilus influenzae and penicillin resistance in Streptococcus pneumoniae. Three substudies were performed: a) a general susceptibility study, analyzing 550 strains received by the Spanish Laboratorio de Referencia de Neumococos throughout February and March 2005; b) a study on the influence of penicillin resistance on the activity of beta-lactams, analyzing 251 penicillin-susceptible strains (MICor=2 mg/l) randomly chosen among those received by the Spanish Laboratorio de Referencia de Neumococos throughout 2005; and c) an H. influenzae susceptibility study analyzing 150 strains received by Instituto Valenciano de Microbiologia throughout 2005. A total of 71% of S. pneumoniae strains were susceptible to penicillin, 21% exhibited intermediate resistance and 8% strains presented full resistance. H. influenzae beta-lactamase production rate was 18.6%. Of the non-beta-lactamase-producing strains, 3% were not susceptible to ampicillin. Cefpodoxime and cefixime exhibited the highest intrinsic activity against H. influenzae, while amoxicillin and cefpodoxime were the most active compounds against S. pneumoniae. All H. influenzae strains were susceptible to oral cephalosporins and amoxicillin/clavulanic acid. The increase in penicillin resistance in S. pneumoniae influenced cefixime, cefaclor and cefuroxime to a higher degree than amoxicillin and cefpodoxime.

We characterize alterations in the QRDR fragment of gyrA and parC in Salmonella spp. following repeated exposure to different quinolones in two in vitro models. Mutations in QRDR of gyrA were found in only 25% of the mutants. The most frequent mutations were Asp87(R)Asn and Asp87(R)Tyr. Strains with the former mutation had a higher ciprofloxacin MIC than those with the latter. We did not find mutations in parC. Our data confirm that mutation in the QRDR fragment of gyrA is the mechanism that produces the greatest decrease in fluoroquinolone susceptibility. There is a greater reduction in the ciprofloxacin susceptibility of strains that were originally nalidixic acid-resistant than in that of strains that were originally susceptible, and this confirms that there is a greater likelihood of therapeutic failures with such strains.

Salmonella mutants with reduced fluoroquinolone susceptibility were generated in vitro following repeated exposure to subinhibitory concentrations of the drugs and the alterations in the gyrA gene were characterized. Afterwards, the change in bactericidal activity exhibited by ciprofloxacin against the resulting mutants was determined. A decrease in the bactericidal activity of ciprofloxacin was found in all the mutants, but was more pronounced in mutants of nalidixic acid-resistant strains. This may help to explain the therapeutic failures sometimes described when fluoroquinolones are used in the treatment of these strains. In addition, our model evaluates the first interactions produced between the microorganism and the antibiotics, and demonstrates that the loss of bactericidal activity of ciprofloxacin occurs following exposure to all of the fluoroquinolones tested.

Estudiamos la influencia de la produccion de betalactamasas en Haemophilus influenzae y del grado de sensibilidad a la penicilina en Streptococcus pneumoniae sobre la actividad intrinseca de distintos betalactamicos orales. Realizamos tres subestudios: 1) un estudio general de sensibilidad, analizando 550 aislamientos consecutivos recibidos en el Laboratorio de Referencia de Neumococos durante los meses de febrero y marzo de 2005; 2) un estudio de la influencia de la sensibilidad a la penicilina sobre la actividad del resto de los betalactamicos, analizando la sensibilidad de 251 cepas sensibles a la penicilina (CMI iU0,06 mg/l), 165 cepas con resistencia intermedia (CMI 0,12-1 mg/l) y 139 resistentes (CMI iÝ2 mg/l), elegidas aleatoriamente entre todos los aislamientos recibidos durante el ano 2005; y 3) un estudio de sensibilidad de H. influenzae, analizando 150 cepas recibidas por el Instituto Valenciano de Microbiologia a lo largo del ano 2005. El 71% de las cepas de S. pneumoniae fueron sensibles a la penicilina, el 21% presentaron baja resistencia o resistencia intermedia, y un 8% alta resistencia. La tasa de produccion de betalactamasas fue del 18,6% en H. influenzae. El 3% de las cepas no productoras de betalactamasas fueron no sensibles a la ampicilina. La cefpodoxima y la cefixima presentaron la mayor actividad intrinseca frente a H. influenzae, mientras que frente a S. pneumoniae esta correspondio a la amoxicilina y la cefpodoxima. Mientras que el 100% de las cepas de H. influenzae fueron sensibles a las cefalosporinas orales y a amoxicilina-acido clavulanico, el aumento de la resistencia a la penicilina en S. pneumoniae afecto en mayor grado a la actividad de la cefixima, el cefaclor y la cefuroxima que a la amoxicilina y la cefpodoxima.

