Ticking the clock: Modified Kirby-Bauer’s technique for accelerated antibiotic susceptibility reporting 

References

1. method. Clinical isolates demonstrated 97.48% CA for EDD 6-hour testing and 97.1% CA for EDD 10-hour testing, with minimal discrepancies (VME ≤ 0.63%, ME ≤ 0.58%, mE ≤ 1.94%). Early growth (EDD 10-hour) from primary isolation plates of urine and positive flagged blood cultures showed >99% CA, with no major or very major discrepancies. Conclusion: EDD 10-hour testing demonstrated high accuracy and concordance with the standard 18-24 hour method, offering a cost-effective alternative method for faster AST reporting. Early reporting using EDD can enhance timely antibiotic administration, reduce AMR, and improve patient outcomes without additional costs. This method aligns with FDA-recommended thresholds for AST systems and provide significant advantage in clinical settings. Keywords: Antimicrobial susceptibility testing, Early disc diffusion, Kirby Bauer’s disc diffusion, Turnaround time. Received: 15-05-2025; Accepted: 19-07-2025; Available Online: 20-09-2025 This is an Open Access (OA) journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms. For reprints contact: reprint@ipinnovative.com
2. Introduction Antibiotic resistance refers to a bacterium's capacity to genetically encode resistance genes that overturn the inhibitory effects of antibiotics in order to thrive. It may be acquired through horizontal gene mutation processes like conjugation, transformation, and transduction or it may arise intrinsically through integration and spontaneous recombination into the bacterial genome. 1 Antimicrobial resistance (AMR) is now a global health emergency. We are moving towards a post-antibiotic age where our current arsenal antibiotics won't work because of the overuse and injudicious prescription of antibiotics. Globally, AMR causes 700,000 deaths annually, but if the above-mentioned urgent steps to address drug-resistant infections are not taken, this number is expected to rise to 10 million deaths annually by
3. 2,3 Antimicrobial susceptibility testing (AST) of microorganisms can be performed using various methods, one of which is the disc diffusion technique, first standardized by Bauer et al. in 1966. 4 Kirby-Bauer disc diffusion is the most common routine traditional method used for AST reporting. Kirby-Bauer disk diffusion method is easier, reproducible dependable and materials required are Content available at: https://www.ipinnovative.com/open-access-journals Indian Journal of Microbiology Research Journal homepage: www.ijmronline.org Gowtham et al. / Indian Journal of Microbiology Research 2025;12(3):346–356 347 cost effective and allows visibility of growth and can be used for isolates from mixed cultures. This method provides the laboratory with a great deal of flexibility and antibiotic panels can be created based on antibiogram and the presence of antibiotic-resistant strains in that particular area. 5,6 Prompt administration of the appropriate antibiotic treatment is essential for saving lives in severe infections and can also help lower healthcare costs. 7-10 Studies have demonstrated that timely and effective antibiotic therapy significantly improves clinical outcomes and reduces mortality. 11 Fast access to accurate results from antimicrobial susceptibility tests (AST) is seen as a major unaddressed need in treating infectious diseases. Immediate and accurate diagnostic approaches for AST are essential for lowering dependence on broad-spectrum antibiotics and addressing the increasing challenge of resistance. 12 Delayed release of Microbiological culture reports leads to starting of empirical treatment with broad spectrum antibiotics leading to emergence of multidrug resistance. There are published data on shortened incubation period for disc diffusion testing, but these protocols are yet to be standardized for recommendations by CLSI or EUCAST. In this study, an approach to decrease the turnaround time for disc diffusion testing was made by reducing the incubation period required for the growth of the bacteria from clinical sample that precedes disk diffusion testing. This study was performed to evaluate the disc diffusion susceptibility test results on early growth of isolates i.e., at 6 and 10 hours of incubation by comparing with the susceptibility test results performed on 18-24 hours growth. Early disc diffusion (EDD) testing will decrease the turnaround time (TAT) of AST reports and this method would be the most cost-effective method to accelerate AST results. 13
4. Materials and Methods A laboratory based cross sectional study was conducted in the tertiary care hospital, Staphylococcus aureus [ATCC 29213], Staphylococcus aureus [ATCC 25923], Enterococcus faecalis [ATCC 29212], Escherichia coli [ATCC 25922] and Pseudomonas aeruginosa [ATCC 27853] were used as quality control (QC) strains.
