Comparative antimicrobial activity of aspirin, paracetamol, flunixin meglumine, tolfenamic acid, diclofenac sodium and pheniramine maleate

• Some of the tested drugs (NSAIDs including aspirin, diclofenac, meloxicam & flunixin meglumine) possess broad-spectrum antimicrobial activity but not in therapeutically achievable non-toxic concentrations in the body. Paracetamol was the least effective as antimicrobial similar to pheniramine maleate.
• A few of the NSAIDs may be evaluated as topical use antimicrobials (aspirin, diclofenac & flunixin) being microbial growth inhibitors at 0.128% concentration, similar levels of antimicrobial drugs including antibiotics are often used in topical antiseptic preparations (gels/ ointments/creams/powders/ lotions).
• Studies should be conducted on synergism/ antagonism of NSAIDs with commonly used antibiotics and herbal antimicrobials so that antimicrobial potential of NSAIDs can be therapeutically utilized.
• Susceptibility of bacteria to NSAIDs may be an aid in the differentiation of a few bacteria like Klebsiella and Raoultella; Enterococcus and Streptococcus (meloxicam); Bacillus and Paenibacillus (Diclofenac). However, multicentric studies on a greater number of strains are essential to establish the difference.

1. Comparative Antimicrobial Activity of Aspirin, Paracetamol , Flunixin- Meglumine , Tolfenamic Acid, Diclofenac Sodium and Pheniramine Maleate Bhoj R Singh Principal Scientist & Head of Epidemiology Indian Veterinary Research Institute, Izatnagar-243122, India Main Resource: Acta Scientific Veterinary Sciences 3.9 (2021): 30-42 Available at https://actascientific.com/ASVS/pdf/ASVS-03-0200.pdf 3rd International Conference on Virtual Pharma 4th December 2021

2. Source: Epidemiology Data, ICAR-IVRI, 2021) DOI :  10.13140/RG.2.2.24249.08809 3rd International Conference on Virtual Pharma 4th December 2021

3. Globally about 35 billion doses of antibiotics (3500-5000 tons) are used for therapy and disease prevention in human medicine, and about 50000 tons for treatment and growth promotion in the livestock and agriculture sectors. New estimates are >100 000 tonnes of antibiotic uses in livestock (OIE, 2019) Globally, about 10% of antimicrobials are used in humans and 90% in livestock, but in India, it is not that skewed? Worldwide, each year about 700,000 people die from antimicrobial-resistant infections, and this mortality has been projected to reach 10 million annully by 2050 ( O'Neill, 2014 ). The crude infectious disease mortality rate in India is more than 400 per 100,000 persons and at least 23,000 deaths in adults and 58,000 neonatal deaths per year are caused by AMR bacteria ( Laxminarayan & Chaudhury , 2016 ). >2.8 million antibiotic-resistant infections occur in the U.S. each year, and more than 35,000 people die (2019 AR Threat Report) By 2030, antimicrobial resistance could force up to 24 million people into extreme poverty ( UN Ad hoc Interagency Coordinating Group on Antimicrobial Resistance , 2019). 3rd International Conference on Virtual Pharma 4th December 2021

4. Therapeutic alternatives to antibiotics Vaccines & antibodies ( Monoclonals ) Herbal antimicrobials Bacteriophages Predatory bacteria, Bacteriocins , Synbiotics , Probiotics and prebiotics NSAIDs Antimicrobial peptides Faecal Transplant Therapy (FTT) Antibacterial Oligonucleotides & Crisper Cas-9 Homeopathy Acupuncture and Acupressure Generally recognized as safe (GRAS) chemicals Bhabhut / Ashes/ Fumes/ Mantras Source: DOI :  10.13140/RG.2.2.24249.08809 3rd International Conference on Virtual Pharma 4th December 2021

