Curr Microbiol (2011) 62:10281033 DOI 10.

1007/s00284-010-9819-7

Effect of Different Antibiotics and Non-Steroidal Anti-Inammatory Drugs on the Growth of Lactobacillus casei Shirota
Alade Jimenez-Serna Humberto Hernandez-Sanchez

Received: 21 June 2010 / Accepted: 5 November 2010 / Published online: 21 November 2010 Springer Science+Business Media, LLC 2010

Abstract The purpose of this study was to investigate whether some non-steroidal anti-inammatory drugs (NSAIDs) could cause inhibition of the growth of Lactobacillus casei Shirota (LcS) or whether this microorganism is able to use some of them as the sole carbon source, considering that the simultaneous consumption of NSAIDs and a dairy drink fermented with LcS could help to prevent the appearance or improve the healing of gastric ulcers. Acetylsalicylic acid (ASA), sodium acetylsalicylate (SAS), acetaminophen, sodium naproxen, and sodium ibuprofen were added as the sole carbon source to a basal medium and tested for biodegradation by LcS. The same NSAIDs were added in different concentrations to disks and plated on MRS Agar to test the possible inhibitory effect of these compounds on LcS. Also, the resistance of LcS to 12 different antibiotics was studied on MRS agar. None of the NSAIDs tested could be used by LcS as the sole carbon source at the assayed concentrations. In the case of the disk diffusion method, sodium naproxen showed inhibition zones for the 500-lg disks and sodium ibuprofen was inhibitory for the 250- and 500-lg disks. However, when the macrobroth dilution method was used, the growth of LcS was inhibited by ASA, SAS, acetaminophen, and sodium ibuprofen. This strain showed resistance to the antibiotics sulfamethoxazole with trimethoprim, peoxacin, and gentamicin. This is the rst study on the effect of NSAIDs on probiotic bacteria. The results of the biodegradation test indicate that the simultaneous consumption of

NSAIDs and a dairy beverage with LcS is not likely to change the bioavailability of the drugs.

Introduction Probiotics have been widely used as food components all over the world. The word probiotic derives from the Greek, meaning for life. The Food and Agriculture Organization (FAO) of the United Nations and the World Health Organization (WHO) have stated that there is adequate scientic evidence to indicate that the so-called probiotic foods do provide health benets and that specic studied and proved strains are safe for human use. A panel of experts from FAO and WHO dened probiotics as Live microorganisms which when administered in adequate amounts confer a health benet on the host [21]. Lactic acid bacteria (LAB) are probably the most important probiotic microorganisms associated with the human gastrointestinal tract [10]. The genus Lactobacillus is one of the most important LAB isolated from fermented milks and from the intestinal tract of humans and other animals. Lactobacillus casei Shirota (LcS) is a strain isolated from the human intestine by the late Minoru Shirota at Kyoto University and is resistant to gastric and bile acids; therefore, the bacteria are able to reach the small intestine alive after oral ingestion. The following effects of LcS have been documented on experimental animals: antitumor activity, increase of host immune cells, inhibitory effect on immunoglobulin E production, protection against infection and antihypertensive activity after oral administration [15]. Other strains of Lb. casei have been reported to reduce CD8? T cell-mediated skin inammation when orally administered [6]; however, non-prescription non-steroidal anti-inammatory drugs (NSAIDs) are

A. Jimenez-Serna H. Hernandez-Sanchez (&) Departamento de Graduados e Investigacion en Alimentos, Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional, Carpio y Plan de Ayala, CP. 11340 Mexico, D.F., Mexico e-mail: hhernan1955@yahoo.com

