Antimicrobial susceptibility testing (AST) of clinically significant isolates of Nocardia
species is recommended because certain isolates of this genus differ in susceptibility to commonly used antimicrobial agents (2
). Generally, susceptibility or resistance to certain antimicrobial agents can be predicted for the most frequently isolated species of clinical significance, including N. brasiliensis
, N. farcinica
, N. cyriacigeorgica
, N. nova
, and the N. transvalensis
complex (represented in this study by N. wallacei
). However, for other newly described species and some strains of the more common species, susceptibility patterns are unknown or are not predictable (2
). The broth microdilution method is the recommended method for AST of Nocardia
One of the stated goals of this study was to assess the intra- and interlaboratory reproducibility of broth microdilution susceptibility testing of Nocardia isolates. Results showed that the level of reproducibility was partly related to the antimicrobial agent being tested.
There was acceptable reproducibility (≥90% agreement at the mode ± 1 dilution) among all laboratories for several antimicrobial agents, including amikacin, ciprofloxacin, clarithromycin, and moxifloxacin. These drugs gave clear and unambiguous endpoints that were easily interpreted by all laboratories. Selective removal of results from laboratories 5 and 6 resulted in acceptable reproducibility for amoxicillin-clavulanic acid, linezolid, minocycline, and tobramycin.
There were differences among testing sites in the extents of variation of their own results from the modal result. One laboratory (laboratory 3) reported significantly fewer readings outside the mode ± 1 dilution for all antimicrobial-organism combinations compared to the other 5 testing sites, and 2 laboratories (laboratories 5 and 6) reported significantly more readings outside the mode ± 1 dilution compared to the other testing sites (). In addition, one testing site (laboratory 6) reported a significantly higher number of readings spanning more than 3 dilutions (for the 65 antimicrobial-organism combinations) than the remaining laboratories (). The reasons for the lack of reproducibility reported by these laboratories are unclear. Endpoint determination of some antimicrobial agents was more difficult, possibly related to attributes of the agent itself, to the particular isolate tested, or to complexities of the testing methodology.
Our assessment of lot-to-lot variation in the microdilution plates indicated that panel lots were functionally identical and did not contribute to result variation. Likewise, run-to-run variation was minimal, except for laboratory 6, which reported 8 instances of variations among runs.
Considerable deviation from the mode value was seen for several antimicrobial agents, including ceftriaxone, imipenem, sulfamethoxazole, trimethoprim-sulfamethoxazole, and tigecycline. MICs for ceftriaxone were most problematic for N. cyriacigeorgica
and N. wallacei
, with inadequate agreement (<90%) even at the mode ± 2 dilutions. In addition, disk diffusion results were also ambiguous for these two species that are considered susceptible to this drug by in vitro
MIC testing (2
). It is unclear if these discrepancies were due to inadequate inocula or to interpretation inconsistencies due to the growth characteristics of these isolates. Given the wide variability of results from the various laboratories, it appears that the broth microdilution method as described may not allow reliable results for ceftriaxone to be obtained with N. cyriacigeorgica
and N. wallacei
. The addition of disk diffusion testing was not useful for resolving testing inconsistencies.
MIC results for imipenem with N. cyriacigeorgica
and N. farcinica
were only in agreement at the mode ± 2 dilutions; both species are considered susceptible to this drug (2
). However, there was complete agreement among test sites by interpretative categories, and disk diffusion tests for N. cyriacigeorgica
were reproducibly susceptible. For N. farcinica
, the disk diffusion discrepancies from the expected susceptible result may in part be due to the more rapid growth of this species compared to other species of Nocardia
. Moreover, because of the characteristic smooth colony morphology of N. farcinica
(smooth colonies suspend more efficiently and produce a more homogenous inoculum in broth than rough colonies), this species can easily be overinoculated when preparing the test inocula. In addition, imipenem disk diffusion results for N. farcinica
may have been misinterpreted as resistant by one testing site due to the presence of tiny colonies within the zone of inhibition; these colonies are characteristic of this antimicrobial-organism combination and are not indicative of resistance (R. J. Wallace, Jr., and B. Brown-Elliott, unpublished observations). All other testing sites reported imipenem zone diameters within the susceptible or intermediate ranges when testing N. farcinica
. Both MIC and disk diffusion results for N. wallacei
with imipenem were not reproducible; this species shows variable susceptibility to this drug, possibly based on the difficulty in test interpretation or the difficulty in achieving consistent inoculum concentrations.