5. NSAIDs as Alternatives to Antibiotics Numerous non-antibiotic molecules ( anthelmintics , anticancer drugs, antipsychotics, antidepressant drugs, antiplatelets and NSAIDs) have been evaluated for their antimicrobial potential (Khan et al., 2020; Lagadinou et al., 2020). Some of the cyclooxygenase inhibitory anti-inflammatory and antipyretics ( Leão et al., 2020) viz., acetaminophen ( paracetamol ), acetylsalicylic acid (aspirin), diclofenac and ibuprofen, flurbiprofen and similar non-steroidal anti-inflammatory drugs (NSAIDs) are claimed to be antimicrobial also (Singh et al., 2021). Aspirin and paracetamol are reported to enhance the performance of antibiotics either through their synergistic antibacterial action with antibiotics (Ahmed et al., 2016, 2017; Chan et al., 2017; Hadera et al., 2018; Babik et al., 2021; Singh, 2021 DOI:  10.1101/2021.05.21.445232 ) 3rd International Conference on Virtual Pharma 4th December 2021

6. NSAIDs as antimicrobials Tolfenamic acid is shown to inhibit only a strain of S. aureus (MIC = 5 mg/ mL ) but was ineffective against C. albicans , E. coli and P. aeruginosa strains even at 10 mg/ mL concentration ( Kruszewska et al., 2002). Pheniramine maleat e reported inhibiting of growth of S. aureus and S. epidermidis at >20 mg/ mL level ( Gocmen et al., 2009). 100% of strains are shown to be susceptible at a 12.8 mg/ mL concentration of aspirin (Singh et al., 2021). Paracetamol was either almost ineffective or mildly effective as antimicrobial at a 12.8 mg/ mL level (Singh et al., 2021; Audigier et al., 2017). Diclofenac could inhibit E. faecalis strains at 0.75 to ≥50 mg/ mL ( Kruszewska et al., 2002; Salem- Milani et al., 2013; Abd El- Baky et al., 2016). 7.81  μg /ml concentration meloxicam is reported to inhibit biofilm formation by P. aeruginosa (She et al., 2018). 3rd International Conference on Virtual Pharma 4th December 2021

7. Limits of NSAIDs Plasma concentration of aspirin ranges between 4.9-8.9 µg/ mL in therapeutically applicable dosages and get converted to salicylate rapidly which may be in plasma with a concentration of 42-62 µg/ mL , and it may keep on increasing with chronic use of aspirin (NCBI, 2021 ). Aspirin causes acute toxicity if consumed @150 mg/ Kg . At the maximum therapeutically achievable concentration ( 30 µg/ mL ). In supra-therapeutic toxic dosages plasma concentration of paracetamol is reported even up to 1.5 mg/ mL ( Medsafe , 2012). Flunixin meglumine in animals may be given at a maximum dose of 1-1.5 mg/kg body weight ( Papich , 2016) . Due to the lethal nephrotoxicity of diclofenac to vultures (scavengers of dead animals) with LD 50  of 0.1–0.2 mg/ Kg (Swan et al., 2006), its use in animals is prohibited. Even in human being a dose equivalent to 2.5 g may induce lethal kidney failure ( Smolinske et al., 1990; Hunter et al., 2011). The maximum recommended doses of meloxicam are 7.5 to 15 mg/ 60 Kg BW ( https://www.drugs.com/dosage/meloxicam.html ) in higher doses (>1.2 mg/ Kg) it causes liver and kidney toxicity ( Mahaprabhu et al., 2011). Pheniramine maleate is used @ 1mg/Kg and the maximum dose is 3 mg/Kg, it causes acute toxicity over 15 mg/ Kg (Paul et al., 2009) 3rd International Conference on Virtual Pharma 4th December 2021

8. Objective Do NSAIDs may be alternative antimicrobials? If yes, what may be effective concentration? 3rd International Conference on Virtual Pharma 4th December 2021

