Antimicrobial resistance in invasive and colonising Streptococcus pneumoniae in North India

The present study was done to detect the antibiotic resistance in S. pneumoniae. One hundred twenty S. pneumoniae isolates from clinical specimens and 50 from nasopharyngeal sites were subjected to antimicrobial susceptibility testing by Kirby Bauer disk diffusion method and minimum inhibitory concentration (MIC) determination for penicillin and cefotaxime non-susceptible isolates. A total of 22 isolates (18.3%) from clinical sites and eight (16%) from nasopharyngeal sites showed decreased susceptibility to penicillin by oxacillin disk diffusion test. MICs of 26 of these resistant strains ranged from 0.12-1 microg/mL (intermediate resistance) by broth dilution and E test. Only four isolates, two from sputum and two from nasopharyngeal swabs, showed MIC of 2 microg/mL (complete resistance). However, MIC of two cefotaxime resistant isolates (by disk diffusion) was in the susceptible range (0.5 microg/mL). Highest antimicrobial resistance was seen to cotrimoxazole (55.2%) and tetracycline (61.2%). Antimicrobial resistance to cotrimoxazole and tetracycline was much more in clinical isolates than colonizing isolates. Multi-drug resistant phenotype was detected in 76.9% (20 of 26) of isolates that were intermediately sensitive to penicillin and 50% (2 of 4) of penicillin resistant isolates (co-resistant to tetracycline and cotrimoxazole). Routine screening for antibiotic susceptibility is recommended for clinical isolates of pneumococci. Strains with reduced susceptibility to penicillin should be subjected to MIC determination to detect relative resistance or true resistance as such strains are associated with increased virulence.The choice of antibiotics should be guided by the prevalence of local resistance patterns of pneumococci.     

False susceptible penicillin G MICs for Streptococcus pneumoniae with a commercial microdilution system

A commercial minimal inhibitory concentration (MIC) panel that contains a broth of undisclosed formulation intended to support growth of streptococci unable to grow in Mueller-Hinton broth was found to give false susceptible MIC results for pneumococci. The authors encountered several Streptococcus pneumoniae isolates that showed resistance by the oxacillin disk diffusion test, but showed a susceptible penicillin G MIC when tested in an unsupplemented MIC panel. Seven isolates that showed penicillin G resistance by oxacillin disk diffusion testing were retested for penicillin G susceptibility with oxacillin disk diffusion, commercial MIC panels, Mueller-Hinton with blood agar dilution, and Mueller-Hinton with blood broth microdilution. Five of these isolates produced a button of growth in the commercial MIC panel growth control well, but no growth in the penicillin G-containing wells (0.06-4.0 micrograms/mL), suggesting a valid test system and susceptibility to penicillin G. When tested by the three standard methods, these seven isolates showed resistance (R) or relative resistance (I) to penicillin G. These data indicate that frozen MIC panels from this commercial source (American Micro Scan, Campbell, CA) are not reliable for detection of resistance to penicillin G in S. pneumoniae, and if these panels are used, results must be confirmed by another method.     

Comparison of testing methods for detection of decreased linezolid susceptibility due to G2576T mutation of the 23S rRNA gene in Enterococcus faecium and Enterococcus faecalis

E-test, Vitek 2, MicroScan, agar dilution, and disk diffusion were compared for detection of decreased linezolid susceptibility due to 23S rRNA gene G2576T mutation among 32 clinical Enterococcus strains initially reported as intermediate or resistant by E-test alone or Vitek 2 confirmed by E-test. Agar and broth dilution methods were in concordance with PCR detection of the mutation, and disk diffusion was somewhat less sensitive but equally specific.     