Sulfonamides, often in combination with other antimicrobial agents, remain the drugs of choice for treatment of nocardial infections because of the historical in vitro
susceptibility of Nocardia
isolates to these drugs and because of observed clinical effectiveness (12
). In this study, there was considerable lack of reproducibility of sulfonamide MIC results, especially when testing N. farcinica
and N. wallacei
. Because of the unique growth characteristics of Nocardia
species, the correct endpoint may be difficult to determine; this is most probably the reason for the discrepant sulfonamide MIC results observed in this study. Guidelines state that the endpoint is the well exhibiting 80% inhibition of growth compared to the positive control well (5
). Wells containing low concentrations of sulfonamide may also be used as a comparison for 80% inhibition, as better growth at the lowest drug concentration compared to the single growth control is frequently observed (R. J. Wallace, Jr., and B. Brown-Elliott, unpublished observations). Determination of the correct endpoint is critical. Because of a lack of intermediate breakpoints for both sulfamethoxazole and trimethoprim-sulfamethoxazole, a 1-dilution endpoint difference can mean the difference between a susceptible and a resistant result. It should be noted that current CLSI guidelines recommend testing only trimethoprim-sulfamethoxazole; interestingly our results show more consistency with trimethoprim-sulfamethoxazole testing than with sulfamethoxazole alone.
Analysis of sulfonamide MIC results by interpretive category indicates that a considerable number of isolates were unexpectedly reported as resistant to these drugs. Of a total of 1,681 MIC results for both sulfamethoxazole and trimethoprim-sulfamethoxazole with all species, 1,436 (85.4%) isolates were reported as susceptible and 245 (14.6%) were reported as resistant (data not shown). In contrast, by disk diffusion, only 1.7% of 296 total Nocardia sulfisoxazole results were interpreted as resistant, with 78.7 and 19.6% reported as susceptible and “intermediate,” respectively. Because this resistance was seen inconsistently among testing sites and species, we conclude that “resistant” results are more an indication of inaccurate sulfonamide endpoint determination than of intrinsic resistance to the drugs tested.
Results of this study indicate that the use of the disk diffusion test for sulfonamides is useful for validation of MIC results (see below). In addition, because of the difficulty of interpretation of sulfonamide MICs, it is possible that susceptibility to sulfonamides may be more reliably predicted by disk diffusion testing. Zones of inhibition seen with disk diffusion may be easier to interpret; CLSI guidelines for interpretation of sulfonamides indicate that slight growth surrounding the disk (20% or less of the lawn of growth) should be disregarded and the more obvious margin of growth should be measured to determine the zone diameter (4
In a recent retrospective study of 765 Nocardia
isolates, Uhde et al. reported unusually high levels of in vitro
sulfonamide resistance, as determined by MIC testing, with 61% of isolates showing resistance to sulfamethoxazole and 42% showing resistance to trimethoprim-sulfamethoxazole (14
). As the authors note, the set of isolates may have included many that were unresponsive to the usual treatment provided. However, no data were available to those authors regarding the antimicrobial treatment or the clinical outcomes. Particularly, it is not known how many patients were treated successfully with sulfonamides. In contrast, in an additional retrospective study of sulfonamide susceptibility results of 552 recent clinical Nocardia
isolates from various regions of the United States, Brown-Elliott et al. reported only 2% of isolates to have MICs reported indicating resistance to trimethoprim-sulfamethoxazole or sulfamethoxazole (1
). Given the data presented by Brown-Elliott et al. and our own data demonstrating the difficulty of sulfonamide susceptibility test interpretation with these organisms, we are concerned that the in vitro
results reported by these Uhde et al. may not accurately reflect in vivo
The reasons for poor agreement among laboratories at the mode ± 1 dilution for tigecycline with N. cyriacigeorgica and N. brasiliensis are unclear. At least for N. brasiliensis, inconsistencies may be due to the growth characteristics of this species and the difficulties associated with inoculum preparation.
