Interlaboratory variation of antibiograms of methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains with conventional and commercial testing systems.

Laboratory-prepared (conventional) and commercial susceptibility testing systems were compared by using a group of methicillin-resistant (MR) and methicillin-susceptible (MS) strains of Staphylococcus aureus. A group of 25 MR and 15 MS S. aureus strains were coded and tested blindly by disk diffusion, agar dilution, broth microdilution, Sensititre, Micro-Media, Sceptor, API 3600S, MicroScan, Autobac I, and MS-2 systems. All systems were incubated at 35 degrees C and read with either a manual or automated reader at the recommended times. Where applicable, systems were also read at 48 h. Among the conventional assays, the broth and agar dilution methods were comparable, both detecting 88% of the MR strains at 24 h and detecting 92 and 96%, respectively, at 48 h. The disk diffusion method was less efficient, detecting only 36 and 72% at 24 and 48 h, respectively. Detection of cephalothin resistance was low for all systems at both time periods, with agar dilution and disk diffusion being the most and least efficient, respectively. Some variability was also seen with detection of resistance to clindamycin and gentamicin. Among the MS strains, variability among the conventional systems occurred with methicillin, gentamicin, ampicillin, and penicillin. Comparison of the commercial systems with manual readers with the broth microdilution method (reference method) showed that for MR strains, the Sceptor system gave identical results at 24 and 48 h. Sensititre detected 68 and 88% of the MR strains, whereas Micro-Media was least effective detecting 12 and 80% at 24 and 48 h, respectively. None of the commercial systems detected cephalothin resistance well, with only one strain being indicated by the Sceptor and Sensititre systems at 48 h. Slight differences were also seen among the systems with clindamycin and gentamicin. With regard to the MS strains, variability among the systems was seen with methicillin, penicillin, ampicillin, clindamycin, and gentamicin. Among commercial systems with automated readers, the API system detected a greater number of MR strains than did the reference method at 24 and 48 h, 96 and 100%, respectively. The MicroScan method was comparable to the reference method detecting 80 and 88% of the MR strains at both time periods, respectively. Both Autobac I and MS-2 were much less effective in detecting MR strains, noting only 32 and 16%, respectively, at the 3- to 6-h readings. Poor detection of cephalothin resistance among MR strains was evident in all systems. Variability also occurred among the systems with clindamycin, gentamicin, and ampicillin. A single strain of the MR group was reported to be vancomycin resistant by the API system. Among the MS group, the greatest variability was seen with methicillin. Less variability occurred with penicillin, ampicillin, gentamicin, and vancomycin.     

Evaluation of three techniques for detection of low-level methicillin-resistant Staphylococcus aureus (MRSA): a disk diffusion method with cefoxitin and moxalactam,the Vitek 2 system,and the MRSA-screen latex agglutination test

Very-low-level methicillin-resistant Staphylococcus aureus (MRSA), or class 1 MRSA, is often misdiagnosed as methicillin-susceptible S. aureus (MSSA). We evaluated the performances of three methods for detection of low-level methicillin resistance: the disk diffusion method using the cephamycin antibiotics cefoxitin and moxalactam, the Vitek 2 system (bioMérieux), and the MRSA-screen test (Denka). Detection of the mecA gene by PCR was considered to be the "gold standard." We also determined the sensitivity of the oxacillin disk diffusion method with 5- and 1-microg disks and that of the Oxascreen agar assay with 6 mg of oxacillin liter(-1) for detection of MRSA. We compared the distributions of MICs of oxacillin and cefoxitin by the E-test (AB Biodisk), and those of moxalactam by dilutions in agar, for MRSA and MSSA isolates. The 152 clinical isolates of S. aureus studied were divided into 69 MSSA (mecA-negative) and 83 MRSA (mecA-positive) isolates, including 63 heterogeneous isolates and 26 class 1 isolates (low-level resistance). The cefoxitin and moxalactam disk diffusion tests detected 100% of all the MRSA classes: cefoxitin inhibition zone diameters were <27 mm, and moxalactam inhibition zone diameters were <24 mm. The Vitek 2 system and the MRSA-screen test detected 94 and 97.6% of all MRSA isolates, respectively. The sensitivities of the 5- and 1-microg oxacillin disks were 95.2 and 96.4%, respectively, whereas that of the Oxascreen agar screen assay was 94%. All of the tests except the 1-microg oxacillin disk test were 100% specific. For the class 1 MRSA isolates, the sensitivity of the Vitek 2 test was 92.3%, whereas those of the MRSA-screen test and the disk diffusion method with cefoxitin and moxalactam were 100%. Therefore, the cefoxitin and moxalactam disk diffusion methods were the best-performing tests for routine detection of all classes of MRSA.     

