Practical disk diffusion method for detection of inducible clindamycin resistance in Staphylococcus aureus and coagulase-negative staphylococci

Resistance to macrolides in staphylococci may be due to active efflux (encoded by msrA) or ribosomal target modification (macrolide-lincosamide-streptogramin B [MLSB] resistance; usually encoded by ermA or ermC). MLSB resistance is either constitutive or inducible following exposure to a macrolide. Induction tests utilize closely approximated erythromycin and clindamycin disks; the flattening of the clindamycin zone adjacent to the erythromycin disk indicates inducible MLSB resistance. The present study reassessed the reliability of placing erythromycin and clindamycin disks in adjacent positions (26 to 28 mm apart) in a standard disk dispenser, compared to distances of 15 or 20 mm. A group of 130 clinical isolates of Staphylococcus aureus and 100 isolates of erythromycin-resistant coagulase-negative staphylococci (CNS) were examined by disk approximation; all CNS isolates and a subset of S. aureus isolates were examined by PCR for ermA, ermC, and msrA. Of 114 erythromycin-resistant S. aureus isolates, 39 demonstrated constitutive resistance to clindamycin, while 33 showed inducible resistance by disk approximation at all three distances. Only one isolate failed to clearly demonstrate induction at 26 mm. Of 82 erythromycin-resistant CNS isolates that contained ermA or ermC, 57 demonstrated constitutive clindamycin resistance, and 25 demonstrated inducible resistance, at 20 and 26 mm. None of the 42 S. aureus isolates or 18 CNS isolates containing only msrA and none of the erythromycin-susceptible isolates yielded positive disk approximation tests. Simple placement of erythromycin and clindamycin disks at a distance achieved with a standard disk dispenser allowed detection of 97% of S. aureus strains and 100% of CNS strains with inducible MLSB resistance in this study.     

Agar dilution method for detection of inducible clindamycin resistance in Staphylococcus spp

We describe the development and validation of an agar dilution method for the detection of inducible clindamycin resistance by using 227 previously characterized erythromycin-resistant, clindamycin-susceptible Staphylococcus sp. isolates. Mueller-Hinton agar with defibrinated horse blood containing a range of erythromycin concentrations (1 to 8 mg/liter) combined with clindamycin at 0.5 mg/liter was used to determine the optimal concentration that produced growth of inducible isolates while inhibiting that of isolates without the inducible phenotype. A concentration of clindamycin of 0.5 mg/liter with erythromycin at 1 mg/liter was the optimal combination for detection of inducible resistance and resulted in a sensitivity of 100% (95% confidence interval [CI], 97.9 to 100) and a specificity of 100% (95% CI, 93.0 to 100). Attention must be paid to ensuring that a sufficient inoculum has been used, since an inoculum below the standard 10(7) bacteria/ml may result in false-negative results. This method has been incorporated into routine use in our laboratory.     

Collaborative evaluation of an erythromycin-clindamycin combination well for detection of inducible clindamycin resistance in beta-hemolytic streptococci by use of the CLSI broth microdilution method

Constitutive or inducible clindamycin resistance can occur in beta-hemolytic streptococci due to the presence of an erm gene. The Clinical and Laboratory Standards Institute (CLSI) has recommended a disk approximation test (D-zone test) with erythromycin and clindamycin disks and a single-well broth test combining erythromycin and clindamycin for detection of inducible clindamycin resistance in staphylococci, but only a disk approximation test for the beta-hemolytic streptococci. This collaborative study assessed two different erythromycin and clindamycin concentration combinations in single wells (1 μg/ml + 0.25 μg/ml [erythromycin plus clindamycin] and 1 μg/ml + 0.5 μg/ml) with three different brands of Mueller-Hinton broth supplemented with 3% lysed horse blood for testing of frozen panels prepared for this study. All labs performed the D-zone test as described by the CLSI. A total of 155 nonduplicate streptococcal isolates (50 group A, 48 group B, 28 group C, and 29 group G isolates) were tested; 99 isolates showed inducible resistance by the D-zone test. There were some differences noted based upon the test medium. The sensitivity of the erythromycin plus clindamycin combination of 1 μg/ml + 0.25 μg/ml was 91 to 100%, while the sensitivity of the combination of 1 μg/ml + 0.5 μg/ml was 95 to 100%. Specificity overall was 98%. The slightly higher sensitivity of the combination of 1 μg/ml + 0.5 μg/ml is recommended. This study has demonstrated that a single-well microdilution test incorporating erythromycin and clindamycin in combination is a sensitive and specific indicator of inducible clindamycin resistance and could be included in routine test panels.     