Background: Mutant prevention concentration (MPC) is a new parameter that may be of aid in determining the risk of resistant mutants being selected. Methods: The MPCs of 224 Mycobacterium tuberculosis clinical isolates were estimated by plating more than 1010 cells on drug-containing agar and determining the concentration that allowed no colony growth. Antibiotics used were isoniazid, rifampicin and rifabutin. Results: The MPC90 of clinical isolates in our setting is 2.4, 2.2 and 0.4 mg/l for isoniazid, rifampicin and rifabutin, respectively. Conclusions: Isoniazid and rifampicin are two drugs that present a low risk of selection of resistant mutants when used in monotherapy. However, determination of the MPC of each strain can provide data to minimize this risk and thus enable treatment to be optimized.

This study looked the selection of resistant mutants in Mycobacterium avium-intracellulare during antibiotic treatment. The mutant prevention concentration (MPC) of 20 Mycobacterium avium and 12 Mycobacterium intracellulare isolates was determined. Fifty percent of Mycobacterium avium strains had MPC (MPC50) values lower than 16, 64, 40, 55 and 60 mg/L for rifabutin, rifampicin, ciprofloxacin, levofloxacin and moxifloxacin, respectively. In the case of Mycobacterium intracellulare, 50% had MPC (MPC50) values below 60, 30, 35, 16, 2.5 and 14 mg/L for linezolid, rifabutin, levofloxacin, gatifloxacin, moxifloxacin and clarithromycin, respectively. The high capacity for selecting resistant mutants of all the antibiotics studied emphasises the need to restore the immune system if necessary and to administer combined treatments in order to cure patients.

INTRODUCTION. In the last years, we have verified the increasing emergence of bacteria, specially Escherichia coli, that produce expanded spectrum beta-lactamases (ESBL), enzymes which confer resistance to all cephalosporins (except cephamycins) and aztreonam. These bacteria are frequently resistant also to non-beta-lactam antibiotics, a fact which poses an important clinical problem.METHODS. Descriptive study of ESBL-producing strains of E. coli isolated in all kind of specimens in two hospitals of Southern Alicante (Spain), throughout a period of 57 months (January 1999 to September 2003), paying a close attention to their origin (outpatients or admitted patients), co-resistance to non beta-lactam antibiotics and evolution of their incidence.RESULTS. Respectively, 3% and 2.25% of E. coli strains isolated in each hospital produce ESBL (3.83% and 2.85% of strains from admitted and 2.74% and 2.1% from outpatients). 30.73% and 24.58% of strains ESBL were isolated in admitted patients. We found in both hospitals much higher percentages of co-resistance to ciprofloxacin, gentamicin and trimetoprim-sulfamethoxazole in ESBL-producing strains.CONCLUSION. The percentage of ESBL-producing E. coli is high in our environment, but it is even more noteworthy its clear trend to increase. It is very remarkable the high percentage of ESBL-producing strains isolated from outpatients. Finally, we emphasize the high percentages of co-resistance to non-beta-lactam antibiotics.

Background: Westudied the importance of the efflux pump mechanisms in Salmonella spp. mutants with reduced fluoroquinolone susceptibility generated in vitro. Methods: The efflux pump was studied using MC-207,110 as an inhibitor of these systems. Results: Wild strains with mutations in gyrA exhibit greater activity of the efflux pump systems thannalidixic-acid-susceptible strains (30-fold). When we evaluate the respective mutants, in those of susceptible strains there is seen to be greater elimination of the antibiotic (13-fold), whereas in mutants of nalidixic-acid-resistant strains these systems are not modified. When evaluating the influence of the antibiotic generating the mutants, ciprofloxacin is seen to be the quinolone that activates the efflux pump systems the most. Conclusions: Repeated exposure to low concentrations of all the fluoroquinolones studied leads to activation of the efflux pump systems and a reduction in susceptibility, even when there are no mutations in gyrA. Activation of these mechanisms is greatly influenced by the chemical structure of the antibiotic. The capacity of these systems to eliminate fluoroquinolones is limited and therefore, for the microorganism to acquire high-level fluoroquinolone resistance, they must be complemented by other mechanisms.

The effect of applying various criteria for elimination of repeated isolates in the same patient on the prevalence and antibiotic susceptibility of microorganisms frequently associated with urinary infections were analyzed. When time criteria were applied, there was a decrease in the number of isolates of 13.4–27.5%, whereas when variations in antibiotic susceptibility were considered, between 4.3 and 20.3% of the isolates were eliminated. The absolute number of isolates varied considerably depending on which of these two criteria were used, but neither criterion was seen to modify significantly the antibiotic susceptibility of these microorganisms and this confirms the recommendations of the NCCLS for this group of pathogens.

Objectives: The mutant prevention concentration (MPC) has recently been defined to characterize the capacity for severely restricting the selection of resistant mutants during antibiotic treatment. We determined this parameter for ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin and linezolid in Mycobacterium tuberculosis clinical isolates in our setting.Methods: We determined the antibiotic concentration that prevents the selection of resistant mutants following inoculation with a high mycobacteria inoculum on Middlebrook 7H11 plates with serial dilutions of the antibiotics in 224 M. tuberculosis isolates.Results: Fifty percent of the strains exhibited values of MPC (MPC50) lower than 0.8, 0.6, 0.4, 0.4 and 0.6 mg/L for ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin and linezolid, respectively. If 90% of the strains are considered (MPC90), the values are seen to rise to 2, 1.8, 1, 1.2 and 1.2 mg/L, respectively.Conclusions: When we compare this parameter with the drug levels in serum and tissue, it can be seen that ciprofloxacin is the least useful of the fluoroquinolones studied, whereas moxifloxacin appears to be the most active. Linezolid exhibits excellent activity against this microorganism (MPC90 1.2 mg/L and AUC 140.3 mg.h/L) and this makes us consider that its usefulness in the treatment of this pathology should be thoroughly evaluated.