A secondary goal of this study was to select a candidate clinical isolate to serve as a quality control organism for susceptibility testing of the Nocardia
species. Although none of the isolates evaluated was a perfect candidate, N. nova
was closest to optimal, with the most reproducible, although highly susceptible, results. Laboratories should obtain results similar to those obtained in this study with this organism (). The inclusion of this strain in the quality control battery is useful to show the particular attributes of Nocardia
strains grown in the presence of antimicrobial agents. This strain has been deposited in the American Type Culture Collection (ATCC) as ATCC BAA-2227. Additional quality control organisms should also be tested, as outlined in the most recent CLSI document (5
Summary of MIC results for N. nova from six test sitesa
As with other organisms, inoculum preparation is an important factor in AST of Nocardia. Because of the clumping properties of these organisms, preparation of an adequately homogeneous organism suspension is especially challenging and important. In this study, we tested the use of pellet pestles to grind clumps of organisms in a small volume of water, thereby creating a dense suspension with fewer clumps. For N. cyriacigeorgica, N. farcinica, and N. nova, the supernatant resulting from this technique was adequately dense to allow preparation of a suspension corresponding to a 0.5 McFarland standard, as determined by nephelometer reading. For N. brasiliensis and N. wallacei, the clumps were more difficult to disperse, and the supernatant was less dense than with other species, and therefore a larger volume of supernatant was necessary to achieve the target suspension density. Interestingly, results obtained in this study for N. wallacei were the least reproducible with several antimicrobial agents, possibly because of the difficulty of inoculum preparation.
The CLSI procedure states a target organism concentration of 1 × 105
to 5 × 105
CFU/ml as the goal for accurate susceptibility testing (5
). Our results show that the achievement of an adequate colony count was directly related to the ease with which the organism suspension was obtained. Overall, colony counts obtained with N. cyriacigeorgica
, N. farcinica
, and N. nova
were more frequently in the expected range, while colony counts obtained with N. brasiliensis
and N. wallacei
were lower than expected (data not shown). Because in most cases, isolate identification is unknown at the time AST is initiated and because of the differences observed in colony count for the various species, inocula may best be prepared at the middle of the acceptable range of nephelometer readings for all organisms in an attempt to achieve an adequate organism concentration for all species.
The relationship between Nocardia concentration (as determined by colony count) and MIC results has not previously been explored. In this study, participating laboratories reported colony counts either within the target range or lower than the target range for each isolate tested and MIC interpretation discrepancies occurred regardless of the colony count reported (data not shown). Some laboratories that reported organism concentrations within the target range reported MICs 2 or more dilutions greater or less than the mode value. Likewise, some laboratories that reported colony counts less than the target range reported MICs that were within ±1 dilution of the mode value. This suggests that colony counts did not give a true representation of the inocula, and we recommend that they not be performed. Variables other than colony count appear to more significantly affect the breakpoint determination.
More experienced investigators recommend the use of the agar disk diffusion method in combination with the broth MIC to help determine the adequacy of the inoculum (17
). Until more work is performed to optimize inoculum standardization, we recommend that an agar disk diffusion plate, preferably with a sulfisoxazole disk, be incorporated in the test setup. This can be prepared from the 0.5 McFarland suspension initially prepared for microdilution testing. If, after incubation, the growth on the Mueller-Hinton plate is confluent (inoculum too heavy), or if isolated colonies are present (inoculum too light), the entire test should be repeated with careful attention to inoculum density. An appropriate inoculum would show streaks of growth with spaces between the streaks (). Based on previous studies comparing zone diameter on disk diffusion testing to MIC results, this growth appearance is different from that expected from Kirby Bauer testing of other bacteria but has been determined to be indicative of adequate Nocardia
). This may be due to the colony size and growth characteristics of Nocardia
species compared to other bacteria.
Example of an appropriate inoculum for susceptibility testing: N. wallacei on Mueller-Hinton agar with a sulfisoxazole disk.
As mentioned above, the use of disk diffusion with a sulfisoxazole disk is also useful to check the accuracy of the sulfonamide MIC, especially if the MIC reading indicates that the isolate is sulfonamide resistant. Results obtained in this study showed disk diffusion results to be more indicative of susceptibility for some organisms. A discrepancy between the sulfonamide MIC and disk diffusion interpretive category would indicate that the test should be repeated or sent to a referral laboratory for result confirmation. The disk diffusion result should not be reported; it is solely intended to verify the accuracy of the sulfonamide MIC result.
Comparison of MIC results with expected results for a particular species is especially useful for drugs that are difficult to test, which may give ambiguous results and which are potentially useful in a clinical situation (such as the sulfonamides and, if tested at all, ceftriaxone, for N. cyriacigeorgica and N. wallacei). MICs that differ from the expected results should be repeated to verify those results.
