Rapid identification of enterococci by reduction of litmus milk.
Of 100 strains of enterococci, 83% reduced litmus milk within 4 h. None of the 100 non-enterococcal streptococci tested were positive.
Animals↗
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Preliminary evaluation of a rapid colorimetric method for the presumptive identification of group A streptococci and enterococci.
A rapid colorimetric method for the presumptive identification of group A streptococci and enterococci based upon pyroglutamyl aminopeptidase activity is described. Of 76 group A streptococcal isolates from primary plates, 83 gave positive reactions, and the remaining 7 were positive on retesting in pure culture. Of the 31 enterococcal isolates tested, all gave positive reactions. Despite occasional positive reactions with staphylococci and Klebsiella pneumoniae, the test could be useful and cost-effective in the clinical laboratory.
Bacteriological Techniques↗
Enterococci highly resistant to penicillin and ampicillin: an emerging clinical problem?
Sixteen clinical isolates of ampicillin-resistant enterococci (ARE) were recovered from the microbiology laboratory of a 450-bed rehabilitation medical center from January 1981 to September 1987. These isolates were detected when a disk diffusion test using 10 micrograms of ampicillin on a blood agar plate revealed no zones of inhibition. Tube macrodilution tests yielded an MIC of greater than or equal to 16 micrograms of ampicillin per ml. None of the isolates were penicillinase producers by the chromogenic cephalosporin disk test. Ten isolates were Enterococcus faecium, four isolates were E. raffinosus, one isolate was E. gallinarum, and one isolate was not identified (lost). There were 6 male and 10 female patients. The sources of isolates were urine (n = 7), wound (n = 5), ascitic fluid (n = 2), blood (n = 2), peritoneal catheter tip (n = 1), Bartholin's cyst abscess (n = 1), rectal swab (n = 2), and pancreatic abscess (n = 1). The organism was isolated from multiple sites in 4 patients, was a pure culture isolate in 5 patients, and was part of a polymicrobial flora in 11 patients. Six patients were diabetic, and four had liver cirrhosis. All but four patients had received at least one antibiotic within 3 weeks of ARE isolation. The MICs (micrograms per milliliter) for 50 and 90% of isolates tested, respectively, were as follows: ampicillin, 64 and 64; penicillin, 128 and greater than 128; vancomycin, 1 and 2; gentamicin, 4 and 16; ciprofloxacin, 1.6 and 3.2; imipenem, 128 and greater than 128; and daptomycin (LY146032), 1.6 and 6.4. ARE may be an emerging pathogen in the hospitalized patient population.
Adolescent↗
Problems with the disk diffusion test for detection of vancomycin resistance in enterococci.
A total of 53 strains of enterococci, including recently isolated strains with high-level resistance to vancomycin, were tested for vancomycin susceptibility by broth microdilution and disk diffusion using Mueller-Hinton media with and without supplementation with 5% blood. By using currently published parameters of the National Committee for Clinical Laboratory Standards for the disk diffusion test, we found that strains for which MICs were 8 to 32 micrograms/ml were incorrectly placed in the susceptible or intermediate category, which caused both very major (1.9%) and minor (11.5%) errors. When we used newer, recently proposed breakpoints for vancomycin, we found 13.5% minor errors but no very major errors. Changing disk diffusion breakpoints to less than or equal to 14 mm for resistant [corrected] and greater than or equal to 15 mm for susceptible [corrected] would eliminate the problem for the strains with MICs of 32 micrograms/ml but not for those with MICs of 8 micrograms/ml. For those strains, it is necessary to perform an MIC test to differentiate them from strains with MICs of less than or equal to 4 micrograms/ml.
Drug Resistance, Microbial↗
Species identities of enterococci isolated from clinical specimens.
