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Received Date : 31-Jan-2014 Revised Date : 13-Apr-2014 Accepted Date : 10-May-2014 Article type
: Original Article
Corresponding author mail id: [email protected]
In vitro evaluation of antimicrobial activity of Lactobacillus rhamnosus IMC 501®, Lactobacillus paracasei IMC 502® and SYNBIO® against pathogens
Maria Magdalena Comana,b*, Maria Cristina Verdenellib,c, Cinzia Cecchinib,c, Stefania Silvib,c, Carla Orpianesib,c, Nadiya Boykod, Alberto Crescib,c
a
School of Advanced Studies, University of Camerino, Camerino, Italy
b
c
Scuola di Bioscienze e Medicina Veterinaria, University of Camerino, Camerino, Italy
Synbiotec S.r.l., Spin-off of UNICAM, Camerino, Italy
d
Medical Faculty, Uzhhorod National University, Uzhhorod, Ukraine
Running headline: Lactobacillus antimicrobial asset versus pathogens
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jam.12544 This article is protected by copyright. All rights reserved.
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*Corresponding author Maria Magdalena Coman Tel: +39 0737402737 Fax: +39 0737402418 e-mail address: magda.coman @unicam.it Full postal address: School of Advanced Studies, c/o Scuola di Bioscienze e Medicina Veterinaria, Via Gentile III da Varano, Camerino, 62032, Italy Synbiotec S.r.l., Via D’Accorso, n.30, Camerino, 62032, Italy
Abstract Aims: Probiotic lactobacilli have a great potential to produce antimicrobial compounds that inhibit and control the microbial pathogen growth. The antimicrobial and antifungal activities of two probiotic strains, Lactobacillus rhamnosus IMC 501® and Lactobacillus paracasei IMC 502®, and their 1:1 combination, named SYNBIO® were studied using four different methods. Methods and Results: Using two modified streak methods and a well diffusion method, the inhibitory activity of the probiotics and their metabolites towards six Gram+, nine Grampathogenic bacterial strains and eight Candida strains, was tested. Antagonistic effect of probiotic Lactobacillus strains was also investigated by co-culturing assay highlighting a significant inhibition of most of the pathogens tested in this study. The combination SYNBIO® showed a microbicidal activity against most of the strains tested in the study. Conclusions: Compared to the control, most of the pathogenic bacteria and yeast were inhibited by all probiotic strains tested to various degrees. Significance and Impact of the Study: Screening Lactobacillus strains according to their activity in various environmental conditions could precede the clinical efficacy studies for
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adjunct treatment with probiotics in cure of different gastrointestinal and vaginal tract infections.
Keywords Probiotics, Lactobacillus rhamnosus IMC 501®, Lactobacillus paracasei IMC 502®, antimicrobial activity, modified streak methods, co-culture method
Introduction The diversity of microbiota species residing in the gastrointestinal tract is dependent upon the host’s age, diet and health status. It was suggested that the intestinal mucosa may play a central role in host-microbiota-pathogen interactions (Thirabunyanon, 2011). The interactions between bacteria and the human host involve several microbiota that occupy varied environments and represent a continuum from symbiosis and commensalism to pathogenesis. Among the variable microbial components of the human gut microbiota health-promoting, mucosa-adherent species are included. Probiotics are “live microorganisms, which when administered in adequate amounts confer a health benefit on the host” (FAO/WHO, 2002). Although not all probiotic cultures confer identical benefits to the hosts, the potential mechanisms of action include competitive exclusion, maintenance of barrier function, metabolic and antimicrobial effects, enhancement of a balanced microbial flora, modulation of signal transduction, and immunomodulation of innate or adaptive immunity (Sherman et al., 2009; Travers et al., 2011). Specific roles and benefits of probiotics in the gastrointestinal tract include protection against infection, lowered cold and influenza-like symptoms in children, lowering of blood cholesterol levels, and suppression of allergic asthma. Despite strong evidence for the functional claims of probiotics, poor characterization of the specific molecular mechanisms by which these probiotic microbes elicit health benefits underlies the
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skepticism of the validity of these findings in the biomedical community (Baugher and Klaenhammer, 2011). Infectious diseases are the biggest problem in human being and every year gastrointestinal infections are responsible for significant morbidity and mortality worldwide. World Health Organization (WHO, 2004) estimates there to be more than four billion episodes of diarrhoeal disease annually, while there were 2.2 million deaths attributable to enteric infection, making it the fifth leading cause of death at all ages worldwide. Enteric bacteria include Salmonella species, Shigella species, Proteus species, Klebsiella species, Escherichia coli, Pseudomonas species, Vibrio cholerae and Staphylococcus aureus which are major etiologic agents of enteric infection (Tambekar and Bhutada, 2010).
Consumption of non-pathogenic bacterial species, such as probiotics, can contribute to barrier function by decreasing paracellular permeability, providing innate defence against pathogens and enhancing the physical impediment of the mucous layer, which may help protection against infections, prevention of chronic inflammation, and maintenance of mucosal integrity (Ohland and MacNaughton, 2010). Probiotic cultures have long been considered to exert protective effects against pathogens via direct antagonism or competitive exclusion. The rise in antibiotic resistant bacteria has awakened the scientific community to the prophylactic and therapeutic uses of probiotics, and to reconsider them as alternatives to antibiotics (Tambekar and Bhutada, 2010). These organisms can function as microbial barriers against gastrointestinal pathogens through competitive exclusion of pathogen binding, modulation of the host’s immune system and production of inhibitory compounds such as organic acid (e.g. lactic acid and acetic acid), oxygen catabolites (e.g. hydrogen peroxide), proteinaceous compounds (e.g. bacteriocins), fat and amino acid metabolites and other compounds (e.g. reuterin) (Marianelli et al., 2010).
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Previous studies have clearly shown that the production of antibacterial organic acid molecule(s) by Lactobacillus strains is strain-specific and that specific probiotic strains are active against selected enteric pathogens. The antimicrobial activity of lactobacilli depends on multiple factors: metabolic characteristics such as their mode of sugar fermentation (homofermentative or heterofermentative), as well as environmental growth conditions such as oxygen availability (aerobic, microaerobic and anaerobic conditions) (Annuk et al., 2003; Hu¨tt et al., 2006).
The selection of probiotics is frequently based on the ability to adhere to the gastrointestinal mucosa and competitive exclusion of pathogens. Adhesion to and colonization of the mucosal surfaces are possible protective mechanisms against pathogens through competition for binding sites and nutrients or immune modulation (Ouwehand and Salminen, 2003). The protective role of probiotic bacteria against gastrointestinal pathogens and the underlying mechanisms have received special attention. Pathogen inhibition by probiotic may provide significant protection against pathogen infection via a natural barrier against pathogen exposure in the gastrointestinal tract and this would enhance human health (Collado et al., 2007).
There are some evidences that lactic acid bacteria have an anti-oxidative potential (Martarelli et al., 2011). This property could be helpful in allowing lactobacilli to colonize the intestines, as well as in the course of inflammation to protect the intestinal mucosa against excessive oxidative stress (Hu¨tt et al., 2006). In addition to compete with pathogens for niches and nutrients, “competitively excluding” disease causing microbes from the host, certain probiotic bacteria have also been shown to produce potent antimicrobial peptides (bacteriocins) which specifically target the invading pathogen (Sleator, 2010). Bacteriocins have specific inhibitory
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activity against Gram-positive bacteria, whereas organic acids are more effective against Gram-negative pathogens (Marianelli et al., 2010). There are several methods used to detect antimicrobial activity of probiotic strains. Generally, tests for antagonism are performed on solid media and involve the detection of growth inhibition of an indicator strain caused by the test culture. Methods, that will be used in this study, to demonstrate the antagonistic potential are generally referred to simultaneous/direct antagonism procedures based on diffusion of inhibitory substances in agar medium (PolakBerecka et al., 2009). The first aim of this study was to evaluate the antagonistic properties of the two probiotic strains Lactobacillus rhamnosus IMC 501® and Lactobacillus paracasei IMC 502®, and also their 1:1 combination, named SYNBIO® against several pathogenic bacteria: 6 Gram-positive bacterial strains, 9 Gram-negative bacterial strains and 8 yeast strains. The contemporaneous use of four different assays (modified cross streak method, radial inhibition method, well diffusion method and co-culture method) has allowed to identify the most reliable technique for the detection of antimicrobial activity of probiotics against several pathogen strains.
