Determination of the In Vitro Susceptibility of 220 Mycobacterium fortuitum Isolates to Ten Antimicrobial Agents

Abstract

The authors investigated the In Vitro susceptibility to antimicrobial agents of 220 Mycobacterium fortuitum isolates originating from clinical samples (14) of patients attending the Hospital Universitario de Canarias and Hospital del Tórax, and from environmental sources (206): 3 from sea water, 10 from the water supply and 193 from sewage. The Minimum Inhibitory Concentration (MIC) was calculated using the broth microdilution method with Mueller-Hinton Broth without supplement. Amikacin was the most efficacious antimicrobial agent against all the isolates of M. fortuitum with an MIC which was considerably lower than its critical concentration. The good results achieved with amikacin In Vitro are confirmed by those obtained in vivo, with patients infected with M. fortuitum. No significant difference was found in the efficacy of amikacin and ofloxacin against all the isolates assayed.     

In Vitro Testing of Aspergillus fumigatus Clinical Isolates for Susceptibility to Voriconazole,Amphotericin B and Itraconazole: Comparison of Sensititre versus NCCLS M38-A Using Two Different Inocula

Abstract

Voriconazole, amphotericin B and itraconazole were tested In Vitro against 18 strains of Aspergillus fumigatus isolated from cystic fibrosis patients. Susceptibility was tested with the broth microdilution method (M38-A protocol- NCCLS). Results of this reference method were compared with those of an experimental commercial microdilution broth method (Sensititre). Two different inocula, prepared from 2- and 7-day cultures, were used. Minimum inhibitory concentrations (MICs) of the reference method ranged from 0.25 to 2 μg/ml for voriconazole, 0.06 to 1 μg/ml for amphotericin B, 0.016 to >16 μg/ml for itraconazole. There were no significant differences in the MIC ranges or MIC₉₀ values obtained with the two testing methods or with the two types of inocula. These findings confirm the good In Vitro activity of voriconazole, itraconazole and amphotericin B against A. fumigatus. They also indicate that reliable susceptibility data can be generated more rapidly by commercial systems and use of 2-day cultures for inoculum preparation.     

Suppression of adjuvant arthritis in rats by intraperitoneal Mycobacterium butyricum

Abstract

The In Vitro activity of fluoroquinolones, including lomefloxacin, ofloxacin, ciprofloxacin, sparfloxacin, moxifloxacin and gatifloxacin, was evaluated against 55 clinical isolates of Mycobacterium tuberculosis by absolute concentration method on Lowenstein-Jensen (L-J) and Middlebrook's 7H11 media. Both ofloxacin susceptible and ofloxacin resistant strains of M. tuberculosis isolates were tested. The in vitro activities of these fluoroquinolones on the M. tuberculosis isolates were in the order: lomefloxacin相似文献   

In Vitro Susceptibility of Trichosporon beigelii to Antifungal Agents

Abstract

The minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of amphotericin B, flucytosine, miconazole, fluconazole and itraconazole against 21 isolates of Trichosporon beigelii in RPMI-1640 medium were determined using National Committee for Clinical Laboratory Standards (NCCLS) methodology in microdilution method. Most isolates were sensitive to miconazole (MIC₉₀ 0.78 μ/ml), fluconazole (MIC₉₀ 6.25 μ/ml), and itraconazole (MIC₉₀ 0.19 μ/ml), with the former being the most active agent tested (MFC₉₀ 3.12 μ/ml). Although amphotericin B inhibited most strains (MIC range, 0.78-3.12 μ/ml), poor fungicidal activity was observed (MFC range, 1.56 -12.5 μ/ml) showing a pattern of relative resistance In Vitro. Flucytosine showed generally poor activity against most isolates tested. These In Vitro findings confirm the resistance of T.beigelii to amphotericin B and suggest that azoles may be an alternative to the former for the treatment of disseminated trichosporonosis. However, in vivo studies would better validate these In Vitro findings.     

