Etest Methodology Research Articles

MODIFIED PAP METHOD TO DETECT HETERORESISTANCE TO VANCOMYCIN AMONG METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS ISOLATES AT A TERTIARY CARE HOSPITAL

This study was an attempt at developing, establishing, validating and comparing the modified PAP method for detection of hetero-vancomycin resistant Staphylococcus aureus (h-VRSA) with the routine antimicrobial susceptibility testing (using the BSAC standardized disc diffusion method), minimum inhibitory concentrations of vancomycin using standard E-test methodology and the Hiramatsu’s screening method. A total of 50 methicillin resistant Staphylococcus aureus obtained from various clinical specimens, along with the Mu 3 and Mu 50 strains as controls, were studied. No VRSA isolates were obtained. However, four of the test strains were positive by the Hiramatsu’s screening method, of which only one isolate could be confirmed by the modified PAP analysis method. This isolate was a coloniser from the drain fluid of a liver transplant recipient. The sensitivity, specificity, positive predictive value and the overall efficiency of the Hiramatsu’s screening method with the modified PAP analysis as the gold standard were found to be 100, 93.8, 25 and 94%, respectively. It is very essential for clinical laboratories to screen for h-VRSA, given the increasing use of glycopeptide antibiotics in therapy and the potential for failed therapy in patients infected with these strains.

In vitro synergistic activity of antibiotic combinations against Brucella melitensis using E-test methodology

The treatment of brucellosis is still problematic, because of high rates of treatment failure or relapses. As the microorganism is an intracellular pathogen, treatment requires combined regimens. However, limited existing data on in vitro combinations are avaliable for Brucellae. The aim of this study was to investigate the in vitro efficacy of various traditional and new antibiotic combinations against 16 Brucella melitensis strains. The combination effect of antimicrobial agents was evaluated by E-test synergy method to obtain a fractional inhibitory concentration (FIC) index. Co-Trimoxazole (SXT) and moxifloxocin (MXF) exhibited the lowest MIC, while Rifampin (RIF) had the highest MIC in the study. Combinations with RIF showed the best synergistic activity (100% of RIF-tetracycline (TET), and 87.5% of RIF-SXT). Synergistic activity was also detected at seven (43.7%) of ciprofloxocin (CIP)-SXT, four (25%) of TET-MXF, and two (12.5%) of TET-SXT combinations. The combinations that demonstrated additivity were TET-SXT, CIP-SXT and TET-MXF. Antagonism was observed only with the TET-Streptomycin (STR) combination in three strains (18.8%). Further work including randomized controlled clinical trials is required to fully evaluate the usefulness of these data.

Antibiotic synergy profiles of multidrug-resistantPseudomonas aeruginosain a nosocomial environment

Pseudomonas aeruginosa is one of the most common nosocomial pathogens in intensive care units (ICUs), with an increased prevalence of multidrug resistance in nosocomial infections. This study aimed to determine antibiotic synergy profles of multidrug-resistant P. aeruginosa originating from ICU patients in a teaching hospital in Pretoria, South Africa. Susceptibility studies and in vitro synergy testing were performed, utilising agar dilution and Etest methodology, respectively. Susceptibility studies revealed 94% of isolates exhibiting resistance to more than three anti-pseudomonal agents tested. Results from antibiotic synergy studies suggested that cefepime (p‹0.0001) or meropenem (p‹0.0001) in combination with amikacin are possible treatment options available in this specifc setting.