Materials and methods Bacterial strains and culture conditions The probiotic strains subject of this study, Lact. rhamnosus IMC 501® and Lact. paracasei IMC 502®, and their combination, named SYNBIO®, were provided by Synbiotec S.r.l. (Camerino, Italy) (Verdenelli et al., 2011). Standard pathogenic bacteria were purchased from ATCC and DSM, while the clinically isolated bacterial strains were provided by IMV NASU (Institute of Microbiology and Virology, National Academy of Science of Ukraine, Kiev, Ukraine) and Candida strains by
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ISS (Istituto Superiore di Sanità, Rome, Italy). The Listeria strains were isolated from foods by IZS (Istituto Zooprofilattico Sperimentale, Umbria-Marche, Italy) (Table 1). Strains were maintained at -80°C in 15% (w/w) glycerol. De Man Rogosa and Sharpe (MRS) broth (Oxoid Ltd., Basingstoke, Hampshire, UK) for lactobacilli, Triptic Soy Broth (TSB, Oxoid) for pathogens and Sabouraud Dextrose broth (SAB, Oxoid) for Candida strains were inoculated from the stock culture collection and incubated for 24-48 h at 37°C under aerobic conditions.
Assessment of the antagonistic activity In order to choose the best methodology for detection of antimicrobial activity of Lactobacillus strains the following methods were examined. Modified cross-streak method Antimicrobial activity of the selected strains was tested against pathogenic strains using a modified “deferred cross-streak” technique (Fang et al., 1996). Briefly, MRS agar plates were streaked with the probiotic strain tested (106 CFU ml-1) in the centre of the plate covering a 1cm×2cm area and then incubated anaerobically at 37°C until grown to confluence. After incubation, the probiotic growth was outlined and then removed. Each plate was incubated again over chloroform for 1 h to inactivate any remaining cells and air dried for 45 min. The plate was then spread with 100 µl of potential pathogen tested at 107 CFU ml-1 and incubated at 37°C for 24 h. The inhibition activity of the probiotic strains was evaluated measuring the zone of inhibition around probiotic growth (Verdenelli et al., 2009). Radial streak method MRS agar plates were prepared and inoculated with 0.5 McFarland (1.5×108 CFU ml-1) of each probiotic bacterial suspension by covering a circle area in the centre of the Petri dish.
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After 48 h of incubation at 37°C the plates were seeded with pathogen indicator strains (0.5McF) by radial lines of inoculum from the border to the centre of the plate (Fig. 1). The microbial interactions were analyzed after 24 h of incubation at 37°C by the observation of the inhibition zone size. The growth inhibitory activity (GI) was calculated subtracting the circle diameter (CD, cm) of the Lactobacillus spreading zone from the inhibition zone diameter observed (IZD, cm) as follows GI = (IZD-CD)/2 (Bosch et al., 2012). Agar well diffusion method Potential mechanisms involved in the inhibition of pathogenic bacterial growth were investigated by a well diffusion assay. This set of experiments was run in order to test if the inhibitory effect of the culture supernatants was exclusively due to its acidic pH or whether other mechanisms were involved. In order to prepare Cell Free Supernatant (CFS), each probiotic Lactobacillus strains was cultivated in MRS broth for 24 h at 37°C. CFS was obtained by centrifuging the culture at 12000g for 20 minutes and sterilised by filtration using 0.20 µm porous membranes (Sigma, St. Louis, United States). Inhibitory activity of CFS of probiotic lactobacilli was investigated by well diffusion method (Mami et al., 2008). MRS agar plates were inoculated with an overnight culture of the indicator pathogen strains in the stationary phase (107-108 CFU ml-1). Wells of 10 mm in diameter were cut into agar plates with a sterile metal cylinder and 100 μl of each CFS was placed into each well. The plates were incubated for 24 h at 37°C and antimicrobial activity recorded as growth-free inhibition zones around the wells. Inhibition zones were measured in mm from the edge of the wells. Liquid co-culture assay The capability of Lact. rhamnosus IMC 501®, Lact. paracasei IMC 502® and SYNBIO® to interfere with the growth of several pathogens was evaluated also by inoculating both kind of strains, probiotic and pathogen, simultaneously before incubation. The co-culture method was
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performed to rank the antagonistic potential of the probiotics when grown with the target pathogen concurrently. The Lactobacillus strains and the pathogen strains were grown on MRS broth and TSB, respectively (or SAB broth for Candida strains). The co-culture experiments was performed in a modified MRS medium, mMRS, (MRS broth and TSB/SAB broth 1:1) capable of sustaining the growth of both types of microorganisms, probiotics and pathogens. A 2 ml aliquot of mMRS was inoculated with 100 µl of Lactobacillus suspension and pathogen strains (108 CFU ml-1) and incubated at 37°C in aerobic conditions. Positive controls were prepared by inoculating the same medium either with the Lactobacillus strain or with the pathogen one. To check whether the pathogens were inhibited or killed, 50 µl of coculture suspension was seeded on specific agar medium and incubated at 37°C for 24-48 h. Comparing the growth with a negative (-) and a positive control (++++), presence of growth of pathogenic bacteria on the agar plate was interpreted as an inhibitory activity (+++, ++ or + corresponding to 25, 50 or 75% inhibition), while no growth (-) was interpreted as microbicidal activity (100% inhibition).
Statistical analysis All experiments were carried out in triplicate, and each sample was analysed in triplicate. The results were expressed as mean ± standard deviation (SD). For each experiment and where applicable a negative and a positive control were prepared.
Results Antimicrobial activity on agar plates by modified cross-streak method Table 2 shows that Lact. rhamnosus IMC 501® has inhibitory activity against both Gram+ and Gram- bacteria. The major effect was registered against Staph. aureus ATCC 25923 and E. coli DSM 1103 and also against the two C. albicans strains (ATCC 10261 and ISS7). Lact.
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paracasei IMC 502® demonstrates an inhibition effect on the most Gram+ and Grambacteria, especially towards Staph. aureus ATCC 25923 and Pr. mirabilis IMV4. Moreover all the Candida strains are strongly inhibited, except C. glabrata and C. tropicalis. Some examples of the inhibitory potential of the two probiotic strains against Candida strains are showed in Fig. 2. The combination SYNBIO® gave a bactericidal and fungicidal activity against most of the strains used in the study, in particular C. albicans ATCC 10261, ISS1, ISS2, ISS7 and C. krusei ISS4 showed a strong inhibition. In general, Lact. paracasei IMC 502® showed a higher activity towards all the pathogens tested, especially towards Candida strains and the same strong inhibition was also registered for SYNBIO®.