123

 A. Jimenez-Serna, H. Hernandez-Sanchez: Effect of NSAIDs on Lb. casei

1029

usually the pain and inammation medication of choice for most people due in part to their relatively quick action [26]. NSAIDs are a miscellaneous group of compounds with the capacity to reduce or eliminate erythema, swelling, fever and pain caused by different stimuli [23]. The main action of this kind of drugs is the inhibition of cyclooxygenase (COX) enzymes. There are two classes of enzymes: COX-1 which is thought to be expressed constitutively and COX-2 which is induced during inammation. Non-selective COX inhibition with agents, such as aspirin, acetaminophen, ibuprofen, indomethacin, and naproxen, which inhibit both enzymes, provides effective pain relief for inammatory situations, but carries with it a risk for erosive gastritis and gastrointestinal bleeding [2]. Inammatory responses generated by activation of the lipopolysaccharide (LPS)/Toll-like receptor (TLR) 4 signaling pathway are fundamental in NSAID-induced gastrointestinal pathologies [28]. As mentioned before, probiotics have been used for the prevention and treatment of various gastrointestinal disorders including the enhancing of gastric ulcer healing by means of probiotic Lb. rhamnosus GG in rats. This effect is thought to be the result of attenuation of cell apoptosis to cell proliferation ratio and increase in angiogenesis [14]. There is also a report on the prevention of indomethacin-induced small intestine injury in rats by means of LcS [28]. In this case, a 1-week treatment with viable LcS showed a preventive effect on indomethacininduced enteropathy by suppressing the LPS/TLR4 signaling pathway and this probiotic effect of LcS could be produced by L-lactic acid. A similar clinical trial was performed in humans in a double-blind, cross-over study in which 20 healthy volunteers ingested a daily dose of a probiotic mixture of Bidobacterium and Lactobacillus strains or a placebo and 50 mg/day of indomethacin for 21 days. The treatment with probiotics decreased the indomethacin-induced intestinal inammation [16]. These studies indicate that probiotic administration could help in the treatment of gastrointestinal ulceration associated with NSAIDs use. With this possibility in mind and considering that the simultaneous consumption of NSAIDs and a dairy drink fermented with LcS could help to prevent the appearance of some of the ulceration process and intestinal injury symptoms, the effects of some NSAIDs on the in vitro growth of LcS were investigated to determine whether the drugs can cause inhibition of the microorganism or whether LcS is able to use some of the NSAIDs as the sole carbon source. Probiotics have also been used for the prevention of antibiotic-associated diarrhea [12]. During antibiotic therapy, taking probiotics as well maintains the intestinal biota in adequate balance [3]. However, if the probiotic organisms are sensitive to the prescribed antibiotic, the benet would be questionable; therefore, a resistance test is advisable before recommending this option.

Materials and Methods Bacterial Strain Pure culture of the probiotic bacteria LcS was isolated from Yakult (Yakult Mexico, Mexico City, DF, Mexico). The microorganism was cultured at 37C for 24 h in anaerobic jars (BBL GasPak Anaerobic System, Cockeysville, USA) in Lactobacilli MRS broth (Acumedia Manufactures, Inc., Lansing, USA). Cells were preserved frozen at -70C in 50% glycerol until used. Biochemical Tests They were used to conrm that the microorganism isolated from Yakult was Lb. casei. These tests included the fermentation of galactose, glucose, fructose, mannitol, cellobiose, maltose, lactose, trehalose, rafnose, arabinose, and xylose as described by Cai et al. [5]. Antibiotic Resistance Test Study of the resistance of LcS to different antibiotics (tetracycline, ampicillin, erythromycin, cephalothin, cefuroxime, cefotaxime, penicillin, dicloxacillin, ceftazidime, sulfamethoxazole with trimethoprim, peoxacin, and gentamicin) was done by means of Multidiscs (Bio-Rad, Mexico City, Mexico) against Gram (?) microorganisms on MRS agar. NSAIDs Acetylsalicylic acid (ASA) and acetaminophen were purchased from Central de Drogas SA de CV (Naucalpan, Mexico). Sodium naproxen and sodium ibuprofen were purchased from Sigma (Sigma-Aldrich, St. Louis, USA). Sodium acetylsalicylate (SAS) was prepared by the stoichiometric neutralization of ASA with sodium hydroxide. Use of NSAIDs as the Sole Carbon Source Each NSAID was added as sole carbon source in basal medium according to the methodology of Su et al. [25]. The basal medium specic for Lb. casei was prepared according to the report of Morishita et al. [17], and each NSAID was added as the carbon source instead of glucose. The medium was sterilized by ltration. The concentrations used for each NSAID were the following (mg ml-1): ASA 1 and 2, SAS 10 and 20, acetaminophen 2 and 5, sodium naproxen 0.5 and 1, and sodium ibuprofen 0.5 and 1. The concentrations were selected based on the maximal solubility of each drug. Basal medium without a carbon source was used as negative control and with the addition of 1%