Influence of disk separation distance on accuracy of the disk approximation test for detection of inducible clindamycin resistance in Staphylococcus spp

We undertook this study to assess the accuracy of the clindamycin-erythromycin disk approximation test (D-test) for detection of inducible clindamycin resistance in Staphylococcus spp. One hundred sixty-three Staphylococcus aureus and 68 coagulase-negative Staphylococcus (CoNS) spp. which were erythromycin nonsusceptible but clindamycin susceptible were tested using the D-test performed at both 15-mm and 22-mm disk separations and compared with genotyping as the "gold standard." The rate of inducible clindamycin resistance was 96.3% for S. aureus and 33.8% for CoNS spp. The sensitivities of the D-tests performed at 15 mm and 22 mm were 100% and 87.7%, respectively, and specificities were 100% for both. The use of 22-mm disk separation for the D-test to detect inducible clindamycin resistance results in an unacceptably high very major error rate (12.3%). All isolates with false-negative results harbored the ermA gene, and the majority were methicillin-resistant Staphylococcus aureus. False-negative results were associated with smaller clindamycin zone sizes and double-edged zones. We recommend using a disk separation distance of 相似文献   

Evaluation of disk approximation and single-well broth tests for detection of inducible clindamycin resistance in Streptococcus pneumoniae

This study evaluated an agar disk diffusion D-zone test and an erythromycin-clindamycin (ERY + CLI) single-well broth test for inducible CLI resistance in Streptococcus pneumoniae. The standard CLSI disk approximation test and a single-well combination test incorporating 1 plus 0.5 μg/ml ERY + CLI detected >96% of isolates containing the ermB determinant.     

Incidence of inducible clindamycin resistance in staphylococci: first results from Turkey

In total, 408 staphylococcal isolates were tested for inducible clindamycin resistance (ICR) by the disk-diffusion induction test (D-test). ICR was detected in 5.7% of 105 methicillin-resistant Staphylococcus aureus (MRSA) isolates, 3.6% of 111 methicillin-susceptible S. aureus isolates, 30.8% of 94 methicillin-resistant coagulase-negative staphylococcal (CoNS) isolates, and 11.2% of 98 methicillin-sensitive CoNS isolates. All MRSA isolates that were erythromycin-resistant and clindamycin-susceptible were positive by the D-test. The same results were obtained with an azithromycin instead of an erythromycin disk. All isolates were susceptible to quinupristin-dalfopristin. The cost-benefit of the d-test should be evaluated locally after determining the incidence of the different resistance phenotypes.     

Prevalence of inducible clindamycin resistance in gram positive organisms in a tertiary care centre

Gram positive organisms are one of the leading pathogens causing skin and soft tissue infections. For these infections, clindamycin is a useful alternate drug in penicillin-allergic patients. This study was conducted to investigate the prevalence of erythromycin-induced clindamycin resistance in gram positive organisms in the southern part of the country. A total of 522 consecutive clinical isolates from blood, CSF, sputum, throat, pus, and urine were collected between November 2006 and April 2007 and tested for erythromycin resistance and inducible clindamycin resistance. There was a relatively higher incidence of inducible clindamycin resistance among the MRSA isolates. We conclude, therefore, that clindamycin is not a suitable alternative antibiotic for use in staphylococcal skin and soft tissue infections.     