Antimicrobial activity on agar plates by radial streak method The results of radial streak method showed that the two probiotic strains and their 1:1 combination exhibit a significant inhibition of Gram+ and Gram- pathogen strains. Considering both Gram+ and Gram- bacteria, the highest growth inhibitory activity (GI) was observed in the case of Lact. paracasei IMC 502® against B. cereus after 48 h preincubation of the Lactobacillus under aerobic conditions (24.08 ± 3.85 mm). After 48 h preincubation of Lact. rhamnosus IMC 501®, the lowest activity against Ent. faecium DSM 13590 was detected (12.05 ± 1.27 mm). All the other strains mostly displayed intermediate inhibitory activity towards target bacteria (Fig. 3). There was no inhibition activity against four Grambacteria (E. coli EPEC IMV1, Kl. oxytoca IMV2, Ps. aeruginosa IMV5 and Sh. sonnei IMV6). It is remarkable the high inhibitory activity of Lact. paracasei IMC 502® against Ps. aeruginosa and E. coli from culture collection and any activity on the clinically isolated strains.
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The same inhibitory trend of Lact. paracasei IMC 502® was also registered for SYNBIO®. All the Candida strains growth was inhibited by the probiotics used in this study (Fig. 3 and 4). It is remarkable to note (Fig. 3) the highest inhibitory activity of SYNBIO® against C. krusei ISS4 respect to the single strains.
Antimicrobial activity by agar well diffusion method Antimicrobial effect of cell free supernatant (CFS) of probiotic lactobacilli by well diffusion method is shown in Table 3. The diameter of inhibition zones varied among the lactobacilli strains; however, differences were not significant. A wide inhibition zone of Ent. faecium DSM 13590 and Kl. pneumoniae IMV3 was obtained by Lact. rhamnosus IMC 501®, Lact. paracasei IMC 502® and SYNBIO® revealing a strong activity against these two pathogen strains. No inhibition was detected for Candida strains (data not shown). Listeria gray 309, Staph. aureus ATCC 25923, Ps. aeruginosa ATCC 27853 and clinical isolated, and Pr. mirabilis IMV4 are resistant to all the three cell free supernatants.
Antimicrobial activity by in liquid co-culture assay The growth inhibition values of pathogens caused by testing probiotics in liquid media were estimated, being ranked as expressing high (100%), intermediate (50%), and no inhibitory effectiveness (0%), looking at the growth on agar plates of the co-culture after the 24 h incubation in broth. The effect of co-culturing probiotic bacteria with pathogenic bacteria (Gram+ and Gram- bacteria, and also Candida strains) in mixed MRS/TSB media is shown in Fig. 5. Compared to the control (growth without any probiotic bacteria co-cultured), most of the pathogenic bacteria and Candida strains were inhibited by all probiotic strains tested to various degrees. An example of growth checking is indicated in the Fig. 6.
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In co-culture growth, a considerable activity (>50%) of the tested probiotic strains was registered only against Gram+ bacterial strains (B. cereus DSM 345, Staph. aureus ATCC 25923 and L. grey 309). Regarding the Gram- pathogens, the Lactobacillus strains and their combination, SYNBIO® give a high antagonistic activity against all the pathogens, with a percentage of antagonistic effectiveness between 75% and 100%. E. coli DSM 1103 and Ps. aeruginosa clinical isolated was also inhibit, but the effectiveness was much lower. The inhibitory activity of probiotic bacteria against Candida strains was less of 50% in some cases and absent in other cases, in particular against C. albicans ISS1, ISS2, ISS7 and C. tropicalis ISS6.
Discussion Lactobacilli are known for their production of various antimicrobial compounds (Ouwehand and Vesterlund, 2004; Pangallo et al., 2008 Bilkova et al., 2011). In vitro experimental studies have demonstrated that selected lactic acid strains are effective against diarrhoeagenic bacteria. By producing metabolites such as acetic and lactic acids, and thus lowering the pH, a large number of Lactobacillus strains inhibit the growth of bacterial pathogens (Servin and Coconnier, 2003). The antagonistic activity of probiotic Lactobacillus strains depends on the environmental growth conditions, e.g. aerobic/anaerobic conditions (Annuk et al., 2003). However, the comparisons between solid and liquid media in specific conditions help to rank different lactobacilli according to their antagonistic activity versus potential pathogens. The present study aimed to determine whether the two probiotic strains Lact. rhamnosus IMC 501® and Lact. paracasei IMC 502® and their 1:1 combination SYNBIO® were able to exert an antagonistic effect against a range of pathogenic bacteria assessed by different methods. The pathogens strains (Gram-positive and Gram-negative bacteria and yeasts) were
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purposefully selected to represent the target area of the study, which included the microbial inhibition
of
enteropathogens,
food-borne
pathogens,
spoilage
microorganisms
(contaminants) and also clinical isolated pathogens. Nowadays, there are not many studies that have tested the antipathogenic activity of probiotic strains against clinically isolated pathogen strains, pathogens with a high incidence to develop diseases in humans. The probiotic bacteria tested showed to possess varying degrees of inhibition towards pathogenic bacteria. Spore formers and Gram positive bacteria were affected more than Gram negative bacteria by the two Lactobacillus strains and their 1:1 mix, namely SYNBIO®. In fact, there is some evidence that lactobacilli are not efficient in the case of Gram- bacteria compared with Gram+ bacteria (Piard et al., 1990; Ravaei et al., 2013).
Anyway results are different with the different methods used, revealing different antimicrobial mechanisms. The presented results show that Lact. rhamnosus IMC 501®, Lact. paracasei IMC 502® and SYNBIO® can strongly inhibit the growth of potential pathogens. This attribute is advantageous when considering the use of these strains in food preservation as well as feed supplements or in alternative medicine. The agar diffusion method was used to demonstrate the antagonistic potential of the two Lactobacillus strains and SYNBIO® against at least one of the pathogen indicator strains used in this assays. The inhibition zone diameter produced by spot on the cross-streak and radial methods of assay was higher than the observed diameter in agar well diffusion and was, anyway, in line with the reports from other studies (Cadirci and Citak, 2010). According to Con and Gokalp (2000), this inhibitory effect was because of all or same metabolite such as lactic acid, acetic acid, diacetyl, bacteriocin, etc. which was produced during the assay period. The results indicated that both cross-streak methods were comparatively more efficient in the study of antimicrobial activity using live cells of probiotic strains, in comparison with the
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supernatant. This could be explained by a good diffusion of metabolites from Lactobacillus strains and thus this may probably be resulted in the growth inhibition of the pathogenic microorganisms. There was a significant inhibition of bacterial and fungal growth as depicted by the zones of inhibition. The antagonistic activity in liquid media is favoured by rapidly diffusing antimicrobial compounds, including organic acids and co-aggregation of different indigenous bacteria with Lactobacillus strains used in this study. Researchers have associated high antagonistic activity of lactobacilli with production of organic acids resulting in pH decrease (Ouwehand and Vesterlund, 2004).
Administration of preparations containing a well-characterized probiotic strain to humans could be used to prevent or cure both bacterial vaginitis and gastrointestinal disorder (Ndesendo et al., 2008). This approach may overcome problems relating to drug-resistant strains, chronic toxicity, as well as the loss of normal microbiota. Further investigations could contribute significantly to our understanding of the complex triangular relationship of probiotics, pathogens and gut/vaginal microbiota within the human gastrointestinal/vaginal tract. So, more elaborated tests (analysis) working with complex microbiota and environmental conditions similar to the gastrointestinal/vaginal tract are required to get a more accurate view of antimicrobial properties of probiotics. Moreover, the composition of the medium and culture conditions may indirectly affect the sensitivity of the indicator strain (Polak-Berecka et al., 2009). It may be stated that the size of inhibition zones depends not only on sensitivity of the target strain on antimicrobial compounds produced by Lactobacillus but also on the method used for detection.
