Disk Diffusion Test Results Research Articles

Evaluation and comparison of tests to detect methicillin resistant S. aureus

Phenotypic methods for detection of methicillin resistant Staphylococcus aureus (MRSA) have been compared with the gold standard which, as of now, is by the detection of mecA gene and femA gene by polymerase chain reaction (PCR). Discrepancies in detection have an adverse effect on patient management, thereby highlighting the importance of accuracy in detection. Our study aims to evaluate the efficacy of cefoxitin disk diffusion test to detect MRSA and compare it with other phenotypic and molecular methods. The study was conducted from June 2006 to December 2007 and included 610 Staphylococcus aureus (S. aureus) isolates obtained from clinical samples. All isolates were tested for MRSA using oxacillin screen agar plates with 6 microg/ml of oxacillin, cefoxitin disk diffusion using 30 microg disk and MIC of oxacillin. Selected isolates (55) were tested for presence of mecA gene and Fem A gene by PCR. Out of 610 isolates, MRSA was identified in 34.09% by cefoxitin disk diffusion, 34.9% by oxacillin screen agar, 34.4% by MIC and 37.3% by oxacillin disk diffusion. When selected isolates were tested with molecular methods, the cefoxitin disk diffusion and PCR tests were comparable. Prevalence of MRSA (34.09%) is quite high as in other studies. The oxacillin disk diffusion test which was used routinely earlier is showing low specificity (56%). Among all phenotypic methods, cefoxitin disk diffusion and PCR alone have similar sensitivity and specificity. Results of cefoxitin disk diffusion test are in concordance with the PCR for mecA gene. Thus, the test can be an alternative to PCR for detection of MRSA in resource constraint settings.

Comparison of results of fluconazole and voriconazole disk diffusion testing for Candida spp. with results from a central reference laboratory in the ARTEMIS DISK Global Antifungal Surveillance Program

The accuracy of antifungal susceptibility testing is important for reliable resistance surveillance and for the clinical management of patients with serious infections due to Candida spp. Our primary objective was to compare the results of fluconazole and voriconazole disk diffusion testing of 3227 Candida spp. performed by 47 centers participating in the ARTEMIS program with disk diffusion and MIC results obtained by the central reference laboratory. The overall categoric agreement between participant disk diffusion test results and reference MIC results was 87% for fluconazole and 95.2% for voriconazole. Likewise good agreement was observed between participant disk diffusion test results and reference laboratory disk diffusion test results, with an agreement of 90.5%, 1% very major error (VME), and 3.4% major error (ME) for fluconazole and 94.2%, 1.1% VME, and 2.5% ME for voriconazole. The disk diffusion test was reliable for detecting those isolates of Candida spp. that were characterized as resistant to fluconazole and voriconazole by MIC testing. External quality assurance data obtained by surveillance programs such as the ARTEMIS Global Antifungal Surveillance Program ensure the generation of useful surveillance data and result in the continued improvement of antifungal susceptibility testing protocols.

COMPARISON OF CEFOXITIN DISC DIFFUSION TEST, OXACILLIN SCREEN AGAR, AND PCR FOR mecA GENE FOR DETECTION OF MRSA

Cefoxitin is a potent inducer of the mecA regulatory system. It is being recommended for detection of methicillin resistance in Staphylococcus aureus (MRSA) when using disk diffusion testing. The aim of our study was to evaluate the efficacy of cefoxitin disc diffusion test to characterize MRSA and compare it with oxacillin agar screening and detection of mecA gene by PCR. Materials and Methods: Fifty strains of S. aureus isolated from clinical samples were used in the study. Routine antibiotic susceptibility testing was performed including oxacillin disk. Oxacillin screen agar plates with 4% NaCl and 6 µg/ml of oxacillin were inoculated and interpreted as per standard guidelines. Cefoxitin disc diffusion test was performed using 30 µg disc and zone sizes were measured. PCR for amplification of the mecA gene was performed. Results: Out of the 50 isolates, 28 were found to be methicillin resistant by oxacillin disc diffusion test, 30 were resistant by oxacillin screen agar method, and 32 were resistant with cefoxitin disc diffusion. For these 32 isolates mecA gene was positive. Conclusion: Results of cefoxitin disc diffusion test is in concordance with the PCR for mecA gene. Thus, the test can be an alternative to PCR for detection of MRSA in resource constraint settings.

