Evaluation of antimicrobial activity of Lactobacillus species associated with dentures

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1 235 Evaluation of antimicrobial activity of Lactobacillus species associated with dentures Bharathi Prakash, Malathi Shekar, Indrani Karunasagar UNESCO-MIRCEN Center for Marine Biotechnology, Department of Fishery Microbiology, College of Fisheries, Karnataka Veterinary, Animal and Fisheries Sciences University, Mangalore, Karnataka, , India; ABSTRACT Lactobacilli are the most common Gram positive bacilli found associated with dentures. Diet especially dairy products are the major source of oral lactobacilli. Various species of oral lactobacilli are known to produce antimicrobial substances like bacteriocins and lantibiotics whose action on microbes of different genera has been reported but their inhibitory activity on other members of the Lactobacillus species associated with oral environment is sparse. In this study we isolated Lactobacillus species from denture samples and evaluated their inhibitory activity against other oral lactobacilli isolates by cross streak method. Out of the seven Lactobacillus species isolated, casei and plantarum were more prevalent than other species. plantarum presented a broad spectrum of antimicrobial activity by inhibiting all the other species of Lactobacillus. casei inhibited plantarum, salivarius and acidophilus. salivarius had limited activity and showed moderate sensitivity of fermentum and acidophilus. delbruckii was sensitive to all lactobacilli tested except plantarum. fermentum activity was very restricted in that it was inhibitory only to acidophilus and that was observed as growth thinning along the streak. Results demonstrate the susceptibility of fermentum to all lactobacilli. plantarum, casei and delbruckii had good inhibitory activity against salivarius while minimal inhibitory activity was shown by salivarius towards other lactobacilli. Keywords: Lactobacillus spp., denture, antimicrobial effect, bacteriocin INTRODUCTION Human oral flora is highly diverse with approximately 700 bacterial species, majority of which have not yet been cultured in vitro [1]. Insertion of prosthesis brings about changes in the oral environment resulting in easy colonization of oral microbes on these inert and non-shading surfaces. Dentures (artificial teeth set) accumulate plaque, stain and calculus in a way similar to natural teeth [2]. Though Lactobacillus species comprise a small percentage of the normal oral flora of humans, they are found to be predominant denture colonizers [3]. Lactobacillus species are Gram positive bacilli that are catalase negative and recognized for their ability to produce antimicrobial substances and bacteriocins [4]. Bacteriocins are antibacterial proteins. They are highly species specific and produced by strains of bacteria active against other strains of the same or related species [5, 6]. This phenomenon of microbial interference is well known as lactic antagonism [4]. Nisin, diplococcin, acidophilin, bulgaricin, lactacin and plantaricin are some of the important bacteriocins that have been identified and characterized [7]. The bactericidal activity of bacteriocins is attributable to the alteration in the permeability of the cell membrane [8]. Further, the inhibitory activity of these substances is confined to Gram-positive bacteria [9]. Oral lactobacilli with antimicrobial spectrum has been reported earlier and most of them are found to be active against streptococci, staphylococci and micrococci, but not against Candida [10,11]. plantarum, paracasei, salivarius and rhamnosus with high antimicrobial activity of their bacteriocins has Research Article, Biotechnol. Bioinf. Bioeng. 2011, 1(2): Society for Applied Biotechnology. Printed in India.