9. Study materials and Methods Pure compounds from Sigma Aldrich were solubilised in Mueller Hinton broth (MHB, BBL Difco ) to the required concentration viz. aspirin 51.2 mg/ mL , meloxicam 1.28 mg/ mL , paracetamol 10.24 mg/ mL , flunixin meglumine 12.8 mg/ mL , diclofenac sodium 10.24 mg/ mL , tolfenamic acid 12.8 mg/ mL and pheniramine maleate 12.8 mg/ mL. MIC was determined through serial broth dilution method in micro (96 well) plates (Singh et al., 2021) for 499 microbial strains in the repository of Division of Epidemiology with a known history of Carbapenem drug ( imipenem / meropenem/ ertapenem ) susceptibility. 3rd International Conference on Virtual Pharma 4th December 2021

10. Test strains belonging to 117 species under 26 genera of Gram-negative (G- ve ) bacteria (322 strains), nine genera of Gram-positive ( G+ve ) bacteria (173 strains) and four strains of Candida species. Major groups of Gram-Negative bacteria tested Strains Tested Acinetobacter (A. alcaligenes 1, A. aclcoaceticus 3, A. lwoffii 3) 7 Aeromonas (A. bestiarum 5, A. caviae 2, A. eucranophila 2, A. hydrophila 2, A. media 2, A popoffii 4, A. salmonicida 2, A. schubertii 3, A. trota 4) 26 Enterobacter (E. agglomerans 13, E. gregoviae 1) 14 Erwinia (E. amylovora 2, E. aphidicola 1, E. carotovora 1, E. cyperipedii 1, E. nimipressuralis 1, E. stuartii 1, E. tasmaniensis 1) 8 Escherichia coli 101 Hafnia alvei 17 Klebsiella (K. pneumoniae 31, K. oxytoca 2) 33 Moraxella (M. bovis 1, M. ovis 2, M. osloensis 1, M. phenylpyruvica 1) 5 Pasteurella (P. canis 7, P. multocida 2) 9 Proteus (P. mirabilis 12, P. penneri 2, P. vulgaris 1) 15 Pseudomonas (B. cepacia 1, P. aeruginosa 9, P. alcaligenes 1, P. diminuta 1, P. paucimobilis 1, P. pseudoalcaligenes 2, P. stutzeri 1) 16 Raoultella terrigena 12 Salmonella enterica ssp. enterica 24 Serratia (S. fonticola 1, S. grimaceae 4, S. marce scens 3, S. odorifera 3, S. plymuthica 1, S, proteomaculans 2, S. rubidaea 1) 15 3rd International Conference on Virtual Pharma 4th December 2021

11. Major groups of Gram-positive bacteria tested Strains Tested Bacillus (B. badius 1, B. amyloliquifaciens 1, B. brevis 1, B. cereus 5, B. licheniformis 3, B. megaterium 2, B. mycoides 2, B. sphaericus 1) 16 Enterococcus (E. durans 2, E. faecalis 8, E. faecium 9, E. malodoratus 1, E. solitarus 4) 24 Paenibacillus (P. lactis1, P. larvae 2, P. pantothenticus 14) 17 Staphylococcus (of 21 species) 83 Streptococcus (S. equi 1, S. milleri 8, S. phocae 1, S. pneumoniae 3, S. porcinus 1, S. pyogenes 1, S. salivaris 2, S. suis 5) 22 Candida C. albicans 3 & C. famata 1 4 Of the 495 bacterial strains, 85 were carbapenem-resistant ( CR+ve ) and 410 were carbapenem-sensitive (CR- ve ). 3rd International Conference on Virtual Pharma 4th December 2021

12. Results The study revealed that 92.79%, 44.09%, 54.91% and 30.26% bacterial strains were sensitive to 2.56 mg/ mL aspirin3.2 mg/ mL flunixin meglumine , 2.56 mg/ mL diclofenac and 1.28 mg/ mL meloxicam , respectively. Pheniramine maleate and paracetamol could inhibit many of the microbial strains tested at 12.8 mg/ mL (1.28%) concentration. Tolfenamic acid showed no antimicrobial activity even at a 12.8 mg/ mL concentration. No significant difference in susceptibility to NSAIDs was detected among CR+ve & CR- ve and G+ve & G- ve bacterial strains. Oxidase positive (102) strains were significantly more often susceptible to NSAIDs than oxidase negative (393) bacterial strains. Among Oxidase positive bacteria Pseudomonads were significantly (p <0.001) more often resistant to aspirin, meloxicam and flunixin but no difference for susceptibility to diclofenac . 3rd International Conference on Virtual Pharma 4th December 2021

