The E-Test and Campylobacter jejuni.

The E-Test is a recently introduced method for performing antimicrobial susceptibility tests. We compared the E-Test to the broth microdilution test and to the standard agar dilution test by using five antimicrobial agents tested against 55 clinical isolates of Campylobacter jejuni from 11 locations in USA (group 1). Later, we selected 30 strains (group 2), which were more resistant than the original survey isolates. Erythromycin, tetracycline, and ciprofloxacin were tested on both groups of organisms. When the three test methods were compared with each other at +/- 1 log2 dilution, the E-test gave the best overall agreement, with 85% of all strains being within acceptable limits. Category interpretation of erythromycin (drug of choice) results was a problem using current NCCLS guideline breakpoints. For the E-Test and the agar dilution method, 82% of the strains were in the intermediate category; but with the broth microdilution method, only 16.4% of the isolates were interpreted as intermediate. If the susceptible category breakpoint was raised to less than or equal to 2 micrograms/ml, then only 3% of the C. jejuni isolates would be interpreted as intermediate by any of the three methods. Our preference for antimicrobial susceptibility testing of C. jejuni is either E-Test or agar dilution at 42 degrees C for 16 hr.

Antimicrobial activity of LY164846, a new oral cephalosporin, and recommendations for disk diffusion tests.

LY164846 is a new oral cephalosporin with a limited spectrum of antimicrobial activity that includes staphylococci (other than methicillin-resistant), streptococci (other than enterococci), Haemophilus influenzae (beta-lactamase-negative and beta-lactamase-positive), Branhamella catarrhalis (beta-lactamase-negative and beta-lactamase-positive), and Neisseria species (beta-lactamase negative and beta-lactamase-positive). The tentative recommendations for susceptibility breakpoints are less than or equal to 4 micrograms/ml and greater than or equal to 19 mm for susceptible, 8 micrograms/ml and 15-18 mm for intermediate, and greater than or equal to 16 micrograms/ml and less than or equal to 14 mm for resistant.

Optimizing testing of methicillin-resistant Staphylococcus species.

Selection of the appropriate NaCl concentration for test medium for oxacillin susceptibility testing of Staphylococcus aureus and coagulase-negative staphylococci has been problematic when using different antimicrobial susceptibility testing methods. Broth microdilution, using cation-adjusted Mueller-Hinton broth + 2% NaCl, is the currently recommended reference method. There is currently no recommendation for the addition of NaCl to agar for dilution susceptibility tests when Staphylococcus species are tested with oxacillin. We examined the effects of adding 0, 2%, 4%, and 5% NaCl to Mueller-Hinton agar and broth for agar dilution, Etest, and broth microdilution tests. The results of these tests were compared with the reference broth microdilution results and with the results of a hybridization assay using a mec gene probe. We tested 223 strains of staphylococci, 128 of which were mec gene positive and had oxacillin minimum inhibitory concentrations (MICs) > or = 4 micrograms/ml. Seven strains of S. aureus were mec probe negative but were oxacillin resistant. Seven coagulase-negative strains (three S. epidermidis, one S. haemolyticus, and three S. simulans) were mec probe positive and were oxacillin susceptible. The MICs for oxacillin-resistant strains increased two- to fourfold with the addition of 2% NaCl, but the MICs for oxacillin-susceptible strains were unchanged. Major and very major interpretative rates ranged from 18.2% to 20.2% for agar dilution and Etest without NaCl added to the medium, and these rates decreased to < 1% with the addition of 2% NaCl to the medium. The addition of 4% or 5% NaCl caused major error rates of > 17% for all test methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Antimicrobial susceptibility of Streptococcus mutans isolated from patients with endocarditis.

Forty-one strains of Streptococcus mutans (34 from blood specimens from patients with endocarditis and 7 from stock cultures) were tested for susceptibility to penicillin, ampicillin, methicillin, erythromycin, cephalothin, vancomycin, chloramphenicol, tetracycline, gentamicin, streptomycin, and kanamycin. Minimal inhibitory and bactericidal concentrations were determined by a broth microdilution procedure. Most of the strains were very susceptible to ampicillin, penicillin, and erythromycin, with most strains having minimal inhibitory concentrations of 0.08 mug/ml or less. Most of the strains were also susceptible to cephalothin, methicillin, chloramphenicol, tetracycline, and vancomycin. Gentamicin was the most effective aminoglycoside. The antimicrobial susceptibility patterns are similar to those of other viridans streptococci. S. mutans strains have proven to be difficult for some microbiologists to identify. But when organisms suggesting S. mutans are isolated from patients with endocarditis, they should be at least identified as nonenterococcal streptococci so that appropriate therapy can be initiated.