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.     

Screening for methicillin resistance in Staphylococcus aureus and coagulase-negative staphylococci: an evaluation of three selective media and Mastalex-MRSA latex agglutination

Laboratory confirmation of MRSA is important for the implementation of infection control; conventional screening culture methods take up to five days for confirmation. The purpose of this study is to ascertain the efficiency of three selective media for growth of methicillin-resistant Staphylococcus aureus (MRSA) before and after enrichment in salt broth, and to evaluate the Mastalex-MRSA latex agglutination kit for detection of methicillin resistance. Screening swabs were collected from 63 patients, yielding 125 S. aureus isolates and 40 coagulase-negative staphylococcus (CNS) isolates. Selective media used were mannitol salt agar (MSA), Baird-Parker agar with ciprofloxacin (BPC) and bromocresol purple (BCPA). Polymerase chain reaction (PCR) for mecA gene detection was used as the reference standard for evaluation of the Mastalex-MRSA assay, which was also evaluated on colonies of S. aureus from the selective media. No significant difference was found in efficiency of MRSA isolation among the selective media. Pre-enrichment in the salt broth did not enhance isolation of MRSA. Methicillin-sensitive S. aureus and CNS were significantly inhibited in all selective media (P<0.05). Only BPC significantly selected out methicillin-resistant CNS (P<0.01). Mastalex-MRSA was 97% specific and sensitive for the detection of MRSA. It was 65% sensitive and 100% specific in detecting methicillin resistance in CNS. In conclusion, all selective media performed equally well (MRSA isolation rate of approximately 80%). Mastalex-MRSA provided rapid and reliable detection of MRSA from selective media, reducing the time required for confirmation of this organism.     

Improved and rapid detection of methicillin-resistant Staphylococcus aureus nasal carriage using selective broth and multiplex PCR

To improve efficiency in detecting nasal methicillin-resistant Staphylococcus aureus (MRSA), we evaluated a multiplex PCR using pre-enrichment of the specimen in selective broths, and compared it with detection performed by routine tests in hospital laboratories. Nasal swab specimens from 311 patients were inoculated onto mannitol-salt agar (MSA) at the hospital laboratories and in two Mueller-Hinton broths with 7% NaCl containing oxacillin at concentrations of 2 and 4 micro g/ml. Isolates on MSA were identified as MRSA by classical laboratory tests (coagulase and oxacillin disk diffusion tests). Oxacillin broth cultures were subcultured on blood agar and MRSA isolates were identified by coagulase and susceptibility tests, including agar dilution and the oxacillin-screening method (gold standard method). Simultaneously, multiplex-PCR was performed from the selective broths to detect S. aureus species-specific and mecA gene segments (OxMPCR method). Thirty-two S. aureus isolates were recovered: 29 (90.6%) were MRSA strains and 3 (9.4%) were oxacillin-susceptible isolates. Twenty-eight (96.5%) MRSA isolates were detected by OxMPCR, while 17 (58.6%) were identified by routine tests (P=0.002). This new method for detection of MRSA nasal carriers showed higher sensitivity and led to faster reporting--i.e., within 24 h--of results.     

Detection of the Staphylococcal mecA gene by chemiluminescent DNA hybridization.

A new solution-phase DNA hybridization capture assay for the rapid detection of the mecA gene in clinical isolates of Staphylococcus was compared with multiplex PCR and disk diffusion methods. The assay uses a DNA capture probe immobilized on paramagnetic particles and a second DNA probe labeled with an acridinium ester. Bacteria from 24-h cultures are lysed, and the lysates are hybridized with the DNA probes. After magnetic separation to remove unhybridized labeled probe, the mecA gene is detected by the chemiluminescence of the hybridized probe. Four hundred consecutive staphylococcal isolates were assayed, including 147 S. aureus and 253 coagulase-negative Staphylococcus isolates. Among the S. aureus isolates, 14 of 147 were MecA+ by both the hybridization capture assay and PCR; 133 of 147 were MecA negative by both assays (positive and negative predictive values, 100%). Comparison of disk diffusion results with those obtained by genotypic methods indicated that 14 of 147 S. aureus isolates judged to be resistant were positive by both methods; 119 of 147 were Oxs and negative by both genotypic methods (positive and negative predictive values, 50 and 100%, respectively). The remaining 14 S. aureus isolates were MecA- Oxr; among these, 13 were beta-lactamase hyperproducers. For coagulase-negative staphylococci, 130 of 253 were MecA+ by the hybridization capture assay; 129 of 130 of these isolates were positive by PCR (positive and negative predictive values, 99.2 and 100%, respectively). Comparison with the disk diffusion assay showed that 128 of the coagulase-negative MecA+ isolates were Oxr; 111 of 253 were MecA- and Oxs (positive and negative predictive values, 90.8 and 99.1%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of new Vitek 2 card and disk diffusion method for determining susceptibility of Staphylococcus aureus to oxacillin