Linezolid-ResistantStaphylococcus cohnii,Greece

To the Editor: Since 2003, linezolid has typically been used to treat infections caused by multidrug-resistant gram-positive cocci such as vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus (1). In Greece, a major problem is nosocomial dissemination of vancomycin-resistant enterococci. Use of linezolid for the treatment of such infections led to the emergence of linezolid–vancomycin resistant E. faecium; however, linezolid resistance of staphylococci is still relatively low in this country (2). We describe an outbreak caused by a linezolid-resistant S. cohnii in an intensive care unit (ICU) in Greece. From July through October 2007, nonrepetitive coagulase-negative staphylococci that exhibited resistance to linezolid were isolated from blood cultures from 4 separate patients hospitalized in the ICU at Sismanoglion General Hospital of Athens, a 450-bed tertiary care hospital. The ICU is a 10-bed, level II unit, comprising 2 rooms with 1 bed each and 2 rooms with 4 beds each. Each isolate was recovered in 2 of 2 blood culture sets per patient, indicating true bacteremia. The demographic and clinical information for the patients is described in the Table. The mean duration of time preceding linezolid therapy was 22 days. Table Clinical characteristics of 4 patients from whom linezolid-resistant Staphylococcus cohnii was isolated, Greece, 2007* Isolates were first identified to the species level by using an API Staph system (bioMerieux, la Balme les Grottes, France) and a molecular method based on the tuf gene followed by sequencing analysis (3). Susceptibility testing for various antimicrobial agents was performed by disk diffusion and using Clinical Laboratory Standards Institute criteria; susceptibilities were interpreted according to Institute guidelines (4). In addition, MICs to oxacillin, vancomycin, teicoplanin, quinupristin-dalfopristin, linezolid, daptomycin, and tigecycline were determined by Etest (AB Biodisk, Solna, Sweden) according to manufacturer’s instructions. Resistance genes mecA, vat, vga, erm, aac(6´)-Ie+aph(2″), ant(4′)-Ia, and aph(3′)-IIIa, as markers for resistance to β-lactams, dalfopristin, macrolides, and aminoglycosides, were identified by PCR as previously reported (5,6). The presence of G2576T in domain V of the 23S rRNA, which is mainly associated with linezolid resistance in clinical isolates, was detected by using PCR and digestion of the product with NheI (2). The number of mutated versus nonmutated alleles was determined as described by Pillai et al. (7). In addition, isolates were examined for the presence of the cfr gene, which was found to be correlated with linezolid resistance in some coagulase-negative staphylococci and for mutations of ribosomal protein L4, L22 genes (8,9). Clonality of isolates was assessed by pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with SmaI (2). The molecular method identified the isolates as S. cohnii subsp. ureolyticus. The API Staph system has correctly identified 2 of them; the remaining 2 isolates were falsely characterized as S. xylosus. According to disc diffusion test results, the isolates were resistant to cefoxitin, oxacillin, penicillin, rifampin, quinupristin-dalfopristin, erythromycin, clindamycin, fusidic acid, tobramycin, gentamicin, and linezolid. MICs were linezolid 32, oxacillin 256, quinupristin-dalfopristin 8, vancomycin 2, teicoplanin 2, tigecycline 0.125, and daptomycin 0.5 mg/L. Molecular methods detected the following resistance genes: mecA, ermA, aac(6´)-Ie+aph(2″), and aph(3´)-IIIa. The isolates, despite their resistance to streptogramins, were negative for vat and vgaA genes. In addition, all isolates carried the G2576T mutation and had 4 of 5 mutated alleles. No isolate carried the cfr gene or any mutation on ribosomal protein L4 and L22 genes. PFGE results indicated that all isolates were clonally related, belonging to the same clone. Outbreaks caused by linezolid-resistant staphylococci are rare worldwide (10); in Sismanoglion Hospital, before the outbreak period, no linezolid-resistant staphylococci and enterococci had been isolated. However, in the ICU, a statistically significant increase in the use of linezolid was observed in 2004 and in 2007 (0.58 vs. 1.34 defined daily doses/100 patient-days, respectively); heavy use of linezolid may have created substantial selection pressure in favor of the linezolid-resistant isolates. The 4 patients were treated in the same room by the same personnel; thus, a potential explanation for this outbreak is patient-to-patient transmission of linezolid-resistant strains on the hands of healthcare personnel. However, cultures of ICU personnel (nasal cavity and hands) grew only methicillin-resistant S. aureus and methicilllin-resistant S. epidermidis. In addition, environmental samples taken from the beds and the equipment of these patients were negative for S. cohnii. Strict control measures were taken (e.g., isolation of infected patients, increased environmental cleaning, and reinforcement of proper glove and gown use and hand disinfection with alcohol gel), and the outbreak strain was not recovered from other patients in the ICU or in other departments of the hospital after the initial outbreak. In conclusion, to avoid spread of staphylococcal resistance in ICUs, measures such as hand hygiene and adequate central venous catheter handling should be taken, and policies regarding antimicrobial drug use must be applied.