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For this reason In conclusion, this study point out that it is not enough to test the antimicrobial activity of the probiotic strains by one method, but more than two simultaneous techniques must be applied in order to obtain reliable and significant results. In vitro screening of Lactobacillus strains according to their activity in various environmental conditions may be a valuable method that could precede clinical efficacy studies for adjunct treatment with probiotics in cure of different gastrointestinal and vaginal tract infections.
Acknowledgements The authors wish to thank Dr. Francesca Mondello and Mrs. Antonietta Girolamo from Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Rome, Italy for providing Candida strains.
Conflict of interest No conflict of interest declared.
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Bosch, M., Nart. J., Audivert, S., Bonachera, M.A., Alemany, A.S., Fuentes, M.C. and Cuñé, J. (2012) Isolation and characterization of probiotic strains for improving oral health. Archives of Oral Biology, 57(5), 539-549. Cadirci, B.H. and Citak, S. (2010) Antagonistic Effects of Some Lactobacilli On Some GramNegative Bacteria. GU J Sci, 23(2), 119-123. Collado, M.C., Meriluoto, J. and Salminen, S. (2007) Role of commercial probiotic strains against human pathogen adhesion to intestinal mucus. Letters in Applied Microbiology, 45, 454-460. Con, A.H. and Gokalp, H.Y. (2000) Production of bacteriocin-like metabolites by lactic acid cultures isolated from Sucuk samples. Meat Sci., 55, 89-96. Fang, W., Shi, M., Huang, L. and, Wang, Y. (1996) Antagonism of lactic acid bacteria towards Staphylococcus aureus and Escherichia coli on agar plates and in milk. Vet. Res. 27, 3-12. FAO/WHO Report of a Joint (2002) Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food London Ontario, Canada. Hu¨tt, P., Shchepetova, J., Loivukene, K., Kullisaar, T. and Mikelsaar, M. (2006) Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens. Journal of Applied Microbiology, 100, 1324-1332. Mami, A., Henni, J.E. and Kihal, M. (2008) Antimicrobial Activity of Lactobacillus species Isolated from Algerian Raw goat’s Milk against Staphylococcus aureus. World Journal of Dairy & Food Sciences, 3(2), 39-49. Marianelli, C., Cifani, N. and Pasquali, P. (2010) Evaluation of antimicrobial activity of probiotic bacteria against Salmonella enterica subsp. enterica serovar typhimurium 1344 in a common medium under different environmental conditions. Research in Microbiology, 161, 673-680.
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Martarelli, D., Verdenelli, M.C., Scuri, S., Cocchioni, M., Silvi, S., Cecchini, C. and Pompei, P. (2011) Effect of a probiotic intake on oxidant and antioxidant parameters in plasma of athletes during intense exercise training. Curr. Microbiol., 62(6), 1689-96. Ndesendo, V.M.K., Pillay, V., Choonara, Y.E., Buchmann, E., Bayever, D.N. and Meyer, L.C.R. (2008) A review of current intravaginal drug delivery approaches employed for the prophylaxis of HIV/AIDS and prevention of sexually transmitted infections. AAPS PharmSciTech, 9(2), 505-520. Ohland, C.L. and MacNaughton, W.K. (2010) Probiotic bacteria and intestinal epithelial barrier function. Am. J. Physiol. Gastrointest. Liver Physiol., 298, 807-819. Ouwehand, A.C. and Salminen, S. (2003) In vitro adhesion assays for probiotics and their in vivo relevance: a review. Microb Ecol Health Dis., 15, 175-184. Ouwehand, A.C. and Vesterlund, S. (2004) Antimicrobial components from lactic acid bacteria. In: Salminen S, Ouwehand A, von Wright A (eds.), Lactic Acid Bacteria: Microbial and Functional Aspects, 3rd ed. Marcel Dekker, New York., 375-395. Piard, J.C., Delome, F., Giraffa, G., Commissaire, J. and Desmazeaud, M.J. (1990) Evidence for a bacteriocin produced by Lactococcus lactis CNRZ 481. Neth. Milk. Dairy. J., 44, 143-158. Polak-Berecka, M., Waśko, A. and Koston, D. (2009) Comparison of different methods for detection of antimicrobial activity of probiotic strains of Lactobacillus rhamnosus against some food spoilage microorganisms. Annales UMCS, Biologia. 64(1), 15-24. Ravaei, A., Heshmati, Z., Salehi, T.Z., Tamai, I.A., Ghane, M. and Derakhshan, J. (2013) Evaluation of Antimicrobial Activity of Three Lactobacillus spp. against Antibiotic Resistance Salmonella typhimurium. Advanced Studies in Biology, 5(2), 61-70.
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Servin, A.L. and Coconnier, M.H. (2003) Adhesion of probiotic strains to the intestinal mucosa and interaction with pathogens. Best Pract Res Clin Gastroenterol. 17(5), 741754. Sherman, P.M., Ossa, J.C. and Johnson-Henry, K. (2009) Unraveling mechanisms of action of probiotics. Nutr Clin Pract, 24, 10-14. Sleator, R.D. (2010) Probiotic therapy - recruiting old friends to fight new foes. Gut Pathogens, 2, 1-5. Tambekar, D.H. and Bhutada, S.A. (2010) An Evaluation Of Probiotic Potential Of Lactobacillus Sp. From Milk Of Domestic Animals And Commercial Available Probiotic Preparations In Prevention Of Enteric Bacterial Infections. Recent Research In Science And Technology, 2(10), 82-88. Thirabunyanon, M. (2011) Biotherapy for and protection against gastrointestinal pathogenic infections via action of probiotic bacteria. Maejo International Journal of Science and Technology, 5, 108-128. Travers, M.A., Florent, I., Kohl, L. and Grellier, P. (2011) Probiotics for the Control of Parasites: An Overview. Journal of Parasitology Research, ID 610769. Verdenelli, M.C., Ghelfi, F., Silvi, S., Orpianesi, C., Cecchini, C. and Cresci, A. (2009) Probiotic properties of Lactobacillus rhamnosus and Lactobacillus paracasei isolated from human faeces. European Journal of Nutrition, 48, 355-363. Verdenelli, M.C., Silvi, S., Cecchini, C., Orpianesi, C. and Cresci, A. (2011) Influence of a combination of two potential probiotic strains, Lactobacillus rhamnosus IMC 501® and Lactobacillus paracasei IMC 502® on bowel habits of healthy adults. Letters of Applied Microbiology, 52, 596–602. WHO - The World Health Organization (WHO) website [http://www.who.int/healthinfo/ global_burden_disease/GBD_report_2004update_part2.pdf]
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Table 1. Pathogen strains used in the study. Pathogens Strain code Origin Gram positive Bacillus cereus DSM 345 culture collection Enterococcus faecium DSM 13590 culture collection Listeria gray 309 isolated from food Listeria monocytogenes 306 isolated from food Staphylococcus aureus ATCC 25923 culture collection Streptococcus mutans ATCC 20523 culture collection Gram negative Escherichia coli DSM 1103 culture collection Escherichia coli EPEC IMV1 clinical isolated Klebsiella oxytoca IMV2 clinical isolated Klebsiella pneumoniae IMV3 clinical isolated Proteus mirabilis IMV4 clinical isolated Pseudomonas aeruginosa ATCC 27853 culture collection Pseudomonas aeruginosa IMV5 clinical isolated Salmonella enterica DSM 14221 culture collection Shigella sonnei IMV6 clinical isolated Yeast Candida albicans ATCC 10261 ATCC 10261 culture collection Candida albicans ISS2 ISS2 clinical isolated Candida albicans ISS7 ISS7 clinical isolated Candida albicans resistant ISS1 ISS1 clinical isolated Candida glabrata ISS3 ISS3 clinical isolated Candida krusei ISS4 ISS4 clinical isolated Candida parapsilosis ISS5 ISS5 clinical isolated Candida tropicalis ISS6 ISS6 clinical isolated ATCC-American Type Culture Collection; DSM-German Collection of Microorganisms and Cell Cultures; IMV-Institute of Microbiology and Virology, Ukraine; ISS-Istituto Superiore di Sanità, Italy.