Detection of methicillin resistance in Staphylococcus aureus is a challenge, especially low-level resistance, which is often misdiagnosed. The aim of this study was to compare the diagnostic accuracies of the automated Vitek 2 system and disk diffusion tests, using cefoxitin and moxalactam, for the detection of methicillin resistance in S. aureus strains. Four sets of genotypically diverse isolates were selected from a national reference collection, including mecA-negative S. aureus isolates (n = 56), hospital-acquired (n = 88) and community-acquired (n = 40) S. aureus isolates, and heterogeneous methicillin-resistant S. aureus isolates (n = 29). Oxacillin susceptibility was tested by the Vitek 2 system with the AST P549 card and by disk diffusion methods using 10, 30, and 60 microg cefoxitin and 30 microg moxalactam. Oxacillin resistance was confirmed by PCR for the mecA gene. The overall sensitivities for oxacillin resistance detection were 97.5% for the Vitek 2 automated system, 98.7% for 60-microg cefoxitin and moxalactam disk diffusion, and 99.6% for 10- and 30-microg cefoxitin disks, respectively. Methicillin-susceptible S. aureus isolates were correctly reported as susceptible by all methods. The median times for methicillin testing were 7 h for the Vitek 2 system versus 24 h for disk diffusion methods. In conclusion, the cefoxitin and moxalactam disk diffusion methods and the Vitek 2 automated system are highly accurate methods for methicillin resistance detection, including a range of representative Belgian methicillin-resistant S. aureus strains and unusual strains exhibiting cryptic or low-level oxacillin resistance.     

Evaluation of a cefoxitin disk diffusion test for the routine detection of methicillin-resistant Staphylococcus aureus

Two oxacillin disk methods were compared with a cefoxitin disk diffusion test for detection of methicillin-resistant Staphylococcus aureus (MRSA), with PCR for mecA as the reference method. When tested with 115 MRSA and 350 methicillin-susceptible S. aureus isolates, the cefoxitin disk test (specificity 100%, sensitivity 96.5%) was superior to the oxacillin disk methods (specificity 99.1%, sensitivity 90.4%). Testing with both oxacillin and cefoxitin disks would give better sensitivity (100%) than the cefoxitin test alone, but at the expense of specificity (99.1%). The cefoxitin disk test required no special test conditions and would improve the reliability of routine tests for detection of MRSA.     

Validation of multiplex PCR strategy for simultaneous detection and identification of methicillin resistant Staphylococcus aureus

Multiplex polymerase chain reaction (PCR) strategy is described for rapid identification of clinically relevant methicillin resistant Staphylococcus aureus (MRSA) that targets mecA and coagulase genes. In this study, 150 staphylococcal clinical isolates were used that included 40 isolates of MRSA, 55 isolates of methicillin susceptible S. aureus (MSSA), 44 isolates of methicillin susceptible coagulase negative Staphylococcus spp. (MS-CoNS) and 11 isolates of methicillin resistant coagulase negative Staphylococcus spp. (MR-CoNS). Out of 55 S. aureus strains, three strains demonstrated mecA gene, which appeared to be oxacillin sensitive by disc diffusion. When (MS-CoNS) were evaluated, 10 isolates classified as oxacillin sensitive phenotypically, yielded positive results in PCR method. The results for mecA detection by PCR were more consistent with disk susceptibility tests in case of MRSA (100%) and MSSA (95%) isolates. In contrast to above results with MRSA and MSSA, mecA detection by PCR in MS-CoNS showed less correlation with disk susceptibility tests (77%). The results for coag detection by PCR were consistent with phenotypic tests in all isolates.     