Evaluation of Alamar colorimetric broth microdilution susceptibility testing method for staphylococci and enterococci.

We compared the results of the Alamar broth microdilution susceptibility testing method with the results of the National Committee for Clinical Laboratory Standards reference broth microdilution method for 119 gram-positive organisms. The strains were tested for their susceptibilities to 20 antimicrobial agents. Only appropriate antimicrobial agents were evaluated for each species of bacteria. Absolute categorical agreement between the reference method and the test method was 91.5% for enterococci, 99.8% for oxacillin-susceptible staphylococci, and 97.4% for oxacillin-resistant staphylococci. Essential agreement (percent complete agreement plus percent minor errors) was > 99% for all organisms tested. The results for enterococci showed no very major errors, one major error with ofloxacin, and numerous minor errors with the quinolones. However, all except one of the minor errors were within +/- 1 log2 dilution of the reference result. For staphylococci, only 2 very major errors (one each with chloramphenicol and oxacillin), 1 major error (chloramphenicol), and 15 minor errors (multiple drugs) were observed. The Alamar colorimetric system was easy to use and the results were easy to read. It appears to be an acceptable method for antimicrobial susceptibility testing of staphylococci and enterococci.

Development of provisional disc diffusion breakpoints for testing quinupristin/dalfopristin.

Quinupristin/dalfopristin is an injectable streptogramin with broad activity against many Gram-positive bacteria, including Streptococcus pneumoniae, Staphylococcus aureus and Enterococcus faecium. Although a number of studies have reported the MICs of this compound against a variety of bacteria, there are no published reports of disc diffusion testing. We tested total disc masses of 7.5 and 15 micrograms with varying ratios of the component compounds, quinupristin and dalfopristin, combined in the following quinupristin: dalfopristin ratios (in microgram): 7.5:0, 0:7.5, 10:5, 5:10, 5:2.5 and 2.5:5. Zone diameters and MICs were determined in parallel for 44 isolates of staphylococci, 47 isolates pneumococci and 64 isolates of enterococci. Control strains were included for each species tested, including a strain of Enterococcus faecalis with known resistance to quinupristin/dalfopristin. Using tentative definitions for quinupristin/dalfopristin of < or = 2 mg/L as susceptible and > or = 4 mg/L as resistant, each of the four discs containing ratios of both component compounds separated presumptive susceptible organisms from resistant ones better than either quinupristin or dalfopristin alone. The best correlation of zone sizes and MICs for predicting susceptibility to quinupristin/dalfopristin at an MIC of < or = 2 mg/L was achieved with the 5 micrograms:10 micrograms disc and a zone diameter of > or = 18 mm. These criteria may be useful for identifying organisms that are presumptively susceptible to quinupristin/dalfopristin.

Antibiotic susceptibility of Streptococcus bovis and other group D streptococci causing endocarditis.

Seventy-four strains of Streptococcus bovis and 35 strains of enterococci (Streptococcus faecalis and its varieties, Streptococcus faecium and Streptococcus durans), most of which were isolated from patients with endocarditis, were tested for their susceptibility to penicillin, ampicillin, erythromycin, cephalothin, vancomycin, methicillin, tetracycline, chloramphenicol, kanamycin, streptomycin, and gentamicin. Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) were determined by a microtiter broth dilution technique. All of these organisms are group D streptococci, but the S. bovis strains are not enterococci. On the basis of both MIC and MBC, the S. bovis strains were much more susceptibile in general to antibiotics then were the enterococcal strains. For the S. bovis strains, the lowest MICs were obtained with penicillin, ampicillin, and erythromycin, and the lowest MBCs with penicillin and ampicillin. Although these antibiotics were also the most active against the enterococci, the MICs and MBCs were much higher than obtained with the S. bovis strains. Gentamicin was the most active aminoglycoside. On the basis of in vitro susceptibility results, the S. bovis strains resemble the viridans streptococci rather than enterococci.