Use of an oxacillin disk screening test for detection of penicillin- and ceftriaxone-resistant pneumococci

In a context of worldwide emergence of resistance among Streptococcus pneumoniae strains, early detection of strains with decreased susceptibility to beta-lactam antibiotics is important for clinicians. If the 1-microgram oxacillin disk diffusion test is used as described by the National Committee for Clinical Laboratory Standards, no interpretation is available for strains showing zone sizes of /=2.0 microgram/ml) to penicillin. For ceftriaxone, among 98 strains with no zone of inhibition in response to oxacillin, 68 had intermediate resistance (MIC, 1.0 microgram/ml), and 22 were resistant (MIC, >/=2.0 microgram/ml). To optimize the use of the disk diffusion method, we propose that the absence of a zone of inhibition around the 1-microgram oxacillin disk be regarded as an indicator of nonsusceptibility to penicillin and ceftriaxone and recommend that such strains be reported as nonsusceptible to these antimicrobial agents, pending the results of a MIC quantitation method.     

Coagulase-negative staphylococci: comparison of phenotypic and genotypic oxacillin susceptibility tests and evaluation of the agar screening test by using different concentrations of oxacillin

This study evaluated the oxacillin susceptibilities of 152 coagulase-negative staphylococcal (CoNS) strains of 12 species by disk diffusion; agar dilution; E-test; the slide latex agglutination test (Slidex MRSA Detection test; bioMérieux S/A, Paris, France); the agar screening test with 1, 2, 4, or 6 microg of oxacillin per ml and incubation for 24 or 48 h; and detection of the mecA gene by PCR. The results revealed that the agar screening test with 4 micro g of oxacillin per ml and incubation for 48 h was superior to any single phenotype-based susceptibility assay, presenting a sensitivity and a specificity of 100% each. For the different methods evaluated, the sensitivities and specificities were as follows: for disk diffusion, 94.2 and 91.8%, respectively; for the agar dilution test 100 and 73.5%, respectively; for E-test, 100 and 71.4%, respectively; and for the slide latex agglutination test, 97.1 and 98%, respectively. A good correlation was observed between oxacillin susceptibility testing results and PCR results for Staphylococcus epidermidis, S. haemolyticus, S. hominis subsp. hominis, and all mecA-positive strains. However, at least 60% of the mecA-negative isolates of the species S. saprophyticus, S. cohnii subsp. urealyticum, S. lugdunensis, and S. sciuri were erroneously classified as oxacillin resistant by the agar dilution test. Conversely, the slide latex agglutination test presented a high sensitivity (97.1%) and a high specificity (98%) for all CoNS species. Our results demonstrated the accuracy of the agar screening test with 4 micro g of oxacillin per ml and incubation for 48 h and the slide latex agglutination test for the appropriate detection of the oxacillin susceptibilities of CoNS isolates. Both assays are technically simple and can be easier to perform in routine laboratories than PCR.     

Assessment of metronidazole susceptibility in Helicobacter pylori: statistical validation and error rate analysis of breakpoints determined by the disk diffusion test

Metronidazole susceptibility of 100 Helicobacter pylori strains was assessed by determining the inhibition zone diameters by disk diffusion test and the MICs by agar dilution and PDM Epsilometer test (E test). Linear regression analysis was performed, allowing the definition of significant linear relations, and revealed correlations of disk diffusion results with both E-test and agar dilution results (r2 = 0.88 and 0.81, respectively). No significant differences (P = 0.84) were found between MICs defined by E test and those defined by agar dilution, taken as a standard. Reproducibility comparison between E-test and disk diffusion tests showed that they are equivalent and with good precision. Two interpretative susceptibility schemes (with or without an intermediate class) were compared by an interpretative error rate analysis method. The susceptibility classification scheme that included the intermediate category was retained, and breakpoints were assessed for diffusion assay with 5-microg metronidazole disks. Strains with inhibition zone diameters less than 16 mm were defined as resistant (MIC > 8 microg/ml), those with zone diameters equal to or greater than 16 mm but less than 21 mm were considered intermediate (4 microg/ml < MIC 相似文献   