5. Clinical isolates One hundred clinical isolates, both sensitive and resistant strains, isolated from various clinical samples were included in the study. They included 25 isolates each of Staphylococcus aureus, Enterococcus species, Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae) and Pseudomonas aeruginosa identified with standard biochemical reactions. 14 Both Quality control (QC) strains and clinical isolates were inoculated onto peptone water and turbidity adjusted to
6. 5 McFarland standard. A loopful of this inoculum was then inoculated by quadrant streaking onto Blood Agar (BA) and MacConkey Agar (MA) plates. These plates were incubated at 37°C in ambient air for 6, 10, and 18-24 hours. Bacterial inoculum for Kirby Bauer’s disc diffusion was prepared by taking similar type of colonies to be tested using sterile inoculation straight wire. 3-5 individual colonies from the culture growth of clinical isolates and QC strains were selected from plates incubated for 6, 10, and 18-24 hours of incubation (Figure 1). The colonies were suspended in peptone broth, the turbidity of this suspension was adjusted to 0.5 McFarland standard. A sterile swab was soaked into the inoculum tube and rotated against the sides of the tube to remove excess fluid. The dried surface of a cation adjusted Muller Hinton Agar (MHA) plate was inoculated by streaking the swab three times over the entire agar surface rotating the plates approximately 60 degree each time and lastly along the rim of the plate with the swab to pick up any excess inoculum. The plates were allowed to dry for 3-5mins to allow evaporation of excess moisture and appropriate antibiotic discs were applied onto the agar surface depending on the organism (Table 1) using sterile forceps. The MHA plates were incubated aerobically at 37ᵒC for 18-24 hours as per CLSI guidelines. The size of the zone of inhibition from all the plates inoculated with growth at 6 hours, 10 hours and 18-24 hours was measured manually and interpreted as Sensitive (S), Intermediate (I) and Resistant (R) based on CLSI (16) guidelines 2024 (Figure 2 and Figure 3). Figure 1: Growth of S. aureus on blood agar at 6, 10 and 18-24 hours of incubation 348 Gowtham et al. / Indian Journal of Microbiology Research 2025;12(3):346–356 Table 1: Displays the number of organisms, quality control strains with ATCC numbers, clinical isolates distribution of resistance patters and clinical samples Staphylococcus Enterococcus Enterobacterales Pseudomonas Clinical Isolates (Count) S. aureus (25) Enterococcus species (25) E.coli (12) P. aeruginosa (25) K. pneumoniae (13) QC Strains (Triplets) S. aureus (ATCC 25923) E. faecalis (ATCC 29212) E.coli (ATCC 25922) P. aeruginosa (ATCC 27853) S. aureus(ATCC 29213) 100 Clinical Isolates (Distribution of resistance pattern) Methicillin - resistance Staphylococcus aureus (MRSA) - 20 Vancomycin Resistant Enterococcus (VRE) - 02 E.coli (Carbapenem-resistant) - 03 E.coli (Sensitive-) - 04 E.coli (Variable -) - 05 P. aeruginosa(Multi-drug resistant) – 05 P. aeruginosa (Sensitive) - 14 P. aeruginosa (Variable) - 06 Methicillin - sensitive Staphylococcus aureus (MSSA) – 05 Vancomycin sensitive Enterococcus - 23 K. pneumoniae (Multi-drug resistant) - 05 K. pneumoniae (Sensitive) - 03 K. pneumoniae (Variable) - 05 25 Urine samples (Count) S. aureus - 0 Enterococcus species - 7 E.coli -11 P. aeruginosa -3 K. pneumoniae -04 25 Positive flagged blood samples(Count) S. aureus -10 Enterococcus species -0 E. coli -08 P. aeruginosa -0 K. pneumoniae -07 Figure 2: EDD Susceptibility testing of P. aeruginosa at 6, 10 and 18-24 hours growth Gowtham et al. / Indian Journal of Microbiology Research 2025;12(3):346–356 349