In vitro antimicrobial activity of cefoperazone, cefotaxime, moxalactam (LY127935), azlocillin, mezlocillin, and other beta-lactam antibiotics against Neisseria gonorrhoeae and Haemophilus influenzae, including beta-lactamase-producing strains.

Minimum inhibitory concentrations and agar disk diffusion tests were determined on clinical isolates of beta-lactamase-positive and beta-lactamase-negative Neisseria gonorrhoeae and Haemophilus influenzae with the newer beta-lactam antibiotics, cefoperazone, cefotaxime, moxalactam (LY127935), azlocillin, mezlocillin, and piperacillin, and with seven older beta-lactam antibiotics. All the drugs were active against beta-lactamase-negative strains of N. gonorrhoeae and H. influenzae. The drug most active against beta-lactamase-positive N. gonorrhoeae was cefotaxime, followed closely by cefoperazone, moxalactam, piperacillin, and mezlocillin. The drugs most active against beta-lactamase-positive strains of H. influenzae were cefotaxime, moxalactam, cefoperazone, and cefamandole.

Relationship between in vitro susceptibility test results for chloramphenicol and production of chloramphenicol acetyltransferase by Haemophilus influenzae, Streptococcus pneumoniae, and Aerococcus species.

Haemophilus influenzae, Streptococcus pneumoniae, and Aerococcus species were tested for susceptibility to chloramphenicol by standard broth microdilution and disk-diffusion methods. MICs and zone diameter breakpoints were correlated with production of chloramphenicol acetyltransferase (CAT). A comparison of MICs and zone diameters indicated that the interpretative criteria for H. influenzae and S. pneumoniae should be an MIC of less than or equal to 4 micrograms/ml or a zone diameter greater than or equal to 25 mm for susceptible strains and an MIC of greater than or equal to 8 micrograms/ml or a zone diameter of less than or equal to 20 mm for resistant strains; for Aerococcus species, interpretative criteria should be an MIC of less than or equal to 8 micrograms/ml or a zone diameter of greater than or equal to 20 mm for susceptible strains and an MIC of greater than or equal to 32 micrograms/ml or a zone diameter of less than or equal to 12 mm for resistant strains. All but four strains of H. influenzae and one strain of S. pneumoniae that were resistant to chloramphenicol by these criteria produced CAT. For Aerococcus species, however, chloramphenicol-resistant strains were negative for CAT as determined by a commercially available disk test. When comparing susceptibility results with CAT production, thiamphenicol was a better indicator of the presence of the enzyme than chloramphenicol and may be useful in assaying resistance to chloramphenicol.

Antimicrobial susceptibility testing of Francisella tularensis with a modified Mueller-Hinton broth.

A modified Mueller-Hinton broth was developed to perform antimicrobial susceptibility tests on Francisella tularensis. Adequate growth of the organism was obtained within 24 h of inoculation, and MICs could be read at that time. We tested 15 selected strains of F. tularensis and five reference quality control strains in this medium with 36 antimicrobial agents. The MICs of the aminoglycosides and tetracycline increased 1 to 3 dilutions in this medium compared with those in the usual medium, but the other antimicrobial agents were not consistently affected by the medium. Even though the medium caused an increase in MICs, the aminoglycosides and tetracyclines remained very active in vitro against F. tularensis. Other antimicrobial agents effective in vitro were chloramphenicol, erythromycin, ceftazidime, moxalactam, cefotaxime, ceftriaxone, and Sch 29482 (a cephalosporin).

Evaluation of Alamar colorimetric MIC method for antimicrobial susceptibility testing of gram-negative bacteria.

The Alamar (Alamar Biosciences, Inc., Sacramento, Calif.) colorimetric antimicrobial susceptibility testing method is a new approach to the determination of broth microdilution MICs. The method uses a color indicator to detect growth of microorganisms within the wells of a microdilution tray. The color changes can be read visually or with a fluorometer. The system contains growth and sterility control wells and 20 antimicrobial agents per MIC tray with eight twofold dilutions for each antimicrobial agent. We tested 186 multiresistant, gram-negative bacterial isolates against 33 antimicrobial agents and compared the results to those obtained by agar dilution. Categorical agreement for all agents was 90.9% and ranged from 78.2% for ampicillin-sulbactam to 98.1% for amikacin. Percent agreement for MIC results (within +/- 1 log2 dilution) was 91.0% for all agents and ranged from 69.1% for gentamicin to 97.9% for ciprofloxacin. Most of the disagreements were with the penicillins and cephalosporins for beta-lactamase-producing strains. The Alamar MIC system is very easy to read visually and appears to be a satisfactory addition to currently used MIC determination methods.