13. At 1.28 mg/ mL concentration Parcetamol * , Tolfenamic acid and Pheniramine maleate not inhibited any of the strains. ** No significant difference in susceptibility of G+ve and G- ve bacteria to any of the NSAIDs tested. 3rd International Conference on Virtual Pharma 4th December 2021

14. No significant difference in susceptibility to NDAIDs was detected among CR+ve (85) and CR- ve (410) bacterial strains, though 3rd International Conference on Virtual Pharma 4th December 2021

15. Oxidase -positive (102) strains were significantly (p <0.001) more often susceptible to NSAIDs than oxidase -negative (393) bacterial strains. Among Oxidase -positive bacteria Pseudomonads were significantly (p <0.001) more often resistant to aspirin, meloxicam and flunixin but no difference for susceptibility to diclofenac than other oxidase -positive bacteria. 3rd International Conference on Virtual Pharma 4th December 2021

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18. Genus of bacteria Percent of strains with MIC (mg/ mL ) of Aspirin ≤1.28 Meloxicam ≤1.28 Paracetamol ≤1.28 Flunixin ≤0.80 Diclofenac ≤1.28 Acinetobacter 85.71 57.14 0.00 14.29 0.00 Aeromonas 96.15 73.08 0.00 61.54 0.00 Enterobacter 35.71 0.00 0.00 7.14 0.00 Erwinia 100.00 25.00 0.00 12.50 0.00 Escherichia 85.15 24.75 0.00 3.96 0.99 Hafnia 76.47 0.00 0.00 11.76 11.76 Klebsiella 21.21 9.09 0.00 0.00 0.00 Moraxella 100.00 100.00 0.00 0.00 80.00 Pasteurella 100.00 77.78 0.00 11.11 100.00 Proteus 53.33 0.00 0.00 0.00 0.00 Pseudomonas 25.00 25.00 0.00 12.50 12.50 Raoultella 16.67 100.00 0.00 41.67 16.67 Salmonella 8.33 29.17 0.00 12.50 25.00 Serratia 6.67 26.67 0.00 20.00 20.00 3rd International Conference on Virtual Pharma 4th December 2021

19. Genera of Bacteria Percent of strains with MIC (mg/ mL ) of Aspirin ≤1.28 Meloxicam ≤1.28 Paracetamol ≤1.28 Flunixin ≤0.80 Diclofenac ≤1.28 Bacillus 87.50 100.00 0.00 68.75 62.50 Enterococcus 50.00 0.00 0.00 12.50 4.17 Paenibacillus 100.00 100.00 0.00 82.35 5.88 Staphylococcus 71.08 1.20 0.00 65.06 16.87 Streptococcus 36.36 45.45 0.00 27.27 4.55 Candida 25.0 50.0 100.0 Max Plasma Con. therapeutically achievable 4.9-8.9 µg/ mL 0.8-3.3 μ g/ mL 12.5-20 µg/ mL 3.3±2.2 µg/ mL 2.9±2.9 µg/ mL Minimum MIC observed in the study 10 µg/ mL S. pyogenes 10 µg/ mL A. media Y. enterocolitica 160 µg/ mL Aerococcus sp. 12.5 µg/ mL Staph. hominis 40 µg/ mL Strept . suis Maximum recommended dose/ day 65 mg/ Kg 1 mg/ Kg 60 mg/ Kg 2.2 mg/ Kg 2.5 mg/ Kg 3rd International Conference on Virtual Pharma 4th December 2021