7. Clinical sample Fifty clinical samples (25 turbid Urine samples and 25 Positive flagged blood culture samples) were also inoculated. Urine samples were inoculated onto chromogenic Urichrome agar (UCA) (Figure 4A) and Positive flagged blood cultures were sub cultured onto Blood agar (BA) and MacConkey agar (MA) (Figure 4 B&C). The BA, MA and UCA plates were incubated aerobically at 37℃. Those clinical samples which yielded single type of growth were included in the study. The growth at 10 hour and 18-24 hours of incubation were used to perform Kirby Bauer’s disc diffusion as per CLSI guidelines. 15 The size of the zone of inhibition of 10- hour old growth was compared with the standard 18-24 hour old growth and interpreted as Sensitive (S), Intermediate (I) and Resistant (R) based on Clinical and Laboratory Standards Institute (CLSI) guideline
8. 15 Growth at 6 hour of incubation Growth at 10 hour of incubation Growth at 18-24 hour of incubation A B C D E Row A: Susceptibility testing of QC strain of Staphylococcus aureus [ATCC 29213] at 6, 10 and 18-24 hours growth. Row B: Susceptibility testing of QC strain of Staphylococcus aureus [ATCC 25923] at 6, 10 and 18-24 hours growth. Row C: Susceptibility testing of QC strain of Enterococcus faecalis [ATCC 29212] at 6, 10 and 18-24 hours growth. Row D: Susceptibility testing of QC strain of Pseudomonas aeruginosa [ATCC 27853] at 6, 10 and 18-24 hours growth. Row E: Susceptibility testing of QC strain of Escherichia coli [ATCC 25922] at 6, 10 and 18-24 hours growth. Figure 3: EDD Susceptibility testing of QC strains at 6h, 10h and 18-24h growth 350 Gowtham et al. / Indian Journal of Microbiology Research 2025;12(3):346–356 Figure 4: A): Culture growth of urine sample inoculation on Urichrome agar after 10 hour and 18- 24 hours of incubation; B): Culture growth of Gram Positive Cocci on blood agar from Positive flagged blood culture sample after 10 hour and 18-24 hours of incubation; C): Culture growth of Gram Negative Bacilli on Mac Conkey agar from Positive flagged blood culture sample after 10 hour and 18-24 hours of incubation The following antibiotic disks panels were used for AST testing as per CLSI guidelines.
9. Staphylococcus aureus: Cefoxitin (30µg), Ciprofloxacin (5µg), Gentamicin (10µg), Tetracycline (30µg), Linezolid (30µg), Cotrimoxazole (23.5/1.25µg), Erythromycin (15µg), Clindamycin (2µg)
10. Enterocccus spp: Ampicillin (10µg), Penicillin (10µg), Tetracycline (30µg), Erythromycin (15µg), high level Gentamicin (120µg), Linezolid (30µg), Vancomycin (30µg), Teicoplanin (30µg).
11. Escherichia coli and Klebsiella pneumoniae: Ampicillin (30µg), Ceftriaxone (30µg), Cefepime (30µg), Amoxycillin clavulanicacid (20/10µg), Pipericillin tazobactam (100/10µg), Imipenem (10µg), Amikacin (30µg), Ciprofloxacin (5µg), Cotrimoxazole (10µg).