2 236 been earlier reported [12]. However, studies on inhibitory activity of lactobacilli isolated from dentures are lacking. Hence this study was undertaken to evaluate antimicrobial effect of Lactobacillus species isolated from the dentures to see if there is an effect of dental material such as that is used for denture on the biological activity. MATERIALS AND METHODS The subjects who visited the Prosthodontic Department of KVG Dental College, Sullia, Dakshina Kannada district, Karnataka and local dental clinics were included in this study. A total of fifty healthy subjects (28 men and 22 women) in the age group of 35 to 80 years, wearing complete removable dentures for minimum one year who were not on any immunosuppressive, antibiotics and antifungal drugs for at least a year were included in the study. Palatal mucosa was examined for any signs of denture induced stomatitis and oral lesions. Sample collection The samples were collected by wiping the tissue facing surface of the upper (maxillary) dentures using a sterile swab and processed within an hour. Each swab was immersed in an aliquot (4.5 ml) of physiological saline and vortexed to disperse the adhering bacteria to the swab. A loopful of this suspension was streaked on the Rogosa agar (HiMedia, Mumbai) and incubated for 48 h at 37 C. Based on the morphology, colony characteristics and a battery of biochemical tests, the phenotypic identification of Lactobacillus species was confirmed according to Hadadji et al. [13]. The list of tests carried out to identify and speciate is given in the table 1. Antimicrobial activity Methods such as agar well diffusion [14], cross streak [15], well dilution [16] and colony count assay [17] have been used for the detection of bacteriocin of which cross streak method has been found be an acceptable technique to study the inhibition by bacteriocin [18]. Seven representative Lactobacillus species isolated from the denture samples were used as both producer and indicator species in the antimicrobial assay. The Lactobacillus isolates were grown in MRS broth (HiMedia, Mumbai), centrifuged at 14,000g for 15 min and supernatant discarded. Cells were rinsed twice with sterile 0.85% NaCl and were adjusted to ~ 10 8 cfu/ml, using Mc Farland No.0.5 as a standard solution [19]. All producer and indicator species were prepared as above. Cross streak method with slight modification was used to evaluate the antibacterial assay [15, 16]. A loopful of each Lactobacillus species was streaked on a fresh Rogosa agar plate as a strip for half a centimeter wide from end to end at the center to serve as producer species. Plates were allowed to dry and incubated for 48 h at 37 C. After incubation, growth was scraped using a sterile glass slide and plates were fumigated with chloroform for 30 min. The indicator Lactobacillus species were cross streaked perpendicular to the streak of producer species on either side one below the other. The plates were further incubated for 48 h at 37 C. RESULTS AND DISCUSSION Seven species of lactobacilli that included plantarum, casei, salivarius, delbruckii, fermentum, brevis and acidophilus were isolated from the denture samples (data on distribution of species not shown). Each of the seven species was studied as a producer and also served as an indicator species. The antimicrobial activity of each producer species was evaluated. The inhibition

3 237 zones produced by each of the producer species against all their indicator species were measured and graded as sensitive, moderately sensitive and resistant. The results of the inhibition patterns produced by Lactobacillus species are shown in table 2. Out of the seven Lactobacillus species, plantarum was found to have broad spectrum of inhibitory activity against all the species isolated in this study. casei showed moderate sensitivity of plantarum, salivarius and acidophilus. salivarius showed inhibition of fermentum and acidophilus with the activity being weaker in the case of plantarum, casei, delbruckii and brevis. delbruckii showed moderate sensitivity of other species but hardly any inhibition of plantarum was observed. fermentum was resistant to all the Lactobacillus species except acidophilus in which growth thinning was observed. brevis showed good inhibition of all the lactobacilli with the exception of salivarius which was least sensitive. acidophilus showed minimal inhibition of plantarum and delbruckii but good inhibition of other Lactobacillus species tested. Table 1. Biochemical tests for the identification of Lactobacillus isolates. Biochemical Tests plantarum casei salivarius delbruckii fermentum brevis acidophilus Growth at 15 C + + _ + _ Growth at 45 C _ + Gas from glucose + _ + + _ Acid production from the following carbohydrate fermentation Arabinose + _ + + _ Galactose Lactose Maltose Mannitol Melezitose + + _ Melibiose + _ + _ Raffinose + _ + _ Salicin Sorbitol Trehalose _ + Aesculin hydrolysis _ Nitrate reduction _ Arginine hydrolysis _ _ The Lactobacillus species isolated from dentures showed noticeable antimicrobial activity to other lactobacilli. Their inhibitory activity is probably due to the combination of factors including acids, hydrogen peroxide, antibiotics, diacetyl, and bacteriocins in addition to ph depression and nutrient depletion. They exert their lethal activity through adsorption to specific receptors located on