Suscetibilidade antimicrobiana in vitro dos Staphylococcus coagulase negativa oculares

To assess the in vitro susceptibility of conjunctival and corneal coagulase negative Staphylococcus (CoNS) to methicillin, fluoroquinolones and aminoglycosides. A total of 707 conjunctival and corneal CoNS disk diffusion test results were retrospectively analyzed, from January 2000 through December 2003. From 2000 to 2003, there was an increase in number of CoNS isolated from conjunctiva (n=57 to n=153) and cornea (n=28 to n=78). The frequency of conjunctival and corneal methicillin-resistant CoNS also increased (1.8 to 19.6% and 14.3 to 29.3%, respectively). There was no statistically significant difference between fluoroquinolones-resistant CoNS percentages in conjunctiva (ofloxacin: 1.8 to 7.8% and ciprofloxacin: 3.5 to 9.2%) and cornea (ofloxacin: 14.3 to 9.0% and ciprofloxacin: 14.3 to 10.3%). Evaluating the results of the conjunctival samples, there was increased resistance to tobramycin (15.8 to 34.0%) and to gentamycin (10.5 to 25.5%). There was no change in resistance of corneal isolates to tobramycin (28.6 to 26.9%) and to gentamycin (21.4 to 23.1%). there was a decrease in in vitro CoNS susceptibility to methicillin, tobramycin and gentamycin. Fuoroquinolones represented by ofloxacin and ciprofloxacin demonstrated stable in vitro susceptibility.

Comparison of cefoxitin and moxalactam 30 µg disc diffusion methods for detection of methicillin resistance in coagulase-negative staphylococci

To compare cefoxitin and/or moxalactam 30 microg disc diffusion (DD) methods to detect methicillin resistance in coagulase-negative staphylococci (CoNS) using both high- and low-density (HD/LD) inoculum techniques. A challenge set of 192 CoNS was tested. DD test results were compared with PBP2a detection. With the LD inoculum, the sensitivity/specificity of cefoxitin and moxalactam were 94.4%/100% and 100%/92.4%, respectively, using the DD breakpoints of the Comité de l'Antibiogramme de la Société Française de Microbiologie. With the HD inoculum, the sensitivity/specificity of cefoxitin and moxalactam were 93.7%/100% and 100%/96.9%, using the cefoxitin DD breakpoints of the CLSI and a resistant/susceptible breakpoint of < 20 mm/>or=20 mm for moxalactam. Comparison of receiver operating characteristic AUCs did not show significant difference between studied assays, but the overlapping zone where both PBP2a-positive and PBP2a-negative isolates were observed concerned a lower number of strains with moxalactam than with cefoxitin (P < 0.001). Combination of cefoxitin and moxalactam DD methods demonstrated that all isolates with a concordant cefoxitin/moxalactam phenotype were correctly classified. Interestingly, all isolates misclassified by each DD method used alone were cefoxitin-susceptible and moxalactam-resistant. Although all DD methods studied here performed well for detecting methicillin resistance in CoNS, moxalactam had a higher accuracy than cefoxitin to differentiate heteroresistant isolates from PBP2a-negative strains. Identification of isolates that should be submitted to a confirmatory test to conclude on methicillin resistance can be easily obtained by combining cefoxitin and moxalactam DD methods.