Susceptibility testing of multidrug-resistant Staphylococcus aureus with the Sceptor microdilution system.

The antimicrobial susceptibilities of Staphylococcus aureus isolates were concurrently determined by the Sceptor system (BBL Microbiology Systems, Cockeysville, Md.) and by the standard disk diffusion method. For the methicillin-resistant isolates, there was greater than 98% agreement between the two test results with penicillin G, erythromycin, clindamycin, tetracycline, gentamicin, and tobramycin. Major disagreements (susceptible by one method and resistant by the other) were 7% for methicillin, 13.5% for cephalothin, 3.5% for cefamandole, and 27% for amikacin. The major discrepancies for methicillin were eliminated by supplementing the inoculum broth with salt. For methicillin-susceptible isolates, agreement between the two methods was 96 to 100% for all antibiotics except amikacin.     

Performance of different methods of oxacillin resistance detection in atypic strains of Staphylococcus aureus

Seventy-three of aminoglycoside-susceptible methicillin-resistant Staphylococcus aureus (AS-MRSA) and 12 kanamycin-tobramycin-resistant methicillin-susceptible S. aureus (KTR-MSSA) isolates were phenotypically and genotypically examined for methicillin susceptibility. The AS-MRSA profile represents 8.3% of MRSA strains and the KTR-MSSA profile represents 1.38% of MSSA strains. The diffusion method using the 5 microg oxacillin and 30 microg cefoxitin discs on Mueller-Hinton Agar (MHA) with and without NaCl, the incubation at 35 degrees C or 30 degrees C for 24 or 48 hours respectively, and the determining oxacillin MICs by E-test (AES, Combourg, France) were performed and used as phenotypic methods. We also used the mecA gene PCR which was considered as the "gold standard" for methicillin resistance detection, and the Slidex MRSA Detection (bioMérieux) that detect the presence of mecA gene product (PBP 2a). To increase the level of PBP 2a expression, the 30 microg cefoxitin disc was used as an inducer. All the AS-MRSA strains (100%) were detected by the cefoxitin disc in all conditions and by the oxacillin disc on MHA with 2% of NaCl at 35 degrees C. Without NaCl, the sensitivity fell to 97,2% by oxacillin disc. The oxacillin MICs for these isolates ranged from 2 to 128 mg/l. The mecA gene determinant and its product PBP 2a were detected in all AS-MRSA strains. All KTR-MSSA strains were phenotypically methicillin-susceptible and oxacillin MICs were below or borderline of breakpoint (< or =2 mg/l). The mecA gene determinant and its product were detected in one strain which was considered to be the most heterogeneous of those tested.     

Antibiotic resistance of Staphylococcus aureus at north Lebanon: place of the methicillin resistance and comparison of detection methods

The aim of this study was to evaluate the susceptibility of 100 Staphylococcus aureus strains isolated from the laboratory of Microbiology of the Islami Hospital of Tripoli (Lebanon) to 19 antibiotics, and to determine the prevalence of methicillin resistant strains. 30% of strains studied were methicillin resistant, 96% were resistant to the penicillin G. Clavulanic acid restaurated the amoxicillin activity to 29%. The resistance level was 34% for amikacin, 3% for gentamycin and tobramycin, 10% for chloramphenicol, 44.33% for tetracyclin, 7% for erythromycin, 4.04% for clindamycin, 20% for trimethoprim-sulfametoxasol and 0% for vancomycin and teicoplanin. The methicillin-resistant Staphylococcus aureus possess more important resistant level in comparison with the methicillin sensitive strains. We compared the ability of latex agglutination test (Slidex(R) SARM, bioMérieux, France) to detect the production of penicillin-binding protein 2' (PBP 2') in 100 clinical isolates of S. aureus with two reference methods: the oxacillin disk diffusion test and the MIC determination by the E-test (AB BIODISK, Sweden). The two reference methods give the same results for the detection of methicillin resistant S. aureus. The Slidex test was positive for all 30 isolates determined to be methicillin resistant by the reference methods (sensitivity 100%). The latex test was negative for 42 of 70 isolates determined to be methicillin susceptible by the reference methods, and the latex test was positive for 28 isolates determined to be susceptible (specificity 60%).     

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.     

Detection of intrinsically resistant (heteroresistant) Staphylococcus aureus with the Sceptor and AutoMicrobic systems.