20. Antimicrobial activity of Aspirin Most of the Erwinia, Moraxella and Pasteurella spp. strains were susceptible (MIC ≤1.28 mg/ mL ) to aspirin. Among G- ve bacteria, Erwinia, Moraxella and Pasteurella spp. strains were more often (p,< 0.05) susceptible than Enterobacter , Klebsiella , Proteus, Pseudomonas, Raoultella , Salmonella and Serratia species . Salmonella s trains were the most resistant (MIC >1.28 mg/ mL ) G- ve bacteria to aspirin; and more often resistant (p, <0.04) than Acinetobacter , Aeromonas , Enterobacter , Erwinia, E. coli, H. alvei , Moraxella & Proteus species strains. Strains of the Panenibaccilus spp. were the most often susceptible, & strains of Streptococcus spp. were the most resistant. More of the S. aureus strains were resistant to aspirin (MIC >1.28 mg/ mL ) than S. epidermidis (p, 0.03) strains. Among strains of different species of other genera of bacteria, no significant difference was observed with for their susceptibility to aspirin. Among Oxidase -positive bacteria strains of Pseudomonas species were the most resistant ones. 3rd International Conference on Virtual Pharma 4th December 2021

21. Antimicrobial activity of Meloxicam All Morexella and Raoultella spp. strains were susceptible (MIC ≤1.28 mg/ mL ) and significantly more (<0.05) often than strains of all other bacteria but Pasteurella strains. All Enterobacter , H. alvei and Proteus spp. strains were resistant (MIC >1.28 mg/ mL ), significantly more often (<0.05) than Acinetobacter , Aeromonas , Erwinia, Moraxella , Pasteurella , Pseudomonas, Raoultella , Salmonella & Serratia species strains. All Bacillus and Paenibacillus species strains were susceptible but all enterococci were resistant to meloxicam . Among strains of different species of genera of bacteria, no significant difference was observed for their susceptibility to aspirin. 3rd International Conference on Virtual Pharma 4th December 2021

22. Flunixin meglumine & Diclofenac , Tolfenamic acid & Pheniramine maleate >61% Aeromonads were susceptible to flunixin meglumine , and was the group of significantly (p <0.03) more susceptible genus of G- ve bacteria tested in the study. All strains tested of Klebsiella & Proteus species were refractory (MIC >1.6 mg/ mL ) to flunixin. All Paseteurella and 80% of Moraxella spp. strains were susceptible (MIC ≤1.28 mg/ mL ) to diclofenac but on other bacterial strains diclofenac was often non-inhibitory except for 25% S. enterica and 20% of Serratia species strains. All 4 Candida strains were susceptible to diclofenac . At 1.28 mg/ mL concentration none of the 495 strains of bacteria tested was inhibited by paracetamol , pheniramine maleate and tolfenamic acid except a strain of Aerococcus species susceptible to 160 µg/ mL of paracetamol . 3rd International Conference on Virtual Pharma 4th December 2021

23. Conclusions and future perspectives Some of the tested drugs (NSAIDs including aspirin, diclofenac, meloxicam & flunixin meglumine ) possess broad-spectrum antimicrobial activity but not in therapeutically achievable non-toxic concentrations in the body. Paracetamol was the least effective as antimicrobial similar to pheniramine maleate. A few of the NSAIDs may be evaluated as topical use antimicrobials (aspirin, diclofenac & flunixin) being microbial growth inhibitors at 0.128% concentration, similar levels of antimicrobial drugs including antibiotics are often used in topical antiseptic preparations (gels/ ointments/creams/powders/ lotions). Studies should be conducted on synergism/ antagonism of NSAIDs with commonly used antibiotics and herbal antimicrobials so that antimicrobial potential of NSAIDs can be therapeutically utilized. Susceptibility of bacteria to NSAIDs may be an aid in the differentiation of a few bacteria like Klebsiella and Raoultella ; Enterococcus and Streptococcus (meloxicam); Bacillus and Paenibacillus (Diclofenac). However, multicentric studies on more number of strains are essential to establish the difference. 3rd International Conference on Virtual Pharma 4th December 2021

24. Thanks to the audience and Organizers For an opportunity to interact 3rd International Conference on Virtual Pharma 4th December 2021