Over-the-counter antibiotics compromising aminoglycoside activity

Abstract

Introduction: Antimicrobial resistance (AMR) is a global issue that needs addressing. While antibiotic stewardship has improved often by restricting antibiotic use, some antibiotics that are still sold legally over the counter (OTC), notably in sore throat medications. Recent findings suggest OTC antibiotics could trigger cross-resistance to antibiotics used in clinical treatments, whether systemic or topical. Here we investigated the impact of three antibiotics contained in OTC sore throat medicines on emerging AMR in vitro.

Methods: Bacterial pathogens were exposed to a bactericidal concentration of an aminoglycoside in the presence or absence of a during-use concentration of bacitracin, gramicidin or tyrothricin in a time-kill assay. Damage to the bacterial membrane was also investigated by measuring potassium leakage and membrane potential alteration post-OTC antibiotic exposure.

Results: Gramicidin (15 µg/mL) significantly decreased the bactericidal activity of amikacin, tobramycin or gentamicin in Acinetobacter baumannii. It also decreased gentamicin bactericidal activity in Enterobacter cloacae, Escherichia coli and Klebsiella pneumoniae, while tyrothricin decreased the aminoglycoside efficacy in E. cloacae and E. coli. Gramicidin significantly decreased bacterial membrane potential and caused significant potassium leakage.

Conclusion: Gramicidin and to some extent tyrothricin impacted aminoglycoside efficacy by affecting membrane potential, which is essential for aminoglycosides uptake. Thus, some OTC antibiotics can interfere with aminoglycoside activity, which could in turn affect treatment efficacy. Although the likelihood of OTC antibiotics and aminoglycosides being used at the same time might not be common, this research highlights one potential reason for OTC antibiotics' usage to result in treatment failure and their contribution to AMR development.

Figure 1.

Bactericidal efficacy of aminoglycoside co-exposed OTC antibiotics in A. baumannii. (a) A. baumannii treated with amikacin (2 µg/mL) for 3 hours. There was a significant difference in log₁₀ reduction when co-exposed with BZK (4 µg/mL; P < 0.0001) or gramicidin (15 µg/mL; P < 0.0001). When co-exposed to bacitracin (5 IU/mL) the difference in bactericidal efficacy to amikacin alone was not significant (P > 0.9999). (b) A. baumannii treated with tobramycin (2 µg/mL) for 3 hours. Tobramycin efficacy was significantly reduced when co-exposed to either BZK (4 µg/mL; P = 0.0401) or gramicidin (15 µg/mL; P = 0.0004) but not with bacitracin (5 IU/mL; P = 0.2085). (c) A. baumannii treated with gentamicin (2 µg/mL) for 3 hours. Gentamicin bactericidal efficacy was significantly decreased when exposed to BZK (4 µg/mL; P < 0.0001) or gramicidin (15 µg/mL; P < 0.0001) but not with bacitracin (5 IU/mL; P = 0.5136). ns, not significant (P > 0.05), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 (raw data are available in Table S1a–c, available as Supplementary data at JAC Online). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 2.

Bactericidal efficacy of aminoglycoside co-exposed OTC antibiotics in Enterobacteriaceae. (a) E. cloacae treated with gentamicin for 3 hours. Co-exposed to gramicidin or tyrothricin significantly decreased gentamicin efficacy when compared to gentamicin treatment alone (P = 0.0002 and P = 0.0074 respectively). (b) E. coli cultures treated with gentamicin for 3 hours. There was a significant decrease in gentamicin efficacy when cultures were co-exposed to BZK (P = 0.0217), gramicidin (P < 0.0001) or tyrothricin (P = 0.0001). (c) K. pneumoniae treated with gentamicin for 3 hours. Co-exposure to gramicidin significantly decreased efficacy when compared to gentamicin alone (P = 0.0172). ns, not significant (P > 0.05), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 (raw data available in Table S2a–c). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 3.

A. baumannii membrane potential following exposure to OTC antibiotics. CCCP (5 µM) was used as a positive control. ns, not significant (P > 0.05), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, **** P ≤ 0.0001. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 4.

Potassium concentration in solution following exposure to OTC antibiotics in A. baumannii. ns, not significant (P > 0.05), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 5.

Mechanism of protection during co-exposure with gramicidin or tyrothricin. (a) When bacteria are exposed to only aminoglycosides, the potassium homeostasis in maintained and the cells remain polarized. This allows aminoglycosides to enter bacteria and kill them. (b) When co-exposed with either gramicidin or tyrothricin, the OTC antibiotics create pores in the bacterial membranes. This cause intracellular potassium to leak out and therefore depolarizes cells. Aminoglycosides cannot enter depolarized cells and therefore the gramicidin or tyrothricin will ‘protect’ the bacteria from aminoglycoside activity. The battery represents the bacterial membrane potential and the pill capsule represents aminoglycoside treatments. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.