123

1030

A. Jimenez-Serna, H. Hernandez-Sanchez: Effect of NSAIDs on Lb. casei

glucose was used as a positive control. Growth was followed as the change in absorbance was at 550 nm and the inoculum was added to give an initial absorbance of 0.15 in all cases, which is equivalent to a concentration of 6 9 107 cfu ml-1. Measurement of absorbance has been shown to be an adequate method to evaluate the use of different carbon sources by Lb. casei in basal media [25]. MRS Agar Disk Diffusion Method The possible antibacterial activity of each NSAID was evaluated by this method according to the indications of Wanger [27]. Bacterial suspensions with a concentration of 106 cfu ml-1 were prepared and plated using double layer of MRS agar [19]. Subsequently, 5-mm disks containing 50, 100, 250, and 500 lg sodium ibuprofen; 50, 100, 250, and 500 lg sodium naproxen; 250, 500, 1000, and 2000 lg acetaminophen; 500, 1000, 1500, and 3000 lg ASA; and 500, 1000, 1500 and 3000 lg SAS were applied. Plates were incubated under anaerobic conditions for 48 h at 37C followed by measuring the diameters of the inhibition zones, including the diameter of the disk (mm). The concentrations were selected based on the maximal solubility of SAS, sodium naproxen, and sodium ibuprofen in water, ASA in ethanol, and acetaminophen in methanol. Disks with the same amount of solvent were simultaneously applied to the plates as controls. MRS Macrobroth Dilution Method This method is an alternative to the agar disk diffusion method which could provide additional information on the possible inhibitory effect of NSAIDs. The diffusion method could have limitations such as the possible alteration of the physiologic status of the cell due to internal accumulation of NSAIDs and the poor diffusion of these compounds in solid media. The assay was performed in 10-ml test tubes containing MRS broth and different concentrations of the NSAIDs. Two test tubes containing the broth without the NSAIDs were included in each test, one was inoculated with LcS (positive control) and the other was left uninoculated (negative control) as a check for media sterility. The minimal inhibitory concentration (MIC), dened as the lowest concentration of the antimicrobial compound that will inhibit the growth of the microorganism detected by a lack of turbidity at 590 nm matching the negative control after 24 h of incubation at 37C, was evaluated for each NSAID [20]. Statistical Analyses All the experiments were performed in triplicate, and the results were expressed as the average standard

deviation. Statistical differences among groups were determined by One-way ANOVA using MS-Excel 2002.