Conventional tests and commercially available systems were used to determine the species identities of clinical isolates of enterococci. Strict adherence to the conventional test scheme of Facklam and Collins (R. R. Facklam and M. D. Collins, J. Clin. Microbiol. 27:731-734, 1989) resulted in the misidentification of lactose-negative Enterococcus faecalis isolates as Enterococcus solitarius, but this problem was overcome by the application of additional tests. The commercially available systems tested were unable to recognize some of the more recently described enterococcal species. E. faecalis accounted for 87.1% of 302 consecutive isolates. Enterococcus faecium (8.6%), Enterococcus avium (0.7%), Enterococcus durans (0.3%), Enterococcus gallinarum (1.0%), Enterococcus casseliflavus (1.0%), Enterococcus hirae (0.3%), and Enterococcus raffinosus (0.3%) isolates were also identified. None of the isolates produced beta-lactamase, but 15.4% of 235 isolates tested, including 1 strain of E. gallinarum, displayed high-level resistance to gentamicin.
Culture Media↗
Vancomycin susceptibility and identification of motile enterococci.
Thirty-seven clinical isolates of Enterococcus gallinarum and Enterococcus casseliflavus and three type or reference strains of the species were studied with respect to vancomycin susceptibility and key identification characteristics. With the exception of one clinical isolate of E. casseliflavus (MIC, 4 micrograms/ml), MICs of vancomycin were 8 to 32 micrograms/ml. The type strain of E. gallinarum, NCDO 2313, and five of the clinical isolates had similar penicillin-binding protein profiles and shared 90 to 100% DNA homology. Two isolates, identified as E. gallinarum by conventional tests, were shown to be non-pigment-producing E. casseliflavus on the basis of penicillin-binding protein profile and DNA homology. The type and reference strains of E. casseliflavus, ATCC 25788 and ATCC 25789, were nonmotile in our experiments. However, both shared 65 to 100% DNA homology with each other and with five clinical isolates of E. casseliflavus. These data suggest that the MICs of vancomycin observed for strains of E. gallinarum and E. casseliflavus are higher than those usually associated with other enterococci and may be a common property of these species. Additionally, pigment production and motility may occasionally be misleading criteria for definitive identification of these organisms.
Bacterial Proteins↗
Detection of high-level aminoglycoside resistance in enterococci other than Enterococcus faecalis.
The ability of six screening methods to detect high-level aminoglycoside resistance in enterococcal species other than Enterococcus faecalis was investigated. The 85 Enterococcus isolates, which included 55 E. faecium, 11 E. gallinarum, 9 E. casseliflavus, 5 E. raffinosus, 4 E. avium, and 1 E. mundtii, were tested by using aminoglycoside-supplemented brain heart infusion agar (BHI), Remel EF Synergy Quad plates, high-content aminoglycoside diffusion disks, standard (prepared in-house) microdilution panels, Pasco MIC Gram Positive microdilution panels, and Vitek GPS-TA cards. When tested on BHI, 32 and 35 strains showed resistance to gentamicin and streptomycin, respectively. Resistance profiles obtained with Remel EF Synergy Quad plates were in complete agreement with those obtained on BHI. However, growth on Mueller-Hinton agar-based plates was not as heavy. Some isolates showed only weak growth and required 48 h for resistance to become evident, especially with swab inoculation of quadrants containing 2,000 micrograms of gentamicin per ml. Profiles obtained by use of the agar-based screens were used as the basis for evaluating the other methods. Disk diffusion showed complete agreement. No false resistance occurred by either microdilution method, but 48 h of incubation was needed for detection of some gentamicin-resistant isolates, and 14% of the streptomycin-resistant strains were not detected by standard microdilution. The Vitek GPS-TA card detected 81 and 100% of the gentamicin- and streptomycin-resistant isolates, respectively. In general, most methods used to detect high-level aminoglycoside resistance in E. faecalis appear to be reliable for the testing of the other enterococcal species. However, further investigations with a greater number of resistant E. raffinosus, E. avium, and E. mundtii isolates, when they are available, will be useful for establishing the full range of enterococci that can reliably be tested by the various methods.
