Agar Dilution MIC Research Articles

Development of a meropenem susceptibility disc: drug content, preliminary interpretive and quality control criteria and potency bioassay.

Laboratory studies were conducted to develop an appropriate susceptibility disc for meropenem. Laboratory-prepared paper discs containing 5, 10, and 20 micrograms of meropenem were investigated. Agar dilution MICs and agar disc diffusion tests were performed with 367 rapidly growing clinical isolates by methods recommended by the National Committee for Clinical Laboratory Standards (NCCLS). Agar disc diffusion tests with 10 micrograms meropenem discs acceptably defined organisms as susceptible or resistant. The preliminary interpretive zone size criteria for 10 micrograms meropenem discs were: susceptible, greater than or equal to 14 mm, and resistant, less than or equal to 10 mm, by application of the NCCLS imipenem MIC interpretive susceptible and resistant breakpoints to the meropenem data. A total of 270 agar disc diffusion assays were performed with each of four ATCC reference strains, using prototype discs (Becton Dickinson (BBL] containing 10 micrograms meropenem. The preliminary quality control limits for agar disc diffusion tests with these discs are: Escherichia coli ATCC 25922, 28-32 mm; Pseudomonas aeruginosa ATCC 27853, 26-31 mm; Staphylococcus aureus ATCC 25923, 33-39 mm; and Enterococcus faecalis ATCC 29212, 19-23 mm. Further studies are necessary to re-evaluate both the preliminary interpretive and quality control criteria before definitive limits can be established. A reproducible, large plate bioassay with Enterococcus faecalis ATCC 29212 was developed to monitor meropenem disc potency.

In-vitro interactions of FCE 22101 with aminoglycosides against Gram-negative rods

Agar dilution MICs of FCE 22101 were measured for 894 consecutively-isolated Gram-negative rods from clinical specimens (413 Escherichia coli, 104 Klebsiella spp., 54 Enterobacter spp., 19 Citrobacter spp., 131 Proteus spp., 43 Acinetobacter spp., 9 Serratia spp., 3 Providencia spp., 4 Yersinia spp., 3 Hafnia spp. and 111 Pseudomonas spp.). Excluding Pseudomonas spp., 98% of these isolates were susceptible to 8 mg/l FCE 22101. Resistance (MIC greater than 8 mg/l) varied from 11% in Serratia spp. to 0% in Citrobacter spp. Interactions between FCE 22101 and gentamicin, tobramycin and amikacin were tested by the chequerboard method for 150 of the isolates. The geometric mean sigma FICs were 0.80 for FCE + gentamicin, 0.78 for FCE + tobramycin and 0.79 for FCE + amikacin. Synergy, defined at FIC index (sigma FIC) less than 0.5, for at least one combination, was found in only 16/150 (11%) of the isolates. Most other isolates showed an additive response (sigma FIC = greater than 0.5-1). No antagonism was detected. No significant difference was observed between the various species tested, except that sigma FIC values were lower (geometric mean sigma FIC = 0.61-0.68) for non-fermentative species than for fermentative species (geometric mean sigma FIC = 0.83-0.86). Likewise, geometric sigma FIC values were lower (0.67-0.69) for isolates resistant to FCE and/or aminoglycosides than for those susceptible to both components (sigma FIC 0.85-0.88).

Laboratory- and species-specific interpretive breakpoints for disk diffusion tests of chloramphenicol susceptibility of Haemophilus influenzae.

A total of 601 clinical isolates of Haemophilus influenzae isolated in six different regions of Sweden were tested for chloramphenicol susceptibility by using agar dilution MIC determinations and disk diffusion tests. For seven strains MICs were 4 micrograms/ml or higher, and for one strain the MIC was 2 micrograms/ml. All eight strains produced chloramphenicol acetyltransferase. For the remaining 593 strains, MICs were less than or equal to 1 microgram/ml, and the MICs for 50% and 90% of the strains were both 0.5 microgram/ml. Disk diffusion tests carried out by using revised interpretive criteria introduced in 1984 by the Swedish Reference Group for Antibiotics correctly identified the 593 strains as susceptible and the 8 strains as resistant. Quality assessments were performed in 29 clinical microbiology laboratories. The revised criteria for chloramphenicol disk diffusion testing gave rise to false resistance results in some laboratories. The interpretive accuracy improved when the interlaboratory variation was compensated for by using adjusted breakpoints. Such revision was possible through peak correction, single-strain regression analysis, and standard curve regression analysis. Peak-corrected breakpoints improved the accuracy from an overall incidence of false-resistant isolates of 4.4% to 2.3%. Single-strain regression analysis and standard curve regression analysis provided laboratory- and species-specific breakpoints which reduced false resistance rates of 0.14% and 0%, respectively.