In vitro activity of pristinamycin against Streptococcus pneumoniae

The activity of the pristinamycin was investigated using disk diffusion agar or ATB PNEUMO system and MIC determination using reference liquid medium method (NCCLS) on 749 S. pneumoniae strains isolated in Aquitaine in 1999. We have realized the killing curves against 10 isolates selected from erythromycin-susceptible and resistant S. pneumoniae strains. All the strains tested by ATB PNEUMO system were susceptible to pristinamycin, using disk diffusion agar, 6.8% of strains were intermediate or resistant. However the MIC's of pristinamycin determined by liquid dilution method against these strains were < 1 mg/L. These data suggest that the zone of inhibition around the disk was not correlated with MIC for erythromycin pneumococci and MIC testing must be performed. The results of killing curves showed a very good and rapid bactericidal activity of pristinamycin within two hours for concentration equal to 4 x MIC and four hours for 2 x MIC.     

Development of interpretive criteria and quality control limits for broth microdilution and disk diffusion antimicrobial susceptibility testing of Streptococcus pneumoniae.

A five-center collaborative study was undertaken to develop quality control and specific interpretive criteria for susceptibility testing of Streptococcus pneumoniae against 12 antimicrobial agents. MICs were determined for 248 pneumococcal clinical isolates (with an emphasis on resistant strains) by use of the National Committee for Clinical Laboratory Standards (NCCLS)-recommended broth microdilution procedure incorporating lysed horse blood-supplemented Mueller-Hinton broth. NCCLS disk diffusion testing was also performed for each isolate by using Mueller-Hinton sheep blood agar incubated in 5% CO2. Repetitive testing of S. pneumoniae ATCC 49619 with different sources and lots of media and disks allowed development of quality control ranges which encompassed approximately 95% of MIC and zone size values observed in the study. Good intra- and interlaboratory reproducibilities were seen with these testing methods and all of the drugs examined. On the basis of the results of this study, MIC interpretive criteria are proposed for 11 agents. Comparisons of MICs and disk diffusion zone sizes allowed disk diffusion zone size interpretive criteria to be proposed for five drugs and confirmed the use of the oxacillin disk test for prediction of penicillin susceptibility among pneumococci. Excessive numbers of minor-category interpretive errors precludes recommendation at this time of the disk diffusion method for testing of pneumococci against five of the drugs. Use of these proposed quality control and interpretive criteria should provide for reproducible test results and allow recognition of recently emerging resistance among pneumococcal clinical isolates.     

Determination of Penicillin MICs for Streptococcus pneumoniae by Using a Two- or Three-Disk Diffusion Procedure

The potential for the use of the disk diffusion method to accurately predict penicillin MICs for Streptococcus pneumoniae was investigated with penicillin (6 μg), methicillin (5 μg), and oxacillin (1 μg) disks. A total of 183 S. pneumoniae isolates were tested by three MIC procedures (agar dilution, microdilution, and E-test). Regression analyses of the geometric mean of the three MIC results against (i) the sum of the zone diameters for methicillin, penicillin, and oxacillin disks; (ii) the sum of the zone diameters for methicillin and penicillin disks; and (iii) each of the three individual zone diameters were performed. Calculated MICs were determined from each of these regression analyses and compared to the mean reference MICs. A high level of correlation was obtained with both the two- and the three-disk procedures (r = 0.97), with essential agreement rates (±1 doubling dilution) between MICs calculated by the three-disk procedure and the two-disk procedure and the mean reference MICs of 98.4 and 98.9%, respectively. No major or very major errors were obtained with the two- or three-disk procedures. The accuracy of the disks used individually was lower (r = 0.84 to 0.93). However, oxacillin and methicillin disk testing remain excellent for screening strains, with all penicillin-susceptible strains having zones of >21 and >22 mm, respectively. The combination disk procedure, which involves the use of three disks (methicillin, oxacillin, and penicillin) or two disks (methicillin and penicillin) for testing S. pneumoniae, can provide accurate penicillin MICs and qualitative category results that are comparable to results obtained by the E-test, agar, and microdilution MIC methods.     