Results The pattern of carbohydrate fermentation of the strain isolated from Yakult was identical to that of Lb. casei and to that observed for most strains of Lb. casei [29]. The strain was able to ferment galactose, glucose, fructose, sucrose, mannitol, mannose, cellobiose, maltose, lactose, and trehalose, and as expected, it was not able to utilize rafnose, arabinose, and xylose [5]. Based on these results, it is concluded that the strain isolated from Yakult is indeed Lb. casei. The growth expressed as absorbance at 550 nm of LcS in a basal medium with the different NSAIDs as carbon sources compared with the growth in a basal medium with no carbon source (blank) and in a basal medium with glucose as the sole carbon source (control) can be observed in Fig. 1. No signicant differences (P [ 0.05) could be detected by One-way ANOVA among the curves (with the exception of the control medium), indicating that LcS cannot use the NSAIDs as carbon sources since all the growth curves were similar to blank. Maximal absorbance values in the range 0.440.51 (equivalent to 1.4 and 3 9 108 cfu ml-1) were obtained in all cases after 6 h of incubation. After this period, the cultures reached the stationary phase and the absorbance started to decrease. The ANOVA results also indicate that under these conditions, the NSAIDs at the concentrations used are not inhibitory to the growth of LcS since the curves are similar to the blank curve. These concentrations are equivalent to the ingest of 267 mg of aspirin or 668 mg of acetaminophen or 134 mg of sodium naproxen or ibuprofen simultaneously with an 80-ml bottle of a dairy fermented beverage containing 1 9 108 cfu ml-1 of LcS. The assay was limited by the low water solubility of the drugs. The adequate growth to a maximal absorbance of 0.95 (equivalent to 7.8 9 108 cfu ml-1) after 9 h in the control medium is indicative of the capability of the basal medium with the original carbon source (glucose) to support the development of LcS. When the disk diffusion method was used, sodium naproxen showed inhibition zones with diameters of 15 2 mm (Fig. 2a) for the highest concentration (500 lg) disks. Sodium ibuprofen was inhibitory only at the higher concentration disks (250 and 500 lg) showing halos with diameters of 10 1 and 13 2 mm, respectively (Figs. 2b, c). In the case of acetaminophen, ASA and SAS, they were not inhibitory for LcS even at the highest tested concentrations (2,000 lg/disk for acetaminophen and 3,000 lg/disk for ASA and SAS) used in the experiment. It is worth noticing that these concentrations were

123

 A. Jimenez-Serna, H. Hernandez-Sanchez: Effect of NSAIDs on Lb. casei Fig. 1 Growth expressed as absorbance at 550 nm of LcS in a basal medium with different NSAIDs as carbon sources compared with the growth in a basal medium with no carbon source (negative control) and with 1% glucose (positive control)
1 0,9 0,8 0,7

1031

As (550 nm)

0,6 0,5 0,4 0,3 0,2 0,1 0 0 1 2 3 4 5 6 7 8 9 10

Time (h)
Negative control (no carbon source) Acetylsalicylic acid 0.2% Sodium naproxen 0.1% Positive control (Glucose 1%) Acetaminophen 0.5% Sodium acetylsalicylate 2% Sodium ibuprofen 0.1%

Fig. 2 Inhibition zones in MRS Agar for Lactobacillus casei Shirota in the presence of a sodium naproxen (500 lg/disk), b sodium ibuprofen (250 lg/disk), and c sodium ibuprofen (500 lg/disk). The four disks in the periphery contain the NSAID, and the disk in the center is the negative control without the drug

four and six times higher than the inhibitory concentrations for sodium naproxen and sodium ibuprofen. In the case of the macrobroth dilution method, MIC values of 400 lg ml-1 for sodium ibuprofen and of 4,000 lg ml-1 for ASA and acetaminophen were measured. In the case of SAS and sodium naproxen, a decrease in absorbance was detected (see Fig. 4), though a complete inhibition could not be observed to be able to calculate an MIC value. The results of the antibiotic resistance test are shown in Fig. 3. LcS proved to be a strain resistant to sulfamethoxazole with trimethoprim, peoxacin, and gentamicin, and sensitive to tetracycline, ampicillin, erythromycin, cephalothin, cefuroxime, cefotaxime, penicillin, dicloxacillin, and ceftazidime.
Fig. 3 Inhibition zones in MRS Agar for Lactobacillus casei Shirota in the presence of the antibiotics tetracycline (TE), ampicillin (AM), erythromycin (E), cephalothin (CF), cefuroxime (CXM), cephotaxime (CTX), penicillin (PE), dicloxacillin (DC), ceftazidime (CAZ), sulfamethoxazole with trimethoprim (SXT), peoxacin (PEF), and gentamicin (GE)

Discussion The NSAIDs are classied in several chemical groups with a broad range of pharmacokinetic characteristics; however,