12. Pseudomonas aeruginosa: Ceftazidime (30µg), Cefepime (30µg), Cefoperazone sulbactam (75/30µg), Piperacillin tazobactum (100/10µg), Aztreonam (30µg), Meropenem (10µg), Imipenem (10µg), Amikacin (30µg), Levofloxacin (5µg). Categorical agreement (CA), minor error (mE), major error (ME), and very major error (VMEs) of the Early disc diffusion (EDD) test methods were calculated in reference to the approved 18-24 hours growth and quality control isolates antibiotic susceptibility testing as per CLSI guidelines 2024 (16). Categorical agreement (CA) means similar interpretive criteria (susceptible/intermediate/resistant) was agreed upon between the two methods. Minor error (mE) means a susceptible or resistant result was shown as intermediate and vice versa. Major error (ME) denotes a susceptible isolate shown as resistant and calculated only for susceptible isolates. Very major error (VME) suggests a resistant isolate shown as susceptible and calculated only for resistant isolates. Gowtham et al. / Indian Journal of Microbiology Research 2025;12(3):346–356 351
13. Results In this study, the new methodology, early disc diffusion (EDD) method from 6- and 10-hour growth of the clinical isolates and quality control isolates were studied in comparison to the established 18-24 hours growth Kirby- Bauer antimicrobial susceptibility test method. But for the isolates directly from clinical samples, only 10-hour early growth culture were included in the study and 6-hour early growth were not included due to heterogeneous culture yield and extremely small colonies that made it difficult to distinguish the mixed type of growth. Quality control strains Staphylococcus aureus [ATCC 29213], Staphylococcus aureus [ATCC 25923], Enterococcus faecalis [ATCC 29212], Escherichia coli [ATCC 25922] and Pseudomonas aeruginosa [ATCC 27853] were tested in triplicates for early disc diffusion (EDD) method at 6hour and 10hour of growth. The susceptibility results showed 100% agreement with 18-24h growth standard method, with no major or minor discrepancies. The susceptibility testing results of clinical isolates from 6-hour growth (EDD6) compared with the Standard 18-24 hour growth (St24) reference method showed 97.48% categorical agreement (CA) with 0.63% very major error (VME), 0.58% major error (ME) and 1.94% minor error (mE) discrepancies. A linear regression analysis of inhibitory zones from EDD6 and St24 revealed an r 2 value of 0.87 and a slope value of 0.94 which suggests a high level of correlation between the two methods (Figure 5). EDD for 6h growth of S. aureus showed 96% categorical agreement with 1.29% VME, 0.87% ME, 3% mE, Enterococcus showed 99% with 1% mE discrepancy, P. aeruginosa showed 96.4% CA with 2.94% VME, 0.59% ME,
14. 66% mE, E. coli showed 97.5% CA with 2.55% mE and K. pneumoniae showed the highest rate of 99.2% CA with
15. 43% ME compared to the 24h standard reference method respectively (Table 2). EDD for 10h growth of S. aureus showed 94.5% CA with 0.87% ME, 5% mE, Enterococcus showed 98.5% CA with 1.5% mE, P. aeruginosa showed 96% CA with 5.88% VME, 0.59% ME, 2.66% mE, E. coli showed 100% CA with no errors and K. pneumoniae showed 98.46% CA with 2.43% ME and 0.76% mE compared to the 18-24h standard reference method respectively (Table 3). Figure 5: Linear regression analysis of inhibitory zones from EDD 6-hour growth compared to standard 18-24 hours growth of clinical isolates 352 Gowtham et al. / Indian Journal of Microbiology Research 2025;12(3):346–356 Table 2: Comparison of AST testing by disk diffusion from 6-hours old growth method versus 18-24 hours standard growth with clinical isolates and QC strains. Clinical isolates 6-hour incubation 6-hour incubation AST report Reference method (18-24 hour) AST report Discrepancies (n/n %) Organism S I R Total S I R Total Agreement (CA) Very major (VME) Major (ME) Minor (mE) S.aureus 113 11 76 200 114 9 77 200 192/200 (96%) 1/77 (1.29%) 1/114 (0.87%) 6/200 (3%) Enterococcus 129 