Effect of temperature on the in vitro susceptibility of Staphylococcus aureus to penicillinase-resistant penicillins.

Heteroresistant (methicillin-resistant) and nonheteroresistant strains of Staphylococcus aureus were tested for their susceptibility to penicillinase-resistant penicillins at incubation temperatures of 37, 35, and 30 C. Susceptibilities were determined by agar dilution and by the standard Kirby-Bauer agar diffusion tests. Minimal inhibitory concentrations were higher at 35 and 30 C than at 37 C. Heteroresistance could be detected with the Kirby-Bauer test if the incubation temperature was 30 or 35 C instead of 37 C, when tests were performed against methicillin, oxacillin, or nafcillin, because the resistant organisms grew up to the disks even though the susceptible organisms were inhibited. At 37 C, the resistance was detectable with some strains but not with others. When cloxacillin disks were used, the temperature effect was not seen. The incubation temperature did not affect results with nonheteroresistant strains. Therefore, it is recommended that all Kirby-Bauer tests be incubated at a temperature of 35 C to insure detection of methicillin-resistant S. aureus strains. Detection of these strains is of increasing importance because the incidence of infections with these organisms is increasing, particularly in hospitalized patients.

In vitro susceptibilities of aerotolerant Campylobacter isolates to 22 antimicrobial agents.

We evaluated the in vitro activities of 22 antimicrobial agents against 78 human and animal isolates belonging to two aerotolerant Campylobacter species, C. cryaerophila and C. butzleri, using a broth microdilution technique. An additional 10 antimicrobial agents were included at concentrations found in selective Campylobacter media. Strains of C. cryaerophila belonged to two DNA hybridization groups: DNA hybridization group 1A, which includes the type strain of C. cryaerophila, and DNA hybridization group 1B. The aminoglycosides, fluoroquinolones, and one tetracycline (minocycline) demonstrated the most activity against all DNA hybridization groups (C. cryaerophila DNA groups 1A and 1B and C. butzleri). Most isolates were resistant to cephalosporin antibiotics, with the exception of cefotaxime, and were variably susceptible to trimethoprim-sulfamethoxazole. C. cryaerophila DNA hybridization group 1A isolates were generally susceptible to the tetracyclines, chloramphenicol, nalidixic acid, azithromycin, erythromycin, and roxithromycin and moderately susceptible to clindamycin, trimethoprim-sulfamethoxazole, ampicillin, and ampicillin-sulbactam. The MICs of tetracyclines were higher for C. butzleri and C. cryaerophila DNA hybridization group 1B isolates than for C. cryaerophila DNA hybridization group 1A isolates, but most strains were still susceptible to doxycycline and tetracycline; all isolates were susceptible to minocycline. C. butzleri and C. cryaerophila DNA hybridization group 1B isolates were generally resistant to the macrolide antibiotics (including erythromycin), chloramphenicol, clindamycin, nalidixic acid, ampicillin, and trimethoprim-sulfamethoxazole. Differences in antimicrobial susceptibility between aerotolerant Campylobacter species and more common Campylobacter species, e.g., C. jejuni, suggest that different treatment strategies may be necessary. Strains of all three DNA hybridization groups of aerotolerant Campylobacter isolates were susceptible to colistin, polymyxin B, and rifampin at concentrations commonly used in selective media. These results suggest that primary isolation methods for Campylobacter species may need to be modified to include aerotolerant Campylobacter strains.

A simplified method for testing Bordetella pertussis for resistance to erythromycin and other antimicrobial agents.