Anti-Bacterial Agents↗
Evaluation of the Vitek GPS-TA card for laboratory detection of high-level gentamicin and streptomycin resistance in enterococci.
The Vitek GPS-TA card (Vitek Systems, Hazelwood, Mo.) was compared with single-concentration broth microdilution and disk diffusion methods using high-content disks for the detection of high-level resistance to gentamicin and streptomycin in 99 isolates of enterococci (81 Enterococcus faecalis isolates and 18 Enterococcus faecium isolates). The GPS-TA card accurately detected high-level resistance to gentamicin, but not streptomycin, in E. faecalis. When streptomycin is being considered for therapy, either disk diffusion or time-kill studies should be used to confirm susceptible results obtained by Vitek testing. Additional studies are needed to determine the best method for testing E. faecium isolates.
Drug Resistance, Microbial↗
Evaluation of MicroScan rapid panels for detection of high-level aminoglycoside resistance in enterococci.
The ability of MicroScan rapid panels to detect high-level aminoglycoside-resistant enterococci was evaluated. By agar dilution, 46 of 139 isolates were susceptible to gentamicin (GNT) and streptomycin (STRP); the rest were highly resistant to one or both agents. Rapid panels detected 97.5% of STRP- and GNT-resistant isolates and had a specificity of 95.6%. Detection of resistance by conventional panels at 18 h was 64.6% for STRP and 90.2% for GNT.
Drug Resistance, Microbial↗
Effects of blood medium supplements on activities of newer cephalosporins tested against enterococci.
This comparative study determined the effect of blood on the antienterococcal activities of the newer cephalosporins. Standardized disk diffusion susceptibility tests were performed with 57 strains of enterococci (30 Enterococcus faecalis strains) on Mueller-Hinton agar with and without 5% sheep blood supplementation. Twelve cephalosporins representing five different structural groups (based on the 7-alpha position substitution) were tested. The greatest frequency of activity enhancement by blood was observed with cefdaloxime and cefdinir (7-alpha hydroxyimino group) against E. faecalis. Cephalosporins with a 7-alpha methoxyimino group (cefpodoxime, cefepime, and cefpirome) had marked increases in zone diameters (3 to > 9 mm) when tested with the blood supplement. Cephems with 7-alpha amino or carboxy substitutions did not demonstrate any enhanced activity. Awareness of this phenomenon is important for the interpretation and accuracy of cephalosporin susceptibility testing.
Animals↗
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.
Culture Media↗
DNA hybridization and contour-clamped homogeneous electric field electrophoresis for identification of enterococci to the species level.
In this study, 113 Enterococcus faecium, 37 Enterococcus faecalis, 24 Enterococcus gallinarum, 15 Enterococcus raffinosus, and 13 Enterococcus casseliflavus clinical isolates and American Type Culture Collection (ATCC) strains were evaluated by contour-clamped homogeneous electric field electrophoresis. Thirty-one of the E. faecium, 22 of the E. faecalis, 24 of the E. gallinarum, 15 of the E. raffinosus, and 13 of the E. casseliflavus isolates were also evaluated by DNA-DNA hybridization. Genomic DNAs from type strains E. faecalis ATCC 19433, E. faecium ATCC 19434, E. gallinarum ATCC 49573, E. raffinosus ATCC 49427, and E. casseliflavus ATCC 25788 were labeled with biotin for use as probes. E. faecalis differed from all other species in always having a largest fragment of > 400 kb. E. gallinarum was different from all other species in having all SmaI fragments of < 200 kb. Biotin-labeled probes showed a high degree of hybridization with genomic DNA from the same species and a low degree of hybridization when hybridized to genomic DNA from different species for all isolates tested except for four isolates identified as E. faecium by conventional biochemical methods. The DNA from these four isolates hybridized strongly to DNA from E. gallinarum ATCC 49573 and weakly to E. faecium ATCC 19434 DNA and had all SmaI fragments of < 200 kb in size. These data suggest that these isolates are nonmotile E. gallinarum. DNA from each ATCC type strain hybridized strongly with itself and had only a low degree of hybridization with DNA from other ATCC type strains tested. These results suggest that contour-clamped homogeneous electric field electrophoresis patterns and DNA-DNA hybridization with biotin-labeled probes may be of use for species differentiation of some enterococci.
