Agar Screening Method Research Articles

Characteristics and trends in macrolide resistance among Helicobacter pylori strains isolated in Bulgaria over four years

Macrolide resistance trends were examined among Helicobacter pylori strains from 154 patients between 1994 and 1998. Applicabilities of screening agar method (SAM) and modified disk diffusion method (MDDM) were evaluated. Overall primary resistance rates to erythromycin and clarithromycin were 14.8% and 8.7%, respectively. No association was found with age, sex, and diseases. Clarithromycin-resistance rate reached 12.5% in the last 2 years. Secondary resistance to erythromycin occurred more often (in 62.5%) than to clarithromycin (in 42.9%). Therapy with spiramycin or erythromycin in four cases induced no clarithromycin resistance. These data show a considerable prevalence of H. pylori resistance to macrolides, which exhibited a tendency to increase and was often associated with metronidazole resistance. By comparing the MDDM with SAM, an overall agreement was obtained in 81 (94.2%) of 86 results. MDDM and SAM are reliable techniques for testing H. pylori susceptibility to macrolides.

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 </=0.5 microg/ml by agar dilution tests, and the strains were oxacillin susceptible when tested by the BBL Crystal MRSA ID and a Vitek machine with GPS-101 cards. None grew on oxacillin-salt agar screening plates. None were positive for mecA gene sequences by PCR. When S. aureus strains tested by Vitek GPS-SA or GPS-BS cards appear resistant to only penicillin and oxacillin, a confirmatory test such as the oxacillin-salt agar screening method should be performed.

Methicillin-Resistant Staphylococcus aureus and Methicillin-Resistant Coagulase-Negative Staphylococci From Conjunctivas of Preoperative Patients

To evaluate the incidence and characteristics of carriers of conjunctival methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative Staphylococci (MRCNS) among preoperative patients at an eye clinic, bacterial growth was studied in 978 conjunctival specimens of 628 preoperative patients without signs of ocular infection. Specimens were evaluated for growth of bacteria on nutrient agar plate, blood agar plate, or chocolate agar plate for 2 days at 37°C. Methicillin resistance was confirmed by disk diffusion and agar screening methods. Susceptibilities of the bacterial strain to ampicillin (ABPC), cefazolin (CEZ), minocycline (MINO), gentamicin (GM), erythromycin (EM), vancomycin (VCM), and ofloxacin (OFLX) were determined by the disk diffusion method. Of the 628 patients (978 eyes), 352 patients (580 eyes) had positive bacterial growth. Among them, 8 (13 eyes) had MRSA growth and 2 (4 eyes) had MRCNS growth. The rate of nasolacrimal duct obstruction was significantly higher in eyes with positive bacterial growth than in eyes with negative growth. Of the 10 patients (17 eyes) with MRSA or MRCNS growth, 8 were older than 80 years, 5 had the same bacterial strains in the anterior nares and throat, 6 had nasolacrimal duct obstruction, 3 had dry eye, and 9 had been recently hospitalized. All 10 bacterial strains were resistant to ABPC, CEZ, and EM, but were sensitive to MINO and OFLX. Five strains were resistant and 5 were sensitive to GM. Two strains examined were sensitive to VCM. After topical antibiotic use for 0.5 to 6 months, the bacterial strains were eliminated. Two of the 17 eyes with MRSA or MRCNS growth did not undergo surgery. No postoperative endophthalmitis developed in 976 of the 978 eyes. Ophthalmologists should be aware that about 50% of preoperative patients without signs of ocular infection may have bacterial growth on the conjunctiva, and that elderly patients with nasolacrimal duct obstruction, dry eye, and recent hospital stays may be carriers of MRSA or MRCNS.