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Table 2. Degree of inhibition on tested potential human pathogens by Lact. rhamnosus IMC 501®, Lact. paracasei IMC 502® and SYNBIO®. Indicator (pathogen) strains
Inhibition of growth* by Lact. rhamnosus Lact. paracasei IMC 501® IMC 502® SYNBIO®
Gram positive Bacillus cereus DSM 345 ++ ++ Enterococcus faecium DSM 13590 + ++ + Listeria gray 309 ++ + Listeria monocytogenes 306 ++ ++ ++ Staphylococcus aureus ATCC 25923 +++ +++ +++ Streptococcus mutans ATCC 20523 ++ + ++ Gram negative Escherichia coli DSM 1103 +++ ++ ++ Escherichia coli EPEC IMV1 + ++ ++ Klebsiella oxytoca IMV2 + + Klebsiella pneumoniae IMV3 ++ + ++ Proteus mirabilis IMV4 ++ +++ ++ Pseudomonas aeruginosa ATCC 27853 ++ ++ ++ Pseudomonas aeruginosa IMV5 + ++ ++ Salmonella enterica DSM 14221 ++ + ++ Shigella sonnei IMV6 + + Yeasts Candida albicans ATCC 10261 +++ ++++ ++++ Candida albicans ISS2 ++++ ++++ Candida albicans ISS7 +++ ++++ ++++ Candida albicans resistant ISS1 ++++ ++++ Candida glabrata ISS3 Candida krusei ISS4 ++++ ++++ Candida parapsilosis ISS5 ++ +++ +++ Candida tropicalis ISS6 * – no inhibition, + zone of inhibition
Received Date : 31-Jan-2014 Revised Date : 13-Apr-2014 Accepted Date : 10-May-2014 Article type
: Original Article
Corresponding author mail id: [email protected]
In vitro evaluation of antimicrobial activity of Lactobacillus rhamnosus IMC 501®, Lactobacillus paracasei IMC 502® and SYNBIO® against pathogens
Maria Magdalena Comana,b*, Maria Cristina Verdenellib,c, Cinzia Cecchinib,c, Stefania Silvib,c, Carla Orpianesib,c, Nadiya Boykod, Alberto Crescib,c
a
School of Advanced Studies, University of Camerino, Camerino, Italy
b
c
Scuola di Bioscienze e Medicina Veterinaria, University of Camerino, Camerino, Italy
Synbiotec S.r.l., Spin-off of UNICAM, Camerino, Italy
d
Medical Faculty, Uzhhorod National University, Uzhhorod, Ukraine
Running headline: Lactobacillus antimicrobial asset versus pathogens
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*Corresponding author Maria Magdalena Coman Tel: +39 0737402737 Fax: +39 0737402418 e-mail address: magda.coman @unicam.it Full postal address: School of Advanced Studies, c/o Scuola di Bioscienze e Medicina Veterinaria, Via Gentile III da Varano, Camerino, 62032, Italy Synbiotec S.r.l., Via D’Accorso, n.30, Camerino, 62032, Italy
Abstract Aims: Probiotic lactobacilli have a great potential to produce antimicrobial compounds that inhibit and control the microbial pathogen growth. The antimicrobial and antifungal activities of two probiotic strains, Lactobacillus rhamnosus IMC 501® and Lactobacillus paracasei IMC 502®, and their 1:1 combination, named SYNBIO® were studied using four different methods. Methods and Results: Using two modified streak methods and a well diffusion method, the inhibitory activity of the probiotics and their metabolites towards six Gram+, nine Grampathogenic bacterial strains and eight Candida strains, was tested. Antagonistic effect of probiotic Lactobacillus strains was also investigated by co-culturing assay highlighting a significant inhibition of most of the pathogens tested in this study. The combination SYNBIO® showed a microbicidal activity against most of the strains tested in the study. Conclusions: Compared to the control, most of the pathogenic bacteria and yeast were inhibited by all probiotic strains tested to various degrees. Significance and Impact of the Study: Screening Lactobacillus strains according to their activity in various environmental conditions could precede the clinical efficacy studies for
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adjunct treatment with probiotics in cure of different gastrointestinal and vaginal tract infections.
Keywords Probiotics, Lactobacillus rhamnosus IMC 501®, Lactobacillus paracasei IMC 502®, antimicrobial activity, modified streak methods, co-culture method
Introduction The diversity of microbiota species residing in the gastrointestinal tract is dependent upon the host’s age, diet and health status. It was suggested that the intestinal mucosa may play a central role in host-microbiota-pathogen interactions (Thirabunyanon, 2011). The interactions between bacteria and the human host involve several microbiota that occupy varied environments and represent a continuum from symbiosis and commensalism to pathogenesis. Among the variable microbial components of the human gut microbiota health-promoting, mucosa-adherent species are included. Probiotics are “live microorganisms, which when administered in adequate amounts confer a health benefit on the host” (FAO/WHO, 2002). Although not all probiotic cultures confer identical benefits to the hosts, the potential mechanisms of action include competitive exclusion, maintenance of barrier function, metabolic and antimicrobial effects, enhancement of a balanced microbial flora, modulation of signal transduction, and immunomodulation of innate or adaptive immunity (Sherman et al., 2009; Travers et al., 2011). Specific roles and benefits of probiotics in the gastrointestinal tract include protection against infection, lowered cold and influenza-like symptoms in children, lowering of blood cholesterol levels, and suppression of allergic asthma. Despite strong evidence for the functional claims of probiotics, poor characterization of the specific molecular mechanisms by which these probiotic microbes elicit health benefits underlies the
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skepticism of the validity of these findings in the biomedical community (Baugher and Klaenhammer, 2011). Infectious diseases are the biggest problem in human being and every year gastrointestinal infections are responsible for significant morbidity and mortality worldwide. World Health Organization (WHO, 2004) estimates there to be more than four billion episodes of diarrhoeal disease annually, while there were 2.2 million deaths attributable to enteric infection, making it the fifth leading cause of death at all ages worldwide. Enteric bacteria include Salmonella species, Shigella species, Proteus species, Klebsiella species, Escherichia coli, Pseudomonas species, Vibrio cholerae and Staphylococcus aureus which are major etiologic agents of enteric infection (Tambekar and Bhutada, 2010).
Consumption of non-pathogenic bacterial species, such as probiotics, can contribute to barrier function by decreasing paracellular permeability, providing innate defence against pathogens and enhancing the physical impediment of the mucous layer, which may help protection against infections, prevention of chronic inflammation, and maintenance of mucosal integrity (Ohland and MacNaughton, 2010). Probiotic cultures have long been considered to exert protective effects against pathogens via direct antagonism or competitive exclusion. The rise in antibiotic resistant bacteria has awakened the scientific community to the prophylactic and therapeutic uses of probiotics, and to reconsider them as alternatives to antibiotics (Tambekar and Bhutada, 2010). These organisms can function as microbial barriers against gastrointestinal pathogens through competitive exclusion of pathogen binding, modulation of the host’s immune system and production of inhibitory compounds such as organic acid (e.g. lactic acid and acetic acid), oxygen catabolites (e.g. hydrogen peroxide), proteinaceous compounds (e.g. bacteriocins), fat and amino acid metabolites and other compounds (e.g. reuterin) (Marianelli et al., 2010).