Evaluation of Antimicrobial Resistance Among Gram-Negative Isolates Collected from Intensive Care Units and Reliability of Routine Disc Susceptibility Tests at a Teaching Hospital in Tehran

Antibiotic resistance of Intensive Care Unit (ICU) born aerobic gram-negative bacteria was evaluated during a six months period at a teaching hospital in Tehran, and determination of the validity of the results obtained from disc diffusion tests, using discs manufactured in Iran. Disc susceptibility tests using Iranian and standard discs (diffusion discs available in international markets) were performed on 108 aerobic gram-negative isolates obtained from the clinical samples of patients with at least 72 hours of stay in the ICU. The Minimum Inhibitory Concentrations (MIC) was subsequently determined by collaborators not involved with the disc testing evaluation. Acinetobacter was the most frequently isolated gram negative species (26%). High resistance rates were obtained for all antibiotics studied except for imipenem (98% sensitive). Results of disc diffusion tests performed by the Iranian discs were in moderate to strong agreement with those obtained from the standard discs. When comparing disc results with the MIC results, it was noted that the total number of very major, and minor discrepancies were approximately the same with both sets of discs. The total number of major discrepancies was higher for the Iranian discs (more false positive results). The total number of very major discrepancy rates was more than the acceptable 1.5% limits for each antibiotic tested, using both Iranian and standard discs. High rates of resistance in aerobic gram-negative isolates studied, leaves imipenem as the only reliable agent for the empirical treatment of ICU infections. The high rates of very major discrepancies with both sets of discs show that physicians cannot rely on disc diffusion tests only, in order to guide therapy for the treatment of very serious infections in the ICUs, even if standard discs are used instead of the Iranian discs, and there is great need to establish a fast and easy way to determine the MIC values. Although it is better to repeat the study with a much larger sample size in order to make good judgment.

Daptomycin tested against 915 bloodstream isolates of viridans group streptococci (eight species) and Streptococcus bovis

To evaluate the activity of daptomycin tested against numerous species of viridans group streptococci and Streptococcus bovis, which are associated with wound infections, sepsis, cellulitis, endocarditis, abscesses and dental caries. The incidence of penicillin-resistant (non-susceptible) and MLS(B)-resistant strains among viridans group streptococci often varies by species. The activity of daptomycin was compared with seven other antimicrobial classes using reference broth microdilution and disc diffusion methods tested against 915 bacteraemic isolates of streptococci (815 viridans group strains; 100 S. bovis). Among all species of viridans group streptococci and S. bovis, 99.9% of isolates were susceptible to daptomycin (MIC values, < or = 0.016-2 mg/L). In contrast, penicillin, erythromycin and tetracycline susceptibility varied widely between species. Erythromycin susceptibility was in the range 48.6-88.7%, penicillin susceptibility in the range 65.5-98.1% and tetracycline in the range 35.0-93.9%. The inter-method agreement between daptomycin and linezolid resistance (comparison agent) disc diffusion and broth microdilution test results was high, each showing near complete susceptibility (99.9%). Daptomycin is an active antimicrobial agent that has a usable potency against eight species of viridans group streptococci, as well as S. bovis, with all MIC values at < or =2 mg/L.

