(H-320) Instant Antimicrobial susceptibility test (iAST)

Introduction:: Growing antibiotic resistance has been a major global health threat for decades. This issue is largely attributed to the inappropriate use of antibiotics. Antimicrobial susceptibility tests (AST) can help to mitigate this situation by determining the minimal inhibitory concentration (MIC) of the drug to the pathogen, thereby providing guidance for a more accurate antibiotic prescription. Speed, however, is the main issue with conventional phenotypic ASTs. [1] The process requires several hours for overnight culture to determine the minimal inhibitory concentration (MIC). And the time needed for sample preparation also extended the time spent. In infection cases like sepsis, the risk of death increases swiftly by the hour.[2] Various new phenotypic methods have been developed in recent years to shorten the test time [3,4]. However, overnight culturing is still inevitable for most of the methods. Shih and Shih laboratory at Drexel University has developed an instant AST (iAST) using piezoelectric plate sensor (PEPS) that can determine the minimum inhibitory concentration (MIC) in 30 min. The goal of this study is to use iAST to test Cefoxitin against Gram-positive Staphylococcus aureus (SA, ATCC-29213), Ampicillin against Gram-negative Escherichia coli (EC, ATCC-25922), and particularly Gentamicin against hard-to-test Gram-negative Pseudomonas aeruginosa (PA, ATCC-27583) and Klebsiella pneumoniae (KP, ATCC-700603)[5,6]. The MICs obtained will be compared to those obtained by microdilution and those accepted by the Clinical & Laboratory Standards Institute (CLSI)


Materials and Methods:: The PEPSs used for iAST were 1 mm long and 1 mm wide (insert in Fig. 1(a)) made of 127µm thick lead zirconate-titanate (PZT) (PSI-5H4E, Piezo Systems). For bacteria attachment, the PEPS was first coated with (3-Aminopropyl)trimethoxysilane ) (APS) followed by placing 1 mL of a live bacterial suspension on a PEPS for 10 min followed by DI water rinsing. iAST was carried out by immersing bacteria-attached PEPS in DI water (see Insert of Fig. 1(a)). Charges generated by the metabolism of live bacteria changes the electrical impedance of the PEPS, which manifests as a broadening of the top of a resonance peak in a phase angle (of the electrical impedance) versus frequency spectrum (Figs. 1(a)-1(b)) as measured by an AIM 4710 electrical impedance analyzer. Increasing the antibiotic concentration in the DI water instantly reduces the broadening of the top of resonance peak. By increasing the antibiotic concentration 2-fold every 5 min, the MIC can be determined as the antibiotic concentration that at and above which the width of the top of the resonance peak ceases to change with the antibiotic concentration (Figs. 1(c)-1(d)).





Results, Conclusions, and Discussions:: Results: All MICs determined by iAST along with the MIC ranges of CLSI and the MICs of microdilution are listed in Table 1. As can be seen, the MICs determined by iAST were all within the range of the MICs accepted by CLSI. Indicating that the MICs determined by the iAST results were reliable and repeatable for both Gram-positive bacteria such as SA but also Gram-negative bacteria such as EC, PA, and KP. Note PA and KP are known for producing inaccurate MICs by Vitek2 and other methods. It is worth noting that iAST produced 6/6 MICs that were within the CLSI range for both PA and KP, indicating that iAST is reliable even for difficult-to-test bacteria like PA and KP.

Conclusions: We have successfully demonstrated accurate MIC determination for both Gram-positive bacteria SA and Gram-negative bacteria EC, PA, and KP in 30 min. Particularly, 6/6 MIC values were all within the CLSI MIC ranges, indicating the reliability of the iAST. In addition, the iAST requires only a small number of bacteria (10³ CFU) per test which could greatly reduce the time needed for growing enough bacteria for testing, which could further shorten the sample to result in time.


Acknowledgements (Optional): : The author would like to thank the support of NIH grants # R43AI152716-01 and R03EB030215.


References (Optional): :

1. van Belkum, A., et al., Nat Rev Microbiol 18, 299–311 (2020).


2. Kumar, A., et al. Crit. Care Med. 34, 1589–1596 (2006).


3. Spencer, D. C., et al., Nature communications, 2020, 11(1): 5328.


4. Kandavalli V., et al., Nature Communications, 2022, 13(1): 6215.


5. Bulik, C., et al., J. Clinic. Microbio, 2010, 48(7), 2402-06.


6. Sader, H.S., et al., J. Clinical. Microbiol, 2006, 44(3), 1101-04.