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Previous studies have clearly shown that the production of antibacterial organic acid molecule(s) by Lactobacillus strains is strain-specific and that specific probiotic strains are active against selected enteric pathogens. The antimicrobial activity of lactobacilli depends on multiple factors: metabolic characteristics such as their mode of sugar fermentation (homofermentative or heterofermentative), as well as environmental growth conditions such as oxygen availability (aerobic, microaerobic and anaerobic conditions) (Annuk et al., 2003; Hu¨tt et al., 2006).
The selection of probiotics is frequently based on the ability to adhere to the gastrointestinal mucosa and competitive exclusion of pathogens. Adhesion to and colonization of the mucosal surfaces are possible protective mechanisms against pathogens through competition for binding sites and nutrients or immune modulation (Ouwehand and Salminen, 2003). The protective role of probiotic bacteria against gastrointestinal pathogens and the underlying mechanisms have received special attention. Pathogen inhibition by probiotic may provide significant protection against pathogen infection via a natural barrier against pathogen exposure in the gastrointestinal tract and this would enhance human health (Collado et al., 2007).
There are some evidences that lactic acid bacteria have an anti-oxidative potential (Martarelli et al., 2011). This property could be helpful in allowing lactobacilli to colonize the intestines, as well as in the course of inflammation to protect the intestinal mucosa against excessive oxidative stress (Hu¨tt et al., 2006). In addition to compete with pathogens for niches and nutrients, “competitively excluding” disease causing microbes from the host, certain probiotic bacteria have also been shown to produce potent antimicrobial peptides (bacteriocins) which specifically target the invading pathogen (Sleator, 2010). Bacteriocins have specific inhibitory
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activity against Gram-positive bacteria, whereas organic acids are more effective against Gram-negative pathogens (Marianelli et al., 2010). There are several methods used to detect antimicrobial activity of probiotic strains. Generally, tests for antagonism are performed on solid media and involve the detection of growth inhibition of an indicator strain caused by the test culture. Methods, that will be used in this study, to demonstrate the antagonistic potential are generally referred to simultaneous/direct antagonism procedures based on diffusion of inhibitory substances in agar medium (PolakBerecka et al., 2009). The first aim of this study was to evaluate the antagonistic properties of the two probiotic strains Lactobacillus rhamnosus IMC 501® and Lactobacillus paracasei IMC 502®, and also their 1:1 combination, named SYNBIO® against several pathogenic bacteria: 6 Gram-positive bacterial strains, 9 Gram-negative bacterial strains and 8 yeast strains. The contemporaneous use of four different assays (modified cross streak method, radial inhibition method, well diffusion method and co-culture method) has allowed to identify the most reliable technique for the detection of antimicrobial activity of probiotics against several pathogen strains.
Materials and methods Bacterial strains and culture conditions The probiotic strains subject of this study, Lact. rhamnosus IMC 501® and Lact. paracasei IMC 502®, and their combination, named SYNBIO®, were provided by Synbiotec S.r.l. (Camerino, Italy) (Verdenelli et al., 2011). Standard pathogenic bacteria were purchased from ATCC and DSM, while the clinically isolated bacterial strains were provided by IMV NASU (Institute of Microbiology and Virology, National Academy of Science of Ukraine, Kiev, Ukraine) and Candida strains by
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ISS (Istituto Superiore di Sanità, Rome, Italy). The Listeria strains were isolated from foods by IZS (Istituto Zooprofilattico Sperimentale, Umbria-Marche, Italy) (Table 1). Strains were maintained at -80°C in 15% (w/w) glycerol. De Man Rogosa and Sharpe (MRS) broth (Oxoid Ltd., Basingstoke, Hampshire, UK) for lactobacilli, Triptic Soy Broth (TSB, Oxoid) for pathogens and Sabouraud Dextrose broth (SAB, Oxoid) for Candida strains were inoculated from the stock culture collection and incubated for 24-48 h at 37°C under aerobic conditions.
Assessment of the antagonistic activity In order to choose the best methodology for detection of antimicrobial activity of Lactobacillus strains the following methods were examined. Modified cross-streak method Antimicrobial activity of the selected strains was tested against pathogenic strains using a modified “deferred cross-streak” technique (Fang et al., 1996). Briefly, MRS agar plates were streaked with the probiotic strain tested (106 CFU ml-1) in the centre of the plate covering a 1cm×2cm area and then incubated anaerobically at 37°C until grown to confluence. After incubation, the probiotic growth was outlined and then removed. Each plate was incubated again over chloroform for 1 h to inactivate any remaining cells and air dried for 45 min. The plate was then spread with 100 µl of potential pathogen tested at 107 CFU ml-1 and incubated at 37°C for 24 h. The inhibition activity of the probiotic strains was evaluated measuring the zone of inhibition around probiotic growth (Verdenelli et al., 2009). Radial streak method MRS agar plates were prepared and inoculated with 0.5 McFarland (1.5×108 CFU ml-1) of each probiotic bacterial suspension by covering a circle area in the centre of the Petri dish.
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After 48 h of incubation at 37°C the plates were seeded with pathogen indicator strains (0.5McF) by radial lines of inoculum from the border to the centre of the plate (Fig. 1). The microbial interactions were analyzed after 24 h of incubation at 37°C by the observation of the inhibition zone size. The growth inhibitory activity (GI) was calculated subtracting the circle diameter (CD, cm) of the Lactobacillus spreading zone from the inhibition zone diameter observed (IZD, cm) as follows GI = (IZD-CD)/2 (Bosch et al., 2012). Agar well diffusion method Potential mechanisms involved in the inhibition of pathogenic bacterial growth were investigated by a well diffusion assay. This set of experiments was run in order to test if the inhibitory effect of the culture supernatants was exclusively due to its acidic pH or whether other mechanisms were involved. In order to prepare Cell Free Supernatant (CFS), each probiotic Lactobacillus strains was cultivated in MRS broth for 24 h at 37°C. CFS was obtained by centrifuging the culture at 12000g for 20 minutes and sterilised by filtration using 0.20 µm porous membranes (Sigma, St. Louis, United States). Inhibitory activity of CFS of probiotic lactobacilli was investigated by well diffusion method (Mami et al., 2008). MRS agar plates were inoculated with an overnight culture of the indicator pathogen strains in the stationary phase (107-108 CFU ml-1). Wells of 10 mm in diameter were cut into agar plates with a sterile metal cylinder and 100 μl of each CFS was placed into each well. The plates were incubated for 24 h at 37°C and antimicrobial activity recorded as growth-free inhibition zones around the wells. Inhibition zones were measured in mm from the edge of the wells. Liquid co-culture assay The capability of Lact. rhamnosus IMC 501®, Lact. paracasei IMC 502® and SYNBIO® to interfere with the growth of several pathogens was evaluated also by inoculating both kind of strains, probiotic and pathogen, simultaneously before incubation. The co-culture method was
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performed to rank the antagonistic potential of the probiotics when grown with the target pathogen concurrently. The Lactobacillus strains and the pathogen strains were grown on MRS broth and TSB, respectively (or SAB broth for Candida strains). The co-culture experiments was performed in a modified MRS medium, mMRS, (MRS broth and TSB/SAB broth 1:1) capable of sustaining the growth of both types of microorganisms, probiotics and pathogens. A 2 ml aliquot of mMRS was inoculated with 100 µl of Lactobacillus suspension and pathogen strains (108 CFU ml-1) and incubated at 37°C in aerobic conditions. Positive controls were prepared by inoculating the same medium either with the Lactobacillus strain or with the pathogen one. To check whether the pathogens were inhibited or killed, 50 µl of coculture suspension was seeded on specific agar medium and incubated at 37°C for 24-48 h. Comparing the growth with a negative (-) and a positive control (++++), presence of growth of pathogenic bacteria on the agar plate was interpreted as an inhibitory activity (+++, ++ or + corresponding to 25, 50 or 75% inhibition), while no growth (-) was interpreted as microbicidal activity (100% inhibition).