Antifungal susceptibility testing of Malassezia yeast: comparison of two different methodologies

All Malassezia species are lipophilic; thus, modifications are required in susceptibility testing methods to ensure their growth. Antifungal susceptibility of Malassezia species using agar and broth dilution methods has been studied. Currently, few tests using disc diffusion methods are being performed. The aim was to evaluate the in vitro susceptibility of Malassezia yeast against antifungal agents using broth microdilution and disc diffusion methods, then to compare both methodologies. Fifty Malassezia isolates were studied. Microdilution method was performed as described in reference document and agar diffusion test was performed using antifungal tablets and discs. To support growth, culture media were supplemented. To correlate methods, linear regression analysis and categorical agreement was determined. The strongest linear association was observed for fluconazole and miconazole. The highest agreement between both methods was observed for itraconazole and voriconazole and the lowest for amphotericin B and fluconazole. Although modifications made to disc diffusion method allowed to obtain susceptibility data for Malassezia yeast, variables cannot be associated through a linear correlation model, indicating that inhibition zone values cannot predict MIC value. According to the results, disc diffusion assay may not represent an alternative to determine antifungal susceptibility of Malassezia yeast.     

Susceptibility of Ureaplasma urealyticum to Tetracycline,Doxycycline, Erythromycin,Roxithromycin, Clarithromycin,Azithromycin, Levofloxacin and Moxifloxacin

Abstract

The in vitro activity of tetracycline, doxycycline, erythromycin, roxithromycin, clarithromycin, azithromycin, levofloxacin and moxifloxacin was tested against 63 clinical isolates of Ureaplasma urealyticum. The minimal inhibitory concentrations (MICs) and the minimal bactericidal concentrations (MBCs) were determined by the broth microdilution method in A7 medium. The miC50 and miC90 of the tested agents after 24 h of incubation were as follows: Tetracycline, 0.5 and 2.0 μg/ml; doxycycline, 0.125 and 0.25 μg/ml; erythromycin, 2.0 and 8.0 μg/ml; roxithromycin, 2.0 and 4.0 μg/ml; clarithromycin, 0.25 and 1.0 μg/ml; azithromycin, 2.0 and 4.0 μg/ml; levofloxacin, 1.0 and 2.0 μg/ml; and moxifloxacin, 0.5 and 0.5 μg/ml, respectively. The MIC values after 24 h and 48 h incubation differed by no more than one dilution for all the agents with the exception of doxycycline (two dilution difference for MIC₉₀). Overall, moxifloxacin was the most active agent in vitro against U. Urealyticum, with the narrowest difference between MIC and MBC values, followed closely by levofloxacin. Clarithromycin was the most active macrolide.     

Comparative Evaluation of the In Vitro Antimycobacterial Activities of Six Aminoglycoside Antibiotics Using an Agar Dilution Method

Abstract

Given the increasing prevalence of mycobacterial resistance to aminoglycoside antibiotics, we examined the susceptibility of 76 clinical isolates of mycobacteria to arbekacin, amikacin, gentamicin, kanamycin, tobramycin and streptomycin using an agar dilution method. Only arbekacin and amikacin showed excellent therapeutic potential (minimum inhibitory concentrationis (MICs) ≤0.25-4 μg/ml) against 30 isolates of rapidly growing mycobacteria, including Mycobacterium fortuitum M. chelonae and a related organismNocardia asteroides. The MIC₉₀ of tobramycin against 23 isolates of M. avium complex was 8 μg/ml, while that of the other 5 aminoglycosides ranged from 64-256 μg/ml. Of the 23 M. tuberculosis isolates tested, 5 showed aminoglycoside resistance (MICs 128 to ≥1,024 μg/ml), while the others were variably susceptible (MICs ≤0.25-32 μg/ml) to all 6 aminoglycosides. The chemotherapeutic potential of arbekacin, amikacin and streptomycin as treatment of tuberculosis was apparent; however, proper patient management would be required to control against the emergence of the drug-resistant strains during prolonged treatment.     

In- Vitro Susceptibility of Clinical Isolates of Pseudomonas aeruginosa to β-Lactam and Aminoglycoside Antibiotics

Summary

The in-vitro susceptibilities of 198 isolates of Pseudomonas aeruginosa from clinical human specimens were determined by an agar dilution technique against (β-lactams and aminoglycosides. These isolates were susceptible to imipenem, aztreonam and ceftazidime with the minimum inhibitory concentration (MIC) for 90% of the strains tested being 8, 16 and 8 μg/ml, respectively. Aminoglycosides, except amikacin, had low activity (MIC90 > 128 μg/ml).     

«Nonantibiotics» - Their Relevance in Research and Potential Place in Future Antimicrobial Chemotherapy

Summary

The susceptibilities of gentamicin- and carbenicillin-resis-tant clinical isolates of Pseudomonas aeruginosa to newer β-lactams, netilmicin and ciprofloxacin were studied by a broth microdilution technique. Imipenem, aztreonam and ceftazi-dime were active against most of the P. aeruginosa strains with minimum inhibitory concentration (MIC) for 90% of the isolates at clinically achievable levels. Piperacillin, azlocillin, cefotaxime, ceftriaxone, cefsulodin, cefoperazone and netilmicin showed poor activity against these organisms, ciprofloxacin exhibited poor activity, inhibiting only 30% of these strains.     