Observatoire du pneumocoque de la région Centre, du 1 er avril 1999 au 31 mars 2000

Seven hundred and ninety six strains of pneumococcus were collected in the Centre region, from 15 laboratories, between 1 st April 1999 and 31 st of March 2000. Data were processed, using 4 th dimension software, and concerned age, file number, consultation/hospitalisation, sample type, susceptibility to oxacillin (5 μg), results of the E-test ® for benzylpenicillin, amoxicillin, cefotaxime and results of the routine disc diffusion test. Strains with reduced susceptibility to benzylpenicillin (PRSP) were collected by the co-ordinating centre to perform MICs by the reference agar dilution test and serotyping. Out of 796 strains, 450 strains (56.7%) were categorised as PRSP and 400 of them were studied by the co-ordinating centre. Forty two percent of the samples originated from lungs, followed by 19.5% from blood samples, 15% from ear pus (85.7% PRSP) and 2.5% from CSF. Thirty nine percent of the patients were female. 36.6% were children under sixteen (70.1% PRSP) and 62.4% were adults (49.2% PRSP). Out of 400 PRSP 106 (26.5%) were characterised as resistant and 294 (73.5%) as intermediate to benzylpenicillin. Compared to the agar dilution test, 90% of the PRSP studied by E-test ® had a MIC value for benzylpenicillin within ±1 dilution. Thirty six strains of PRSP were resistant to amoxicillin (9% of the PRSP) and 10 (2.5% of the PRSP) to cefotaxime. Serotyping was done on 375 strains. The serotypes encountered were the following: 23 (26.9%), 14 (22.1%), 19 (19.5%), 6 (12.8%), 9 (9.9%) and 15 (5.1%).

Patterns of phenotypic resistance to the macrolide-lincosamide-ketolide-streptogramin group of antibiotics in staphylococci.

Phenotypes of resistance to the macrolide-lincosamide-ketolide-streptogramin (MLKS) group of antibiotics have been determined in 540 clinical isolates of staphylococci (210 Staphylococcus aureus and 330 coagulase-negative species). Results of disc diffusion tests using erythromycin A, oleandomycin, rokitamycin, clindamycin, telithromycin, quinupristin and dalfopristin delineated four main groups corresponding to those defined classically using erythromycin and clindamycin only, but with sub-divisions. Resistance to erythromycin was more common in coagulase-negative strains (56%) than in S. aureus (16%); telithromycin, clindamycin, quinupristin-dalfopristin and rokitamycin were active against >97% of S. aureus strains and >88% of the coagulase-negative strains. The commonest resistance phenotype was 'inducible MLS(B)' (12% in S. aureus, 31% in coagulase-negative strains); this group could be divided in terms of the different inducing abilities of erythromycin and oleandomycin. 'Constitutive MLS(B)' and 'MS' phenotypes were more often found in coagulase-negative strains (11 and 13%, respectively) than in S. aureus (2 and 1%). Novel phenotypes were found during the isolation of constitutively resistant mutants from inducible strains, and of resistant mutants from 'MS' strains. This extended phenotyping scheme has revealed further complexities and evolutionary possibilities in patterns of resistance to this group of antibiotics.

In vitro susceptibility of porcine respiratory pathogens to tilmicosin.

Bacterial isolates obtained from swine with various clinical diseases were tested for susceptibility to tilmicosin by minimum inhibitory concentration (MIC) and Kirby-Bauer disk diffusion tests using National Committee on Clinical Laboratory Standards methodology. The tilmicosin MIC90 was < or =0.125 microg/ml for Erysiopelothrix rhusiopathiae, < or = 1 microg/ml for Haemophilus parasuis isolates, 8 microg/ml for Actinobacillus suis and Pasteurella multocida type A, 16 microg/ml for toxigenic and nontoxigenic P. multocida type D, 64 microg/ml for Bordetella bronchiseptica, and >128 microg/ml for Staphylococcus hyicus and Streptococcus suis. The results of disk diffusion testing matched well with the MIC results for each pathogen. This in vitro survey of tilmicosin activity against various swine isolates suggests that further clinical evaluation of tilmicosin in swine may be warranted for disease associated with E. rhusiopathiae, H. parasuis, and A. suis but not B. bronchiseptica, S. suis, or S. hyicus.