Modified procedures for the Sceptor Gram-Positive MIC Panel and the Vitek AutoMicrobic System GPS-M Card were evaluated for their ability to detect methicillin-resistant (heteroresistant) Staphylococcus aureus. A total of 398 clinical isolates (including 222 methicillin-resistant S. aureus) obtained from 10 hospitals were tested. Both systems had 2% NaCl in the oxacillin wells. Sceptor MIC panels were inoculated with an organism suspension prepared from an 18- to 24-h blood agar plate and were inoculated for a full 24 h at 35 degrees C before MICs were read. All methicillin-resistant S. aureus isolates were detected as resistant to oxacillin at greater than or equal to 8 micrograms/ml by the Sceptor method and at greater than 2 micrograms/ml by the Vitek method. All 176 oxacillin-susceptible, methicillin-susceptible S. aureus isolates were correctly distinguished from methicillin-resistant S. aureus isolates by Sceptor. However, with the Vitek system 29 methicillin-susceptible S. aureus isolates tested as falsely resistant to oxacillin and four isolates tested as falsely resistant to vancomycin. The modified testing procedure with the Sceptor system can be used reliably for accurate susceptibility testing of methicillin-resistant and methicillin-susceptible S. aureus. The Vitek GPS-M card does not accurately discriminate between methicillin-resistant and methicillin-susceptible S. aureus with an oxacillin breakpoint of greater than 2 micrograms/ml.     

Comparison of susceptibility test methods to detect penicillin susceptibility in Streptococcus pneumoniae isolates

The increasing prevalence of penicillin-resistant Streptococuus pneumoniae urges for fast and accurate susceptibility testing methods. This study evaluated the comparability of three commonly used techniques; disk diffusion, E-test and agar dilution, to detect penicillin susceptibility in clinical isolates of S. pneumoniae. Fifty pneumococcal isolates, obtained from patients at the University of Malaya Medical Centre, were selected to include both penicillin-susceptible strains and those that had decreased susceptibility (resistant and intermediate) to penicillin. The minimum inhibitory concentration (MIC) values of penicillin to serve as the reference was determined by the agar dilution method in which, based on the MIC breakpoints recommended by the National Committee for Clinical Laboratory Standards (NCCLS), 27 strains had decreased susceptibility to penicillin with 17 strains resistant and 10 intermediate. Comparing to the agar dilution method, oxacillin disk diffusion test detected all strains with decreased penicillin susceptibility as such while E-test showed a close agreement of susceptibility (92%) of the isolates to penicillin. This confirmed that oxacillin is a good screening test for S. pneumoniae isolates with decreased susceptibility to penicillin while E-test is very reliable for rapid and accurate detection of penicillin susceptibility.     

Evaluation of differential inoculum disk diffusion method and Vitek GPS-SA card for detection of oxacillin-resistant staphylococci.

This study was conducted in order to compare the accuracy of detection of oxacillin-resistant staphylococci, defined by microdilution MICs, population analyses, and mec gene hybridization, with the Vitek GPS-SA Susceptibility Card with that of the standard inoculum (10(7) CFU) and high-inoculum (10(9) CFU) disk diffusion tests. By the standard inoculum disk diffusion test, 10 of 67 (15%) isolates of oxacillin-resistant Staphylococcus aureus and 3 of 47 (6%) isolates of Staphylococcus epidermidis were falsely susceptible after 24 h of incubation at 35 degrees C. By the high-inoculum disk diffusion test (10(9) CFU), 4 of the 10 isolates of S. aureus remained falsely susceptible, whereas none of the isolates of S. epidermidis was falsely susceptible. Of the 10 isolates of S. aureus falsely susceptible by the standard disk test, only one remained falsely susceptible after an additional 24 h of incubation at 22 degrees C. All four isolates of S. aureus that were falsely susceptible by the high-inoculum disk diffusion test after 24 h of incubation at 35 degrees C became resistant after an additional 24 h of incubation at 22 degrees C. Thus, extended incubation of both the standard and high-inoculum disk diffusion tests increased their accuracy in detecting oxacillin resistance. All isolates of oxacillin-resistant staphylococci were accurately detected with the Vitek software upgrades (6.1 and 7.1) of the GPS-SA card.     

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.     