Blotting, Southern↗
Cost-effective, clinically relevant method for rapid identification of beta-hemolytic streptococci and enterococci.
Currently popular agglutination and coagglutination methods for the identification of beta-hemolytic streptococci, although rapid and simple to perform, are costly. Furthermore, they fail to distinguish between clinically relevant species and normal flora of the same serogroup. We investigated the use of a series of four physiologic tests to differentiate beta-hemolytic streptococci and enterococci into five clinically relevant groups. We also investigated the use of a new product, Visi-Spot, and evaluated an alternate method for the detection of beta-D-glucuronidase production. Our results suggest that for most routine processing of beta-hemolytic streptococci, physiologic tests are sufficiently rapid, more accurate, and far less costly to perform than serologic methods. The facility of our scheme is enhanced by the use of the Visi-Spot test and the substitution of a commercially available product for more traditional methods of detecting beta-D-glucuronidase.
Agglutination Tests↗
Comparison of rectal and perirectal swabs for detection of colonization with vancomycin-resistant enterococci.
Patients whose gastrointestinal tracts are colonized with vancomycin-resistant enterococci (VRE) may serve as a reservoir for nosocomial transmission. We compared the sensitivities and concordance of several methods used to detect VRE colonization. Eighty-two paired rectal and perirectal swabs were obtained from 13 patients over a 9-day period. The sensitivity of both rectal and perirectal swabs was 79%. There was 100% concordance of culture results between simultaneously obtained rectal and perirectal swabs, and the quantities of growth were similar by these two methods of detection. Our data suggest that rectal and perirectal swabs are equally sensitive for the detection of VRE colonization.
Anti-Bacterial Agents↗
Factors influencing the vitek gram-positive susceptibility system's detection of vanB-encoded vancomycin resistance among enterococci.
Studies were conducted to identify factors contributing to the inability of the Vitek Gram-Positive Susceptibility system (GPS; bioMerieux, Vitek, Inc., Hazelwood, Mo.) to reliably detect vanB-mediated vancomycin resistance among enterococci. To some extent the accuracy of the GPS depended on a particular strain's level of resistance, as all isolates for which vancomycin MICs were > or = 128 mu g/ml were readily detected but detection of resistance expressed by several strains for which MICs were < or = 64 mu g/ml was sporadic. Factors besides the level of resistance were studied in two vanB strains. For one strain (Enterococcus faecium U8304), the ability of GPS to detect resistance was accurate and consistent, while for the other (Enterococcus faecalis V583), GPS results were inconsistent and unreliable. Using these isolates, we established that growth medium had the most notable effect on the detection of resistance. In the absence of vancomycin, Vitek GPS broth supported growth comparable to that obtained with brain heart infusion broth for both E. faecium U8304 and E. faecalis V583. However, in the presence of vancomycin the growth patterns changed dramatically so that neither VanB strain grew well in Vitek broth, and growth of V583 was barely detectable after 8 h of incubation. In contrast, good growth of both strains was observed in brain heart infusion broth supplemented with vancomycin. Additionally, the same medium effect was observed with other inducibly resistant VanB strains. In conclusion, although Vitek broth can support good enterococcal growth, this medium does not sufficiently support expression of vancomycin resistance by certain strains to allow them to be detected by the Vitek automated system. Furthermore, this observation establishes that the type of growth medium used can substantially influence the expression of vancomycin resistance and indicates that medium-based strategies should be explored for the enhancement of resistance detection among commercial systems.