Prevalence of inducible clindamycin resistance in staphylococcal isolates at a Korean tertiary care hospital

Clindamycin resistance in Staphylococcus species can be either constitutive or inducible. Inducible resistance cannot be detected by the conventional antimicrobial susceptibility test. In this study, we determined the prevalence of inducible clindamycin resistance in staphylococcal isolates at a Korean tertiary care hospital. Between February and September 2004, 1,519 isolates of Staphylococcus aureus and 1,043 isolates of coagulase-negative staphylococci (CNS) were tested for inducible resistance by the D-zone test. Overall, 17% of MRSA, 84% of MSSA, 37% of MRCNS, and 70% of MSCNS were susceptible to clindamycin. Of the erythromycin non-susceptible, clindamycin-susceptible isolates, 32% of MRSA, 35% of MSSA, 90% of MRCNS, and 94% of MSCNS had inducible clindamycin resistance. Inducible clindamycin resistance in staphylococci was highly prevalent in Korea. This study indicates importance of the D-zone test in detecting inducible clindamycin resistance in staphylococci to aid in the optimal treatment of patients.     

Testing for induction of clindamycin resistance in erythromycin-resistant isolates of Staphylococcus aureus

Disk diffusion and broth microdilution (BMD) were used to perform clindamycin (CLI) induction testing on 128 selected nonduplicate isolates of Staphylococcus aureus. Disk diffusion testing involved placing CLI and erythromycin (ERY) disks approximately 12 mm apart (measured edge to edge) on a Mueller-Hinton agar plate that had been inoculated with an S. aureus isolate; the plate was then incubated for 16 to 18 h. Two distinct induction phenotypes (labeled D and D(+)) and four noninduction phenotypes (designated as negative [Neg], hazy D zone [HD], resistant [R], and susceptible [S]) were observed in disk diffusion results. A clear, D-shaped zone of inhibition around the CLI disk was designated as the D phenotype and was observed for 21 isolates while a D-shaped zone containing inner colonies growing up to the CLI disk was designated as D(+) (17 isolates). In addition, 10 isolates were CLI susceptible and ERY resistant but were not inducible and showed no blunting of the CLI zone (Neg phenotype). Isolates that were CLI and ERY resistant (constitutive macrolide-lincosamide-streptogramin B resistance) demonstrated either a double zone of inhibition with an inner ring of reduced growth up to the edge of the disks (HD phenotype; 33 isolates) or solid growth around the CLI and ERY disks (R phenotype; 16 isolates). Finally, 31 isolates were susceptible by disk testing to both CLI and ERY (S phenotype). PCR results showed that isolates with a D phenotype harbored ermA, isolates with a D(+) phenotype contained either ermC (16 isolates) or ermA and ermC (one isolate), and all 10 isolates with a Neg phenotype contained msrA. All isolates with an HD or R phenotype harbored at least one erm gene. Isolates showing the D(+) phenotype by disk diffusion were also detected by BMD using a variety of CLI and ERY concentrations; however, isolates with the D phenotype were more difficult to detect by BMD and will likely require optimization of ERY and CLI concentrations in multilaboratory studies to ensure adequate sensitivity. Thus, at present, disk diffusion is the preferred method for testing S. aureus isolates for inducible CLI resistance.     

Phenotypic detection of inducible clindamycin resistance among Staphylococcus aureus isolates by using the lower limit of recommended inter-disk distance

Context: Clindamycin is one of the important alternative antibiotics in the therapy of Staphylococcus aureus, particularly in methicillin-resistant S. aureus (MRSA) infections. Inducible clindamycin resistance (iMLS B - inducible Macrolide-Lincosamide-Streptogramin B resistance) is a critical factor in antimicrobial susceptibility testing. Aims: To know the rate of inducible clindamycin resistance among clinical isolates of Staphylococcus aureus in our hospital by Disk approximation test (D-test) using the average recommended inter-disk distance and comparing the results with that of D-test using the lower limit of recommended inter-disk distance. Materials and Methods: A total of 51 erythromycin-resistant and clindamycin-susceptible S. aureus isolates were subjected to disk approximation testing with 21 +/- 1 mm and 15 mm edge-to-edge distance between the clindamycin and erythromycin disks. Statistical Methods: Z-test levels. Results: Among 51 erythromycin-resistant and clindamycin-susceptible S. aureus isolates, 25 (49%) were recorded as inducible clindamycin resistant by D-test with 21 +/- 1 mm edge-to-edge distance between the clindamycin and erythromycin disks. When we re-tested all the 51 strains by D-test with 15 mm inter-disk distance, we identified 14% more iMLS B strains previously reported as D-test negative. Z-test for MRSA indicates that 15 mm edge-to-edge distance has significant advantage. Conclusions: Since the incidence of inducible clindamycin resistance is high (63% in our study), accurate identification of inducible clindamycin resistance is important to prevent therapeutic failure in infections caused by these strains. We suggest the use of D-test with 15 mm edge-to-edge inter-disk distance for detecting iMLS B .     

