Aims: To evaluate the accuracy of direct disc susceptibility testing performed from positive BACTEC blood culture vials, using a predetermined dilution protocol.
Methods: Direct susceptibility testing was performed from 432 positive blood culture vials, generating 3829 antibiotic-organism results. Results were compared with those obtained by standard disc susceptibility testing according to Clinical Laboratory Standards Institute (CLSI) methods.
Results: When results were compared with the reference method, no very major errors were detected. One (0.03%) major error and 89 (2.3%) minor errors were found. Error rates by organism group ranged from 1.3% for Pseudomonas aeruginosa to 8.2% for β-haemolytic streptococci.
Conclusions: Direct susceptibility testing provided accurate susceptibility results for most organism–antibiotic combinations, with the exception of the β-haemolytic streptococci.
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Early and appropriate treatment of bacteraemia is associated with reduced mortality.1 Although the use of automated blood culture systems has reduced the time-to-detection, conventional processing methods do not provide antimicrobial susceptibility results until two days after initial detection of positive vials. Direct disc susceptibility testing may be practised in some clinical laboratories but without any reference methodology. An early report of direct disc testing in the 1970s from manually incubated blood culture vials showed acceptable results,2 but there are no published data for the newer automated blood culture monitoring systems. In addition, the prevalence of antibiotic resistance phenotypes such as methicillin-resistance in Staphylococcus aureus and extended-spectrum β-lactamase (ESBL) production in Enterobacteriaceae has increased. This study was performed in an 800-bed community hospital over a two-year period to evaluate the accuracy of direct disc susceptibility testing performed directly from positive BACTEC blood culture vials, compared with the results obtained by conventional disc susceptibility methods.
MATERIALS AND METHODS
Blood culture vials were incubated in a BACTEC 9240 system (Becton-Dickinson, USA). Vials with positive growth indexes were processed during working hours (08:30–17:00), while positive vials detected out-of-hours were processed at the start of the next workday. When multiple vials from the same patient episode were present, only the first positive vial was included in the study. Blood culture vials with mixed bacterial morphology on Gram stain were excluded from the study. One of three antibiotic testing panels was selected on the basis of the Gram stain findings.
Dilution of blood–broth mixture, and direct susceptibility testing
If the initial Gram stain showed Gram negative bacilli, two drops (approximately 40 μl) of blood–broth mixture were dispensed from each positive vial using a subculture/venting unit (catalogue no 271056, Becton-Dickinson) into 1 ml of 0.85% saline. For Gram positive cocci, 10 drops (approximately 200 μl) of blood–broth mixture were dispensed from the positive vial into 1 ml of 0.85% saline. The blood–broth saline suspensions were used to perform disc susceptibility testing following Clinical Laboratory Standards Institute (CLSI) methodology.3
Susceptibility testing from overnight subcultures
Positive vials were also subcultured on to bacteriological plate media. Following overnight incubation, cultures with mixed bacterial morphologies were excluded from further analysis.
A suspension was prepared from bacterial colonies, the inoculum was adjusted to 0.5 McFarland using a calibrated photometric device (bioMérieux, France) and disc susceptibility testing performed according to CLSI methods.
Interpretation of results
For both methods, the presence of ESBL was determined by the double-disc approximation method.4 Methicillin resistance in S aureus was detected by both oxacillin and cefoxitin discs. Discrepant results between the two antibiotics were resolved by a pbp2 latex agglutination kit (Oxoid, UK).
Results from the direct susceptibility method were compared against results from the reference method. A very major error denoted a false-susceptible result and a major error denoted a false-resistance result. All other errors were defined as minor errors.
The difference in zone diameters between the reference method and the direct method was calculated for each antibiotic–organism combination. The mean, minimum, maximum and modal values were calculated using Excel (Microsoft, Redmond, Washington, USA).
Validation of testing inoculum for blood–broth saline suspension
Colony counts were performed on a random sample of blood–broth saline dilutions prepared from 19 blood culture vials positive for Enterobacteriaceae, and eight blood culture vials positive for Staphylococcus aureus. For each positive vial, the blood–broth saline suspension was derived as described above, and then serial tenfold dilutions were prepared, resulting in a dilution series ranging from 10−4 to 10−6 of the initial blood–broth saline suspension. A 20 μl aliquot of each tenfold dilution was seeded onto Mueller–Hinton agar (Becton-Dickinson, Franklin Lakes, New Jersey, USA) and colony counts performed as described by Miles and Misra.5
Colony counts performed from the representative blood–broth dilutions showed that the average inoculum for Gram positive cocci in the prepared blood–broth saline suspensions was 3×107 cfu/ml (range 1×106–2×108 cfu/ml). The average test inoculum for Gram negative bacilli was 3×108 cfu/ml (range 2×107–1×109 cfu/ml).