Comparison of Broth Dilution and Semisolid Agar Dilution for In Vitro Susceptibility Testing of Cryptococcus neoformans

Summary

We compared the in vitro activity of amphotericin B, flucytosine, itraconazole, fluconazole, ketoconazole and miconazole against 18 strains of Cryptococcus neoformans by using two methods: microbroth dilution and semisolid agar dilution.

By both of the methods minimum inhibitory concentrations (MICs) showed a wide range for all antifungal agents but not for amphotericin B. Statistically significant differences between the two methods were observed only with amphotericin B and flucytosine, p = 0.048 and p = 0.045 respectively.

Our study suggests that azole susceptibility testing for C. neoformans may be performed by the broth microdilution as well as the semisolid agar test. The choice of the method when testing amphotericin B and flucytosine is more problematic.     

The Minimum Inhibitory Concentration of Seventeen Antimicrobials for Salmonella Isolates from Septic Patients

Abstract

Herein we are reporting, for the first time in Kuwait, the minimum inhibitory concentrations (MICs) of Salmonella blood culture isolates vs. 17 clinically relevant antimicrobial agents. The screening of blood culture specimens was performed with the most advanced Bactec 9240 (Becton Dickinson). From over 20,000 blood cultures, 112 Salmonella isolates were obtained from 67 patients. Their MICs were determined using the automated Vitek microdilution technique (Biomerieux Vitek Inc.). During the whole 1991-1995 study period, the MICs for cefotaxime, ceftazidime, aztreonam, amikacin, gentamicin, ciprofloxacin and imipenem were below their respective susceptibility breakpoints. Resistance to chloramphenicol, ampicillin and cotrimoxazole varied from year to year, from 18% to 50%, except in 1991 when it was nil (1991 was the first year after the Gulf War, with very few newcomers from the Indian subcontinent). All ampicillin-susceptible S. typhi isolates had extremely low MIC values (≤0.25 μg/ml).     

Influence of different susceptibility testing methods and media on determination of the relevant fluconazole minimum inhibitory concentrations for heavy trailing Candida isolates with low‐high phenotype

We investigated the incidence of trailing growth with fluconazole in 101 clinical Candida isolates (49 C. albicans and 52 C. tropicalis) and tried to establish the convenient susceptibility testing method and medium for fluconazole minimum inhibitory concentration (MIC) determination. MICs were determined by CLSI M27‐A2 broth microdilution (BMD) and Etest methods on RPMI‐1640 agar supplemented with 2% glucose (RPG) and on Mueller‐Hinton agar supplemented with 2% glucose and 0.5 μg ml^?1 methylene blue (GMB). BMD and Etest MICs were read at 24 and 48 h, and susceptibility categories were compared. All isolates were determined as susceptible with BMD, Etest‐RPG and Etest‐GMB at 24 h. While all isolates were interpreted as susceptible at 48 h on Etest‐RPG and Etest‐GMB, one C. albicans isolate was interpreted as susceptible‐dose dependent (S‐DD) and two C. tropicalis isolates were interpreted as resistant with BMD. On Etest‐RPG, trailing growth caused widespread microcolonies within the inhibition zone and resulted in confusion in MIC determination. On Etest‐GMB, because of the nearly absence of microcolonies within the zone of inhibition, MICs were evaluated more easily. We conclude that, for the determination of fluconazole MICs of trailing Candida isolates, the Etest method has an advantage over BMD and can be used along with this reference method. Moreover, GMB appears more beneficial than RPG for the fluconazole Etest.     

Very high resistance to amoxicillin in Streptococcus pneumoniae: an epidemiological fact or a technical issue?