123

1032

A. Jimenez-Serna, H. Hernandez-Sanchez: Effect of NSAIDs on Lb. casei

2,5 2

they have some general properties in common. Almost all of the NSAIDs are weak organic acids and most of these drugs are well absorbed, and food does not considerably change their bioavailability [13]. This last statement could not necessarily be true in the case of fermented foods, since they contain live microorganisms, which could somehow interact with the NSAIDs. Most biodegradation studies on pharmaceutical compounds have been done in relation with water treatment, since the occurrence of this kind of compounds in the sewage has received considerable attention recently [4]. Biolm processes have not been successful in the biologic degradation of naproxen; however, trickling through sand resulted in the complete biodegradation of ibuprofen and naproxen [11]. The utilization of ASA as the sole carbon source has been achieved only by the use of very specic microorganisms like Acinetobacter lwofi [8], and acetaminophen degradation has been attained by some strains of Penicillium [9]. These studies indicate that these compounds are not easy to biodegrade; therefore, the negative results in the experiment with the basal medium were not unusual. However, this kind of studies had to be done anyway since some LAB have been reported to degrade some phenolic compounds [22], though not as complex as the NSAIDs. It is then possible to conclude that the consumption of NSAIDs simultaneous with a dairy fermented product with LcS does not affect the bioavailability of the drugs. The MRS Agar disk diffusion method revealed that ASA, SAS and acetaminophen do not have an inhibitory effect on LcS. ASA is an effective anti-inammatory drug, though it may be more effective as an analgesic used for mild to moderate pain of different origins. At the normal dosage, ASAs main adverse effects are gastric distress and gastric and duodenal ulcers which include bleeding [13]. The simultaneous ingest of LcS with ASA or SAS could prevent the appearance or enhance the healing of gastric ulcers. Acetaminophen (paracetamol) is one of the most important non-prescription drugs used for the treatment of mild to moderate pain when an anti-inammatory effect is not necessary [13]. Gastrointestinal bleeding does not occur after its ingestion; therefore, the concurrent use of LcS is not as relevant as in the previous case. Sodium naproxen showed inhibition halos with an average diameter of 15 mm for the highest concentration (500 lg) disks, and sodium ibuprofen was inhibitory only at the higher concentration disks (250 and 500 lg) showing halos with average diameters of 10 and 13 mm, respectively. If these values were compared with the ones reported for some antibiotics for Gram-positive microorganisms, LcS could be classied as a bacterium with intermediate sensitivity to sodium naproxen and low sensitivity to sodium ibuprofen [1].

As (590 nm)

1,5 1 0,5 0 0 5
10 15 20 25

30

Time (h)
Acetylsalicyclic acid (4 mg/ml) Acetaminophen (4 mg/ml) Sodium naproxen (0.4 mg/ml) Sodium acetylsalicylate (9 mg/ml) Sodium ibuprofen (0.4 mg/ml) Positive control (2% glucose)

Fig. 4 Growth expressed as absorbance at 590 nm of LcS in MRS broth in the presence of different NSAIDs compared with the growth in the same broth with 2% glucose (positive control)

The macrobroth dilution method corroborated that LcS is a sensible strain to sodium ibuprofen; however, sodium naproxen showed only a very weak inhibition at a concentration of 0.4 mg ml-1. A possible explanation may involve the presence of an unspecic multidrug efux pump in LcS. ATP-binding cassette (ABC) transporters have been previously described in Lactococcus lactis [18] and Lb. brevis [24]. ABC transporters couple ATP hydrolysis to the uptake and efux of amphiphilic compounds, including antibiotics and aromatic compounds, across the cell membrane [7]. This process decreases the available energy for biomass production, which may lead to an increased glucose consumption rate in an effort to compensate. This in turn may make the culture produce lactic acid faster and reach growth-limiting glucose concentrations earlier than without naproxen. Under these conditions, the transporter will not be able to pump out naproxen effectively, and the drug which under acid conditions is in its protonated neutral form will become membrane permeable and inhibition will occur in about 6 h (Fig. 4). On the other hand, in agar medium, glucose may become limited even before 6 h due to the fact that nutrient diffusion may become an additional growth-limiting factor and after a few generations growth stops around the naproxen disk. The inhibitory effect of ASA, SAS, and acetaminophen (not detectable in the disk method) could be observed due to the higher concentrations attainable in MRS broth, in which diffusion problems are also negligible. It can be concluded that sodium ibuprofen is the strongest growth inhibitor of all the NSAIDs tested against LcS since it has the smallest MIC value. In the case of ibuprofen, gastrointestinal irritation and bleeding occur as adverse effects, though less frequently than with aspirin. The incidence of upper gastrointestinal bleeding in over the counter (OTC) naproxen employ is low but still doubles that of OTC ibuprofen, perhaps due to

