Evaluation of alternative methods for antimicrobial susceptibility testing of fastidious organisms

Abstract

Fastidious organisms such as Haemophilus influenzae and Streptococcus pneumoniae continue to cause considerable morbidity and mortality. In the past antimicrobial susceptibility testing was not needed as susceptibility patterns were predictable. However, considerable resistance has now emerged, and susceptibility testing is required to ensure successful therapy. MICs of fastidious organisms are sometimes needed, when there is no standard diffusion method, or the organism has an MIC close to the susceptibility category breakpoint, or when there is unsuccessful therapy. Conventional dilution methods are intensive, time-consuming, and technically demanding. In addition, they lack precision, especially at high MIC values, since two-fold dilutions of antimicrobial concentrations are performed. Few automated systems for MIC determination of fastidious organisms are available with the exception of the Vitek 2, which only provides a panel for susceptibility testing of S. pneumoniae, after incubation for approximately 8.5 hours. The E test is a robust method and is simple to perform. However, it is quite expensive, especially if several drugs need to be tested, and its use can be effected by storage. There is still no economical and rapid method for MIC determination of fastidious organisms. Spiral Gradient Endpoint test (SGE) utilizes a spiral plater to deposit antibiotic on the agar surface in a decreasing concentration gradient, providing an antimicrobial concentration gradient on the agar for determination of MIC. It is especially suitable for fastidious organisms since agar with different supplements can be used, and it can be incubated in different atmospheres. Some preliminary studies on the use of SGE for susceptibility testing have been performed and the results were good. However, these studies did not use the SGE method for fastidious organisms. In this study, SGE was evaluated for its reproducibility, and the effects of varying the SGE test parameters were determined. A comparison study of the SGE test with the conventional dilution method for susceptibility testing of 4 species of fastidious organisms was conducted. A cost analysis was also performed as shown in Appendix ix. It was found that both the intrabatch and interbatch reproducibility of the SGE method were good, with a range of coefficients of variation (CV) of 6.22 to 14.56% for intrabatch and 11.06 to 18.56% for interbatch reproducibility, which were much lower than those of the standard MIC methods. It was also found that the parameters that would affect the SGE MIC were: incubation time, inoculum density, and antibiotic concentration. Using the optimal conditions obtained, SGE tests were performed on four fastidious organisms, and results were compared with standard methods. There was a high percentage of agreement between the two methods for all organisms. Cost beneficial analysis was also in favour of SGE test, which was found to be cheaper amongst E test and Vitek 2. Early reporting of susceptibility test results can lead to reductions in mortality, morbidity, and shortening of hospital stay. However, all routinely used methods require overnight testing before results can be obtained. The flow cytometer is able to detect viable and non-viable bacterial cells after staining with fluorescent dyes. Its use for AST has been suggested but methods, especially for fastidious organisms have not been developed or evaluated. This study has developed an accurate and precise method for AST of two fastidious organisms, H. influenzae and S. pneumoniae. This FCM-AST was rapid and could provide results within 6 hours. It also had the advantage of being able to analyse non-synchronous heterogeneic cultures, although it was found to be more expensive than more conventional AST methods (Appendix ix). The kinetics of ampicillin and tetracycline were studied and a FCM-AST was evaluated for testing of H. influenzae isolates. Penicillin and erythromycin kinetics were investigated for S. pneumoniae, and FCM-AST was performed on the clinical isolates of the organisms against these antibiotics. The organisms responded well to the lx MIC of antibiotics. Breakpoint antibiotic concentrations were then used for FCM-AST. The results of the AST were found to agree well with those of standard methods. In an era of increasing antibiotic resistance, rapid, accurate, and economical methods for AST are essential. This study has successfully evaluated a simple, economic method, the SGE, and a rapid, though more expensive, method FCM-AST, for the accurate susceptibility testing of fastidious organisms.