To explore reproducibility of high amoxicillin minimum inhibitory concentrations (MIC(AMX) ), isolates received during 2002 and 2003 in the National Reference Laboratory of Streptococcus pneumoniae with an amoxicillin MIC of 16 microg/ml (43 strains) and 8 g/ml (12 strains) when singly determined on a routine basis in this center by agar dilution, were retested 10 times by agar dilution and microdilution following NCCLS guidelines, not only using double dilutions but also dilution steps of 2 microg/ml (i.e, 2, 4, 6, 8, 10, 12, 14 and 16 microg/ml). A significant (p<0.05) shift to a higher MIC(AMX )was obtained with microdilution vs. agar dilution. Routine MIC(AMX )of 16 microg/ml were confirmed in 0 strains by agar dilution and in 6 by microdilution, when retested. These 6 strains presented a modal MIC(AMX )value of 10 microg/ml (5 cases) and of 14 micro g/ml (1 case) when using 2 microg/ml microdilution steps. There is low reproducibility of the highest MIC(AMX )values.     

Promising New Drugs in the Treatment of Tuberculosis and Mycobacteriosis

Summary

The aims of this work were to investigate the possible effect of several antimicrobial agents alone and in combinations against 190 clinical isolates of Mycobacterium tuberculosis and 30 isolates of Mycobacterium avium, 30 Mycobacterium fortuitum and 30 Mycobacterium chelonei. The susceptibility was determined in Müller-Hinton agar and Middlebrook 7H 10 agar. For interpretation of the results the minimum inhibitory concentration (MIC) was determined and for the combinations of antimicrobials we used the Fractional Inhibitory Concentration Index (FIC). The possible clinical efficacy was determined by the Inhibitory Quotient. The synergistic effect of several combinations of these compounds was demonstrated.     

A Comparison Between Disk Diffusion and Microdilution for Susceptibility Testing of Mycobacterium fortuitum Complex

Abstract

We evaluated a disk diffusion method using Mueller-Hinton agar for susceptibility testing of Mycobacterium fortuitum complex organisms. Ninety-five strains were tested both by broth microdilution and disk diffusion. Global results showed good correlation for all antimicrobials except for clarithromycin and erythromycin. However, when the results were analyzed according to species, correlation was poor except for a few antimicrobials. The analysis of the resistant/susceptible results was good for all the antimicrobials tested except azithromycin and erythromycin. In conclusion, the disk diffusion technique could be useful as a screening technique for some antibiotics, but the results must be confirmed by using an accepted reference technique.     

In vitro activities of antimicrobial cationic peptides; melittin and nisin,alone or in combination with antibiotics against Gram-positive bacteria

Abstract

The in vitro activities of two antimicrobial cationic peptides, melittin and nisin alone and in combination with frequently used antibiotics (daptomycin, vancomycin, linezolid, ampicillin, and erythromycin), were assessed against clinical isolates of methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus and Enterococcus faecalis. Using the broth microdilution method, minimum inhibitory concentration (MIC) ranges of melittin and nisin against all strains were 2–8 μg/ml and 2–32 μg/ml respectively. In combination studies performed with the microdilution checkerboard method using a fractional inhibitory concentration index of ?0·5 as borderline, synergistic interactions occurred more frequently with nisin–ampicillin combination against MSSA and nisin–daptomycin combination against E. faecalis strains. The results of the time-killing curve analysis demonstrated that the concentration dependent rapid bactericidal activity of nisin, and that synergism or early synergism was detected in most strains when nisin or melittin was used in combination with antibiotics even at concentrations of 0·5×MIC.     

In Vitro Activity of Cefpirome (HR 810) against Enterococci and Staphylococci

Summary

The inhibitory activity of cefpirome (HR 810), a new cephalosporin derivative for parenteral use, was tested by agar dilution methods against Enterococcus faecatis (100 strains), Staphylococcus aureus (40 strains) and coagulase-negative staphylococcal species (60 strains) in comparison with other beta-lac ta m antibiotics.

For E. faecalls, the cefpirome minimum inhibitory concentration (MIC) range was 2-128 μg/ml, with an MIC,, of 8 μg/ml, and an MIC₉₀, of 64 μg/ml. The optimal bactericidal activity against strains with MICs of ≤ 8 μg/ml occurred at 2-4 times the MIC, and the reduction in the initial inoculum was 99.9-99.7% after 24 h incubation at these concentrations.

Mec gene-negative staphylococci (both S. aureus and coagulase- negative species) had cefpirome MICs of 0.25-2 μg/ml (MIC₅₀ 0.5 μg/ml, MIC₉₀ 1 μg/ml). Mec gene-positive strains had MICs of 0.5-128 μg/ml (MIC₅₀ 2 μg/ml, MIC₉₀ 32 μg/ml). Strains with borderline resistance to oxacillin which did not harbor the mec gene and which were susceptible to cefpirome maintained their susceptibility even when high-density inocula were used and after several passages in media containing the antibiotic.