Present methods of antimicrobial susceptibility testing of Bordetella pertussis are time consuming and require specialized media that are not commercially available. We tested 52 isolates of B. pertussis for resistance to erythromycin, trimethoprim-sulfamethoxazole, chloramphenicol, and rifampin by agar dilution with Bordet-Gengou agar (BGA) containing 20% horse blood (reference method), Etest using BGA and Regan-Lowe agar without cephalexin (RL-C), and disk diffusion using BGA and RL-C. The organisms tested included four erythromycin-resistant isolates of B. pertussis from a single patient, a second erythromycin-resistant strain of B. pertussis from an unrelated patient in another state, and 47 nasopharyngeal surveillance isolates of B. pertussis from children in the western United States. The results of agar dilution testing using direct inoculation of the organisms suspended in Mueller-Hinton broth were within +/-1 dilution of those obtained after overnight passage of the inoculum in Stainer-Scholte medium, which is the traditional method of testing B. pertussis. The Etest method produced MICs similar to those of the agar dilution reference method for three of the four antimicrobial agents tested; the trimethoprim-sulfamethoxazole results were lower with Etest, particularly when the direct suspension method was used. Most of the Etest MICs, except for that of erythromycin, were on scale. Disk diffusion testing using RL-C medium was helpful in identifying the erythromycin-resistant strains, which produced no zone of inhibition around the disk; susceptible isolates produced zones of at least 42 mm. Thus, the antimicrobial susceptibility testing of B. pertussis can be simplified by using the Etest or disk diffusion on RL-C to screen for erythromycin-resistant isolates of B. pertussis.

Evaluation of commercial methods for determining antimicrobial susceptibility of Streptococcus pneumoniae.

Seven commercial systems for antimicrobial susceptibility testing of Streptococcus pneumoniae were evaluated by using a challenge set of 55 pneumococcal isolates with a variety of resistance phenotypes and genotypes. Overall, the results produced by the Pasco and Etest methods were found to be acceptable for all drugs tested except for trimethoprim-sulfamethoxazole testing by the Etest. The Just One system for penicillin MIC testing was also judged to be acceptable (minor error rate, 5.5%). Although the Sensititre and MicroTech methods both produced 12.7% minor errors with penicillin, the Sensititre method classified penicillin-intermediate strains as resistant or vice versa, while four of MicroTech's errors were among intermediate strains that were classified as susceptible. The MicroMedia (minor error rate, 16.4%) and MicroScan Rapid (minor error rate, 63.6%) methods produced unacceptably high levels of errors when testing penicillin. Minor error rates for cefotaxime and ceftriaxone ranged from a low of 12.7% (Etest and Sensititre) to a high of 28% (MicroMedia). Error rates were low for erythromycin, tetracycline, and chloramphenicol by most methods with the exception of the MicroScan method, which had a high very major error rate for erythromycin (34.6%). For testing of beta-lactam drugs, the Pasco, Etest, and Just One tests for penicillin are the most accurate methods; the Sensititre method also provided acceptable results.

Comparison of antistreptolysin O latex screening test with the antistreptolysin O hemolytic test.

This report describes a comparison of the Behringwerke antistreptolysin O (ASO) latex screening test with the ASO hemolytic test. Agreement between the two tests was poor when Difco streptolysin O (SLO) reagent was employed in the hemolytic test; approximately 34% of the sera with ASO titers in the normal range of the hemolytic test gave false-positive latex test reactions. However, the percentage of false-positive latex test reactions was only 5% when Behringwerke SLO reagent was used in the hemolytic test. An assay of the Difco and Behringwerke SLO reagents against an ASO standard indicated that the Difco SLO reagent was more potent than the Behringwerke SLO reagent. The lack of agreement between the Behringwerke latex test and the hemolytic test using Difco SLO reagent is attributed to the potency of the SLO reagents.

"Upper limits of normal" antistreptolysin O and antideoxyribonuclease B titers.

"Upper limits of normal" antistreptolysin O (ASO) and antideoxyribonuclease (ADN) B titers were determined on serum specimens from various age groups of pediatric patients with no history of a recent streptococcal infection and on healthy adult hospital employees. The upper limit of normal value is that level of antibody titer exceeded by no more than 15% of the total subjects in each age group. This value varies with the age of the subject, and the most pronounced differences are between the values of preschool age children, school age children, and adults. The upper limit of normal values for these groups were as follows: preschool age, ASO = 85(100); ADN B = 60(50); school age, ASO = 170(166); ADN B=1 70(166); and adult, ASO = 85(100); ADN B = 85(100).