Antibiotic susceptibility of Listeria monocytogenes in Denmark 1958-2001

In order to see whether the susceptibility of Danish Listeria monocytogenes strains has changed over the years we examined a collection of human isolates from the period 1958-2001. We, furthermore, wanted to compare L. monocytogenes susceptibility testing using a disc diffusion assay with MIC measurements performed by the E-test. 106 strains isolated predominantly from blood cultures and cerebrospinal fluids were examined together with three reference strains. Susceptibility to the following antibiotics was tested by the E-test and by Oxoid discs using Iso-sensitest agar: penicillin G, ampicillin, meropenem, gentamicin, sulphamethoxazole, trimethoprim, ciprofloxacin, erythromycin, vancomycin, linezolid, chloramphenicol and tetracycline. The strains were in the main sensitive to all antibiotics examined using both methods, except for ciprofloxacin, where the strains were intermediate sensitive. However, for penicillin, ampicillin and sulphamethoxazole, while the disc diffusion assay found the strains to be sensitive, MIC measurements generally placed the strains one dilution above the breakpoint for sensitivity in the intermediate sensitive group. Based on the MIC measurements, the antibiotic susceptibility of L. monocytogenes has not changed in Denmark from 1958 to 2001, and the multiresistant strains found in human infections elsewhere have not been found in Denmark.     

Validation of diffusion methods for macrolide susceptibility testing of Helicobacter pylori

Helicobacter pylori resistance to macrolides is increasing, and the need for susceptibility testing has become crucial. The only standardized method is agar dilution, which is not adapted to clinical practice. The present work aimed: (1) to optimize the technical conditions and to assess the reproducibility of the E-test and disk diffusion method for macrolides susceptibility testing of H. pylori, and (2) to assess the performances of these two phenotypic methods in detecting strains harboring a resistance mechanism to macrolides. We used 191 isolates collected in nine centers of France and Belgium. Phenotypic tests were performed on Mueller-Hinton agar supplemented with 10% horse blood, inoculated with a 2-day-old H. pylori suspension (10(8) CFU/ml), and incubated for 72 hr at 37 degrees C under microaerophilic conditions. The reproducibility studied on two randomly selected strains was better for disk diffusion than for the E-test for both clarithromycin and erythromycin. For a subset of 10 strains, the MICs of erythromycin and clarithromycin did not differ from more than one two-fold dilution when determined by E-test or agar dilution method. The breakpoints were for MICs: 1 mg/L for both clarithromycin and erythromycin and for inhibition diameters, 22 mm for clarithromycin and 17 mm for erythromycin. There was a 100% concordance between susceptibility to erythromycin and clarithromycin. However, the susceptible and resistant populations were better separated by testing erythromycin. Of 34 resistant strains, two lacked the A2142G and A2143G point mutations in 23S rRNA by PCR-RFLP. None of 15 tested sensitive strains were positive for one of these two point mutations. For clinical practice, we recommend to assess macrolide susceptibility of H. pylori by using one of these two phenotypic methods under the described technical conditions.     

Interest of the disk diffusion method for screening Clostridium difficile isolates with decreased susceptibility to antibiotics

AimIn vitro determination of Clostridium difficile susceptibility to antibiotics is not routinely performed. The aim of this study was to evaluate the performance of antibiotic susceptibility determination with the disk diffusion method for screening C. difficile isolates with decreased susceptibility to antibiotics.MethodsThirty-six C. difficile isolates (toxigenic or not) isolated in 2005 and 2006 from three hospitals Assistance publique–Hôpitaux de Paris (Jean-Verdier, René-Muret, Beaujon) were studied by disk diffusion method with 14 antibiotics. Mueller-Hinton agar supplemented with sheep blood (Bio-Rad*) were swabbed with a C. difficile suspension at 1 McFarland. To check the results obtained with the disk diffusion method, Minimal Inhibitory Concentration (MIC) were performed respectively with E-test for glycopeptides and metronidazole and with the agar dilution reference method and E-test for new molecules with a potential activity against anaerobes: imipenem, ertapenem, linezolid and moxifloxacin.ResultsThe decreased susceptibility (resistant and intermediate) observed was 40% for amoxicillin–clavulanate, 60% for piperacillin–tazobactam, 100% for ceftriaxone, 81% for imipenem, 61% for ertapenem, 2% for chloramphenicol, 34% for erythromycin, 90% for lincomycin, 2% for linezolid, 98% for levofloxacin, 17% for moxifloxacin and 0% for vancomycin, teicoplanin and metronidazole. The results obtained with the disk diffusion method were compared to MICs obtained with E-test and reference method.ConclusionThe disk diffusion method seems to be a good method to detect isolates suspected to have a decreased susceptibility and consequently to reduce MIC determinations.     