The aim of this study was to determine the importance of the active elimination of antibiotics by active efflux systems, in the decrease in fluoroquinolone sensitivity of clinical isolates of Salmonella spp. as well as the intrinsic antibiotic activity of certain active efflux system inhibitors. The effect of the active efflux system on the decrease in sensitivity to nalidixic acid, ciprofloxacin, ofloxacin and sparfloxacin was studied by investigating the variation in the in vitro activity of these compounds when assayed in association with reserpine and MC 207.110. The active efflux systems inhibited by reserpine displayed low activity in the elimination of these compounds, whereas those inhibited by MC 207.110 showed high activity in the elimination of nalidixic acid and sparfloxacin, but were less effective in the elimination of ofloxacin and ciprofloxacin. These two compounds did not exhibit intrinsic inhibitory activity against Salmonella spp. at the concentrations assayed. These mechanisms of resistance to antibiotics are complex and vary depending on the chemical composition of the antibiotics used, and perhaps the inhibitors of these systems, although they do not exhibit any intrinsic antibiotic activity, may be used as adjuvants to increase the activity of certain antibiotics. These mechanisms complement the mutations in the gyrA gene and this supports the thesis that it is necessary to lower the breakpoint established by the NCCLS for ciprofloxacin, since the strains studied have resistance mechanisms that reduce the activity of this drug and may favour the emergence of resistant mutants during treatment.

The mutant prevention concentration (MPC) of fluoroquinolones, linezolid, rifabutin, rifampicin and macrolides was determined in 16 nontuberculous Mycobacterium species in order to compare the incidence of resistant mutant occurrence for different antibiotics. Linezolid showed good activity against Mycobacterium gordonae and Mycobacterium kansasii. Rifabutin had a lower MPC values than rifampicin. The fluoroquinolones were more active against M. gordonae and Mycobacterium chelonae. Clarithromycin had better MPC values than azithromycin but its intracellular concentration was high. Mutant prevention concentration may help to establish a better clinico-microbiological correlation, the sub-populations of drug-resistant micro-organisms may be detected by that method. It is still important to administer correct drug doses, to use combined therapies and to find new drugs that are useful in the treatment of these infections.

We repeatedly exposed six Salmonella spp. strains of different serotypes (three susceptible to nalidixic acid and three resistant) to constant low concentrations of various fluoroquinolones with the aim of characterising the mutations that produce the first decrease in susceptibility to these agents. The fluoroquinolone-susceptibility of all the strains was reduced after repeated exposure to these agents. However, gyrA mutants were not always produced. Furthermore, the type of mutation produced and the time taken for it to appear varied depending on the initial resistance to nalidixic acid, the antibiotic used and the serotype involved. Therefore, we believe that the initial decrease in quinolone-susceptibility is due to various mechanisms and, in many cases, is not caused by mutations in the gyrA gene

We evaluated the Binax NOW rapid immunochromatographic membrane test (ICT) for detection of Streptococcus pneumoniae urinary antigen in a population-based prospective study of adults with community-acquired pneumonia (CAP). ICT was performed with urine samples obtained from 452 (91.7%) of 493 patients enrolled. Pneumococcal antigen was detected in 19 (70.4%) of 27 patients with pneumococcal pneumonia. The test results were more frequently positive for patients who had not received antibiotics before testing (26.6% vs. 12.1%; P=.002). Only 16 (10.3%) of 156 samples obtained from patients with nonpneumococcal pneumonia yielded a positive result. Of the 269 patients who had pneumonia with no pathogen identified, antigen was detected in 69 (25.7%). With conventional microbiological criteria used as the "gold standard," the test had a sensitivity of 70.4% and a specificity of 89.7%. Testing concentrated urine samples with the ICT may be a useful technique for rapid diagnosis of pneumococcal pneumonia in adults with CAP.

Our objective was to evaluate the usefulness of serotyping, phagotyping, antibiotyping and RAPD (random amplified polymorphic DNA) in the study of the epidemiology of salmonellosis in Elche (Spain). We examined 1232 clinical isolates of non‐typhoid Salmonella serotypes. Serotyping adequately differentiates 12.3% of the isolates and phagotyping raises this percentage to 37%. Antibiotyping is a suitable complement in certain circumstances, such as in the case of specific outbreaks. RAPD is a fast and simple technique which is within the reach of most laboratories, complementing the results obtained by antibiotyping and serotyping, in order to be able to control outbreaks of salmonellosis in everyday practice.