2 69 200 127 4 69 200 198/200 (99%) 0/69 (0%) 0/127 (0%) 2/200 (1%) P.aeruginosa 171 20 34 225 167 24 34 225 217/225 (96.4%) 1/34 (2.94%) 1/167 (0.59%) 6/225 (2.66%) E.coli 62 6 52 120 64 5 51 120 117/120 (97.5%) 0/51 (0%) 0/64 (0%) 3/120 (2.5%) K.pneumoniae 40 5 85 130 41 5 84 130 129/130 (99.2%) 0/84 (0%) 1/41 (2.43%) 0/130 (0%) Total 515 44 316 875 513 47 315 875 853/875 (97.48%) 2/315 (0.63%) 3/513 (0.58%) 17/875 (1.94%) QC Strains 6-hours incubation S.aureus 48 0 0 48 48 0 0 48 48/48 (100%) 0 (0%) 0/48 (0%) 0/48 (0%) Enterococcus 24 0 0 24 24 0 0 24 24/24 (100%) 0 (0%) 0/24 (0%) 0/24 (0%) P.aeruginosa 27 0 0 27 27 0 0 27 27/27 (100%) 0 (0%) 0/27 (0%) 0/27 (0%) E.coli 30 0 0 30 30 0 0 30 30/30 (100%) 0 (0%) 0/30 (0%) 0/30 (0%) Total 129 0 0 129 129 0 0 129 129/129 (100%) 0 (0%) 0/129 (0%) 0/129 (0%) Total clinical isolates and QC isolates All organisms 644 44 316 1004 642 47 315 1004 982/1004 (97.8%) 2/315 (0.63%) 3/642 (0.46%) 17/1004 (1.6%) Table 3: Comparison of AST testing by disk diffusion from 10-hours old growth method versus 18-24 hour standard growth with clinical isolates and QC strains Clinical Isolates 10-hour incubation 10hour incubation AST report Reference method (18-24 hour) AST report Discrepancies (n/n %) Organism S I R Total S I R Total Agreement (CA) Very major (VME) Major (ME) Minor (mE) S.aureus 110 13 77 200 114 9 77 200 189/200 (94.5%) 0/77 (0%) 1/114 (0.87%) 10/200 (5%) Entercoccus 127 3 70 200 127 4 69 200 197/200 (98.5%) 0/69 (0%) 0/127 (0%) 3/200 (1.5%) P.aeruginosa 170 20 35 225 167 24 34 225 216/225 (96%) 2/34 (5.88%) 1/167 (0.59%) 6/225 (2.66%) E.coli 65 5 51 120 64 5 51 120 120/120 (100%) 0/51 (0%) 0/64 (0%) 0/12 (0%) K.pneumoniae 39 6 85 130 41 5 84 130 128/130 (98.46%) 0/84 (0%) 1/41 (2.43%) 1/130 (0.76%) Total 511 47 318 875 513 47 315 875 850/875 (97.1%) 2/315 (0.63%) 3/513 (0.58%) 20/875 (2.28%) Gowtham et al. / Indian Journal of Microbiology Research 2025;12(3):346–356 353 Table 3 Continued… QC Strains 10-hours incubation S.aureus 48 0 0 48 48 0 0 48 48/48 (100%) 0 (0%) 0/48 (0%) 0/48 (0%) Enterococcus 24 0 0 24 24 0 0 24 24/24 (100%) 0(0%) 0/24 (0%) 0/24 (0%) P.aeruginosa 27 0 0 27 27 0 0 27 27/27 (100%) 0 (0%) 0/2 (0%) 0/2 (0%) E.coli 30 0 0 30 30 0 0 30 30/30 (100%) 0 (0%) 0/30 (0%) 0/30 (0%) Total 129 0 0 129 129 0 0 129 129/129 (100%) 0 (0%) 0/1 (0%) 0/129 (0%) Clinical Samples Urine Samples Enterococcus 41 1 14 56 41 1 14 56 56/56 (100%) 0/24 (0%) 0/41 (0%) 0/56 (0%) P.aeruginosa 9 2 16 27 9 2 16 27 27/27 (100%) 0/16 (0%) 0/9 (0%) 0/27 (0%) E.coli 51 4 55 110 52 3 55 110 109/110 (99.09%) 0/55 (0%) 0/52 (0%) 1/110 (0.90%) K.pneumoniae 10 1 29 40 11 2 27 40 39/40 (99.09%) 0/27 (0%) 0/11 (0%) 1/40 (2.5%) Total 113 8 112 233 113 8 112 233 231/233 (99.14%) 0/122 (0%) 0/113 (0%) 2/233 (0.85%) Positive Flagged Blood (culture) samples S.aureus 49 2 29 80 49 3 28 80 79/80 (98.75%) 0/28(0%) 0/49 (0%) 1/80 (1.25%) E.coli 39 2 54 96 40 3 53 96 95/96 (98.95%) 0/53 (0%) 0/40(0%) 1/96 (1.04%) K.pneumoniae 47 4 33 84 47 4 33 84 84/84 (100%) 0/33 (0%) 0/47 (0%) 0/84 (0%) Total 136 8 116 260 136 10 114 260 258/260 (99.23%) 0/114 (0%) 0/136 (0%) 2/260 (0.76%) Total clinical isolates, QC isolates and clinical samples All organisms 884 67 546 1497 891 65 541 1497 1468/1497 (98.06%) 2/551(0.36% ) 3/891(0.33%) 24/1497 (1.60%) Figure 6: Linear regression analysis of inhibitory zones from EDD10 hour growth compared to standard 18-24 hours growth of clinical isolates 354 Gowtham et al. / Indian Journal of Microbiology Research 2025;12(3):346–356
16. 1% categorical agreement (CA) with 0.63% very major error (VME), 0.58% major error (ME) and 2.28% minor error (mE) discrepancies were observed for the susceptibility results of 10-hour growth (EDD10) compared with the standard 18-24 hours (St24) hour growth. A linear regression analysis of inhibitory zones from EDD10 and St24 revealed an r 2 value of 0.89 and a slope value of 0.93 which suggests a high level of correlation between the two methods (Figure 6).