A total of 432 positive blood culture vials were eligible for inclusion in the study. Isolates tested included Escherichia coli (n = 143), Klebsiella spp. (n = 83), Proteus spp. (n = 23), Enterobacter spp. (n = 21), other Enterobacteriaceae (n = 18), Pseudomonas aeruginosa (n = 32), S aureus (n = 66) and large colony-forming β-hemolytic streptococci (n = 46). Twenty per cent of E coli and 32% of Klebsiella spp. were ESBL-producing strains, and 36% of S aureus isolates were methicillin-resistant.
A total of 3829 antibiotic–organism test results were available for analysis. One major error (0.03%) and 89 minor errors (2.3%) were detected, but no very major errors. The proportion of errors varied according to the tested organism.
Direct susceptibility testing for Enterobacteriaceae showed 53 (1.9%) minor errors out of a possible 2828 antibiotic–organism combinations (table 1). Minor error rates ranged from 0% for imipenem to 6.4% for amoxicillin–clavulanic acid. Minor error rates for amoxicillin–clavulanic acid were significantly higher for ESBL-producing isolates (16.4%) when compared with non-ESBL-producing strains (4.0%) (p<0.05).
Direct testing for S aureus showed one major error (0.2%) and 18 (3.0%) minor errors out of 593 antibiotic–organism combinations (table 2), resulting in an overall error rate of 3.2% for S aureus. The major error was reported for one isolate of methicillin-resistant S aureus. This isolate was resistant to oxacillin by direct disc testing, but susceptible to oxacillin when the reference CLSI method was performed. The strain was resistant to cefoxitin when tested by both methods, and positive by latex agglutination for the pbp2 protein. Minor errors were also detected for erythromycin (17.0%), ciprofloxacin (4.5%) and clindamycin (6.1%).
There were three (1.3%) minor errors out of a possible 224 antibiotic–organism combinations for P aeruginosa, occurring in aztreonam (3.1%) and ceftazidime (6.3%).
The highest number of errors was detected in β-hemolytic streptococci, with 15 (8.2%) minor errors out of 184 antibiotic–organism combinations (table 2).
The calculated differences in zone diameters between the reference method and the direct method for the tested organisms are listed in tables 1 and 2 respectively. For Gram negative bacilli and streptococci, the mean difference in zone diameters for most antibiotics was 0 mm with a normal distribution across the zero axis. For S aureus, the average zone diameter by the standard method was 1 mm greater than that produced by the direct method. The distribution of differences in zone diameter for S aureus showed a slight positive skew, suggesting that zone diameters for the direct testing method were slightly smaller than those obtained by the standard method.
Disc susceptibility performed directly from positive blood culture vials may potentially provide rapid susceptibility results, but there is no recent evidence to establish the accuracy of such testing.
This study compared the accuracy of direct disc testing from 432 positive Bactec blood culture vials.
Susceptibility results obtained by the direct disc method were comparable to those obtained by standard Clinical Laboratory Standards Institute methods for most organism–antibiotic combinations, with the exception of erythromycin and clindamycin.
Direct disc testing from Bactec vials provides accurate results for Enterobacteriaceae, S aureus and P aeruginosa.
Direct susceptibility testing performed from positive blood culture vials produced results for most organism–antibiotic combinations that were comparable to those obtained by the reference disc susceptibility method recommended by the CLSI, with the exception of erythromycin and clindamycin. Direct susceptibility testing for the β-haemolytic streptococci also produced unacceptably high rates of minor errors.
The blood–broth dilutions were developed for comparability with the 0.5 McFarland standard required by CLSI methodology. Further optimisation of the blood–broth dilution protocol for S aureus may reduce some of the errors associated with the direct testing method.
Resistance phenotypes that are inherently difficult to detect by disc susceptibility testing (e.g. vancomycin resistance in S aureus) were not assessed in the study. Our results were obtained using the BACTEC system and may not be valid for other blood culture systems. Different dilutions may be required for direct testing to achieve results comparable with those obtained by other methods.
To our knowledge, this is the first study to validate the use of direct disc susceptibility testing on positive blood culture vials incubated in automated blood culture systems. In addition, our study included the testing and successful detection of multi-resistant bacterial phenotypes. Other methods have been published for direct susceptibility testing using Vitek6 7 and Microscan panels,8 with varying degrees of accuracy. Most of these methods require a time-consuming centrifugation step prior to performing susceptibility testing, and reagent acquisition costs are likely to be higher.
In conclusion, this study presents a simple and reproducible method for performing direct disc susceptibility testing of positive blood culture vials, providing results that are comparable with existing methods from the CLSI for Enterobacteriaceae, P aeruginosa and S aureus.
We would like to thank the staff of the Microbiology Laboratory for their participation in this study.
Funding: Internal hospital grant.
Competing interests: None.
Data from the pilot study were presented in part at the 12th International Congress on Infectious Diseases, Lisbon, 15–18 June 2006.
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