Statistical analysis All experiments were carried out in triplicate, and each sample was analysed in triplicate. The results were expressed as mean ± standard deviation (SD). For each experiment and where applicable a negative and a positive control were prepared.
Results Antimicrobial activity on agar plates by modified cross-streak method Table 2 shows that Lact. rhamnosus IMC 501® has inhibitory activity against both Gram+ and Gram- bacteria. The major effect was registered against Staph. aureus ATCC 25923 and E. coli DSM 1103 and also against the two C. albicans strains (ATCC 10261 and ISS7). Lact.
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paracasei IMC 502® demonstrates an inhibition effect on the most Gram+ and Grambacteria, especially towards Staph. aureus ATCC 25923 and Pr. mirabilis IMV4. Moreover all the Candida strains are strongly inhibited, except C. glabrata and C. tropicalis. Some examples of the inhibitory potential of the two probiotic strains against Candida strains are showed in Fig. 2. The combination SYNBIO® gave a bactericidal and fungicidal activity against most of the strains used in the study, in particular C. albicans ATCC 10261, ISS1, ISS2, ISS7 and C. krusei ISS4 showed a strong inhibition. In general, Lact. paracasei IMC 502® showed a higher activity towards all the pathogens tested, especially towards Candida strains and the same strong inhibition was also registered for SYNBIO®.
Antimicrobial activity on agar plates by radial streak method The results of radial streak method showed that the two probiotic strains and their 1:1 combination exhibit a significant inhibition of Gram+ and Gram- pathogen strains. Considering both Gram+ and Gram- bacteria, the highest growth inhibitory activity (GI) was observed in the case of Lact. paracasei IMC 502® against B. cereus after 48 h preincubation of the Lactobacillus under aerobic conditions (24.08 ± 3.85 mm). After 48 h preincubation of Lact. rhamnosus IMC 501®, the lowest activity against Ent. faecium DSM 13590 was detected (12.05 ± 1.27 mm). All the other strains mostly displayed intermediate inhibitory activity towards target bacteria (Fig. 3). There was no inhibition activity against four Grambacteria (E. coli EPEC IMV1, Kl. oxytoca IMV2, Ps. aeruginosa IMV5 and Sh. sonnei IMV6). It is remarkable the high inhibitory activity of Lact. paracasei IMC 502® against Ps. aeruginosa and E. coli from culture collection and any activity on the clinically isolated strains.
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The same inhibitory trend of Lact. paracasei IMC 502® was also registered for SYNBIO®. All the Candida strains growth was inhibited by the probiotics used in this study (Fig. 3 and 4). It is remarkable to note (Fig. 3) the highest inhibitory activity of SYNBIO® against C. krusei ISS4 respect to the single strains.
Antimicrobial activity by agar well diffusion method Antimicrobial effect of cell free supernatant (CFS) of probiotic lactobacilli by well diffusion method is shown in Table 3. The diameter of inhibition zones varied among the lactobacilli strains; however, differences were not significant. A wide inhibition zone of Ent. faecium DSM 13590 and Kl. pneumoniae IMV3 was obtained by Lact. rhamnosus IMC 501®, Lact. paracasei IMC 502® and SYNBIO® revealing a strong activity against these two pathogen strains. No inhibition was detected for Candida strains (data not shown). Listeria gray 309, Staph. aureus ATCC 25923, Ps. aeruginosa ATCC 27853 and clinical isolated, and Pr. mirabilis IMV4 are resistant to all the three cell free supernatants.
Antimicrobial activity by in liquid co-culture assay The growth inhibition values of pathogens caused by testing probiotics in liquid media were estimated, being ranked as expressing high (100%), intermediate (50%), and no inhibitory effectiveness (0%), looking at the growth on agar plates of the co-culture after the 24 h incubation in broth. The effect of co-culturing probiotic bacteria with pathogenic bacteria (Gram+ and Gram- bacteria, and also Candida strains) in mixed MRS/TSB media is shown in Fig. 5. Compared to the control (growth without any probiotic bacteria co-cultured), most of the pathogenic bacteria and Candida strains were inhibited by all probiotic strains tested to various degrees. An example of growth checking is indicated in the Fig. 6.
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In co-culture growth, a considerable activity (>50%) of the tested probiotic strains was registered only against Gram+ bacterial strains (B. cereus DSM 345, Staph. aureus ATCC 25923 and L. grey 309). Regarding the Gram- pathogens, the Lactobacillus strains and their combination, SYNBIO® give a high antagonistic activity against all the pathogens, with a percentage of antagonistic effectiveness between 75% and 100%. E. coli DSM 1103 and Ps. aeruginosa clinical isolated was also inhibit, but the effectiveness was much lower. The inhibitory activity of probiotic bacteria against Candida strains was less of 50% in some cases and absent in other cases, in particular against C. albicans ISS1, ISS2, ISS7 and C. tropicalis ISS6.
Discussion Lactobacilli are known for their production of various antimicrobial compounds (Ouwehand and Vesterlund, 2004; Pangallo et al., 2008 Bilkova et al., 2011). In vitro experimental studies have demonstrated that selected lactic acid strains are effective against diarrhoeagenic bacteria. By producing metabolites such as acetic and lactic acids, and thus lowering the pH, a large number of Lactobacillus strains inhibit the growth of bacterial pathogens (Servin and Coconnier, 2003). The antagonistic activity of probiotic Lactobacillus strains depends on the environmental growth conditions, e.g. aerobic/anaerobic conditions (Annuk et al., 2003). However, the comparisons between solid and liquid media in specific conditions help to rank different lactobacilli according to their antagonistic activity versus potential pathogens. The present study aimed to determine whether the two probiotic strains Lact. rhamnosus IMC 501® and Lact. paracasei IMC 502® and their 1:1 combination SYNBIO® were able to exert an antagonistic effect against a range of pathogenic bacteria assessed by different methods. The pathogens strains (Gram-positive and Gram-negative bacteria and yeasts) were
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purposefully selected to represent the target area of the study, which included the microbial inhibition
of
enteropathogens,
food-borne
pathogens,
spoilage
microorganisms
(contaminants) and also clinical isolated pathogens. Nowadays, there are not many studies that have tested the antipathogenic activity of probiotic strains against clinically isolated pathogen strains, pathogens with a high incidence to develop diseases in humans. The probiotic bacteria tested showed to possess varying degrees of inhibition towards pathogenic bacteria. Spore formers and Gram positive bacteria were affected more than Gram negative bacteria by the two Lactobacillus strains and their 1:1 mix, namely SYNBIO®. In fact, there is some evidence that lactobacilli are not efficient in the case of Gram- bacteria compared with Gram+ bacteria (Piard et al., 1990; Ravaei et al., 2013).