4 238 the external surface of sensitive bacteria, followed by killing of such bacteria [4]. The inhibitory activity of Lactobacillus species against microbes of different genera is well documented. bulgaricus was found to inhibit pathogens such as streptococcal species and actinomycetes, while rhamnosus strains have been reported to have distinct inhibitory activity against oral Porphyromonas gigivalis and Fusobacterium nucleatum [20]. The food pathogen Listeria monocytogence was inhibited significantly by a potent broad spectrum bacteriocin produced by salivarius UCC118, in mice compared to its stable mutant [21]. The antibacterial effect of plantarum on casei has been reported [22]. Our results are in agreement with the previous studies in that similar inhibitory activity by plantarum and acidophilus was observed. acidophilus occupies an important position in controlling undesirable microflora in the intestinal tracts of humans and animals [23]. Studies by other workers and results of our lactobacilli isolates revealed that antimicrobial activity of our isolates could be due to bacteriocin. Table 2. Inhibition patterns produced by denture Lactobacillus species. Producer Species plantarum ND casei 24 ND (S) 10 (R) 10 (R) 21 salivarius 11 (S) 13 (S) ND 11 (S) (S) 24 delbruckii 13 (S) ND fermentum 9 (R) 10 (R) 8 (R) 10 (R) ND 8 (R) 10 (R) brevis (S) ND 24 acidophilus 13 (S) (S) ND Degree of inhibition - zones ranging 8 to 10 mm = Resistant (R), 11 to 13 mm = Sensitive (S), 19 to 25 mm = moderately sensitive ; ND: Not Done. In this study, plantarum, delbruckii, brevis and acidophilus are the potential bacteriocin producers as they have shown broad spectrum of antimicrobial activity compared to casei and salivarius. fermentum did not show any inhibition of other lactobacilli indicating that no bacteriocin was produced. The prevalence of casei and plantarum was higher than other species in the denture samples which could be attributed to the elimination of other competitive flora within the oral environment due to their antimicrobial activity. Further studies are required to fully understand the antimicrobial substances produced by these denture lactobacilli and other contributing factors towards their antimicrobial activity in the oral niche. REFERENCES plantarum casei Indicator species with inhibition zones (mm) salivarius delbruckii fermentum brevis acidophilus [1] Aas JA, Bruce JP, Lauren NS, et al. J. Clin. Microbiol. 2005, 43: [2] Coulthwaite L, Verran J. Br. J. Biomed. Sci. 2007, 64: [3] Marsh PD. Adv. Dent. Res. 1994, 8: [4] James MJ, Martin JL, David AG. Modern Food Microbiology, 7th ed., Springer, New York, 2005, [5] Gaur Y, Narayan K, Chauhan S, et al. Ind. J. Microbiol. 2004, 44:1-30. [6] Daw MA, Falkiner RF. Micron 1996, 27:

5 [7] Nettles CG, Barefoot SF. J. Food Protect. 1993, 56: [8] Jagadeeswari S, Vidya P, MukeshKumar DJ, et al. E- J. Environ. Agric. Food.Chem. 2010, 9: [9] Klaenhammer TR, FEMS Microbiol. Rev. 1993, 12: [10] Topisirovic L, Kojic M, Fira D, et al. Int. J. Food Microbiol. 2006, 112: [11] Gilliland SE, Speck M J. Food Protect. 1977, 40: [12] Cotter PD, Hill C, Ross RP. Nat. Rev. Microbiol 2005, 3: [13] Hadadji M, Benama R, Saidi N, et al. Afr. J. Biotechnol. 2005, 4: [14] Ho SL Won YK. J. Korean Med. Sci. 2009, 24:S57-S62. [15] Madigan MT, Martiko JM, Parker J, Madigan MT., Brock Biology of Microorganisms., (8 th ed) Prentice-Hall International Inc, New Jersey, 1997, [16] Spanggaard B, Huber I, Nielsen J, et al. Env. Microbiol. 2001, 3: [17] Godiosa O, Consignado MD, Adrian C, et al. J. Microbiol. Infect. Dis. 1993, 22: [18] Lertcanawanichakul M, Sawangnop S. Walailak J. Sci. Tech. 2008, 5: [19] Kivanc M. Die Nahrung 1990, 34: [20] Iva S, Kari K, Meurman JH. Int. J. Probiotics Prebiotics 2007, 2: [21] Sinead CC, Yin L, Christian U. Proc. Natl. Acad. Sci. 2007, 104: [22] Todorov SD, Dicks LMT. Process Biochem. 2006, 41: [23] Barefoot SF, Klaenhammer TR. Appl. Environ. Microbiol. 1983, 45:

Research Article. The effects of hyaluronic acid on the morphological physiological differentiation of Lactobacillus

Research Article. The effects of hyaluronic acid on the morphological physiological differentiation of Lactobacillus Available online www.jocpr.com Journal of Chemical and Pharmaceutical Research, 2016, 8(7):368-372 Research Article ISSN : 0975-7384 CODEN(USA) : JCPRC5 The effects of hyaluronic acid on the morphological