Modified PAP method to detect heteroresistance to vancomycin among methicillin resistant <i> Staphylococcus aureus</i> isolates at a tertiary care hospital

This study was an attempt at developing, establishing, validating and comparing the modified PAP method for detection of hetero-vancomycin resistant Staphylococcus aureus (h-VRSA) with the routine antimicrobial susceptibility testing (using the BSAC standardized disc diffusion method), minimum inhibitory concentrations of vancomycin using standard E-test methodology and the Hiramatsu's screening method. A total of 50 methicillin resistant Staphylococcus aureus obtained from various clinical specimens, along with the Mu 3 and Mu 50 strains as controls, were studied. No VRSA isolates were obtained. However, four of the test strains were positive by the Hiramatsu's screening method, of which only one isolate could be confirmed by the modified PAP analysis method. This isolate was a coloniser from the drain fluid of a liver transplant recipient. The sensitivity, specificity, positive predictive value and the overall efficiency of the Hiramatsu's screening method with the modified PAP analysis as the gold standard were found to be 100, 93.8, 25 and 94%, respectively. It is very essential for clinical laboratories to screen for h-VRSA, given the increasing use of glycopeptide antibiotics in therapy and the potential for failed therapy in patients infected with these strains.

In vitro activities of ‘new’ and ‘conventional’ antibiotics against multidrug resistant Gram negative bacteria from patients in the intensive care unit

To identify and collect clinical isolates of multi-drug resistant Gram negative bacteria and to perform antimicrobial susceptibility testing using an extended panel of antibiotics including tigecycline, colistin, aztreonam, piperacillin/tazobactam and ampicillin/sulbactam. Minimum inhibitory concentrations (MICs) using the Epsilometer test (E-test) methodology were determined for 28 distinct multi-drug resistant Gram negative isolates from patients in the intensive care unit (ICU). Tigecycline had good in vitro activity against Acinetobacter species and Enterobacter cloacae, and colistin had potent in vitro activity against Acinetobacter, E. cloacae and Pseudomonas aeruginosa. Enterobacter cloacae and Serratia marcescens but not P. aeruginosa or Acinetobacter species were susceptible to piperacillin/tazobactam. Ampicillin/sulbactam had poor in vitro activity for most isolates tested. The activity of tigecycline and colistin did not appear to be affected by the presence of extended spectrum beta-lactamases (ESBLs) and metallo-beta-lactamases (MBLs) and aztreonam maintained its in vitro activity against the Enterobacteriaceae tested despite the presence of MBLs. Tigecycline and colistin have potent in vitro activity and might have useful therapeutic activity in patients with infections due to multi-drug resistant Acinetobacter species and E. cloacae, including those harbouring ESBLs and MBLs. In addition, colistin demonstrated potent in vitro activity against multi-drug resistant P. aeruginosa.

Synergy testing of multidrug resistant Acinetobacter baumanii against tigecycline and polymyxin using an E-test methodology

The emergence of multi drug resistant nosocomial Acinetobacter baumanii infection has prompted the evaluation of newer antimicrobial agents and older warehoused agents as possible therapeutic candidates. In addition to the carbapenems, two other agents have emerged in clinical use and the in-vitro literature as potential therapeutic agents. In a number of studies tigecycline, a new agent in the glycylcycline class, related to minocycline, and polymyxin B, a polypeptide antibiotic used parenterally in the past as a gram negative rod antimicrobial, have show promising activity against multidrug resistant A. baumanii isolates, including those isolates resistant to the carbapenems [1–3]. Tigecycline appears to be bacteriostatic in vitro and in one published clinical study there has been breakthrough bacteremia. Very little in vitro data exists on the activity of the combination of polymyxin plus tigecycline against multidrug resistant A. baumanii [4, 5]. The E-test methodology has proven reliable for in vitro antimicrobial sensitivity testing [6]. A modified E-test methodology for performing antimicrobial synergy tested has been preliminarily evaluated in the literature and although not extensively validated, has been found to correlate well with traditional checkerboard and time-kill methodologies in several studies [7, 8]. E-test synergy testing is performed by crossing the E-test strips for the 2 test antimicrobials at a 90° angle at their predetermined MIC value on an inoculated Mueller Hinton agar plate. The plates are then incubated for 24 h at 35°C. The MIC of each antimicrobial in combination is interpreted as the value at which the zone of inhibition intersects the E test strip. The fractional inhibitory concentration index (FICI) is calculated and interpreted for synergy, indifference or antagonism. The FICI=MICtigecycline in combination/MICtigecyclinealone+ MICpolymixin in combination/MICpolymixin alone. The FICI is interpreted as follows: 4.0 indicates antagonism. We tested 19 recent clinical isolates of multidrug resistant A. baumanii from a large university teaching hospital for susceptibility to tigecycline and polymyxin B and performed synergy testing on the combination using E-test methodology. Clinical sources of the isolates were as follows: respiratory 9/19 (47.4%), urine 6/19 (31.6%), blood 2/19 (10.5%), wound 2/19 (10.5%). E-test synergy testing, revealed FICI values for tigecycline plus polymyxin between 0.88 and 2.0 for all 19 isolates, with 11/19 isolates having an FICI of 2.0. The FICI distribution was as follows:

Optimising antibiotic dosing regimens based on pharmacodynamic target attainment against Pseudomonas aeruginosa collected in Hungarian hospitals

Owing to increasing resistance rates in Europe, pharmacodynamic analyses were proposed to determine optimal empirical antibiotic therapy against Pseudomonas aeruginosa isolated in Hungary. Minimum inhibitory concentrations for 180 non-duplicate P. aeruginosa collected from 14 hospitals in Hungary were determined by Etest methodology. A 5000-subject Monte Carlo simulation was performed to calculate the bactericidal cumulative fraction of response (CFR) for standard dosing regimens of cefepime, ceftazidime, ciprofloxacin, imipenem, meropenem and piperacillin/tazobactam. In the case of poor CFR, alternative dosage regimens were simulated for selected agents by increasing the infusion time, dose and frequency. Owing to high resistance rates in Hungary, no regimen achieved >90% CFR. CFRs for standard dosing regimens were: meropenem 1 g every 8 h (q8h), 77.1%; ceftazidime 2 g q8h, 75.3%; imipenem 0.5 g every 6 h (q6h), 71.7%; and piperacillin/tazobactam 4.5 g and 3.375 g q6h, 72.4% and 71.0%, respectively. Ciprofloxacin achieved significantly lower bactericidal CFRs than any β-lactam. Prolonged infusion regimens improved the CFR for cefepime, imipenem, meropenem and piperacillin/tazobactam. Overall, the highest CFR (88.1%) was achieved by a 3-h infusion of meropenem 2 g q8h. Given the poor CFR predicted with standard dosage regimens against these isolates, it seems prudent to consider alternative dosage strategies such as increasing doses, frequencies or infusion times as well as combination therapy when empirically treating infections caused by P. aeruginosa in Hungary.

Rhizomucor: a model for the emerging importance of mould susceptibility testing

Opportunistic invasive fungal infections in patients with hematologic malignancies often complicate empiric antifungal treatment. Zygomycetes typically are resistant to antifungal agents routinely used for empiric therapy. Susceptibility testing of these moulds currently employs CLSI (formerly NCCLS) standards for microbroth dilution. We present a case of invasive Rhizomucor sp. infection in which the Etest methodology resulted in clinically compatible susceptibility data. Case Report A 53-year-old male was admitted to our teaching hospital for treatment of relapsed acute myelogenous leukemia.

Activity of Retapamulin against Streptococcus pyogenes and Staphylococcus aureus Evaluated by Agar Dilution, Microdilution, E-Test, and Disk Diffusion Methodologies

