Some drugs that do not belong to the group of antibiotics and antibacterial chemotherapeutics may have direct (Tyski 2003; Kruszewska et al. 2012; Laudy et al. 2017; Lagadinou et al. 2020; Kruszewska et al. 2021) or indirect antibacterial activities, e.g., affect the activity of membrane efflux pumps (Nair et al. 2004; Laudy et al. 2018) or the
Some studies have shown the effects of NSAIDs on the growth and virulence of symbiotic and pathogenic human bacterial strains. Antibacterial activity has been demonstrated for diclofenac, ibuprofen, acetylsalicylic acid (Laudy et al. 2016; Zimmermann and Curtis 2017; Lagadinou et al. 2020), flurbiprofen (Zimmermann and Curtis 2017; Lagadinou et al. 2020), piroxicam, indomethacin, and ketoprofen (Elshaer et al. 2022). Moreover, Laudy et al. (2016) showed that ibuprofen, diclofenac, acetylsalicylic acid, and mefenamic acid were substrates for efflux pumps in Gram-negative rods. Due to the differences in the susceptibility of bacteria to NSAIDs, it is interesting whether they can inhibit the growth of bacteria from the
People often supplement antibiotic therapies with some probiotics or use them for digestive system ailments (Blaabjerg et al. 2017; Zawistowska-Rojek and Tyski 2022). However, the constant anti-inflammatory and analgesic use of NSAIDs (Phillips et al. 2004), also in the prevention and treatment of colorectal cancer (Kmiec et al. 2014), might threaten the intestinal microbiota; moreover, gastrointestinal side effects caused by NSAIDs could be associated with affecting the survivability of the probiotic bacteria.
Therefore, this study’s main goal was to estimate whether commonly available anti-inflammatory and analgesic drugs affect the survivability of bacteria from the
We determined the effects of various active substances of NSAIDs, namely acetylsalicylic acid (Mikromol), diclofenac (Amoli Organics), indomethacin (Dr. Ehrenstorfer), ibuprofen (Mikromol), naproxen (USP), ketoprofen (LGC), dexketoprofen (Menarini), flurbiprofen (Sigma-Aldrich), piroxicam (USP), meloxicam (Ph Eur. CRS), celecoxib (Sigma-Aldrich), etoricoxib (Merck), nabumetone (Sigma-Aldrich) nimesulide (Sigma-Aldrich) and of analgesics, such as paracetamol (Sigma-Aldrich) and tramadol (LGC) on the viability of 49
The assessment of the antibacterial activity of the selected agents was carried out in two stages. For the initial screening, 14 NSAIDs and two analgesics were investigated. The tested substances were dissolved in DMSO (POCH S.A., Poland) and diluted in 0.9% NaCl (bioMérieux, France) to 1 ml, to achieve a final concentration of 3.2 mg/ml in the MRS agar (De Man, Rogosa and Sharpe Agar; Merck Millipore, Germany). As a control, DMSO without active substances was used. The strains were harvested from the MRS agar to prepare the cell suspensions at a density of 0.5 on the McFarland scale. Subsequently, 2-μl aliquots of the suspensions of the tested strains were dripped onto the agar surface. The plates were incubated at 37°C for 72 h in an atmosphere with 5% CO2. The compounds for which no growth was observed for at least one of the tested strains were selected for further investigation. In the second stage of antibacterial activity testing, the minimal inhibitory concentrations of diclofenac, ibuprofen, ketoprofen, dexketoprofen, and flurbiprofen in the MRS liquid medium (MRS broth; Merck Millipore, Germany), were determined in triplicate according to the CLSI guidelines (CLSI 2015).
The following NSAIDs: nabumetone, celecoxib, nimesulide, meloxicam, etoricoxib (all belonging to COX-2 inhibitors), naproxen, piroxicam and indomethacin, and the analgesics tramadol and paracetamol at a concentration of 3.2 mg/ml, did not inhibit the growth of all strains of the family
For compounds showing activity against both COX-1 and COX-2, the degree of growth inhibition depended on the used NSAID.