17. Clinical samples Twenty-five clinical isolates isolated turbid urine samples showed greater rate of categorical agreement of 99.14% with only 0.85% minor discrepancy for 10-hour growth (EDD10). Enterococcus and P. aeruginosa showed complete agreement to the 18-24h standard reference method. E. coli and K. pneumoniae both showed 99.09% CA with only 0.90% and
18. 5% mE respectively. The EDD 10-hour growth of 25 isolates isolated from positive flagged blood cultures showed much higher rate of
19. 23% CA with only 0.76% minor discrepancy when compared to 18-24 hours growth reference method. S. aureus showed 98.75% CA with 1.25% mE, E. coli showed 98.95% CA with 1.04% mE and K. pneumoniae showed absolute CA to the 18-24h standard reference method. Overall clinical samples isolate linear regression analysis of inhibitory zones from EDD10 and St24 revealed an r 2 value of 0.91 and a slope value of 0.95 and this indicates a significantly higher correlation between the two methodologies (Figure 7). In the study, it was also observed that D zone test in S. aureus for detection of inducible to clindamycin also showed 100% agreement in 10h EDD to standard 24-hour growth reference method. Detection of EBSL production also accurately matched with 10hour growth susceptibility testing. All resistance mechanisms detection could also be done accurately with 10h EDD method.
20. Discussion This study highlights the importance of using the early growth of the culture isolates in testing antibiotic sensitivity pattern and hence the reducing the turnaround time for culture and sensitivity report. The AST results of the QC isolates were accurate and showed 100% agreement with both EDD 6h and 10h compared to the standard reference 18-24 hours AST results. In the study conducted by Weber et al., the QC strains showed only 98.3% CA. QC strains of S. aureus 6-hour EDD AST reports showed 96.7% agreement with 3.3% major discrepancy. QC strains of E. faecalis 6-hour AST reports showed 95.8% CA and 4.7% mE. QC strain for EDD 6h of E. coli, K. pneumoniae and P. aeruginosa showed 100% agreement to the standard reference 24-hour AST results. QC strain for EDD 10h showed 96.6% CA with 0.5% ME and
21. 4% mE and S. aureus showed 98.3% CA with 1.7% ME. E. faecalis showed 87.5% CA with 12.5% mE. E. coli has
22. 6% CA with 1.4 mE. K. pneumoniae and P. aeruginosa showed 100% agreement to the standard reference 18-24 hours AST results which was comparable with the present study. 5 Figure 7: Linear regression analysis of inhibitory zones from EDD10 hour primary growth compared to standard 18-24 hours growth from clinical samples Gowtham et al. / Indian Journal of Microbiology Research 2025;12(3):346–356 355 A study conducted by Biswas et al., evaluating this EDD testing for QC strains, they observed a similarly high level of correlation between EDD6 and standard 24h as well as between EDD10 and St24. For the 20 quality control strains, they observed the categorical agreement of EDD6 with standard incubation was 94.38% with 5.10% minor error (mE) and 0.69% major errors (MEs). Likewise, 10-h growth comparisons yielded a same pattern of 5.10% mE and 0.69% MEs and no VMEs with 94.38% categorical agreement with standard incubation. 13 In the study conducted by Weber et al., EDD 6h testing of clinical isolates showed 95.6% categorical agreement with
23. 5% very major, 1.6% major and 2.5% minor errors. EDD 10h testing of clinical isolates showed 96.7% categorical agreement with 0.8% major and 2.7% minor errors. 5 In the study conducted by Biswas et al., evaluating this EDD testing for the 48 clinical isolates they observed the categorical agreement of EDD6 with standard incubation was
24. 15% with 1.22% major error and 1.29% minor errors and no very major errors. 5 In our study for clinical samples for EDD 6h with the 18- 24 hours reference method showed 97.48% agreement with