123

 A. Jimenez-Serna, H. Hernandez-Sanchez: Effect of NSAIDs on Lb. casei

1033

a dose effect [13]. Again, the concomitant use of LcS could be recommended but the effect in ulcer prevention or healing could be diminished due to the growth inhibitory effect of the NSAIDs with the possible exception of sodium naproxen which showed just a slight inhibitory effect on LcS in the macrobroth dilution method. It would be advisable then to drink the LcS preparation at least 60 min before the NSAID ingest to avoid this inhibitory effect. The use of probiotics during and immediately after antibiotic treatment usually helps minimize the imbalance in the ecosystem of the intestinal biota, resulting in diarrhea, due to this treatment [3]. In the case of LcS, good results should be obtained in patients treated with sulfamethoxazole with trimethoprim, peoxacin, and gentamicin, since the probiotic is resistant to them.
Acknowledgments This research was supported by Instituto Polit ecnico Nacional (Grants SIP-20070658 and SIP-20080955) and Yakult Mexico (Grant Minoru Shirota 2006).

References
1. Barry AI, Thornsberry C (1985) Susceptibility tests: diffusion test procedures. In: Lennette E (ed) Manual of clinical microbiology, 4th edn. American Society for Microbiology, Washington, pp 978987 2. Bennett JS, Daugherty A, Herrington D, Greenland P, Roberts H, Taubert KA (2005) The use of nonsteroidal anti-inammatory drugs (NSAIDs): a science advisory from the American Heart Association. Circulation 111:17131716 3. Biradar S, Bahagvati S, Shegunshi B (2005) Probiotics and antibiotics: a brief overview. Internet J Nutr Wellness 2(1):17 4. Boyd GR, Zhang S, Grimm DA (2005) Naproxen removal from water by chlorination and biolm processes. Water Res 39:668676 5. Cai H, Rodrguez BT, Zhang W, Broadbent JR, Steele JL (2007) Genotypic and phenotypic characterization of Lactobacillus casei strains isolated from different ecological niches suggests frequent recombination and niche specicity. Microbiology 153:2655 2665 6. Chapat L, Chemin K, Dubois B, Bourdet-Sicard R, Kaiserlian D (2004) Lactobacillus casei reduces CD8? T cell mediated skin inammation. Eur J Immunol 34:25202528 7. Davidson AL, Chen J (2004) ATP-binding cassette transporters in bacteria. Annu Rev Biochem 73:241268 8. Grant DJW, De Szocs JC, Wilson JV (1970) Utilisation of acetylsalicylic acid as sole carbon source and the induction of its enzymatic hydrolysis by an isolated strain of Acinetobacter lwofi. J Pharm Pharmacol 22:461463 9. Hart A, Orr DLJ (1974) Degradation of paracetamol by a Penicillium species. J Pharm Pharmacol 26(Suppl):7071 10. Holzapfel WH, Haberer P, Guisen R, Bjorkroth J, Schillinger U (2001) Taxonomy and important features of probiotic microorganisms in food and nutrition. Am J Clin Nutr 73(suppl):365S 373S 11. Hua J, An P, Winter J, Gallert C (2003) Elimination of COD, microorganisms and pharmaceuticals from sewage by trickling through sandy soil below leaking sewers. Water Res 37: 43954404