Clonal Dissemination of Vancomycin-Resistant Enterococci and Comparison of Susceptibility Testing Methods

One hundred fifty clinical isolates of Enterococcus faecalis (88 isolates) and Enterococcus faecium (62 isolates) were tested in vitro for their susceptibility to vancomycin and high-level aminoglycosides (HLA). Remel’s Synergy Quad Plates (RSQ) were used as the reference method and compared to Kirby-Bauer disc diffusion test, Vitek® GPS-TA card, MicroScan® Panel (GP-6), and Etest®. Streptomycin susceptibility results for MicroScan GP-6 and RSQ were recorded at 24 and 48 h and all other methods and antibiotics were read at 24 h or less. When compared with the agar screen method, all of the methods demonstrated >99% agreement. One isolate was falsely sensitive to gentamicin at 24 h, but resistant at 48 h, when tested on both MicroScan and RSQ agar screen. Thirty-nine isolates showed resistance to vancomycin with all methods. These isolates were from three different local hospitals and were identified as E. faecium. Pulse-field gel electrophoresis demonstrated that all of the vancomycin-resistant isolates were derived from the same clone. Of interest is the observation that high-level resistance to aminoglycosides varied between the clonally related isolates.

Characterization of staphylococci with reduced susceptibilities to vancomycin and other glycopeptides.

During the last several years a series of staphylococcal isolates that demonstrated reduced susceptibility to vancomycin or other glycopeptides have been reported. We selected 12 isolates of staphylococci for which the vancomycin MICs were > or =4 microg/ml or for which the teicoplanin MICs were > or =8 microg/ml and 24 control strains for which the vancomycin MICs were < or =2 microg/ml or for which the teicoplanin MICs were < or =4 microg/ml to determine the ability of commercial susceptibility testing procedures and vancomycin agar screening methods to detect isolates with reduced glycopeptide susceptibility. By PCR analysis, none of the isolates with decreased glycopeptide susceptibility contained known vancomycin resistance genes. Broth microdilution tests held a full 24 h were best at detecting strains with reduced glycopeptide susceptibility. Disk diffusion did not differentiate the strains inhibited by 8 microg of vancomycin per ml from more susceptible isolates. Most of the isolates with reduced glycopeptide susceptibility were recognized by MicroScan conventional panels and Etest vancomycin strips. Sensititre panels read visually were more variable, although with some of the panels MICs of 8 microg/ml were noted for these isolates. Vitek results were 4 microg/ml for all strains for which the vancomycin MICs were > or =4 microg/ml. Vancomycin MICs on Rapid MicroScan panels were not predictive, giving MICs of either < or =2 or > or =16 microg/ml for these isolates. Commercial brain heart infusion vancomycin agar screening plates containing 6 microg of vancomycin per ml consistently differentiated those strains inhibited by 8 microg/ml from more susceptible strains. Vancomycin-containing media prepared in-house showed occasional growth of susceptible strains, Staphylococcus aureus ATCC 29213, and on occasion, Enterococcus faecalis ATCC 29212. Thus, strains of staphylococci with reduced susceptibility to glycopeptides, such as vancomycin, are best detected in the laboratory by nonautomated quantitative tests incubated for a full 24 h. Furthermore, it appears that commercial vancomycin agar screening plates can be used to detect these isolates.

Evaluation of screening and commercial methods for detection of methicillin resistance in coagulase-negative staphylococci.

The National Committee for Clinical Laboratory Standards recommends 48 h of incubation by the oxacillin salt agar screen (OSAS) method for the detection of methicillin-resistant coagulase-negative staphylococci (CoNS). An earlier identification of methicillin resistance is desirable. The time to detection of the mecA gene by PCR was compared with the times to detection by OSAS, by the oxacillin disk diffusion (ODD) method, and with MicroScan Gram Positive Combo type 6 panels (MicroScan Inc. Sacramento, Calif.) and Vitek GPS-SA cards (bioMérieux Vitek Inc., Hazelwood, Mo.). The combination of the Vitek card and the ODD method detected 92 of 99 methicillin-resistant strains of CoNS at 24 h; however, 6 mecA-positive strains were phenotypically methicillin susceptible. We conclude that most methicillin-resistant CoNS can be detected and the results can be reported after overnight incubation by a combination of methods.