Anti-Bacterial Agents↗
Comparison of five selective media for identifying fecal carriage of vancomycin-resistant enterococci.
There is no uniformly accepted method for detecting colonization with vancomycin-resistant enterococci (VRE). The sensitivities of five culture methods were determined for patients known to harbor VRE. Of 189 inpatients, 101 were found to harbor VRE by at least one method. Three methods detected fewer than half of the cultures. Campylobacter agar identified 70% of patients. Enterococcosel broth (containing vancomycin and aztreonam) identified 88% and may be preferred over other media for routine surveillance.
Anti-Bacterial Agents↗
Are clinical laboratories in California accurately reporting vancomycin-resistant enterococci?
In order to determine whether hospital-based clinical laboratories conducting active surveillance for vancomycin-resistant enterococci in three San Francisco Bay area counties (San Francisco, Alameda, and Contra Costa counties) were accurately reporting vancomycin resistance, five vancomycin-resistant enterococcal strains and one vancomycin-susceptible beta-lactamase-producing enterococcus were sent to 31 of 32 (97%) laboratories conducting surveillance. Each strain was tested by the laboratory's routine antimicrobial susceptibility testing method. An Enterococcus faecium strain with high-level resistance to vancomycin (MIC, 512 microg/ml) was correctly reported as resistant by 100% of laboratories; an E. faecium strain with moderate-level resistance (MIC, 64 microg/ml) was correctly reported as resistant by 91% of laboratories; two Enterococcus faecalis strains with low-level resistance (MICs, 32 microg/ml) were correctly reported as resistant by 97 and 56% of laboratories, respectively. An Enterococcus gallinarum strain with intrinsic low-level resistance (MIC, 8 microg/ml) was correctly reported as intermediate by 50% of laboratories. A beta-lactamase-producing E. faecalis isolate was correctly identified as susceptible to vancomycin by 100% of laboratories and as resistant to penicillin and ampicillin by 68 and 44% of laboratories, respectively; all 23 (74%) laboratories that tested for beta-lactamase recognized that it was a beta-lactamase producer. This survey indicated that for clinically significant enterococcal isolates, laboratories in the San Francisco Bay area have problems in detecting low- to moderate-level but not high-level vancomycin resistance. Increasing accuracy of detection and prompt reporting of these isolates and investigation of cases are the next steps in the battle for control of the spread of vancomycin resistance.
Ampicillin↗
Clinical and epidemiological significance of enterococci intrinsically resistant to vancomycin (possessing the vanC genotype)
Constitutive low-level vancomycin resistance is found intrinsically in certain enterococcal species and is encoded by vanC ligase genes. These intrinsically vancomycin-resistant enterococci (VRE) will be referred to as VANC VRE. A prospective study to determine the clinical and epidemiologic significance of VANC VRE was conducted. VANC VRE were recovered from the stools of 34 of 601 (5.7%) patients, a rate similar to that obtained for the stools of 100 outpatients in the community (5%). VANC VRE were also isolated from the nonstool specimens of 9 of 538 patients (1.7%), including two patients with bacteremia. No VRE of the vanA or vanB genotypes were detected in nonstool specimens. Eighty-two hospital contacts of the first 23 patients found to be colonized or infected with VANC VRE were screened, and 6 contacts were found to be gastrointestinal carriers of VANC VRE. However, typing of isolates from these 6 contacts by pulsed-field gel electrophoresis with SmaI showed the isolates to be unique and different from those recovered from the index patients. In fact, all VANC VRE isolates from different patients in this study were unique. A case-control study with patients who were negative when screened for VANC VRE as controls failed to identify any risk factor associated with colonization or infection with this organism. VANC VRE were infrequently recovered from clinical specimens but were occasionally found as part of the normal stool flora. Since no transmission between patients was documented, additional isolation procedures may not be necessary for patients colonized or infected with VANC VRE.
Adult↗