Sceptor System Research Articles

A comparison of PCR detection of <i> Meca</i> with oxacillin disk susceptibility testing in different media and sceptor automated system for both <i> Staphylococcus aureus</i> and coagulase-negative staphylococci isolates

To evaluate three methods for 406 isolates of Staphylococcus aureus and coagulase-negative staphylococci (CNS) for the detection of methicillin resistance (MR) using National Committee for Clinical Laboratory Standards (NCCLS) new interpretive criteria. We used polymerase chain reaction (PCR) as a gold standard method to evaluate three methods [disk diffusion with Mueller-Hinton agar (MHA) and mannitol salt agar (MSA) and Sceptor system (Becton Dickinson, USA)] for the detection of mecA gene. The isolates that were methicillin-resistant with any of the three tests were evaluated further for MR by E-test. MHA, MSA and Sceptor showed sensitivities of 100, 100 and 99% for S. aureus and 100, 82.6 and 72.1% for CNS, respectively. The specificities of the same methods were found as 100, 90.1 and 99.3% for S. aureus and 79.2, 95.8 and 97.2% for CNS, respectively. E-test showed 100% sensitivity for both S. aureus and CNS. Forty-eight CNS and 16 S. aureus isolates, which presented discrepancies with the three phenotypic methods (MHA disk diffusion method, MSA disk diffusion method and Sceptor), were correctly classified as resistant/susceptible with the E-test when compared with PCR. Only five CNS isolates, which were mecA-negative with PCR were resistant with E-test. Analysis of 248 S. aureus revealed that MHA is superior to other phenotype-based susceptibility testing methods in detecting MR. When we examined the results of 158 CNS, none of the three methods proved efficient in detecting MR. We conclude that although the accuracy of the MHA disk diffusion test for the detection of MR approaches the accuracy of PCR for S. aureus isolates, the need for easy and reliable methods of detecting MR in CNS still remains.

A COMPARISON OF PCR DETECTION OF MECA WITH OXACILLIN DISK SUSCEPTIBILITY TESTING IN DIFFERENT MEDIA AND SCEPTOR AUTOMATED SYSTEM FOR BOTH STAPHYLOCOCCUS AUREUS AND COAGULASE-NEGATIVE STAPHYLOCOCCI ISOLATES

Purpose: To evaluate three methods for 406 isolates of Staphylococcus aureus and coagulase-negative staphylococci (CNS) for the detection of methicillin resistance (MR) using National Committee for Clinical Laboratory Standards (NCCLS) new interpretive criteria. Methods: We used polymerase chain reaction (PCR) as a gold standard method to evaluate three methods [disk diffusion with Mueller-Hinton agar (MHA) and mannitol salt agar (MSA) and Sceptor system (Becton Dickinson, USA)] for the detection of mecA gene. The isolates that were methicillin-resistant with any of the three tests were evaluated further for MR by E-test. Results: MHA, MSA and Sceptor showed sensitivities of 100, 100 and 99% for S. aureus and 100, 82.6 and 72.1% for CNS, respectively. The specificities of the same methods were found as 100, 90.1 and 99.3% for S. aureus and 79.2, 95.8 and 97.2% for CNS, respectively. E-test showed 100% sensitivity for both S. aureus and CNS. Forty-eight CNS and 16 S. aureus isolates, which presented discrepancies with the three phenotypic methods (MHA disk diffusion method, MSA disk diffusion method and Sceptor), were correctly classified as resistant/susceptible with the E-test when compared with PCR. Only five CNS isolates, which were mecA-negative with PCR were resistant with E-test. Analysis of 248 S. aureus revealed that MHA is superior to other phenotype-based susceptibility testing methods in detecting MR. When we examined the results of 158 CNS, none of the three methods proved efficient in detecting MR. Conclusions: We conclude that although the accuracy of the MHA disk diffusion test for the detection of MR approaches the accuracy of PCR for S. aureus isolates, the need for easy and reliable methods of detecting MR in CNS still remains.