The in vitro activity of retapamulin against 106 Staphylococcus aureus isolates and 109 Streptococcus pyogenes isolates was evaluated by the agar dilution, broth microdilution, E-test, and disk diffusion methodologies. Where possible, the tests were performed by using the CLSI methodology. The results of agar dilution, broth microdilution, and E-test (all with incubation in ambient air) for S. aureus yielded similar MICs, in the range of 0.03 to 0.25 microg/ml. These values corresponded to zone diameters between 25 and 33 mm by the use of a 2-microg retapamulin disk. Overall, 99% of the agar dilution results and 95% of E-test results for S. aureus were within +/-1 dilution of the microdilution results. For S. pyogenes, the MICs obtained by the agar and broth microdilution methods (both after incubation in ambient air) were in the range of 0.008 to 0.03 microg/ml, and E-test MICs (with incubation in ambient air) were 0.016 to 0.06 microg/ml. For S. pyogenes, 100% of the agar dilution MIC results were within +/-1 dilution of the broth microdilution results. E-test MICs (after incubation in ambient air) were within +/-1 and +/-2 dilutions of the broth microdilution results for 76% and 99% of the isolates, respectively. E-test MICs for S. pyogenes strains in CO(2) were up to 4 dilutions higher than those in ambient air. Therefore, it is recommended that when retapamulin MICs are determined by E-test, incubation be done in ambient air and not in CO(2), due to the adverse effect of CO(2) on the activity of this compound. Diffusion zones (with incubation in CO(2)) for S. pyogenes were 18 to 24 mm. Retapamulin MICs for all strains by all methods (with incubation in ambient air) were < or =0.25 microg/ml. These results demonstrate that S. pyogenes (including macrolide-resistant strains) and S. aureus (including methicillin-resistant and vancomycin-nonsusceptible strains) are inhibited by very low concentrations of retapamulin and that all four testing methods are satisfactory for use for susceptibility testing.

Genotypic and Phenotypic Analysis ofClostridium difficileCorrelated with Previous Antibiotic Exposure

To analyze Clostridium difficile susceptibility results and genotypes in relation to antibiotic exposures that precipitated C. difficile-associated diarrhea (CDAD), we examined 83 nosocomial C. difficile isolates recovered at a tertiary care center in Boston, Massachusetts. MICs were determined by E-test methodology using modified Brucella agar. Isolates were genotyped by pulsed-field gel electrophoresis and restriction enzyme analysis. Antibiotic susceptibilities were: ciprofloxacin (0%), clindamycin (59%), trovafloxacin (63%), ceftriaxone (73%), piperacillin/tazobactam (100%), metronidazole (100%), and vancomycin (100%). The two most common strain groups, isolated from a total of 33 patients, were much more likely to be resistant to clindamycin, erythromycin, and trovafloxacin than other strain groups [79% (26 of 33) versus 2% (1 of 50), respectively]. Clindamycin exposure was strongly associated with CDAD caused by isolates that exhibited multiple resistance to clindamycin, erythromycin, and trovafloxacin (prevalence odds ratio, 4.2; 95% confidence interval, 1.1-16.8), whereas other antimicrobials did not yield significant associations. Resistance of specific C. difficile strains to clindamycin and other antimicrobial agents may contribute to their hospital dissemination and explain, in part, the propensity of clindamycin to trigger nosocomial outbreaks.

Antibiotic susceptibility patterns of anaerobic bacteria isolated in Pretoria, South Africa, during 2003–2004

Antibiotic resistance amongst previously susceptible an aerobic bacteria has in creased during the last decade, raising concerns about the choice of empirical therapy, in this study, Etest methodology and Clinical Laboratory Standards Institute guidelines were used for processing and interpretation of minimum inhibitory concentration values for anaerobic bacteria obtained over a one-year period from two teaching hospitals in Pretoria, South Africa. From the data obtained, the genera Bacteroides, Clostridium and Peptostreptococus were most frequently isolated. The β-lactam/β-lactamase inhibitor combination, piperacillin/tazohactam seems to be the superior choice as empiric therapy for suspected serious anaerobic infections in this setting. In addition, a definite increase in resistance against metronidazole was exhibited by isolates originating from invasive samples, making the drug an unlikely choice for future empiric therapy.