Currently no proficiency test service (such as that of the College of American Pathologists) is available specifically for Nocardia susceptibility testing. If a laboratory elects to perform in-house testing, test performance should be validated prior to offering the test, which is best accomplished through comparison of results with an experienced accredited reference laboratory. In addition, proficiency testing should be done at least twice per year either by comparing results with a reference laboratory or by repeating the evaluation of isolates previously tested.
While this study examined the reproducibility of susceptibility testing of Nocardia
species, there are other factors unrelated to the susceptibility process itself that may make application to patient care more problematic. Knowledge of the mechanisms of drug resistance, particularly to generally effective agents such as the sulfonamides, can be very useful in clinical practice. Such knowledge would be particularly helpful in the case of drugs for which clinical effectiveness may be difficult to determine in vitro
. (Again, the sulfonamides are an example.) Knowledge of specific resistance-conferring mechanisms such as inducible β-lactamase resistance in N. nova
complex might explain some variable test results with β-lactams and eventually make rapid and unequivocal detection of resistance by molecular methods relatively simple (11
). The detection of inducible β-lactamase was not addressed in this study. Given that such inducibility exists in the N. nova
complex, caution should be exercised in the treatment of patients with isolates of N. nova
complex reported to have MICs to amoxicillin-clavulanic acid in the susceptible range. Unfortunately, to date there has been a lack of data regarding resistance mechanisms in most species of Nocardia
, making AST a continuing necessity.
Recent studies have shown that N. farcinica
contains multiple genes relating to β-lactam, aminoglycoside, and macrolide resistance (10
). These findings have led investigators to speculate on the presence of antimicrobial resistance mechanisms such as aminoglycoside-modifying enzymes seen in N. farcinica
. These mechanisms may play a significant role in the finding of a wide range of results of MICs when testing individual strains of Nocardia
. These mechanisms may, in part, also explain the apparent in vitro
susceptibility of amikacin sometimes seen even with extended incubation in species known to be aminoglycoside resistant in vivo
(e.g., N. transvalensis
complex). (R. J. Wallace, Jr., and B. Brown-Elliott, unpublished observations).
Results obtained in this study illustrate the numerous difficulties that may be encountered in the setup and interpretation of broth microdilution testing of Nocardia species and highlight the variation in test interpretation that may occur even within a particular laboratory. These difficulties may influence the decisions of some laboratories to institute this test. However, one laboratory participating in this study (laboratory 2) had limited previous experience with broth microdilution testing of Nocardia isolates but reported only 6.8% of overall results outside the mode ± 1 dilution and only 6.2% of results spanning greater than 3 dilutions (). All results were read and interpreted by a single individual with no previous Nocardia susceptibility testing experience, who was carefully trained and who referred to the interpretive guidelines provided for the study. The experience of laboratory 2 suggests that a relatively inexperienced person who is well trained, meticulous, and conscientious and who is adequately supervised can perform and accurately interpret MIC results for Nocardia species.
In summary, strict attention to appropriate inoculum preparation, thorough training of technical staff, attention to detail by designated test readers, and willingness to seek help with troublesome interpretations may allow for more accurate Nocardia
MIC result interpretation. Laboratories performing this test should become familiar with endpoint determinations, as illustrated in this document () and in the current CLSI guidelines (5
). Comparison of results with expected results for a particular species is especially useful for drugs that are difficult to test and which are potentially useful in a clinical situation (such as ceftriaxone, imipenem, tigecycline, and the sulfonamides). Any isolate that does not have the expected antimicrobial susceptibility pattern of that species should be retested or sent to a reference laboratory for result confirmation. The data from this study suggest that in vitro
testing of N. cyriacigeorgica
and N. wallacei
(and possibly other Nocardia
species) with ceftriaxone cannot currently be reliably performed using broth microdilution testing. In addition, sulfonamide testing by disk diffusion may provide more reliable results than testing by broth microdilution. The testing of appropriate quality control strains to confirm drug potency, the inclusion of the N. nova
ATCC BAA-2227 strain as a reference for nocardial growth patterns in the microdilution panel, and regular proficiency testing will help ensure that accurate results are reported. In addition, we suggest that colony count plates not be inoculated and that a disk diffusion test for sulfisoxazole be incorporated into the test setup to check inoculum density and to confirm sulfonamide MIC results.