Microbiology of Postoperative Infections

Surgical infections, accounting for approximately one quarter of all nosocomial infections, contribute to longer hospital stays and higher medical costs. Clinical failure of antibiotic treatment often is associated with increasing drug resistance. Non-duplicated microbial isolates obtained at our institution from surgical patients were identified and tested for antimicrobial susceptibility using the Sceptor system in 2001-2002 and the Phoenix system in 2003-2005. Epidemiologic data were extracted using Epicenter and Powerlab. During selected periods, genetic and biochemical characterization was performed by pulsed-field gel electrophoresis, colony blot hybridization, gene amplification and sequencing, isoelectric focusing, and enzyme kinetics. The specimens were most commonly isolated from infections at surgical sites (24.0%), the urinary tract (19.0%), and blood stream (10.6%). Isolates of Escherichia coli showed an increasing prevalence of resistance to penicillins, third-generation cephalosporins, ciprofloxacin, and gentamicin, whereas the prevalence of resistant Staphylococcus aureus generally declined. Highly aggressive multi-drug-resistant (MDR) Pseudomonas aeruginosa strains were widespread, although colistin remained effective in 100% of cases. A significant increase was seen in infections attributable to S. epidermidis and Candida albicans. The overall number of Enterobacteriaceae carrying extended-spectrum beta-lactamases increased continuously, and new resistance traits emerged. It appeared that the TEM-92 determinant moved from Enterobacteriaceae to nonfermenting gram-negative rods such as Acinetobacter baumannii. The number and type of antimicrobial-resistance determinants are increasing.

Antibacterial sensitivity of Acinetobacter strains isolated from nosocomial infections.

Acinetobacter species can cause many types of hospital-acquired infection and play an important role in nosocomial pneumonia in intensive care units, skin and wound infections, and meningitis. They are of increasing importance because of their ability to rapidly develop resistance to the major groups of antibiotics. We aimed to determine the antibiotic sensitivity of Acinetobacter strains isolated from, and determined to be the cause of, hospital-acquired infections. A total of 156 cultures of Acinetobacter (strains of A. baumannii [136; 87.2%] and A. iwoffii [20; 12.8%]), were isolated from clinical samples taken from patients in different units of our hospital. Conventional bacterial identification methods and the Sceptor system were used. In the antibiotic sensitivity tests, A. baumannii was susceptible to imipenem (90.4%), norfloxacin (84.5%) and ciprofloxacin (65.4%), and A. iwoffii to amikacin (80.0%), ticarcillin/clavulanic acid (70.0%) and imipenem (60.0%).

Antimicrobial susceptibility testing of veterinary clinical isolates with the Sceptor System

The Sceptor System (Becton Dickinson) was compared with an agar dilution method for antimicrobial susceptibility testing of veterinary clinical isolates. The results indicate that the Sceptor System may be used to test gram-positive and fastidious gram-negative bacteria.

Evaluation of the Sceptor system for identification of bacteria of veterinary origin

The Sceptor system (Becton Dickinson Diagnostic Instrument Systems, Towson Md.) was assessed for its ability to identify veterinary clinical isolates. A total of 605 bacteria, including 315 isolates of the family Enterobacteriaceae, 191 gram-negative nonenteric bacteria, and 99 gram-positive bacteria, were tested. Overall, 534 (88.3%) were correctly identified, 28 (4.6%) were not identified, 12 (2.0%) were incorrectly identified at the genus levels, and 32 (5.3%) were incorrectly identified at the species level. The Sceptor system correctly identified 292 (92.7%) isolates of Enterobacteriaceae, 165 (86.4%) gram-negative nonenteric bacteria, and 77 (77.8%) gram-positive bacteria. One hundred thirty organisms not contained in the data base were tested with the Sceptor system to assess the possibility of expanding the data base. The Sceptor system was an acceptable method for the identification of isolates of Enterobacteriaceae but not gram-negative nonenteric and gram-positive bacteria of animal origin. Development of a veterinary isolate-specific data base would improve the utility of the Sceptor system in veterinary diagnostic bacteriology.