Quantitative antimicrobial susceptibility testing of Haemophilus influenzae and Streptococcus pneumoniae by using the E-test.

The E-test (PDM Epsilometer; AB Biodisk, Solna, Sweden) is an antimicrobial agent gradient-coated plastic test strip which allows MIC determinations on agar media. The test is performed in a manner similar to the agar disk diffusion procedure. A collection of Haemophilus influenzae and Streptococcus pneumoniae strains possessing various resistance mechanisms was used to evaluate the E-test method. H. influenzae strains were tested with both Haemophilus test medium (HTM) and PDM ASM II chocolate agar, while the S. pneumoniae strains were tested on Mueller-Hinton sheep blood agar. E-test MICs for a total of 10 antimicrobial agents were compared with broth microdilution MICs determined according to National Committee for Clinical Laboratory Standards methods. In general, E-test MICs for both species were quickly and easily interpreted and agreed within one log2 MIC increment in 89.8% of tests with H. influenzae and in 80.4% of pneumococcal tests. The majority of disagreements between the E-test and conventional MICs occurred with trimethoprim-sulfamethoxazole because of trailing and diffuse E-test MIC endpoints with both species. Ampicillin MICs for beta-lactamase-producing H. influenzae determined by the E-test differed at times from those determined by conventional testing because of the vagaries of interpreting colonies growing within the E-test inhibition ellipses. E-test penicillin MICs for pneumococci tended to be 1 to 2 log2 dilutions lower than those determined by using Mueller-Hinton broth supplemented with lysed horse blood. Nevertheless, strains of both species with documented resistance to the study drugs were detected by E-tests, i.e., 0.7% of the tests had very major errors with H. influenzae and 0.8% had very major errors with S. pneumoniae. Thus, the E-test represents a potential alternative method for antimicrobial susceptibility testing of these two fastidious bacterial species.     

Evaluation of the E-test for routine testing of the susceptibility of Streptococcus pneumoniae to benzylpenicillin, amoxicillin and cefotaxime

Objective: To study the routine use of the E-test for susceptibility testing of penicillin-resistant Streptococcus pneumoniae.
Methods: A multicenter study of penicillin-resistant S. pneumoniae (PRSP) was carried out in Brittany, France (10 general hospitals, and two university hospitals including a coordinating center). Each hospital detected PRSP by the oxacillin (5-μg) disk method and determined the MICs of penicillin G, amoxicillin and cefotaxime by the E-test under routine conditions. All the PRSP strains were collected in a coordinating center and the MICs were checked by the agar dilution method. The classifications obtained from the MICs determined by the E-test and by the reference method were compared.
Results: Between 1 July 1993 and 30 June 1994, 128 PRSP strains were collected. Agreement within 1 log₂ dilution was obtained for only 62% of strains with benzylpenicillin, 72.5% with amoxicillin and 76% with cefotaxime. These data are well below published values. In addition, 52% of the strains found to be penicillin-resistant by the reference technique were of intermediate resistance according to the E-test. There were major differences in the quality of the results obtained by the participating laboratories.
Conclusions: There are problems of standardization in the routine use of the E-test. Microbiologists should therefore take particular care when performing the test and when reading the results, and ensure that reference strains are included in the assay.     