Evaluation of phenotypic and molecular methods for detection of oxacillin resistance in members of the Staphylococcus sciuri group

In this paper we report on an experimental evaluation of phenotypic and molecular methods as means for the detection of oxacillin resistance in members of the Staphylococcus sciuri group. A total of 109 S. sciuri group member isolates (92 S. sciuri isolates, 9 S. lentus isolates, and 8 S. vitulinus isolates) were tested by the disk diffusion method, the agar dilution method, the oxacillin salt-agar screening method, slide latex agglutination for PBP 2a, and PCR assay for mecA as the reference method. The mecA gene was detected in 29 S. sciuri isolates, and the true-positive and true-negative results of the other tests were defined on the basis of the presence or the absence of the mecA gene. For the different methods evaluated, the sensitivities and specificities were as follows: for the disk diffusion test with a 1-microg oxacillin disk, 100% and 55.9%, respectively; for the disk diffusion test with a 30-mug cefoxitin disk, 93.5% and 100%, respectively; for the agar dilution method, 100% and 50%, respectively; for the oxacillin salt-agar screen test (with 6 microg of oxacillin per ml and 4% NaCl) 100% and 100%, respectively; and for the slide latex agglutination test for PBP 2a, 100% and 100%, respectively. The disk diffusion test with various beta-lactam antibiotics was performed to evaluate their use for the prediction of oxacillin resistance. The results indicate that meropenem, cefazolin, cefamandole, cefuroxime, cefotetan, cefoperazone, cefotaxime, ceftriaxone, moxalactam, cefaclor, and cefprozil may be used as surrogate markers of oxacillin resistance, although further studies of their use for the detection of oxacillin resistance are required.     

ROLE OF AZITHROMYCIN AGAINST CLINICAL ISOLATES OF FAMILY ENTEROBACTERIACEAE: A COMPARISON OF ITS MINIMUM INHIBITORY CONCENTRATION BY THREE DIFFERENT METHODS

Purpose: To determine the effect of azithromycin, a new azalide antibiotic, on clinical isolates of the family Enterobacteriaceae and to determine and compare its minimum inhibitory concentration (MIC) by disk diffusion, agar dilution and E-test methods. Materials and Methods: One hundred fifty-nine bacterial strains belonging to the family Enterobacteriaceae, isolated from different clinical samples, were tested for their susceptibility to azithromycin by disk diffusion, agar dilution and E-test methods. The MIC values were analysed and the percentages of agreement between the different methods were mentioned. Results: Of the 159 isolates of the family Enterobacteriaceae, 60.37% were E. coli followed by Klebsiella species 28.3%, Salmonella and Shigella species 3.77% and Enterobacter and Citrobacter species 1.88% each. Maximum isolates were obtained from urine 117/159 (73.58%). Azithromycin was found to be more active against Salmonella and Shigella species, showing 100% sensitivity the by E-test and 83.33% by the disk diffusion methods. In the agar dilution method, 83.33% of Salmonella and 66.66% of Shigella species were sensitive to azithromycin. The overall agreement between disk diffusion and agar dilution method was 96.8%, between agar dilution and E-test was 88% and between disk diffusion and E-test was 91.2%. Conclusion: Azithromycin may become an important addition to our antimicrobial strategies, especially for the treatment of bacterial diarrhoea and infections caused by Salmonella typhi.     

Comparison of the effects of anaerobic and micro-aerophilic incubation on resistance of Helicobacter pylori to metronidazole.

To assess the influence of incubation conditions on the resistance of Helicobacter pylori this study compared the effect of micro-aerophilic and anaerobic incubation followed by micro-aerophilic incubation on the measurement of metronidazole resistance of 102 H. pylori isolates, by both disk diffusion and Epsilometer (E)-tests. Anaerobic incubation for 24 h before micro-aerophilic incubation for 48 h consistently increased metronidazole activity in both assay methods. Although statistically significant, this was microbiologically less significant, as only 4 of 102 isolates gave discrepant readings (all four were resistant in micro-aerophilic conditions but susceptible in anaerobic/micro-aerophilic conditions). In all four cases variation was by a few millimeters in zone size (i.e., all were close to the cut-off point). There was 100% agreement between disk diffusion and E-test results. Of 104 observations (52 duplicate assays: 13 strains, two atmospheric conditions, two methods of determining resistance) there was 100% intra-observer and inter-observer agreement with regard to susceptibility and resistance status for both E-test and disk diffusion methods. Anaerobic incubation followed by micro-aerophilic incubation had little effect on the estimation of prevalence of metronidazole resistance and seemed to add little, if any, significant advantage over micro-aerophilic incubation alone.