Evaluation of the 10-micrograms clindamycin disk for susceptibility testing of anaerobes by the aerobically incubated thioglycolate broth disk method.

The reliability of the 10-micrograms clindamycin disk was evaluated for susceptibility testing of anaerobes by the aerobic thioglycolate broth disk method. A good correlation between the aerobic thioglycolate broth disk method and the reference agar dilution procedure of the National Committee for Clinical Laboratory Standards was obtained by using a 4-microgram/ml breakpoint. Improved correlation was obtained when the medium of the National Committee for Clinical Laboratory Standards was buffered.     

Enzyme induction in man: a study of the inducible systems of drug elimination

The individual enzyme inducibility was studied with methods of menthol loading and sulphobromophthalein in groups of patients with low and average metabolism. Both methods are suitable for testing inducible conjugation systems by providing indirect information on the rate of drug elimination. In the low-metabolism groups the response in per cent correlated inversely with the initial value, and the changes were significant not only in the low but also in the average-metabolism group at the end of 30 days of phenobarbital treatment. The results are discussed with regard to the avoidability of undesired drug effects.     

Use of a DNA hybridization method to verify results of screening for methicillin resistance in Staphylococci

Tests were performed by the disk diffusion method, agar dilution method and the E test to determine the susceptibility to methicillin and oxacillin of clinical isolates and control strains ofStaphylococcus aureus (n=106) and coagulase-negative species (n=131). Results were compared with those of a dot blot DNA hybridization test, in which themecA gene was detected using an oligonucleotide probe selected from themecA gene. Among theStaphylococcus aureus strains themecA gene was found in all but two strains inhibited by 8 mg/l of methicillin and all but two strains inhibited by 4 mg/l of oxacillin. A disk test using either 1 µg oxacillin or 10 µg methicillin and a tentative resistance breakpoint of 10 mm gave the best agreement with the hybridization test. For coagulase-negative staphylococci 34 of 35 strains inhibited by 8 mg/l methicillin hybridized with the probe as well as 58 of 82 strains inhibited by 1–4 mg/l; 93 of 97 strains inhibited by 0.5 mg/l oxacillin were also positive in the probe test. Using the 1 µg oxacillin disk and a resistance breakpoint of 10 mm good agreement was obtained between results of the disk diffusion and DNA hybridization tests. It is suggested that this genotypic method for detection of methicillin resistance is used as a reference method for routine methods.     

Rapid detection of methicillin resistance in coagulase-negative Staphylococci with the VITEK 2 system

