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

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

28.6
40.9
54.6
53.9
72.8
75.6
10.1
30.5
38.2
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
2011 2016 2021
ESBL production MDR Carbapenem resistance
Source: Epidemiology Data, ICAR-IVRI, 2021)
DOI: 10.13140/RG.2.2.24249.08809

• 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).

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

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)

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 maleate 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).

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 LD50 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)

Objective
• Do NSAIDs may be alternative antimicrobials?
• If yes, what may be effective concentration?

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.

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

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).

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.

67.05
28.90
60.69
17.34
62.11
30.75
18.32
11.18
25.00
0.00
50.00
100.00
-5.00
5.00
15.00
25.00
35.00
45.00
55.00
65.00
75.00
85.00
95.00
105.00
Aspirin 0.128% Meloxicam 0.128% Flunixin 0.16% Diclofenac 0.128%
Susceptibility of microbial strains to NSAIDs
G+ve (173) G-ve (322) Candida (4)
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.

67.06
32.94
25.88
8.24
63.17
30.00
35.12
14.39
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
Aspirin 0.128% Meloxicam 0.128% Flunixin 0.16% Diclofenac 0.128%
Susceptibility of Carbapenem susceptible (CR-ve) and
resistant (CR+ve) microbial strains to NSAIDs
CR+ve CR-ve
No significant difference in susceptibility to NDAIDs was detected among
CR+ve (85) and CR-ve (410) bacterial strains, though

80.39
70.59
60.78
27.45
59.54
20.10
26.72
9.67
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Aspirin 0.128% Meloxicam
0.128%
Flunixin 0.16% Diclofenac 0.128%
Susceptibility of Oxidase positive and Oxidase negative
bacterial strains to NSAIDs
Oxidase+ve
Oxidase-ve
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.

4
1
0
1
4
0
1728
266
146
6
26
0
0
0
0
0
10 0 0 0
40
455
0
0
0
1
0
45 14
46
204 202
6
0
2
0
0
3
08
41
97
0 0
0
50
100
150
200
250
300
350
400
450
500
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 1010.51111.51212.513
Number
of
strains
MIC in mg/ mL
Antimicrobial activity of selected NSAIDs against 495
bacterial and 4 Candia spp. strains
Aspirin
Paracetamol
Diclofenac
Meloxicam

1
2
19
19
34
3532 26
52
62
217
0
0
0
0
00 0 0 1
38
337
0
50
100
150
200
250
300
350
400
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 1010.51111.51212.51313.5
Number
of
strains
MIC in mg/ mL
Antimicrobial activity of selected NSAIDs and
antihistaminic against 495 bacterial and 4 Candida spp.
strains
Flunixin meglumine
Tolfenamic acid
Pheniramine maleate

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

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 0 0 50.0 100.0
Max Plasma Con.
therapeutically
achievable
4.9-8.9 µg/
mL
0.8-3.3 μg/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

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 strains 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.

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.

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.

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.

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

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