Methicillin-Resistant Staphylococcus aureus Epidemiology and Control in Belgian Hospitals, 1991 to 1995

Objectives: To describe the Belgian methicillin-resis-tant Staphylococcus aureus (MRSA) surveillance network, the evolution of methods used in Belgian hospitals for MRSA detection and control, and MRSA incidence from 1994 to1995.Design, Setting, and Participants: Questionnaire surveys; infection control physicians from acute-care hospitals in Belgium.Intervention: Publication of national guidelines for MRSA control in 1993.Results: The participation rate in surveys ranged from 42% to 57% of hospitals. In 1995, 88% of participants detected MRSA strains by disk diffusion tests, with little improvement in standardization since 1991. More centers employed the oxacillin agar screen method (27%), automated systems (29%), or a combination of methods (29%) than in 1991 (P&lt;.005). Between 1991 and 1995, the proportion of hospitals reporting MRSA control measures increased from 68% to 95% (P&lt;.01). Practices that were used increasingly included patient placement in private room (from 50% to 93%, P&lt;.01) and hand decontamination with antiseptic (from 43% to 87%, P&lt;.01). The proportion of centers that reported screening MRSA carriers and treating them topically increased two- and threefold, respectively (P&lt;.05). Surveillance data from 1994 to 1995 showed that MRSA represented a mean of 21.3% of S aureus clinical isolates (range, 1.6% to 62.4%). The median incidence of nosocomial MRSA acquisition was 2.8 per 1,000 admissions, with a wide range (0 to 13.7 per 1,000 admissions) across hospitals of all sizes. The median incidence decreased over the first three semesters of surveillance in hospitals with continuous participation.Conclusion: MRSA detection and control measures have improved in Belgian hospitals after publication of national guidelines. However, MRSA incidence rates show the persistence of nosocomial transmission, with large variations between centers. The national MRSA surveillance network should indicate whether control efforts eventually will curb the problem.

Methicillin-resistant Staphylococcus aureus epidemiology and control in Belgian hospitals, 1991 to 1995. Groupement pour le Dépistage, l'Etude et la Prévention des Infections Hospitalières.

To describe the Belgian methicillin-resistant Staphylococcus aureus (MRSA) surveillance network, the evolution of methods used in Belgian hospitals for MRSA detection and control, and MRSA incidence from 1994 to 1995. Questionnaire surveys; infection control physicians from acute-care hospitals in Belgium. Publication of national guidelines for MRSA control in 1993. The participation rate in surveys ranged from 42% to 57% of hospitals. In 1995, 88% of participants detected MRSA strains by disk diffusion tests, with little improvement in standardization since 1991. More centers employed the oxacillin agar screen method (27%), automated systems (29%), or a combination of methods (29%) than in 1991 (P < .005). Between 1991 and 1995, the proportion of hospitals reporting MRSA control measures increased from 68% to 95% (P < .01). Practices that were used increasingly included patient placement in private room (from 50% to 93%, P < .01) and hand decontamination with antiseptic (from 43% to 87%, P < .01). The proportion of centers that reported screening MRSA carriers and treating them topically increased two- and threefold, respectively (P < .05). Surveillance data from 1994 to 1995 showed that MRSA represented a mean of 21.3% of S aureus clinical isolates (range, 1.6% to 62.4%). The median incidence of nosocomial MRSA acquisition was 2.8 per 1,000 admissions, with a wide range (0 to 13.7 per 1,000 admissions) across hospitals of all sizes. The median incidence decreased over the first three semesters of surveillance in hospitals with continuous participation. MRSA detection and control measures have improved in Belgian hospitals after publication of national guidelines. However, MRSA incidence rates show the persistence of nosocomial transmission, with large variations between centers. The national MRSA surveillance network should indicate whether control efforts eventually will curb the problem.

Methicillin-ResistantStaphylococcus aureusEpidemiology and Control in Belgian Hospitals, 1991 to 1995