Rapid dissemination of Staphylococcus aureus with classic oxacillin resistance phenotype at a new university hospital

To determine the prevalence rates of oxacillin-resistant Staphylococcus aureus (ORSA) at a new university hospital since its opening, the results of disk diffusion tests on all clinical isolates, recovered between 1990 and 1998 at the National Cheng Kung University Hospital, were reviewed. In order, to investigate the mechanisms of oxacillin resistance among strains of S. aureus in Taiwan, MICs were determined by an agar dilution method, and polymerase chain reaction and colony hybridization assays were performed on 288 isolates collected during November 1998 to detect the mecA gene. The prevalence rates of ORSA increased rapidly from 14·1% in 1990 to 61·0% in 1998. The increasing rates were most rapid in the first four-year period, ranging from 11·6 to 106·7% per year, and became steady after 1994, ranging from 1·8% to 11·6%. Of 288 clinical isolates collected in November 1998, 206 (71·5%) were resistant to oxacillin (MIC ≥ 16 mg/L), and four were borderline resistant (MIC 2–8 mg/L). All 210 strains possessed the mec A gene (classic resistance). The present study demonstrated that ORSA could disseminate in a new hospital with great speed, and indicated that all ORSA strains in Taiwan revealed classic resistance phenotype.

Calibration of the disk diffusion test for trovafloxacin susceptibility testing of four anaerobic species

To study trovafloxacin susceptibility among clinical isolates of four anaerobic bacterial species using minimum inhibitory concentrations (MIC) determinations, E test assays and disk diffusion test results and to calibrate the disk diffusion method for these species using single strain regression analysis (SRA). One-hundred and eighty-seven clinical isolates of four anaerobic bacterial species were included. Trovafloxacin MIC determinations were performed using the agar dilution technique and MIC estimations using the E test. The disk diffusion test was performed according to Swedish Reference Group for Antibiotics standardization. NCCLS limits for susceptibility categories were applied. SRA was performed using 1, 3, 10, 30, and 100 microg trovafloxacin disk contents and ATCC control strains. The regression lines obtained permitted the calculation of zone equivalents to MIC limits as well as an evaluation of various disk potencies. Trovafloxacin susceptibility (S + I) was noted in 98.9, 100, 100, and 97% of Bacteroides fragilis, Bacteroides thetaiotaomicron, Clostridium perfringens, and Peptostreptococcus magnus strains, respectively, as judged by MIC determinations. Agar dilution and E test estimations gave the same results, but E test values were consistently lower than MIC values by the reference method. Regression lines calculated for the four species using SRA showed different equation constants indicating species-related differences. Interpretive zone diameter breakpoints were calculated for the four species and used for the interpretation of susceptibility. The disk diffusion test was successfully calibrated for trovafloxacin susceptibility testing of four anaerobic species using single strain regression analysis, SRA. There was a good agreement between the results of MIC-tests and disk testing. Interpretive errors of type I are prone to occur among Bacteroides isolates and might require species-related MIC limits. SRA calculations permitted the testing of the effect of different disk potencies on inhibition zones produced at the interpretive MIC limits. Criteria for the selection of a minimal disk content showed that 5 microg trovafloxacin is sufficient, but a 10 microg disk will safeguard against residual laboratory variation without producing too large inhibition zones for very susceptible strains.