Multiply high-level-aminoglycoside-resistant enterococci isolated from patients in a university hospital.

Enterococci isolated from different body sites were tested for high-level gentamicin resistance. A total of 259 enterococcal isolates were screened for resistance (MIC, greater than 2,000 micrograms/ml) by a broth-tube method. Thirty-nine (15.1%) were found to exhibit resistance and were confirmed by agar screening (1,000 micrograms/ml) and agar dilution MIC determinations. The majority of isolates also showed high-level resistance to kanamycin and streptomycin. The remaining isolates showed high-level resistance to gentamicin and kanamycin but not streptomycin. Synergy testing of several isolates confirmed the correlation between lack of synergy and high-level resistance. A retrospective clinical review was performed. Most patients had a source of definite or likely infection (79%). Serious infections such as endocarditis or meningitis were not observed during the course of this study. Retrospective clinical data suggest that in cases not involving endocarditis or meningitis, neither infection refractory to therapy nor relapse of infection is a common sequela of infection with gentamicin-resistant enterococci in hospitalized patients.

Molecular cloning and mechanism of trimethoprim resistance in Haemophilus influenzae.

We studied 10 trimethoprim-resistant (Tmpr) Haemophilus influenzae isolates for which agar dilution MICs were 10 to greater than 200 micrograms/ml. Trimethoprim resistance was transferred from two Tmpr H. influenzae isolates to a Tmps strain by conjugation or transformation. Wild-type Tmpr strains and Tmpr transcipients did not contain detectable plasmid DNA. The trimethoprim resistance gene was cloned into a cosmid vector, and recombinant plasmids were transduced into Escherichia coli. A 0.50-kilobase intragenic probe derived from a 12.9-kilobase fragment which encoded trimethoprim resistance hybridized with whole-cell DNA from Tmps and Tmpr strains. Southern blot analysis of restricted DNA from isogenic Tmps and Tmpr H. influenzae indicated that acquisition of trimethoprim resistance involved a rearrangement or change in nucleotide sequence. Hybridization was not seen with DNA derived from Tmpr E. coli containing dihydrofolate reductase I, II, and III genes or with Tmpr Neisseria meningitidis, Neisseria gonorrhoeae, and Pseudomonas cepacia. Southern hybridization with 12 multiply resistant encapsulated H. influenzae strains confirmed that the trimethoprim resistance gene was chromosomally mediated. Dihydrofolate reductase activity was significantly greater in cell sonicate supernatants of Tmpr strains in comparison with isogenic Tmps recipients. Differences were not found in the trimethoprim inhibition profile of dihydrofolate reductase activity in Tmps and Tmpr strains. We conclude that the mechanism of trimethoprim resistance in H. influenzae is overproduction of chromosomally located dihydrofolate reductase.

Evaluation of broth disk elution methods for susceptibility testing of anaerobic bacteria with the newer beta-lactam antibiotics

Broth disk elution procedures represent one of the most practical means for clinical laboratories to perform routine antibiotic susceptibility tests on anaerobic bacteria. The accuracy of five disk elution test methods and media (including the one to be proposed by the National Committee for Clinical Laboratory Standards) was evaluated for the testing of newer beta-lactam antibiotics, including cefoperazone, cefotaxime, cefoxitin, ceftazidime, ceftizoxime, moxalactam, and piperacillin. Various numbers of antibiotic disks were used to achieve disk elution test concentrations which approximated the highest MIC termed susceptible by the Food and Drug Administration. A group of 88 anaerobes representing many different species was tested in parallel by the five disk elution methods and the National Committee for Clinical Laboratory Standards reference agar dilution procedure. Overall, full agreement between the reference agar dilution MICs and the disk elution category results was 88.3% for PRAS BHI, 84.5% for Schaedler, 85.7% for thioglycolate, and 87.4% for Wilkins-Chalgren broth. Essential agreement (+/- 1 twofold MIC increment from the disk elution concentration) was achieved with 94.6% of PRAS BHI tests, 94.3% of Schaedler tests, 93.6% of thioglycolate tests, and 95.7% of Wilkins-Chalgren tests. Due to growth failures with a number of isolates and difficulties in interpreting results, the use of Wilkins-West broth was discontinued after approximately one-half of the isolates had been tested. The majority of errors with all of the disk elution methods occurred with isolates (most notably members of the Bacteroides fragilis group) having MICs near the single test concentrations used in the disk methods. With the notable exception of tests for the B. fragilis group, the disk elution methods offered acceptable accuracy with the newer beta-lactam antibiotics tested in this study.

