Do NSAIDs and Other Pain Relief Drugs Can Inhibit the Growth of Lactobacillaceae?

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 quorum sensing systems (Gajdács and Spengler 2019; Elshaer et al. 2022). These drugs are called non-antibiotics, and we determined whether frequently and sometimes chronically taken non-steroidal anti-inflammatory drugs (NSAIDs) and pain relief drugs could affect bacteria of the family Lactobacillaceae present in the microbiota, as well as those administered in probiotic preparations. A group of analgesics and NSAIDs, of which some presented antibacterial activity, was selected for the study. They are widely available (over-the-counter – OTC drugs) and are taken by patients with, among others, osteoarthritis, rheumatoid arthritis, acute gout attack, rheumatic fever, and juvenile chronic arthritis or for blood thinning (Phillips et al. 2004; Bacchi et al. 2012). The basis of the mechanism of action of NSAIDs in humans relies on the inhibition of the activity of cyclooxygenase (COX), an enzyme involved in the synthesis of prostaglandins. COX-1 is an enzyme physiologically active in various sites such as platelets, kidneys, blood vessels, and the stomach, whereas the activity of COX-2 increases rapidly in inflamed tissues (Yu et al. 2016). Due to the frequency and high doses of NSAIDs, gastrointestinal toxicity is likely to be a significant side effect, especially in predisposed patients (Bjarnason et al. 2018). The question is whether these ailments can be reduced using probiotic bacteria and whether NSAIDs will affect their growth.

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 Lactobacillacae family and, in the case of a positive finding, at which concentrations. Lactobacillaceae strains are ubiquitous and can colonize the oral cavity, digestive tract, and vagina, constituting symbiotic protection. Besides, they can be present in soil, water, manure, sewage, and silage. Some bacteria from this family can be classified as probiotic microorganisms that may have local and systemic effects on the host organism. The main benefits resulting from the presence of probiotic microorganisms are stimulation of the immune system, production of antibacterial substances, regulation of the composition of the intestinal microbiota, antimutagenic and anti-cancer effects (Kerry et al. 2018; Klopper et al. 2018; Zawistowska-Rojek and Tyski 2022), and protection of the intestinal tract by the biosynthesis of cytoprotective short-chain fatty acids (Syer et al. 2015; Markowiak-Kopeć and Ślizewska 2020).

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 Lactobacillaceae family, which could impact the feasibility of using probiotic preparations.

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 Lactobacillaceae strains isolated from probiotic products (dietary supplements, foods for special medical purposes, yogurts) and strains isolated from healthy people (cervix or colon swabs). Clinical isolates were derived from the strain collection of the Department of Pharmaceutical Microbiology and Bioanalysis of the Medical University of Warsaw, Poland. The following strains were used: Lactobacillus acidophilus (n = 19), Lacticaseibacillus rhamnosus (10), Lacticaseibacillus casei (6), Lacticaseibacillus paracasei (3), Lactiplanti-bacillus plantarum, and Limosilactobacillus fermentum (1). Three reference strains L. acidophilus ATCC® 4356™, L. rhamnosus ATCC® 53103™, and L. plantarum ATCC® 14917™ were also included.

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 Lactobacillaceae in vitro.

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 L. plantarum and L. acidophilus. Also, the results obtained in this study regarding the activity of nimesulide against lactobacilli strains were consistent with previous research (Kruszewska et al. 2021). In contrast, ibuprofen in our study was more active than dexibuprofen (its S-enantiomer) in all tested strains (Kruszewska et al. 2021). In the study of Laudy et al. (2016), the highest activity showed diclofenac (MICs: 0.2–1.6 mg/ml), followed by ibuprofen (MICs: 1.6–3.2 mg/ml) and acetylsalicylic acid (MICs: 1.6–3.2 mg/ml), whereas naproxen and nimesulide showed no activity against most Gram-negative bacteria. Analyzing Gram-positive cocci, Leâo et al. (2020) showed that for Staphylococcus aureus strains, naproxen and piroxicam were inactive, whereas the MIC of diclofenac and acetylsalicylic acid was 2 mg/ml. According to a review by Zimmermann and Curtis (2017), the MICs of ibuprofen for S. aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus ranged from 0.05–1.25 mg/ml, and that of diclofenac for S. aureus was 0.05 mg/ml.

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 Lactobacillus (Nadatani et al. 2012; Liang et al. 2015). In our study, indomethacin also did not inhibit the growth of lactobacilli. Moreover, two weeks of intragastrical treatment of rats with aspirin changed the oral microbial composition and increased the numbers of Lactobacillaceae (Cheng et al. 2018). However, Montrose et al. (2016) reported that chronic treatment of mice with celecoxib (1,000 ppm) caused a decrease in Lactobacillaceae abundance in the ileal content and feces.