The aim of the present study was to evaluate the accuracy of the new VITEK 2 system (bioMérieux, Marcy l' Etoile, France) for the detection of methicillin resistance in coagulase-negative staphylococci (CoNS) by using AST-P515 and AST-P523 test cards. Analyses of the VITEK 2 oxacillin MIC determination evaluated according to the actual breakpoint (>/=0.5 micro g/ml) of the National Committee for Clinical Laboratory Standards resulted in a high sensitivity of 99.2% but a moderate specificity of 80%. The newly included oxacillin resistance (OR) test of the VITEK 2 system displayed a high sensitivity and a high specificity of 97.5 and 98.7%, respectively. Concordance between the results of the mecA PCR and the VITEK 2 oxacillin MIC was observed for almost all Staphylococcus epidermidis strains, but the reduced specificity was attributable to higher oxacillin MICs for mecA-negative non-S. epidermidis strains, especially S. saprophyticus, S. lugdunensis, and S. cohnii. Evaluation of alternative oxacillin MIC breakpoints of 1, 2, or 4 micro g/ml resulted in improved degrees of specificity of 84, 90.7, and 97.3%, respectively. Only minor changes occurred in the corresponding sensitivity values, which were 98.4, 97.5, and 97.5%, respectively. Methicillin resistance in CoNS was detected after 7 and 8 h in 91.1 and 93.5% of the mecA-positive strains, respectively, by the VITEK 2 OR test and in 86.3 and 89.5% of the mecA-positive strains, respectively, by VITEK 2 oxacillin MIC determination. After 7 and 8 h the VITEK 2 OR test classified 59.2 and 78.9% of the mecA-negative strains, respectively, as susceptible to oxacillin, whereas comparable values were obtained 2 h later by VITEK 2 oxacillin MIC determination. The results of our study encourage the use of the VITEK 2 system, which proved to be a highly reliable and rapid phenotypic method for the detection of methicillin resistance in CoNS.     

Evaluation of the CLSI cefoxitin 30-µg disk-diffusion method for detecting methicillin resistance in staphylococci

Methicillin susceptibility of 415 staphylococcal isolates from Chinese hospitals was assessed using the CLSI disk-diffusion method with a cefoxitin 30-microg disk in comparison with an oxacillin 1-microg disk. PCR-based detection of mecA was the reference standard. The cefoxitin 30-microg disk performed with almost the same high level of accuracy as the oxacillin 1-microg disk in detecting methicillin resistance in Staphylococcus aureus. For coagulase-negative staphylococci (CoNS), the sensitivity of the cefoxitin 30-microg disk was 90.5%, compared with 83.4% for the oxacillin 1-microg disk. Confirmatory testing of isolates with borderline susceptibility and revision of the cefoxitin breakpoint are proposed in order to categorise CoNS more accurately.     

Performance of a New MicroScan WalkAway PC30 panel and disk diffusion method for detection of oxacillin resistance in Staphylococcus spp

The performance of the MicroScan WalkAway PC30 panel for detection of oxacillin resistance was evaluated by use of a collection of 420 staphylococcus isolates. The addition of a cefoxitin test (4 mg/liter) to the oxacillin MIC determination increased its raw performance for Staphylococcus aureus; additional data were required for coagulase-negative staphylococci.     

Incidence of constitutive and inducible clindamycin resistance in Staphylococcus aureus and coagulase-negative staphylococci in a community and a tertiary care hospital

The incidences of inducible clindamycin resistance at two hospitals (an inner-city hospital and a suburban community hospital) were 7 and 12% for methicillin-resistant Staphylococcus aureus, 20 and 19% for methicillin-susceptible S. aureus, and 14 and 35% for coagulase-negative staphylococci, respectively. Given the variability of inducible resistance to clindamycin found in our two hospitals, we conclude that susceptibility testing of staphylococci should include the disk diffusion induction test (D-test).     

Problems with the disk diffusion test for detection of vancomycin resistance in enterococci.

A total of 53 strains of enterococci, including recently isolated strains with high-level resistance to vancomycin, were tested for vancomycin susceptibility by broth microdilution and disk diffusion using Mueller-Hinton media with and without supplementation with 5% blood. By using currently published parameters of the National Committee for Clinical Laboratory Standards for the disk diffusion test, we found that strains for which MICs were 8 to 32 micrograms/ml were incorrectly placed in the susceptible or intermediate category, which caused both very major (1.9%) and minor (11.5%) errors. When we used newer, recently proposed breakpoints for vancomycin, we found 13.5% minor errors but no very major errors. Changing disk diffusion breakpoints to less than or equal to 14 mm for resistant [corrected] and greater than or equal to 15 mm for susceptible [corrected] would eliminate the problem for the strains with MICs of 32 micrograms/ml but not for those with MICs of 8 micrograms/ml. For those strains, it is necessary to perform an MIC test to differentiate them from strains with MICs of less than or equal to 4 micrograms/ml.