Antimicrobial susceptibility of Gram-negative bacteria in Brazilian hospitals: the MYSTIC Program Brazil 2003

Establish the susceptibility pattern of Gram-negative bacteria causing infections in ICU patients, MYSTIC Program Brazil 2003. Gram-negative bacteria (n = 1,550) causing nosocomial infections were collected at 20 Brazilian centers. The central laboratory confirmed the identification and performed the susceptibility tests by Etest methodology (AB Biodisk, Solna, Sweden) for meropenem, imipenem, ciprofloxacin, ceftazidime, cefepime, cefotaxime, piperacillin/tazobactam, gentamicin, and tobramycin. Interpretation criteria used were according to National Committee for Clinical Laboratory Standards (NCCLS). Pseudomonas aeruginosa (30.3%) was the most frequent isolate, followed by E. coli (18.6%), Klebsiella pneumoniae (16.9%), Acitenobacter baumannii (8.8%), and Enterobacter cloacae (7.1%). Pseudomonas aeruginosa (n=470) isolates presented susceptibility rates of 64% to meropenem, 63.8% to piperacillin/tazobactam, 63.4% to amikacin, 58.7% to imipenem. Acitenobacter baumannii presented susceptibility rates to meropenem of 97.1%, and 73% to tobramycin. E. coli and K. pneumoniae were highly susceptible to both carbapenems. Carbapenem resistance among the Enterobacteriaceae is still rare in the region. Acitenobacter baumannii and P. aeruginosa presented elevated resistance rates to all antimicrobials. Since they play an important role in nosocomial infections in this environment, the use of empirical combination therapy to treat these pathogens may be justified.

Erythromycin-Resistant Group A Streptococcal Isolates Collected between 2000 and 2005 in Oahu, Hawaii, and Their emm Types

We examined erythromycin and clindamycin susceptibilities with Etest methodology among 546 group A streptococcal isolates collected in Hawaii between February 2000 and November 2004. Erythromycin resistance was low (3.1%). No isolate was clindamycin resistant. The prevalence of erythromycin resistance in group A streptococci remains low in Hawaii.

Antimicrobial susceptibility in intensive care units: MYSTIC Program Brazil 2002

Establish the susceptibility pattern of Gram-negative bacteria causing infections in ICU patients, MYSTIC Program Brazil 2002. Gram-negative bacteria (n = 503) causing nosocomial infections were collected at seven Brazilian centers. The central laboratory confirmed the identification and performed the susceptibility tests by E-test methodology (AB Biodisk, Solna, Sweden) for meropenem, imipenem, ciprofloxacin, ceftazidime, cefepime, cefotaxime, piperacillin/tazobactam, gentamicin, and tobramycin. Interpretation criteria used were according to National Committee for Clinical Laboratory Standards (NCCLS). Pseudomonas aeruginosa (33%) was the most frequently isolated, followed by A. baumannii (17.1%), K. pneumoniae (12.1%), E. coli (10.5%), and E. cloacae (7.9%). Pseudomonas aeruginosa isolates had susceptibility rates of 67.5% to piperacillin/tazobactam, 59.8% to meropenem, 57.3% to imipenem. A. baumannii presented susceptibility rates to meropenem of 89.5%, 88.4% to imipenem, and 74.4% to tobramycin. E. coli and K. pneumoniae were fully susceptible to both carbapenems. Carbapenem resistance among Enterobacteriaceae is still rare in this region. A. baumannii and P. aeruginosa presented elevated resistance rates to all antimicrobials. Since these two bacterial species play an important role in nosocomial infections, the use of empirical combination therapy to treat these pathogens may be justified.

Mutations in the cyp51A gene and susceptibility to itraconazole in Aspergillus fumigatus serially isolated from a patient with lung aspergilloma