25. 63% very major, 0.58% major and 1.94% minor discrepancies and for clinical isolates of EDD 10h with the 24h-reference method showed 97.1% agreement with 0.63% very major, 0.58% minor and 2.28% minor discrepancies. The novelty of this study is that growth from clinical samples urine and positive flagged blood culture were also included in the study. Even though there are studies using early growth for testing disc diffusion, there are no published data on EDD studies performed directly on isolates isolated from clinical samples. Six-hour old growth was not tested as it was difficult to identify mixed growth as the colonies were extremely small. In this study, EDD for 10h growth isolated from Urine samples showed 99.14% agreement and 0.85% minor error with no very major or major discrepancies. In urine samples E.coli and K. pneumoniae showed more than 99% agreement whereas Enterococcus and P. aeruginosa showed complete agreement to the 18-24h standard reference method. The 10-hour growth of clinical isolates isolated from Positive flagged blood culture samples showed 99.23% agreement with 0.76% minor discrepancy. S. aureus and E. coli showed more than 98% categorical agreement whereas K. pneumoniae showed complete agreement with the 18-24h standard reference method. In this study, EDD for 10hour growth showed more accurate results than 6 hour growth of AST reports. Also, the drug resistance mechanisms such as inducible clindamycin (D-zone) test and ESBL production were also detected in the EDD performed form 10-hour incubated growth and the results correlated well with that performed with 18-24 hours growth standard method. In 10h growth colonies are moderate in size and it’s easy to pick up isolated colonies whereas it was very difficult to pick isolated colonies 6hour growth and also to identify mixed or heterogeneous growth. By performing the AST testing using 10-hour old growth, the reports can be dispatched 10 to 12 hours early decreasing the turnaround time which will have a significant impact on early initiation of patient treatment and antimicrobial stewardship. These values of categorical agreement were well above the threshold (90% or more) provided by the FDA Class II Special Controls Guidance for AST systems. Likewise, AST results from EDD6 and EDD10 met the FDA-recommended threshold of ME of 3% or less and VME upper and lower 95% CIs less than or equal to 7.5 and 1.5%, respectively (Food and Drug Administration). 16 All the statistical analysis indicates that early disc diffusion method using 6- and 10- hour growth is highly reliable and can be considered for routine reporting of antibiotic susceptibility test results.
26. Conclusion Kirby-Bauer disc diffusion is the most common method of performing sensitivity testing in the most of the laboratories. This study shows that the culture isolates as old as 6 and 10 hours have yielded comparable results with that of CLSI approved standard 18-24 hours growth. Hence 6 hours and 10 hours old culture can be used in the routine sensitivity testing and reporting of bacterial isolates such as Staphylococcus aureus, Enterococcus, P. aeruginosa, E. coli and K. pneumoniae. 10hour early disc diffusion is ideal method for replacement for standard method, as it is highly concordant with reports of 18-24 hours standard reference method. Just by reducing growth period without adding extra cost is possible to release early report of AST which in turn helps to reduce AMR and improve antimicrobial stewardship.
27. Financial Support and Sponsorship The study supported by JSS AHER Research grants with approval number JSSAHER/REG/RES/URG/54/2011 - 12/7541.
28. Conflicts of Interest All the authors declare no conflict of interest.
29. Acknowledgements The authors would like to acknowledge the management of JSS AHER for permitting us to write the report. References
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