Antimicrobial susceptibility of invasive Streptococcus pneumoniae in Italy by agar dilution method and E test

Few data on antimicrobial susceptibility of invasive Streptococcus pneumoniae isolated in Italy are available. Ninety-two invasive isolates from all over the country collected from January 1997 to April 1998 were tested for sensitivity to penicillin, erythromycin, ceftriaxone, chloramphenicol, tetracycline, and trimethoprim/sulfamethoxazole by the agar dilution method. Five (5.4%) strains were resistant to penicillin (one highly, four intermediately resistant), 8 (8.7%) to chloramphenicol, 27 (29.3%) to erythromycin, 17 (18.5%) to tetracycline (16 highly, one intermediately), and 21 (22.8%) to trimethoprim/sulfamethoxazole (14 highly, 7 intermediately). All strains were susceptible to ceftriaxone, although the penicillin-resistant strain had the highest minimal inhibitory concentration. (MIC) value (0.5 microg/ml); three penicillin-resistant strains were also resistant to erythromycin. Eight strains were multi-drug resistant, being also resistant to at least three antibiotics. The commercially available E test was compared with the standard agar dilution method for the determination of MIC of penicillin, erythromycin, ceftriaxone, chloramphenicol, and trimethoprim/sulfamethoxazole. E test established the same susceptibility categories for 100% of the strains tested for penicillin and ceftriaxone, 99% for chloramphenicol, 97% for erythromycin, and 74% for trimethoprim/sulfamethoxazole. According to our results, E test was simple to perform, easy to interpret, and a valid method for susceptibility testing of S. pneumoniae. Our study shows that in Italy the rate of penicillin resistance in invasive isolates of S. pneumoniae is one of the lowest in Europe (5.4%), while the rate of erythromycin is very high (29.3%) and is reaching the highest rates of other Southern European countries.     

Rapid method for determining antimicrobial susceptibility of Haemophilus influenzae.

A method was developed to determine the susceptibility of Haemophilus influenzae to ampicillin, cefamandole, and chloramphenicol by using the MS-2 system (Abbott Laboratories) for determining minimum inhibitory concentrations (MIC). The MS-2 results for 132 strains of H. influenzae were compared with the results of agar disk diffusion, agar dilution, and beta-lactamase tests. Twenty-four strains (18.2%) of H. influenzae were resistant to ampicillin by the agar dilution method, as opposed to 25 strains by the MS-2 method. For a beta-lactamase-negative strain, the agar dilution MIC was 4 micrograms/ml, and the MS-2 MIC was 16 micrograms/ml. Twenty-one strains produced beta-lactamase; two beta-lactamase-negative strains were resistant by MS-2, agar dilution, and agar disk diffusion. In addition, one beta-lactamase-negative strain, for which the agar dilution MIC was 32 micrograms/ml and the MS-2 MIC was 16 micrograms/ml, was sensitive by agar disk diffusion. Overall, the MS-2 method compared favorably with the agar dilution method for determining the MIC of ampicillin, cefamandole, and chloramphenicol for H. influenzae.     

E test as susceptibility test and epidemiologic tool for evaluation of Neisseria meningitidis isolates.

The E test (AB Biodisk, Solna, Sweden), a new approach developed to test antimicrobial susceptibility, was compared with the agar dilution method for seven-drug antibiogram analysis of Neisseria meningitidis isolates. The overall E-test quantitative accuracy (+/- 1 log2 dilution) was 93% compared with that of agar dilution testing. The E test was then used to perform the susceptibility tests on a 10-year sample of 102 N. meningitidis isolates, including 5 from a recent epidemic outbreak in the University of Iowa (Iowa City) community. The E test proved to be an efficient methodology for identifying common source clusters of meningococcal disease having resistance to rifampin or sulfonamides. Moreover, the data demonstrated a recent increase in penicillin MICs (MIC for 90% of strains, 0.094 microgram/ml) and an escalation of high-level resistance to trimethoprimsulfamethoxazole (33%) and rifampin (14%). The E test should be considered a simple and accurate susceptibility method for the emerging need to test meningococci and other pathogenic neisserias. Chocolate Mueller-Hinton agar was observed to provide the best support of growth and E-test MIC results that correlated well with results of the reference agar dilution method previously used for neisserias.     