To monitor changes in itraconazole susceptibility of isolates from a patient undergoing treatment for pulmonary Aspergillus infection and relate these changes to genotypic/phenotypic alterations. Six Aspergillus fumigatus isolates were serially recovered from the patient. Itraconazole MICs were determined by Etest and NCCLS methodology. Growth characteristics and phenotype were monitored. Molecular analysis included random amplified polymorphic DNA (RAPD) assay and sequencing of the cyp51A gene. The MIC of itraconazole against the first isolate before treatment was 0.25 mg/L; the MIC against the second isolate, recovered after 6 months of itraconazole therapy, was >16 mg/L; and that against the third isolate, obtained 2 months after discontinuation of the therapy, was 0.5 mg/L. The MIC against the last three isolates, acquired after restoration of itraconazole therapy for 4-7 months, was >16 mg/L. The six isolates shared identical band patterns of RAPD assay using four primers and the same sequence in intertranscribed spacers (ITS). Therefore, the six isolates were likely to be the same strain of A. fumigatus, and mutations involving itraconazole resistance possibly occurred in these isolates after prolonged itraconazole therapy. Sequencing of the cyp51A gene in the coding region revealed a mutation of M220I in cytochrome P450 sterol 14-alpha-demethylase in the second resistant isolate and a mutation of G54R in the last three resistant isolates. Expression changes of some pump genes, such as MDR3, may also, in part, be related to the resistance to itraconazole. We conclude that resistance of A. fumigatus to itraconazole occurred in a patient treated with the drug, and the resistance may result from mutations in the cyp51A gene-the gene encoding the target enzyme for itraconazole.

Antimicrobial susceptibility of invasive isolates of Streptococcus pneumoniae in Ireland

Between January 1999 and June 2002, 646 invasive isolates of Streptococcus pneumoniae were collected in Ireland. MICs of penicillin, ciprofloxacin, cefotaxime, moxifloxacin and linezolid were determined by Etest methodology. Eighty-seven (13.5%) isolates showed intermediate resistance to penicillin, while seven (1.1%) showed high-level resistance. Eighty-seven (13.5%) isolates were resistant to erythromycin, but all isolates were susceptible to cefotaxime, moxifloxacin and linezolid. The prevalence of pneumococcal isolates non-susceptible to penicillin in Ireland is worryingly high, but currently there are alternative agents available to treat invasive infection.

Atividade antimicrobiana in vitro de quinupristina/dalfopristina para cocos gram-positivos isolados de cinco centros brasileiros: resultado do estudo de vigilância L-SMART

Há alguns anos tem-se verificado um aumento progressivo da resistência de alguns cocos gram-positivos a determinados antimicrobianos. Este aumento da resistência tem sido observado principalmente no ambiente hospitalar, e as bactérias mais comumente envolvidas são os Staphylococcus spp. e os Enterococcus spp. Devido a este fato, novos antimicrobianos são avaliados para o tratamento de infecções causadas por estas cepas multirresistentes. A associação quinupristina/dalfopristina (Q/D), também conhecida como Synercid®, é um antibacteriano da classe das estreptograminas, de uso endovenoso, composto por dois derivados semi-sintéticos da pristinamicina. A combinação das estreptograminas B e A na razão de 30:70 tem atividade antimicrobiana voltada para cocos gram--positivos, como Staphylococcus spp., Streptococcus spp., incluindo S. pneumoniae e Enterococcus faecium, sendo o E. faecalis habitualmente resistente. Neste estudo foi avaliada a atividade in vitro de Q/D e outros oito antimicrobianos frente a 631 amostras de cocos gram-positivos isoladas de cinco centros brasileiros, complementadas com outras 20 cepas de E. faecium resistentes à vancomicina, provenientes dos Estados Unidos. Para a avaliação da sensibilidade aos antimicrobianos foi determinada a concentração inibitória mínima (MIC) pelo método do Etest (AB Biodisk, Solna, Suécia) e as cepas testadas foram: Staphylococcus aureus (n = 267), Staphylococcus coagulase negativo (n = 131), Streptococcus pneumoniae (n = 130), Streptococcus beta-hemolíticos (n = 28), Enterococcus faecalis (n = 44) e E. faecium (n = 51). A Q/D demonstrou excelente atividade contra Staphylococcus spp., independente de serem sensíveis ou resistentes à oxacilina. Para S. pneumoniae, a Q/D apresentou igualmente uma ótima atividade, inclusive para as cepas com resistência intermediária ou total para penicilina. Entre as cepas de E. faecium sensíveis à vancomicina, o MIC90 de Q/D obtido foi de 3µg/ml, sendo que 45% das cepas testadas foram sensíveis e 55% apresentaram sensibilidade intermediária à associação. Desta forma, pode-se afirmar que a associação Q/D representa uma nova opção para o tratamento endovenoso de infecções causadas por cocos gram-positivos, principalmente para as cepas multirresistentes, sendo também uma alternativa ao uso de glicopeptídeos.