Comparison of four commercial microdilution systems for susceptibility testing of anaerobic bacteria

The reliability of four commercially available broth microdilution systems (ATB ANA, MHK-Anaerob-Biotest, Dynatech MIC system, Sceptor Anaerobe MIC system) for routine susceptibility testing was evaluated using agar dilution and broth macrodilution as reference methods. Using the categories "susceptible", "moderately susceptible" and "resistant", the rate of essential agreement (complete agreement plus minor errors) of all four systems compared to the two reference methods was satisfactory, ranging from 93% (Dynatech system and agar dilution) to 98% (Sceptor system and agar dilution). The rate of complete agreement compared to the agar dilution and broth dilution method respectively was 61% and 60% for the MHK-Anaerob-Biotest, 65% and 63% for the Dynatech system, 72% and 72% for ATB ANA, and 85% and 80% for the Sceptor system. The Sceptor system was thus superior to the other systems. The systems were easy to operate and inoculate, although arrangement of antimicrobial agents in the Dynatech panels was apt to be confusing.

Commercial identification systems often fail to identify Providencia stuartii.

We tested 145 clinical isolates in an attempt to evaluate some of the most widely used commercial identification systems in Europe in terms of their ability to identify Providencia strains. Two manual miniaturized systems (API 20E and Enterotube II) and three mechanized-automated systems (Cobas-Bact, Sceptor System, and Titertek-Enterobac-Rapid Automated System) were evaluated. Providencia alcalifaciens and Providencia rettgeri strains were correctly identified by all systems in all cases, and in most cases identification was achieved without the aid of supplementary tube tests. By contrast, Providencia stuartii was identified without the aid of supplementary tube tests for only 42.5% (API 20E), 37.5% (Enterotube), 68.7% (Sceptor), and 71.2% (Cobas-Bact) of the isolates. The overall misidentification rates were 16.3, 11.3, 11.3, and 10%, respectively. The Titertek-Enterobac-Rapid Automated System failed to identify only 1 of 80 strains (1.3%) and required supplementary tests in 2 other cases (2.5%). Since four of the multitest systems examined often failed to correctly identify P. stuartii, we conclude that supplementary conventional tube tests should always be used to distinguish this species from the other taxa of the Proteeae tribe.

Rapid determination of antimicrobial susceptibility patterns ofListeria monocytogenes isolates by the autobac AIS and MIC procedures

A total of 34 isolates ofListeria monocytogenes were tested against ampicillin, cephalothin, chloramphenicol, erythromycin, tetracycline, and penicillin-G using the Autobac 3-h AIS and the Autobac 5-h MIC procedures. The results were compared to susceptibility category interpretations and MICs determined using the Sceptor system. With the Sceptor System, all isolates were interpreted to be moderately susceptible to ampicillin and penicillin-G, and susceptible to the four other antibiotics. With the Autobac AIS, all isolates were interpreted to be susceptible to all the antibiotics except penicillin-G. All but one of the 34 isolates were interpreted to be resistant to penicillin-G with the Autobac AIS test. The remaining isolate was interpreted to be indeterminant. The Autobac AIS test was unsatisfactory for determining the susceptibility ofL. monocytogenes isolates to penicillin-G. The Autobac MIC results correlated well with the MIC results of the Sceptor system provided that the Autobac was programmed as though it were testing enterococci. The Autobac MIC reported penicillin-G MICs in units per milliliter and required the use of a conversion factor to obtain micrograms per milliliter, and did not allow for the testing of erythromycin. The Autobac MIC susceptibility category interpretations must not be used, as they were derived from an outdated susceptibility standard. The Autobac MIC test may be used if the limitations given above are observed.

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.

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.

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.