AbstractObjectives:To describe the Belgian methicillin-resis-tantStaphylococcus aureus(MRSA) surveillance network, the evolution of methods used in Belgian hospitals for MRSA detection and control, and MRSA incidence from 1994 to1995.Design, Setting, and Participants:Questionnaire surveys; infection control physicians from acute-care hospitals in Belgium.Intervention:Publication of national guidelines for MRSA control in 1993.Results:The participation rate in surveys ranged from 42% to 57% of hospitals. In 1995, 88% of participants detected MRSA strains by disk diffusion tests, with little improvement in standardization since 1991. More centers employed the oxacillin agar screen method (27%), automated systems (29%), or a combination of methods (29%) than in 1991 (P&lt;.005). Between 1991 and 1995, the proportion of hospitals reporting MRSA control measures increased from 68% to 95% (P&lt;.01). Practices that were used increasingly included patient placement in private room (from 50% to 93%,P&lt;.01) and hand decontamination with antiseptic (from 43% to 87%,P&lt;.01). The proportion of centers that reported screening MRSA carriers and treating them topically increased two- and threefold, respectively (P&lt;.05). Surveillance data from 1994 to 1995 showed that MRSA represented a mean of 21.3% ofS aureusclinical isolates (range, 1.6% to 62.4%). The median incidence of nosocomial MRSA acquisition was 2.8 per 1,000 admissions, with a wide range (0 to 13.7 per 1,000 admissions) across hospitals of all sizes. The median incidence decreased over the first three semesters of surveillance in hospitals with continuous participation.Conclusion:MRSA detection and control measures have improved in Belgian hospitals after publication of national guidelines. However, MRSA incidence rates show the persistence of nosocomial transmission, with large variations between centers. The national MRSA surveillance network should indicate whether control efforts eventually will curb the problem.

Vancomycin-resistant enterococci colonizing the intestinal tracts of hospitalized patients

A point prevalence culture survey was carried out to investigate the prevalence of fecal carriage of vancomycin-resistant enterococci (VRE) among patients admitted to an 800-bed general hospital where no VRE had been isolated previously. Twenty-two of 636 patients (3.5%) were found to be VRE carriers. Eighteen strains were identified as Enterococcus faecium, three were identified as Enterococcus gallinarum, and one was identified as Enterococcus faecalis. The susceptibilities of the enterococci to ampicillin, vancomycin, and teicoplanin were determined by the disk diffusion and the agar dilution methods. High-level resistance (HLR) to gentamicin and streptomycin was determined by the agar screening method. Eighteen strains (82%) were highly resistant to vancomycin, and four strains (18%) were moderately resistant to vancomycin. Five strains were susceptible to teicoplanin (23%; MICs, < or = 8 micrograms/ml). Only one strain (4.5%, E. faecium) showed HLR to gentamicin, and six strains (27%) showed HLR to streptomycin (one E. faecalis and five E. faecium strains). All 18 E. faecium and 1 E. faecalis strain carried the vanA gene, and 3 E. gallinarum strains carried the vanC gene. An epidemiological investigation revealed several risk factors for VRE colonization: hospitalization and duration of stay in the hematology department and prior vancomycin treatment. The study demonstrates that the patient's gastrointestinal tract is a possible reservoir for VRE, even in hospitals where VRE infections have not yet been observed. Therefore, we conclude that infection control precautions and restriction of glycopeptide usage may be key issues in limiting the emergence and spread of nosocomial VRE infections.

Investigation of the reformulated Remel Synergy Quad plate for detection of high-level aminoglycoside and vancomycin resistance among enterococci.

We investigated the accuracy of the recently released Remel Synergy Quad plate, a commercially available agar screening method for detecting high-level aminoglycoside and vancomycin resistance among enterococci that is based on the National Committee for Clinical Laboratory Standards recommended guidelines (National Committee for Clinical Laboratory Standards, M7-A3, 1993). The Synergy Quad correctly determined the gentamicin and streptomycin resistance status for > or = 97% of 147 Enterococcus faecalis and Enterococcus faecium isolates tested. Detection of vancomycin resistance also was reliable, as no false susceptibility occurred with 36 vancomycin-resistant E. faecalis and E. faecium strains and false resistance occurred only once with the 47 susceptible strains tested. One strain each of Enterococcus gallinarum and Enterococcus casseliflavus failed to grow on the screen, but because the true nature and significance of resistance in such isolates is unknown the implication of their screen negativity is uncertain. In summary, the Remel Synergy Quad provides a highly accurate and convenient method for susceptibility testing of enterococci against gentamicin, streptomycin, and vancomycin.