Misclassification of susceptible strains of Staphylococcus aureus as methicillin-resistant S. aureus By a rapid automated susceptibility testing system

Eight Staphylococcus aureus strains initially identified by Vitek GPS-BS or GPS-SA cards as resistant to oxacillin, but susceptible to most non-beta-lactam antibiotics, were found on further testing to be susceptible to oxacillin and ceftizoxime by disk diffusion tests. For all these strains, the MICs of oxacillin were 相似文献   

Detection of methicillin-resistant Staphylococcus epidermidis.

To determine whether methods suggested for detecting methicillin-resistant Staphylococcus aureus apply equally to methicillin-resistant Staphylococcus epidermidis, 135 S. epidermidis isolates were tested by the Vitek AMS gram-positive susceptibility card (Vitek Systems, Inc., Hazelwood, Mo.) and by modifications of agar screen, disk diffusion, and microdilution methods. Modifications included 24- versus 48-h incubation, unsupplemented versus 2% NaCl-supplemented broth, and standard versus direct inoculum. At 24 h, the highest number of resistant strains, 59, was detected by oxacillin (1 microgram) disk diffusion. At 48 h, three additional strains were judged resistant. With one exception, results for oxacillin disk diffusion and agar screen were equivalent at 24 and 48 h. Vitek detected 50 resistant strains. Significantly fewer resistant strains were detected at 24 h by methicillin disk diffusion (5 micrograms) and methicillin microdilution with 2% NaCl. For oxacillin microdilution, neither 2% NaCl supplementation nor the method of inoculum preparation significantly affected the results. Oxacillin microdilution with cation- rather than non-cation-supplemented broth detected significantly fewer (n = 33) resistant strains at 24 h; 51 were resistant at 48 h. To detect methicillin-resistant S. epidermidis, a direct inoculum with either 24-h oxacillin disk diffusion and reincubation of intermediate strains for an additional 24 h or 24-h oxacillin agar screen and reincubation of strains with no growth for a total of 48 h is recommended.     

Determination of penicillin susceptibility of Streptococcus pneumoniae using the polymerase chain reaction.

AIM: To develop a polymerase chain reaction (PCR) based method to detect penicillin susceptibility in isolates of Streptococcus pneumoniae (SP). METHOD: PCR primers were designed to amplify differential nucleotide sequences of the penicillin-binding protein (PBP) genes 2b, 2x, and 1a in penicillin susceptible and resistant strains of SP. Primers derived from the PBP 2x and 2b genes were designed to amplify products from penicillin susceptible S pneumoniae (PSSP), whereas primers derived from the PBP 1a gene were designed to amplify gene sequences of penicillin resistant S pneumoniae (PRSP). RESULTS: Two hundred and thirty clinical isolates of SP from the USA, UK, Kenya, Romania, and the Kingdom of Saudi Arabia were tested. Of the isolates, 116 were penicillin susceptible, 65 were intermediately resistant, and 49 were highly resistant. PCR identified 108 (93%) of 116 of PSSP isolates, 55 (85%) of 65 intermediately resistant isolates, and all of the 49 highly resistant isolates of SP. The susceptibility of 16 (7%) isolates could not be determined using PCR. All of these 16 isolates had a minimum inhibitory concentration (MIC) of penicillin < 1 mg/l. None of the highly resistant isolates was identified as penicillin susceptible by PCR, although two of the isolates with intermediate resistance (MIC = 0.125 mg/l) were. CONCLUSION: Using primers that differentially identify the genotypes of susceptible and resistant strains of SP, PCR provides a rapid method for determining the penicillin susceptibility of SP isolates and could potentially be used for testing clinical samples.