Regarding the present study, it is important to relate the MIC values of the tested compounds obtained in vitro to their concentrations in the body fluids of people taking these drugs. The maximum plasma blood concentration of NSAIDs depends on the pharmaceutical form, and the most popular forms are tablets, capsules, suppositories, drops, syrups, and injections, which are less common. Oral doses of diclofenac are usually 100–150 mg daily in adults (Sarzi-Puttini et al. 2013). According to Willis et al. (1979), the maximum plasma concentration after a single oral dose (100 mg) and rapid intravenous diclofenac sodium injection ranges from 1.4 to 3.0 μg/ml. After administration of suppositories (50 mg) the maximum concentration (C_max) of the plasma was 1.95 ± 0.8 μg/ml (Novartis Pharmaceuticals UK Ltd. 2023). In the case of diclofenac, where the MIC against lactobacilli was 0.05 mg/ml (Table II), which is much higher than the maximum plasma concentration in the human body (0.003 mg/ml), it cannot influence Lactobacillaceae strains.

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) C_max 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 Lactobacillaceae strains.

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, C_max 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 Lactobacillaceae strains.

Dexketoprofen is the (S)-enantiomer of ketoprofen, and the total daily dose should not exceed 75 mg, with a maximum single dose of 25 mg (Aronson 2016). According to González-Canudas et al. (2019), after administration of 25 mg in an oral solution, the blood C_max of dexketoprofen was 4.3 ± 0.9 μg/ml. The MIC against lactobacilli was 0.8 mg/ml (Table II), which is much higher than the maximum blood plasma concentration in the human body, and 0.004 mg/ml of dexketoprofen cannot influence the viability of Lactobacillaceae strains.

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 C_max 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 Lactobacillaceae strains at these doses.

Regarding acetylsalicylic acid, according to Nagelschmitz et al. (2014), after an oral dose of 500 mg, the C_max 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 (Bacteroides, Parabacteroides, Prevotella, Barnesiella, Rikenellaceae), Proteobacteria (Enterobacteriaceae, Pseudomonadaceae, Alphaproteobacteria), Enterococcaceae, and Akkermansia (Wang et al. 2021; Zádori et al. 2023). The increase content of Gram-negative bacteria associated with the use of NSAIDs may cause the intestinal damage (Zádori et al. 2023). However, it should be noted that most of the current knowledge comes from animal studies conducted mainly on rats and mice. There are many differences between humans and rodents in both gastrointestinal anatomy and microbiota composition, so the usefulness of these studies is limited (Wang et al. 2021; Zádori et al. 2023).

In several in vitro studies, compounds such as acetylsalicylic acid, ibuprofen, naproxen, diclofenac, indomethacin, and celecoxib have been found to inhibit the growth of both Gram-positive and Gram-negative bacterial strains at concentrations ranging from tens to thousands of μg/ml (Jiménez-Serna and Hernández-Sánchez 2011; Laudy et al. 2016; Lázár et al 2021), and all these drugs induce intestinal dysbiosis in animals or humans (Zádori et al. 2023). However, the in vitro MICs of NSAIDs found for the tested strains are well above therapeutic plasma concentrations achieved in humans for these compounds. Thus, it is difficult to directly conclude that the antibacterial properties of NSAIDs can induce significant intestinal dysbiosis in humans, unless we consider the long duration of use of these compounds and their pharmacokinetic properties. However, the extent of this phenomenon is still unknown (Maier et al. 2018).

Pharmacological studies on Lactobacillus’ viability after NSAIDs treatment depends on the substance used and the animal species tested. In rat studies, the relative abundance of Lactobacillus strains decreased after treatment with NSAIDs such as diclofenac, indomethacin, celecoxib, on the other hand, the results of other studies indicate no change in the abundance of lactobacilli (Zádori et al. 2023). At present, the reasons for this apparent discrepancy need to be clarified. The loss of lactobacilli likely requires more time, as it has been reported more often after prolonged exposure to the drug (Zádori et al. 2023). In human studies, beneficial effects on L. casei (Endo et al. 2011) and Lactobacillus gasseri (LG) OLL2716 (Suzuki et al. 2017) have been reported in reducing aspirin-induced small intestine injuries. In addition, studies on small intestine damage induced by indomethacin have demonstrated a cytoprotective effect of L. casei (Watanabe et al. 2009). In order to reduce the dysbiosis caused by NSAIDs, introducing the preparations containing probiotic Lactobacillus strains to the therapy is very beneficial. Our in vitro studies showed that it should not cause disturbances in the bacteria viability.

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 Lactobacillaceae family in the human digestive tract. Moreover, taking the probiotic preparations with lactobacilli strains during NSAID therapy to prevent intestinal injury should not affect the viability of these strains, so that they can be taken simultaneously.