Development of a standardized screening method for detection of vancomycin-resistant enterococci

The incidence of vancomycin resistance among enterococci is increasing in the United States and elsewhere in the world, but automated susceptibility testing methods have difficulty detecting resistance expressed by certain strains. The agar screening method described by Willey et al. (B. M. Willey, B. N. Kreiswirth, A. E. Simor, G. Williams, S. R. Scriver, A. Phillips, and D. E. Low, J. Clin. Microbiol. 30:1621-1624, 1992) has been proposed as a reliable method for confirming vancomycin resistance. In this study, we investigated various parameters associated with the agar screening method and, on the basis of the findings, established optimum testing conditions for the method. First, to evaluate media and vancomycin concentrations, one laboratory used Mueller-Hinton and brain heart infusion agars supplemented with 4, 6, and 8 micrograms of vancomycin per ml to test 100 genetically characterized enterococcal strains. On the basis of the results obtained, brain heart infusion agar supplemented with 6 micrograms of vancomycin per ml was selected for further study. Subsequently, eight laboratories used the medium to test both reference and clinical isolates. There was very good performance with the reference strains and, among 158 clinical isolates tested, the method demonstrated sensitivity and specificity of 100% and from 96 to 99%, respectively.

Factors influencing determination of high-level aminoglycoside resistance in Enterococcus faecalis

The ability of seven methods to detect high-level gentamicin (58 strains) and streptomycin resistance (56 strains) among 107 Enterococcus faecalis isolates was investigated at the University of Chicago Medical Center and the University of Nebraska Medical Center. Methods included a standard agar screen plate, high-content disk diffusion, Remel (Lenexa, Kans.) EF Synergy Quad plates, standard microdilution panels prepared in house, Pasco MIC Gram-Positive panels (Difco Laboratories, Detroit, Mich.), MicroScan MIC Type 5 dry panels (Baxter Healthcare Corp., MicroScan Div., West Sacramento, Calif.), and Vitek GPS-TA cards (Vitek Systems Inc., Hazelwood, Mo.). Results indicating false resistance were not obtained by any method, and there was 100% agreement between the results of the disk diffusion and standard agar screen methods. Prolonging incubation from 24 to 48 h increased resistance detection for both agar and microdilution screens. EF Synergy Quad plates inoculated with micropipettes detected 100% of the streptomycin- and gentamicin-resistant isolates. Resistance detection for streptomycin and gentamicin, respectively, was 93 and 96% by standard microdilution, 93 and 98% by Pasco panels, 88 and 89% by MicroScan panels, and 88 and 91% by Vitek GPS-TA cards. False susceptibility occurred more frequently with streptomycin-resistant isolates than it did with gentamicin-resistant strains and appeared to be strain related in some instances. The use of an increased inoculum size enhanced resistance detection with these strains, but it complicated interpretation of results and led to the selection of streptomycin-resistant mutants. Until results of further studies delineate optimum test conditions, a delay in the final interpretation of agar and microdilution screen results until 48 h for isolates showing no or light growth at 24 h may help to minimize the occurrence of false susceptibility reporting.

Evaluation of laboratory methods for the classification of oxacillin-resistant and oxacillin-susceptible Staphylococcus aureus.

The purpose of this study was to examine the efficacy of the oxacillin disk diffusion test and the methicillin and oxacillin agar screen tests as predictors of oxacillin resistance as defined by the reference broth microdilution method. A total of 444 clinical isolates of Staphylococcus aureus collected from individual patients over a four-year period were tested by (1) the oxacillin disk diffusion test, with particular attention to the presence of fine growth of a resistant subpopulation within the zone of inhibition (Ox Grow interpretive criteria); (2) the agar screen method using agar plates with 4% NaCl and either 6 mg/L oxacillin or 10 mg/L methicillin; and (3) the oxacillin and methicillin broth microdilution test methods with 2% NaCl supplementation. Overall, 62 (14%) isolates were resistant and 382 (86%) isolates were susceptible to oxacillin with the use of the reference broth microdilution system. The results indicate that the disk diffusion test with the use of the Ox Grow criteria had a high sensitivity (94%) and negative predictive value (98%) but a low specificity (67%) and positive predictive value (32%) when compared with the reference broth dilution test. Similarly, the agar screen tests had a high sensitivity (95-97%) and negative predictive values (99%) but low specificity (64-74%) and positive predictive values (30-37%). These data indicate that the agar screen tests and the oxacillin disk test with the use of the Ox Grow interpretive criteria may be useful as screening tests for detecting resistance to the penicillinase-resistant penicillins but that all resistant isolates should be confirmed by the reference broth dilution method because of the large number of false-resistant screening test results.

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.