An evaluation of susceptibility testing methods for ampicillin‐sulbactam using a panel of β‐lactamase‐producing bacteria

Bacteria possessing TEM-1-like beta-lactamases are generally regarded as susceptible to ampicillin-sulbactam (SAM), while those harboring OXA-1 enzymes are considered resistant. The current study was undertaken to compare susceptibility testing using various combinations of ampicillin and sulbactam to improve clinical correlation. Members of the Enterobacteriaceae family harboring TEM-1, SHV-1 or OXA-1-like beta-lactamases were tested using the agar dilution method. A substantial proportion of strains harboring OXA-1-like beta-lactamases showed false susceptibility to SAM at the 1:1 ratio or fixed sulbactam concentration of 8 microg/ml. At a fixed sulbactam concentration of 4 microg/ml, the activity of ampicillin-sulbactam appeared to be reduced, with large numbers of TEM-1 producers becoming frankly resistant. Results obtained with the 2:1 ratio exhibited the closest correlation with that obtained by the currently recommended disk diffusion test. However, very major errors were still found between the disk diffusion test and agar dilution test, suggesting the necessity for consideration of a change in criteria for interpretation of disk diffusion test results. In conclusion, SAM susceptibility testing by agar dilution using other than a 2:1 ratio is not recommended and results should be interpreted with caution.

Calibration of disk diffusion antibiotic susceptibility testing: species-related trovafloxacin interpretive zone breakpoints and selection of disk potency.

International comparisons of antibiotic susceptibility require the use of common minimum inhibitory concentration (MIC) limits. Disk diffusion test results are not directly suitable for such comparisons, since different standards are often used and zone breakpoints issued might reflect different MIC limits. We have used single strain regression analysis (SRA) for the calibration of the disk test, both according to species and individual laboratory, and for quality control of trovafloxacin disk diffusion tests in 5 laboratories in Sweden. Preliminary controls using histogram analysis including subtraction histograms of reference strains revealed marked differences between different laboratories. SRA was performed on 4 reference strains, S. aureus, E. faecalis, E. coli and P. aeruginosa, using disks containing 1, 3, 10, 30 and 100 microg trovafloxacin. The results using SRA showed a difference between laboratories using Biodisk PDM medium, which produced smaller zones, and those using Oxoid IsoSensitest. Species-related regression lines for laboratories using either medium were calculated and corresponding interpretive zone breakpoints determined for MIC limits. Rational criteria for the selection of a suitable disk content of an antibiotic were also defined and applied to trovafloxacin. The 10 microg disk selected by NCCLS (National Committee for Clinical Laboratory Standards) proved optimal.

In vitro activity of trovafloxacin against ciprofloxacin-susceptible and -resistant clinical bacterial isolates and assessment of the trovafloxacin disk test

A total of 4241 consecutive clinical bacterial isolates from 10 North American medical centers were tested for susceptibility to trovafloxacin. Trovafloxacin was significantly more active than ciprofloxacin against Gram-positive bacteria, Acinetobacter spp., and Stenotrophomonas maltophilia, and resistance to trovafloxacin occurred in these groups only among isolates with high-level resistance (MIC ≥16 μg/mL) to ciprofloxacin. With other species, the two drugs had comparable activity. Concerns about staphylococci and Pseudomonas aeruginosa with trovafloxacin MICs of 2.0 μg/mL (the upper end of the susceptible category) are discussed. Results of trovafloxacin disk diffusion test on more than 3200 nonfastidious isolates supported the FDA-approved zone size interpretive criteria when the MIC breakpoint of ≤2.0 μg/mL is used to define the trovafloxacin-susceptible category.

Characterization of multiple-antibiotic-resistant Salmonella typhimurium stains: molecular epidemiology of PER-1-producing isolates and evidence for nosocomial plasmid exchange by a clone