False susceptibility of enterococci to aminoglycosides with blood-enriched Mueller-Hinton agar for disk susceptibility testing

Disk diffusion susceptibility tests for enterococci are frequently modified by adding 5% sheep blood (SB) to Mueller-Hinton agar; the performance standards from the National Committee for Clinical Laboratory Standards sanction this addition. Susceptibility testing of aminoglycoside antibiotics is not recommended for enterococci; in actual practice, however, some laboratories do include aminoglycoside antibiotics routinely, and others may test upon request or in selected situations. In examining 50 clinical isolates of enterococci, SB-enriched Mueller-Hinton agar frequently gave enlarged zone sizes that falsely indicated susceptibility (72% for gentamicin and tobramycin), with the average increase in zone size being 6.3 and 7.6 mm, respectively. Comparison agar dilution MICs demonstrated uniform resistance, with or without added SB. The effect was shown to be caused by heme in concentrations as low as 0.03 micrograms/ml, which, when combined with aminoglycoside antibiotics, caused a synergistic growth inhibition of the enterococci, resulting in larger aminoglycoside antibiotic zones. We postulate that the heme effect is related to a catalytic cleavage of intracellular H2O2 and resultant lipid peroxidation. No other organism or antimicrobial agent tested demonstrated a similar effect, although other investigators have shown a similar phenomenon with the broad-spectrum cephalosporins. Because enterococci grow well and give accurate susceptibility results on Mueller-Hinton agar without SB supplementation and because of the spectrum of definable problems with a number of antimicrobial agents, we recommend that enterococci routinely be tested without SB.

In-vitro activity of methicillin against clinical isolates of Staphylococcus aureus.

Methicillin activity against 149 penicillin-resistant, methicillin-susceptible Staphylococcus aureus strains from bacteraemia cases with endocarditis (n = 89) or without endocarditis (n = 60), from the years 1976-1981, was studied with broth dilution and agar dilution. While no differences in methicillin susceptibility were found in relation to the origin of the strains, Staph. aureus of the phage type complex 94,96 showed significantly higher MIC and IC50 by agar dilution than strains of other phage groups/complexes. This difference probably has no clinical importance but is of epidemiological interest. Broth dilution MIC was generally one dilution higher than agar dilution MIC, possibly explained by methodological factors. The MBC/MIC ratios never exceeded two in any of the strains, indicating a lack of tolerance in these clinically important isolates.

Evaluation of the AutoMicrobic system Gram-Negative General Susceptibility-Plus Card

The MICs of 10 antimicrobial agents were determined for 1,000 clinical aerobic and facultatively anaerobic gram-negative bacilli by the 6-h AutoMicrobic system Gram-Negative General Susceptibility-Plus Card (GSC+) (Vitek Systems, Inc., Hazelwood, Mo.) and by an 18-h reference agar dilution method. Results obtained by both systems were evaluated for twofold dilution discrepancies and for interpretive category discrepancies. MICs that differed by more than one twofold dilution between the two test systems were considered to be discrepant. The overall twofold dilution agreement of the GSC+ MICs to agar dilution MICs was 94.0%. The agreement for each drug tested ranged from 91.7% (ampicillin and chloramphenicol) to 96.5% (carbenicillin). Interpretive categories were considered in agreement when the susceptibility interpretations obtained by the two systems were both very susceptible, moderately susceptible, or resistant. The overall interpretive category agreement was 80.5%. This agreement increased to 95.8% when all minor errors (moderately susceptible, one system; very susceptible, the other system) that would not affect therapy were omitted. The AutoMicrobic system GSC+ provides rapid and accurate susceptibility test results for clinical aerobic and facultatively anaerobic gram-negative bacilli.

Variations in susceptibility to latamoxef(moxalactam) and cefoxitin within the Bacteroides fragilis group.

Conventional agar dilution MIC titrations, anaerobic turbidimetric monitoring and viable counting were used to investigate the response to latamoxef (moxalactam) and cefoxitin of four well characterized species within the Bacteroides fragilis group. MIC results indicated that Bact. fragilis and Bact. vulgatus were more susceptible to both antibiotics than were Bact. ovatus and Bact. thetaiotaomicron and that latamoxef was more active than cefoxitin. Continuous turbidimetric monitoring with parallel viable counting confirmed the greater susceptibility of Bact. fragilis and Bact. vulgatus, but also suggested that the differential in activity between latamoxef and cefoxitin was less than MIC titrations had indicated. Moreover, against the more resistant Bact. ovatus and Bact. thetaiotaomicron strains, cefoxitin actually appeared more active than latamoxef in bactericidal and bacteriolytic terms. The interpretation of these results is discussed in the light of the morphological response of the bacteria to the two antibiotics. Because species within the Bact. fragilis group appear to differ considerably in their beta-lactam antibiotic susceptibility there is a need for laboratories to test the antibiotic susceptibility of these organisms or to identify them presumptively to the species level if treatment with beta-lactam agents is contemplated.