Based on the MIC determination, diclofenac, ibuprofen, ketoprofen, dexketoprofen, flurbiprofen, and acetylsalicylic acid inhibited bacterial growth at concentrations of 0.05–3.2 mg/ml, depending on the tested strain (Table I). The highest activity against most of the tested strains was found for diclofenac (MIC = 0.05 mg/ml for two strains and MIC = 0.1 mg/ml for 30 strains). The second most active compounds were ibuprofen (MIC = 0.4 mg/ml for 19 strains) and flurbiprofen (MIC = 0.4 mg/ml for 9 strains). The MIC of ketoprofen and dexketoprofen was 0.8 mg/ml for four strains. The lowest activity against all strains was obtained for acetylsalicylic acid (MIC = 3.2 mg/ml) (Table I). This value agreed with the previous data obtained by Kruszewska et al. (2021) for the probiotic strains
Minimal inhibitory concentration (MIC) of selected NSAIDs against 49
Species | Source/(number of strains) | MIC (mg/ml) | Acetylsalicylic acid | ||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Flurbiprofen | Ketoprofen | Dexketoprofen | Ibuprofen | Diclofenac | |||||||||||||||
0.4 | 0.8 | 1.6 | 3.2 | 0.8 | 1.6 | 3.2 | 0.8 | 1.6 | 3.2 | 0.4 | 0.8 | 1.6 | 0.05 | 0.1 | 0.2 | 0.8 | 3.2 | ||
ATCC* 4356™ | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 1 | |
Probiotic products (n = 10) | 4 | 2 | 4 | 0 | 0 | 9 | 1 | 0 | 9 | 1 | 2 | 5 | 3 | 0 | 5 | 2 | 3 | 10 | |
Clinical isolates (n = 9) | 0 | 4 | 5 | 0 | 0 | 6 | 3 | 0 | 4 | 5 | 1 | 8 | 0 | 0 | 4 | 5 | 0 | 9 | |
ATCC* 53103™ | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 1 | |
Probiotic products (n = 4) | 1 | 3 | 0 | 0 | 0 | 0 | 4 | 0 | 0 | 4 | 0 | 4 | 0 | 0 | 1 | 3 | 0 | 4 | |
Clinical isolates (n = 6) | 0 | 0 | 5 | 1 | 0 | 0 | 6 | 0 | 1 | 5 | 0 | 6 | 0 | 0 | 4 | 2 | 0 | 6 | |
Probiotic products (n = 4) | 2 | 2 | 0 | 0 | 0 | 3 | 1 | 0 | 4 | 0 | 4 | 0 | 0 | 1 | 3 | 0 | 0 | 4 | |
Clinical isolates (n = 2) | 0 | 2 | 0 | 0 | 0 | 2 | 0 | 1 | 1 | 0 | 2 | 0 | 0 | 1 | 1 | 0 | 0 | 2 | |
Probiotic products (n = 3) | 2 | 1 | 0 | 0 | 2 | 1 | 0 | 2 | 1 | 0 | 3 | 0 | 0 | 0 | 3 | 0 | 0 | 3 | |
ATCC* 14917™ | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 1 | |
Probiotic products (n = 3) | 0 | 3 | 1 | 0 | 1 | 2 | 0 | 0 | 3 | 0 | 3 | 0 | 0 | 0 | 3 | 0 | 0 | 3 | |
Clinical isolates (n = 4) | 0 | 2 | 1 | 0 | 0 | 4 | 0 | 0 | 4 | 0 | 1 | 3 | 0 | 0 | 3 | 1 | 0 | 4 | |
Probiotic products (n= 1) | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 1 | |
TOTAL | 49 | 9 | 22 | 17 | 1 | 4 | 29 | 16 | 4 | 29 | 16 | 19 | 27 | 3 | 2 | 30 | 14 | 3 | 49 |
In studies on the effect of indomethacin (10 mg/kg) on the intestinal microbiota in mice, it was found that indomethacin does not affect the viability of
Regarding the present study, it is important to relate the MIC values of the tested compounds obtained
The summary of activity
Active substance | The lowest inhibitory concentration for a minimum 2 strains |
---|---|
Nabumetone | > 3.2 mg/ml |
Celecoxib | > 3.2 mg/ml |
Nimesulide | > 3.2 mg/ml |
Meloxicam | > 3.2 mg/ml |
Etoricoxib | > 3.2 mg/ml |
Naproxen | > 3.2 mg/ml |
Piroxicam | > 3.2 mg/ml |
Indomethacin | > 3.2 mg/ml |
Tramadol | > 3.2 mg/ml |
Paracetamol | > 3.2 mg/ml |
Acetylsalicylic acid | 3.2 mg/ml |
Ketoprofen | 0.8 mg/ml |
Dexketoprofen | 0.8 mg/ml |
Ibuprofen | 0.4 mg/ml |
Flurbiprofen | 0.4 mg/ml |
Diclofenac | 0.05 mg/ml |