We characterized epidemiologic and genetic features of nosocomially originated multiple-antibiotic-resistant Salmonella typhimurium isolates from two hospitals. A total of 32 multiply resistant strains, isolated during a 28-month period, were studied. Four resistance phenotypes were distinguished on the basis of the results of disc diffusion tests. Group 1 was resistant to chloramphenicol, gentamicin, tobramycin, amikacin, and the newer cephalosporins because of the production of an extended-spectrum beta-lactamase (PER-1). Group 2 exhibited the same pattern plus resistance to sulfamethoxazole-trimethoprim (Sxt). Except for Sxt resistance, dominant phenotypes of both groups were transferred on an identical plasmid, pSTI1 (81 MDa). Group 3 was resistant to ampicillin, chloramphenicol, gentamicin, tobramycin, and Sxt. This pattern was also transferred on an 81-MDa plasmid (pSTI2) which differed from pSTI1 on the basis of EcoRI and HindIII restriction fragments. Group 4 was resistant to ampicillin, chloramphenicol, and tetracycline, and a 74-MDa nonconjugative plasmid was detected. Restriction fragment length polymorphism of RNA-encoding DNA and arbitrarily primed PCR tests revealed that bacteria from groups 1, 2, and 3 were clonally related. Epidemiologic data also supported the clonal-dissemination hypothesis. We concluded that S. typhimurium isolates acquire and exchange multiple-resistance plasmids in hospital microflora.

Antimicrobial activity of fusidic acid and disk diffusion susceptibility testing criteria for gram-positive cocci

The in vitro activity of fusidic acid was assessed and was compared with those of cloxacillin, cefamandole, vancomycin, teicoplanin, ofloxacin, ciprofloxacin, pefloxacin, and fleroxacin against 500 gram-positive cocci: 151 Staphylococcus aureus, 197 coagulase-negative staphylococci, and 152 Enterococcus faecalis strains. All clinical isolates were concomitantly tested by disk diffusion and agar dilution procedures as outlined by the National Committee for Clinical Laboratory Standards. The results with fusidic acid were further analyzed by regression line and error rate-bounded methods. With control American Type Culture Collection organisms, the values were within the limits of the National Committee for Clinical Laboratory Standards or published limits. The incidence of resistance to fusidic acid was 0.7% for S. aureus, 2.5% for coagulase-negative staphylococci, and 99.3% for E. faecalis. The correlation coefficient between the results of disk diffusion and agar dilution tests with fusidic acid was 0.90. Current interpretive criteria for susceptibility to fusidic acid (i.e., MIC of < 2 micrograms/ml and inhibitory zone of 20 mm) gave 1% false susceptibility (all strains being E. faecalis). This error rate is practically eliminated if a zone diameter of 21 mm is considered the breakpoint for susceptibility.

Preliminary interpretive criteria for in vitro susceptibility testing of CP-99219 by dilution and disk diffusion methods

CP-99219 is a novel 7-[3-azabicyclo (3.1.0) hexyl] quinolone that possesses activity against a wide variety of aerobic and anaerobic pathogens. The disk diffusion test results (5-μg CP-99219 disks) were compared with CP-99219 minimum inhibitory concentrations (MICs) using 445 contemporary clinical isolates, including 210 strains that were resistant to ciprofloxacin (MICs, ⩾4 μg/ml). A high correlation was observed between methods (r = 0.97), and the proposed conservative zone diameter interpretive criteria were selected primarily from the regression equation as follows: susceptible at ⩾15 mm (⩽2 μg/ml, preferred) and resistant at ⩽10 mm (⩾8 μg/ml); or susceptible at ⩾19 mm (⩽1 μg/ml) and resistant at ⩽14 mm (⩾4 μg/ml). Using these criteria, the occurrence of false-resistant and false-susceptible disk diffusion test results should remain rare (⩽1.5%).

DETECTION OF METHICILLIN-RESISTANT STAPHYLOCOCCI

Susceptibility tests commonly used in the clinical laboratory for detection of methicillin-resistant strains of staphylococci are discussed. The biochemical and genetic basis of methicillin resistance, including heterogeneous expression of resistance, is summarized. The conditions that can affect results of disk diffusion, broth dilution, and agar screen tests and sensitivity and specificity of these tests are reviewed. DNA detection methodologies, which, with their greater accuracy, may replace susceptibility testing, also are discussed.