To evaluate the validity of AST results using NA, QC strains were used for AST in uniform conditions including inoculum density, timing of disk application, temperature of incubation, incubation time, size of the plate, depth of the agar medium, proper spacing of the antibiotic disks, and potency of antibiotic disks. Appropriate QC organisms should be tested daily for all antimicrobial agents routinely included in the antimicrobial battery until a laboratory achieves “satisfactory performance.” CLSI makes the definition of “satisfactory performance” as obtaining unacceptable results in no more than 1 out of 20 or 3 out of 30 results obtained in consecutive test days for each antimicrobial agent/organism combination. Once this satisfactory performance is obtained, a laboratory can convert from daily QC testing to weekly QC testing. If all QC test results are within the acceptable limits, the laboratory can continue weekly testing; however, on occasions when a modification in the test is made, consecutive QC testing is required .
All antimicrobial agent-reference organism combinations using NA were unacceptable compared to MHA. All antibiotic-organism combination showed more than three misreading over 30 successive days; hence, none of the results would be accepted according to CLSI guidelines.
Muller-Hinton agar is a loose agar that allows for better diffusion of the antibiotics than most other media. A better diffusion leads to a truer zone of inhibition . This criterion was missing in NA manifested by smaller IZs with TOB, CN, and P when testing P. aeruginosa ATCC® 27853, AZM and DA when testing S. aureus ATCC® 25923, and AMP, KZ, TOB, and CN when testing E. coli ATCC® 25922 (Table 2). Both the para-aminobenzoic acid (PABA) and thymine/thymidine content in MHA are reduced to a minimum, thus markedly reducing the inactivation of sulfonamides and trimethoprim when the medium is used for testing the susceptibility of bacterial isolates to these antimicrobics . Thirteen errors with smaller inhibition zone over 30 consecutive days were detected on testing SXT for S. aureus ATCC® 25923 using NA (Table 2).
The inability of AST to determine a susceptible result for an organism that is susceptible to the antimicrobial agent being tested is considered a major error (false resistant). Conversely, the inability to detect resistance is assessed as “very major error” (false sensitive). Concerning CLSI guidelines, there is a minimum level of acceptable interpretative errors in susceptibility testing which are quite restrictive. Very major errors should not exceed 1.5%, while major errors should not exceed 3.0%, and overall categorical agreement should equal or exceed 90% for each organism antibiotic challenge [10, 12, 13].
In our study, the discrepancies between the susceptibility results obtained by NA and the standard MHA were obvious when testing clinical isolates with total errors of 27.76%, 22.4%, and 3.6% with P. aeruginosa, S. aureus, and Enterobacteriaceae respectively. Also, very major errors and major errors were beyond the acceptable level of CLSI guidelines (8.98% and 7.6% very major errors and 4.08% and 6% major errors for P. aeruginosa and S. aureus isolates respectively).
Very major error may lead to the initiation of inadequate antimicrobial therapy and may have fatal consequences especially in severely ill patients where these antibiotics are common first-line substances. On the other hand, major errors deprive the patients of treatment with an effective antibiotic and lead to the use of second-choice drugs, usually more recent and expensive, and thus contribute to economic losses and the selection of resistant strains .
Although none of the AST results for E. coli ATCC® 25922 was acceptable when using NA (Table 2), we found a remarkable unexplained agreement in AST results of Enterobacteriaceae on both NA and MHA (96.4%) with no very major error and 0.4% major error (Table 3). Even if the AST results showed full agreement as in the case of Enterobacteriaceae, the issue of lacking data on specific breakpoints concerning the use of NA remains.
Because NA is a general purpose medium rather than standard susceptibility testing medium, there is hardly any data comparing it with MHA in susceptibility testing. Donkor et al. compared NA with MHA in antimicrobial susceptibility testing of Salmonella Typhi and S. aureus isolates. They reported that the overall discrepancy in susceptibility results between NA and MHA was 8.9%, and this discouraged the use of NA in the Kirby-Bauer method as practiced by some laboratories, due to the considerable error margin this medium may introduce into susceptibility results .
Though our results cannot be generalized on the laboratories’ performance as it was a pilot study conducted on a small number of microbiology laboratories and focusing on one factor affecting AST, it highlights the great importance of standardizing every feature in this test to improve the validity of AST reporting of a laboratory.