Comparison of NCCLS microdilution method and Etest in antifungal susceptibility testing of clinical Trichosporon asahii isolates

We investigated the in vitro activity of amphotericin B, fluconazole, and itraconazole against clinical Trichosporon asahii isolates ( n = 43) by NCCLS M27A reference microdilution method and explored the correlation between Etest and NCCLS reference method. Microdilution MIC ranges following 48 h of incubation were 1–8, 0.25–16, and 0.06–4 μg/ml for amphotericin B, fluconazole, and itraconazole, respectively. The corresponding Etest MIC ranges were determined as 0.125- > 8, 0.25- > 64, and 0.03–8 μg/ml. Of interest, Etest tended to produce lower amphotericin B MICs and widen the MIC range compared to microdilution. The influence of Etest on fluconazole and itraconazole MICs was in contrary with that observed for amphotericin B. Etest MICs of fluconazole and itraconazole tended to be higher than microdilution MICs. The wider range of amphotericin B MICs obtained by using Etest methodology may facilitate discrimination of isolates with reduced susceptibility to amphotericin B. However, clinical significance of these findings remain yet unknown and determination of MIC breakpoint values is required.

Evolución de la sensibilidad de 235 cepas de Helicobacter pylori entre 1995 y 1998 e impacto del tratamiento antibiótico

The aim of this study was to investigate the sensitivity of Helicobacter pylori to the antibiotics used in its eradication over a period of four years and to determine the influence of previous treatment on sensitivity. During the period from 1995 to 1998 we determined the sensitivity of 235 consecutive Helicobacter pylori isolates to amoxicillin, metronidazole, clarythromycin and tetracycline by means of E-test methodology. The MIC values found were related with the prior use of eradicating treatment. The percentage of resistant strains were as follows: 23.5% to metronidazole, 12.9% to clarythromycin and 0.7% to tetracycline; none of the strains was resistant to amoxicillin. There were no significant changes in percentage of resistance to the drugs studied over the 4-year period. Resistance to metronidazole and clarythromycin was significantly higher (p 5 0.03 and p < 0.001 respectively) in strains isolated from patients who had received previous treatment. Monitorization of H. pylori sensitivity to the drugs used in its eradication is particularly important in patients who have undergone prior treatment.

In vitro activity of linezolid (U-100766) against Haemophilus influenzae measured by three different susceptibility testing methods

Linezolid has excellent antibacterial activity against a wide range of Gram-positive organisms. Early in vitro investigations suggested that the compound also had activity against some Gram-negative species, including those commonly associated with community-acquired respiratory tract infections ( Haemophilus influenzae and Moraxella catarrhalis). Against 603 recent clinical isolates of H. influenzae from geographically diverse regions of the world, tested by the reference broth microdilution method (HTM), linezolid MIC values ranged from 2–64 μg/ml (MIC 50 and MIC 90 at 16 μg/ml and 32 μg/ml, respectively). A subset of 328 strains was also tested to compare broth microdilution and Etest (AB BIODISK, Solna, Sweden) methodologies. The Etest method produced slightly higher MIC results attributable to a growth-enhancing effect of the 5% CO 2 incubation used in the test procedure and pH changes. Linezolid activity versus H. influenzae was limited as evidenced by reference test results (susceptible breakpoints at ≤2 or ≤4 μg/ml) and variable endpoints were obtained when alternative methods were used such as the Etest or standardized disk diffusion procedures. Clinical laboratories should limit the testing of linezolid against Gram-negative species ( H. influenzae).