12. Johnston BC, Supina AL, Ospina M, Vohra S (2007) Probiotics for the prevention of pediatric antibiotic-associated diarrhea. Cochrane Db Syst Rev 2. Art. No: CD004827 13. Katzung BG (2003) Basic and clinical pharmacology, 9th edn. McGraw-Hill/Appleton & Lange, New York 14. Lam EKY, Yu L, Wong HPS, Wu WKK, Shin VY, Tai EKK, So WHL, Woo PCY, Cho CH (2007) Probiotic Lactobacillus rhamnosus GG enhances gastric ulcer healing in rats. Eur J Pharmacol 565:171179 15. Matsuzaki T (2003) Health properties of milk fermented with Lactobacillus casei strain Shirota (LcS). In: Farnworth ER (ed) Handbook of fermented functional foods. CRC Press, Boca Raton, pp 145175 16. Montalto M, Gallo A, Curigliano V, DOnofrio F, Santoro L, Covino M, Dalvai S, Gasbarrini A, Gasbarrini G (2010) Clinical trial: the effects of a probiotic mixture on non-steroidal antiinammatory drug enteropathya randomized, double blind, cross-over, placebo-controlled study. Aliment Pharm Ther 32: 209214 17. Morishita T, Deguchi Y, Yajima M, Sakurai T, Yura T (1981) Multiple nutritional requirements of Lactobacilli: genetic lesions affecting amino acid biosynthetic pathways. J Bacteriol 148: 6471 18. Nikaido H, Zgurskaya HI (1999) Antibiotic efux mechanisms. Curr Opin Infect Dis 12:529536 19. Orowski A, Bielecka M (2006) Preliminary characteristics of Lactobacillus and Bidobacterium strains as probiotic candidates. Pol J Food Nutr Sci 56:269275 20. Qaiyumi S (2007) Macro- and microdilution methods of antimicrobial susceptibility testing. In: Schwalbe R, Steele-Moore L, Goodwin AC (eds) Antimicrobial susceptibility testing protocols. CRC Press, Boca Raton, pp 7579 21. Reid G, Jass J, Sebulsky MT, McCormick JK (2003) Potential uses of probiotics in clinical practice. Clin Microbiol Rev 16: 668672 22. Rodrguez H, Landete JM, de las Rivas B, Munoz R (2008) Metabolism of food phenolic acids by Lactobacillus plantarum CECT 748T. Food Chem 107:13931398 23. Rosenbloom D, Craven MA (1983) A review of non-steroidal anti-inammatory drugs. Can Fam Physician 29:21212124 24. Sakamoto K, Margolles A, van Veen HW, Konings WN (2001) Hop resistance in the beer spoilage bacterium Lactobacillus brevis is mediated by the ATP-binding cassette multidrug transporter HorA. J Bacteriol 183:53715375 25. Su P, Henrikson A, Mitchell H (2007) Selected prebiotics support the growth of probiotic mono-cultures in vitro. Anaerobe 13:134139 26. Vladislavovna S, Torres-Arreola LP, Reyes-Morales H (2006) Analgesicos antiinamatorios no esteroideos en la terapia del dolor. Rev Med Inst Mex Seguro Soc 44:565572 (in Spanish) 27. Wanger A (2007) Disk diffusion test and gradient methodologies. In: Schwalbe R, Steele-Moore L, Goodwin AC (eds) Antimicrobial susceptibility testing protocols. CRC Press, Boca Raton, pp 5373 28. Watanabe T, Nishio H, Tanigawa T, Yamagami H, Okazaki H, Watanabe K, Tominaga K, Fujiwara Y, Oshitani N, Asahara T, Nomoto K, Higuchi K, Takeuchi K, Arakawa T (2009) Probiotic Lactobacillus casei strain Shirota prevents indomethacin-induced small intestinal injury: involvement of lactic acid. Am J Physiol Gastrointest Liver Physiol 297:G506G513 29. Yakult Central Institute for Microbiological Research (1999) Lactobacillus casei strain Shirota. Yakult Honsha Co., Ltd, Tokyo

123