Ibuprofen is generally dosed from 200 to 600 mg, with a daily dose of up to 2,400 mg (Sarzi-Puttini et al. 2013). According to Janssen and Venema (1985), the maximum plasma concentration after administration of 600 mg tablets of ibuprofen twice, three times, and four times daily ranged from 39.4–66.4 gg/ml. After administration of suppositories (125 mg) Cmax was an average of 23.3 μg/ml (EMA 2003). In this case, where the MIC against lactobacilli was 0.4 mg/ml (Table II), which is much higher than the maximum plasma concentration in the human body (approximately 0.06 mg/ml), ibuprofen cannot influence
For ketoprofen, the average dosage is approximately 150 mg per day, with a maximum of 200 mg (Sarzi-Puttini et al. 2013). According to Roda et al. (2002), after the administration of 200 mg of ketoprofen in the form of oral tablets, Cmax was 5.91 ± 0.66 μg/ml. After rectal administration (100 mg) the mean maximum concentration of the plasma was 6.56 μg/ml (Caillé et al. 1980). The MIC against lactobacilli was 0.8 mg/ml (Table II), which is much higher than the maximum plasma concentration in the human body, and 0.006 mg/ml of ketoprofen cannot impair the viability of
Dexketoprofen is the (
The daily dose of flurbiprofen should not exceed 400 mg (Valentovic et al. 2007). According to Yilmaz and Erdem (2015), after oral administration of a 100 mg formulation tablet, the Cmax of flurbiprofen was 42.1 μg/ml, and after rectal administration (75 mg), the maximum concentration of the plasma was 72.4—77.3 μg/ml (Scaroni et al. 1984). The MIC against lactobacilli was 0.4 mg/ml, which exceeds the maximum plasma concentration in the human body (about 0.04 mg/ml), indicating that flurbiprofen does not influence
Regarding acetylsalicylic acid, according to Nagelschmitz et al. (2014), after an oral dose of 500 mg, the Cmax in blood plasma was 4.84 μg/ml, and after intravenous (iv) administration to 21 volunteers, it was 54.25 μg/ml. In our study, the MIC of acetylsalicylic acid against lactobacilli was 3.2 mg/ml (Table II); it is much higher than in the plasma blood after iv administration (approximately 0.054 mg/ml).
NSAIDs in humans and animals can induce changes in the intestinal microbiome, like dysbiosis (Wang et al. 2021). Potential mechanisms causing NSAIDs-induced dysbiosis can be divided into two groups: NSAIDs-induced changes in the intestinal bacterial environment (such as mucositis, motility, changes in intestinal pH, bile acid metabolome) and direct antibacterial effects of NSAIDs (Maseda and Ricciotti 2020; Zádori et al. 2023). Disturbances in the intestinal microbiome can contribute to the development of many diseases, including irritable bowel syndrome, ulcerative colitis, Crohn’s disease, endocrine, cardiovascular, autoimmune and even neuropsychiatric disorders (Zádori et al. 2023). NSAIDs-induced dysbiosis, in most cases, is characterized by a clear transition from Gram-positive to Gram-negative bacteria, which consequently contributes to the development of intestinal damage. Changes can be observed especially in taxa such as Bacteroidetes (
In several
Pharmacological studies on
In summary, the maximum plasma levels of diclofenac, ibuprofen, ketoprofen, dexketoprofen, flurbiprofen, and acetylsalicylic acid during therapy are much lower than the MIC levels inhibiting the growth of the tested lactic acid bacteria. We, therefore, infer that the tested NSAIDs should not inhibit the growth of bacteria from the