Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Jul 10:13:2205-2222.
doi: 10.2147/IDR.S260766. eCollection 2020.

Small Molecule Adjuvants Potentiate Colistin Activity and Attenuate Resistance Development in Escherichia coli by Affecting pmr AB System

Dipak Kathayat  1 , Linto Antony  2 , Loic Deblais  1 , Yosra A Helmy  1 , Joy Scaria  2 , Gireesh Rajashekara  1
Affiliations

Small Molecule Adjuvants Potentiate Colistin Activity and Attenuate Resistance Development in Escherichia coli by Affecting pmr AB System

Dipak Kathayat et al. Infect Drug Resist. .

Abstract

Background: Colistin is one of the last-resort antibiotics to treat multi-drug resistant (MDR) Gram-negative bacterial infections in humans. Further, colistin has been also used to prevent and treat Enterobacteriaceae infections in food animals. However, chromosomal mutations and mobile colistin resistance (mcr) genes, which confer resistance to colistin, have been detected in bacterial isolates from food animals and humans worldwide; thus, limiting the use of colistin. Therefore, strategies that could aid in ameliorating colistin resistance are critically needed.

Objective: Investigate the adjuvant potential of novel small molecules (SMs) on colistin.

Materials and methods: Previously, we identified 11 membrane-affecting SMs with bactericidal activity against avian pathogenic Escherichia coli (APEC). Here, we investigated the potentiation effect of those SMs on colistin using checkerboard assays and wax moth (Galleria mellonella) larval model. The impact of the SM combination on colistin resistance evolution was also investigated by analyzing whole genome sequences of APEC isolates passaged with colistin alone or in combination with SMs followed by quantitating pmrCAB and pmrH expression in those isolates.

Results: The SM combination synergistically reduced the minimum bactericidal concentration of colistin by at least 10-fold. In larvae, the SM combination increased the efficacy of colistin by two-fold with enhanced (>50%) survival and reduced (>4 logs) APEC load. Further, the SM combination decreased the frequency (5/6 to 1/6) of colistin resistance evolution and downregulated the pmrCAB and pmrH expression. Previously unknown mutations in pmrB (L14Q, T92P) and pmrA (A80V), which were predicted deleterious, were identified in the colistin-resistant (ColR) APEC isolates when passaged with colistin alone but not in combination with SMs. Our study also identified mutations in hypothetical and several phage-related proteins in ColR APEC isolates in concurrent with pmrAB mutations.

Conclusion: Our study identified two SMs (SM2 and SM3) that potentiated the colistin activity and attenuated the development of colistin resistance in APEC. These SMs can be developed as anti-evolution drugs that can slow down colistin resistance development.

Keywords: anti-evolution drug; colistin; pmrCAB; pmrH; resistance; small molecules.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
(A) Survival curve of wax moth larvae treated with colistin alone or in combination with SMs. Larvae (n=15 larva/group) were infected with 6.4×104 CFU of Rifr APEC O78 and then treated with colistin (0.3125 mg/kg) and combination of colistin and SMs (0.3125 mg/kg+12.5 µg) within 30 min of infection. Larval survival was monitored every 12 h for 3 days. (B) APEC load inside wax moth larvae treated with colistin alone or in combination with SMs. APEC load in larvae was quantified by plating the homogenized larval suspension on MacConkey agar plates supplemented with 50 µg/mL rifampicin. PC-infected and buffer mix (DMSO+PBS) treated larvae, ***P<0.0001.
Figure 2
Figure 2
(A) Minimum inhibitory concentration (MIC) of APEC cultures passaged only in presence of colistin or in combination with SMs. Six parallel independent APEC cultures (Col 1–6, Col + SM2 1–6, and Col+ SM3 1–6) were serially passaged (8 passages) in M63 media starting with the sub-inhibitory concentration of colistin (0.0625 µg/mL) to up to 8 µg/mL (beyond resistance breakpoint) in presence and absence of 0.5× MIC of SMs. APEC cultures containing no colistin and no SM (1% DMSO; DMSO 1–6) were used as controls and passaged similarly to cultures containing colistin or colistin and SMs. The black dotted line indicates the MIC breakpoint for colistin resistance. (B) Chemical structure and ChemBridge ID of selected SMs.
Figure 3
Figure 3
Heatmap displaying the non-synonymous mutational comparison between ColR isolates (Col-1, Col-3, Col-5, Col-6, Col + SM2-2 and Col + SM3-5) as compared to ColS (DMSO-1, DMSO-3, Col + SM2-4, Col + SM2-6, Col + SM3-2 and Col + SM3-6) isolates. Phenotype “R” indicates resistant APEC isolate and “S” indicates susceptible APEC isolate. Abbreviation: NA, not available.
Figure 4
Figure 4
(A) Expression (fold change) of pmrC and pmrH in APEC isolates passaged with colistin alone (Col-1, Col-3, Col-5 and Col-6), colistin and SM2 (Col + SM2-2, Col + SM2-4 and Col + SM2-6), and colistin and SM3 (Col + SM3-2, Col + SM3-5 and Col + SM3-6) as compared to control (DMSO-1 and DMSO-3) isolates. (B) Expression (fold change) of pmrA and pmrB in APEC isolates passaged with colistin alone (Col-1, Col-3, Col-5 and Col-6), colistin and SM2 (Col + SM2-2, Col + SM2-4 and Col + SM2-6), and colistin and SM3 (Col + SM3-2, Col + SM3-5 and Col + SM3-6) as compared to control (DMSO-1 and DMSO-3) isolates. Phenotype “R” indicates resistant APEC isolate and “S” indicates susceptible APEC isolate.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kempf I, Jouy E, Chauvin C. Colistin use and colistin resistance in bacteria from animals. Int J Antimicrob Agents. 2016;48(6):598–606. doi:10.1016/j.ijantimicag.2016.09.016 - DOI - PubMed
    1. Poirel L, Jayol A, Nordmann P. Polymyxins: antibacterial activity, susceptibility testing, and resistance mechanisms encoded by plasmids or chromosomes. Clin Microbiol Rev. 2017;30(2):557. doi:10.1128/CMR.00064-16 - DOI - PMC - PubMed
    1. Biswas S, Brunel JM, Dubus JC, Reynaud-Gaubert M, Rolain JM. Colistin: an update on the antibiotic of the 21st century. Expert Rev Anti Infect Ther. 2012;10(8):917–934. doi:10.1586/eri.12.78 - DOI - PubMed
    1. Liu Y, Liu J-H. Monitoring colistin resistance in food animals, an urgent threat. Expert Rev Anti Infect Ther. 2018;16(6):443–446. doi:10.1080/14787210.2018.1481749 - DOI - PubMed
    1. Landman WJM, van Eck JHH. The incidence and economic impact of the Escherichia coli peritonitis syndrome in Dutch poultry farming. Avian Pathol. 2015;44(5):370–378. doi:10.1080/03079457.2015.1060584 - DOI - PubMed

Grants and funding

The research in Dr. Rajashekara’s laboratory was supported by the National Institute for Food and Agriculture (NIFA) Grant # 2015-68004-23131, US Department of Agriculture.

LinkOut - more resources