1055 EMERGENCE OF MULTIPLY RESISTANT PNEUMOCOCCUS

A strain of S. pneumoniae resistant to multiple antibiotics was isolated from an 11-month-old infant with meningitis. CSF obtained after 96 hours of high-dose penicillin-G continued to grow a beta-lactamase negative pneumococcus, type 6. When treatment was changed to ampicillin, chloramphenicol, and rifampin, the child improved; and CSF was sterile within 24 hours. Agar dilution MIC's were: pen-G 1 μg/ml; chloro, 16 μg/ml; ampicillin 0.5 μg/ml; tetracycline, 16 μg/ml; rifampin, 0.03 μg/ml. A cultural survey of day-care center contacts utilized two selective media; one containing pen-G, 0.125 μg/ml, the other chloro, 4.0 μg/ml. 4/14 children <2 years and 5/47 older children and staff had throat carriage of this resistant strain, as did 5/10 of the family contacts of carriers. No carriers were treated. We also cultured 103 children in 4 other day-care centers without detecting the organism. We believe this to be the first report of a penicillin/chloramphenicol resistant pneumococcus in the Western Hemisphere. Due to the seriousness of delaying correct antibiotic therapy, we suggest that significant pneumococcal isolates be screened for both penicillin and chloramphenicol susceptibility. Surveys of contacts, which may yield a high carriage rate, can be performed rapidly using selective media. However, there is no established strategy regarding the treatment and follow-up of carriers.

Methodological implications of testing anaerobe susceptibility to cephalosporins (cefazolin, cefamandole, cefoxitin).

By simultaneously performing broth dilution, agar dilution, and agar diffusion tests with Bacteroidaceae and Peptococcaceae, the influence of methodology upon the outcome of susceptibility testing to cefazolin, cefamandole and cefoxitin was studied. With beta-lactamase positive and negative Bacteroidaceae, the results of the broth dilution and agar dilution tests were in good agreement for cefoxitin. However, when tested with cefaxolin and cefamandole, beta-lactamase positive Bacteroides strains had mostly high broth dilution MICs and relatively low agar dilution MICs. The statistical analysis of the relationship between zone size and broth dilution or agar dilution MICs frequently showed lack of stochastic linearity or relatively low correlation coefficients.

In vitro activity of tiamulin (81.723 HFU), a new pleuromulin derivative, against clinically significant anaerobes.

The susceptibility of more than 40 strains of Gram-negative and Gram-positive anaerobes to tiamulin (Sandoz 81.723 hfu), a new pleuromulin (pleuromutilin) derivative, was determined by broth dilution and agar dilution tests. The influences of density of the inoculum upon MICs was studied by a specially designed pour plate-technique. Bacteroides fragilis, B. vulgatus, B. splanchnicus, B. oralis, B. asaccharolyticus, B. melaninogenicus, Fusobacterium fusiforme (F. nucleatum), Sphaerophorus necrophorus, Clostridium perfringens, C. fallax, Propionibacterium acnes and several species of Peptococcaceae showed broth dilution MICs of 0.03 similar to 1 microgram/ml. Members of B. thetaiotaomicron, B. distasonis and S. freundii (F. mortiferum) were inhibited by 8 similar to 32 microgram/ml and 2 strains of S. varius had a broth dilution MIC of 256 microgram/ml. With most strains, the agar dilution MICs were 2 similar to 4 similar to 8 times the broth dilution MICs. In pour plate-tests, the MICs were not considerably influenced influenced by varying initial concentrations of viable cells. With most anaerobes, the MBCs of tiamulin were more than 100-fold higher than the MICs. The results obtained indicated that, apart from S. varius, B. thetaiotaomicron, B. distasonis and S. freundii (F. mortiferum), members of 16 other anaerobic species including B. fragilis were without exception sensitive to tiamulin.

Penicillin-Resistant Strains of Neisseria Gonorrhoeae in Shahre-Now

Of 921 women examined, 112, or 12.1%, were identified as having gonorrhoea by positive culture; 25.6% of 78 strains isolated from the women were resistant to penicillin at 0.10 mug/ml level and 14.1% resistant at the 0.50 mug/ml level as determined by an agar dilution MIC test. Our results were compatible with a similar but larger study reported in the USA by Jaffe et al. 1976.