Anyway results are different with the different methods used, revealing different antimicrobial mechanisms. The presented results show that Lact. rhamnosus IMC 501®, Lact. paracasei IMC 502® and SYNBIO® can strongly inhibit the growth of potential pathogens. This attribute is advantageous when considering the use of these strains in food preservation as well as feed supplements or in alternative medicine. The agar diffusion method was used to demonstrate the antagonistic potential of the two Lactobacillus strains and SYNBIO® against at least one of the pathogen indicator strains used in this assays. The inhibition zone diameter produced by spot on the cross-streak and radial methods of assay was higher than the observed diameter in agar well diffusion and was, anyway, in line with the reports from other studies (Cadirci and Citak, 2010). According to Con and Gokalp (2000), this inhibitory effect was because of all or same metabolite such as lactic acid, acetic acid, diacetyl, bacteriocin, etc. which was produced during the assay period. The results indicated that both cross-streak methods were comparatively more efficient in the study of antimicrobial activity using live cells of probiotic strains, in comparison with the
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supernatant. This could be explained by a good diffusion of metabolites from Lactobacillus strains and thus this may probably be resulted in the growth inhibition of the pathogenic microorganisms. There was a significant inhibition of bacterial and fungal growth as depicted by the zones of inhibition. The antagonistic activity in liquid media is favoured by rapidly diffusing antimicrobial compounds, including organic acids and co-aggregation of different indigenous bacteria with Lactobacillus strains used in this study. Researchers have associated high antagonistic activity of lactobacilli with production of organic acids resulting in pH decrease (Ouwehand and Vesterlund, 2004).
Administration of preparations containing a well-characterized probiotic strain to humans could be used to prevent or cure both bacterial vaginitis and gastrointestinal disorder (Ndesendo et al., 2008). This approach may overcome problems relating to drug-resistant strains, chronic toxicity, as well as the loss of normal microbiota. Further investigations could contribute significantly to our understanding of the complex triangular relationship of probiotics, pathogens and gut/vaginal microbiota within the human gastrointestinal/vaginal tract. So, more elaborated tests (analysis) working with complex microbiota and environmental conditions similar to the gastrointestinal/vaginal tract are required to get a more accurate view of antimicrobial properties of probiotics. Moreover, the composition of the medium and culture conditions may indirectly affect the sensitivity of the indicator strain (Polak-Berecka et al., 2009). It may be stated that the size of inhibition zones depends not only on sensitivity of the target strain on antimicrobial compounds produced by Lactobacillus but also on the method used for detection.
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For this reason In conclusion, this study point out that it is not enough to test the antimicrobial activity of the probiotic strains by one method, but more than two simultaneous techniques must be applied in order to obtain reliable and significant results. In vitro screening of Lactobacillus strains according to their activity in various environmental conditions may be a valuable method that could precede clinical efficacy studies for adjunct treatment with probiotics in cure of different gastrointestinal and vaginal tract infections.
Acknowledgements The authors wish to thank Dr. Francesca Mondello and Mrs. Antonietta Girolamo from Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Rome, Italy for providing Candida strains.
Conflict of interest No conflict of interest declared.
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Bosch, M., Nart. J., Audivert, S., Bonachera, M.A., Alemany, A.S., Fuentes, M.C. and Cuñé, J. (2012) Isolation and characterization of probiotic strains for improving oral health. Archives of Oral Biology, 57(5), 539-549. Cadirci, B.H. and Citak, S. (2010) Antagonistic Effects of Some Lactobacilli On Some GramNegative Bacteria. GU J Sci, 23(2), 119-123. Collado, M.C., Meriluoto, J. and Salminen, S. (2007) Role of commercial probiotic strains against human pathogen adhesion to intestinal mucus. Letters in Applied Microbiology, 45, 454-460. Con, A.H. and Gokalp, H.Y. (2000) Production of bacteriocin-like metabolites by lactic acid cultures isolated from Sucuk samples. Meat Sci., 55, 89-96. Fang, W., Shi, M., Huang, L. and, Wang, Y. (1996) Antagonism of lactic acid bacteria towards Staphylococcus aureus and Escherichia coli on agar plates and in milk. Vet. Res. 27, 3-12. FAO/WHO Report of a Joint (2002) Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food London Ontario, Canada. Hu¨tt, P., Shchepetova, J., Loivukene, K., Kullisaar, T. and Mikelsaar, M. (2006) Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens. Journal of Applied Microbiology, 100, 1324-1332. Mami, A., Henni, J.E. and Kihal, M. (2008) Antimicrobial Activity of Lactobacillus species Isolated from Algerian Raw goat’s Milk against Staphylococcus aureus. World Journal of Dairy & Food Sciences, 3(2), 39-49. Marianelli, C., Cifani, N. and Pasquali, P. (2010) Evaluation of antimicrobial activity of probiotic bacteria against Salmonella enterica subsp. enterica serovar typhimurium 1344 in a common medium under different environmental conditions. Research in Microbiology, 161, 673-680.
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Table 1. Pathogen strains used in the study. Pathogens Strain code Origin Gram positive Bacillus cereus DSM 345 culture collection Enterococcus faecium DSM 13590 culture collection Listeria gray 309 isolated from food Listeria monocytogenes 306 isolated from food Staphylococcus aureus ATCC 25923 culture collection Streptococcus mutans ATCC 20523 culture collection Gram negative Escherichia coli DSM 1103 culture collection Escherichia coli EPEC IMV1 clinical isolated Klebsiella oxytoca IMV2 clinical isolated Klebsiella pneumoniae IMV3 clinical isolated Proteus mirabilis IMV4 clinical isolated Pseudomonas aeruginosa ATCC 27853 culture collection Pseudomonas aeruginosa IMV5 clinical isolated Salmonella enterica DSM 14221 culture collection Shigella sonnei IMV6 clinical isolated Yeast Candida albicans ATCC 10261 ATCC 10261 culture collection Candida albicans ISS2 ISS2 clinical isolated Candida albicans ISS7 ISS7 clinical isolated Candida albicans resistant ISS1 ISS1 clinical isolated Candida glabrata ISS3 ISS3 clinical isolated Candida krusei ISS4 ISS4 clinical isolated Candida parapsilosis ISS5 ISS5 clinical isolated Candida tropicalis ISS6 ISS6 clinical isolated ATCC-American Type Culture Collection; DSM-German Collection of Microorganisms and Cell Cultures; IMV-Institute of Microbiology and Virology, Ukraine; ISS-Istituto Superiore di Sanità, Italy.
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Table 2. Degree of inhibition on tested potential human pathogens by Lact. rhamnosus IMC 501®, Lact. paracasei IMC 502® and SYNBIO®. Indicator (pathogen) strains
Inhibition of growth* by Lact. rhamnosus Lact. paracasei IMC 501® IMC 502® SYNBIO®
Gram positive Bacillus cereus DSM 345 ++ ++ Enterococcus faecium DSM 13590 + ++ + Listeria gray 309 ++ + Listeria monocytogenes 306 ++ ++ ++ Staphylococcus aureus ATCC 25923 +++ +++ +++ Streptococcus mutans ATCC 20523 ++ + ++ Gram negative Escherichia coli DSM 1103 +++ ++ ++ Escherichia coli EPEC IMV1 + ++ ++ Klebsiella oxytoca IMV2 + + Klebsiella pneumoniae IMV3 ++ + ++ Proteus mirabilis IMV4 ++ +++ ++ Pseudomonas aeruginosa ATCC 27853 ++ ++ ++ Pseudomonas aeruginosa IMV5 + ++ ++ Salmonella enterica DSM 14221 ++ + ++ Shigella sonnei IMV6 + + Yeasts Candida albicans ATCC 10261 +++ ++++ ++++ Candida albicans ISS2 ++++ ++++ Candida albicans ISS7 +++ ++++ ++++ Candida albicans resistant ISS1 ++++ ++++ Candida glabrata ISS3 Candida krusei ISS4 ++++ ++++ Candida parapsilosis ISS5 ++ +++ +++ Candida tropicalis ISS6 * – no inhibition, + zone of inhibition