These studies present some potential advantages of cefpirome over other cephalosporins in the inhibitory activity against Gram-positive cocci.     

Disk Diffusion Method for Fluconazole Susceptibility Testing of Candida albicans Strains

Abstract

This study evaluated the usefulness of the disk diffusion method by using different media for the susceptibility testing of fluconazole against Candida albicans strains. The susceptibility of 108 clinical isolates of C. albicans against fluconazole were determined by microdilution and disk diffusion methods by using RPMI 1640 agar and 25 μg disks. 93 of these isolates were also tested by disk diffusion technique on four different media (yeast nitrogen base agar, Sabouraud dextrose agar, Mueller Hinton agar and Mueller Hinton methylene blue agar). The results of the microdilution method were evaluated visually and optically. The disk diffusion results were determined after 24 and 48 hours of incubation. When the 24-hour zone diameters were compared to the minimal inhibitory concentrations determined visually and optically, the best results were obtained for RPMI 1640 agar and yeast nitrogen base agar. The correlation coefficients were r=?0.34, ?0.41 and r=?0.33, ?0.32 for the first and second media, respectively. The best values (r=?0.29, ?0.39) were obtained for Mueller Hinton methylene blue agar when the 48-hour zone diameters were considered. Agreement between the disk diffusion and microdilution methods was best for RPMI 1640, yeast nitrogen base and Mueller Hinton methylene blue agar after 24 hours of incubation (87-89%, 88-90%, 93-96%, respectively) and for Mueller Hinton methylene blue agar after 48 hours of incubation (89-96%). Disk diffusion method using RPMI 1640, yeast nitrogen base and Mueller Hinton methylene blue agar appears to be a useful, rapid and reliable screening technique for testing the susceptibility of C. albicans strains to fluconazole.     

Comparison of teicoplanin and vancomycin in vitro activity on clinical isolates of Staphylococcus aureus

Abstract

Ninety-one clinical isolates of Staphylococcus aureus have been tested with the Kirby Bauer and the Etest® method to determine the susceptibility to glycopeptides in the 2007–2010 period. Five strains (5·5%) were resistant to vancomycin and nine (9·9%) to teicoplanin. Teicoplanin showed a median minimal inhibitory concentration (MIC) of 1 mg/l (range 0·125–24 mg/l), an MIC50 of 1 mg/l, and an MIC90 of 2 mg/l; vancomycin had a median MIC of 1·5 mg/l (range 0·38–4 mg/l), an MIC50 of 1·5 mg/l, and an MIC90 of 2 mg/l. More isolates were distributed on higher values of MIC for vancomycin. Inhibition halos induced by vancomycin-impregnated paper diskettes were slightly larger than those by teicoplanin. Glycopeptide resistance among methicillin-resistant Staphylococcus aureus in Italy is an underestimated phenomenon, possibly due to the described underestimation of glycopeptides MICs by the automatic broth microdilution method, when compared to agar MIC assays. A teicoplanin MIC creep, as reported for vancomycin, cannot be assumed.     

Daptomycin In Vitro Activity Tested Against Gram-Positive Strains Collected from European and Latin American Medical Centers in 2003

Abstract

Daptomycin, a cyclic lipopeptide, was susceptibility tested against clinical bacterial isolates consecutively collected in hospitals located in Europe (4,731 strains) and Latin America (1,007 strains) in 2003 as part of a continuing surveillance program. The bacterial isolates tested were Gram-positive pathogens that included staphylococci, streptococci and enterococci. The isolates were tested for susceptibility using broth microdilution methods (broth with 50 mg/L Ca⁺⁺ for testing daptomycin). All isolates, except two Enterococcus faecium strains from Europe, were inhibited at daptomycin MIC of ≤4 mg/L. In addition, 99.4 and 97.3% of isolates were inhibited at daptomycin MIC of ≤2 and ≤1 mg/L, respectively. Except for one Staphylococcus aureus and one viridans group streptococci from Europe and one coagulase-negative staphylococci from Latin America, all staphylococcal and streptococcal isolates were inhibited by 1 mg/L of daptomycin. Resistance to other compounds (vancomycin, oxacillin, and penicillin) did not influence daptomycin activity. The activity of daptomycin was very similar in both geographic regions evaluated and demonstrated the same MIC distribution as isolates evaluated in studies in the United States. The results of this study showed that daptomycin continues to be very active against clinical isolates of Gram-positive cocci isolated in Europe and Latin America.