Barbosa, Joana

Teixeira, Paula

English

Repositório Institucional da Universidade Católica Portuguesa

Preparation and inoculation of lettuce Inhibition of Clostridioides difficile by lactic acid bacteria Joana Barbosa & Paula Teixeira  Universidade Católica Portuguesa, CBQF –  Centro de Biotecnologia e Química Fina – Laboratório Associado  Escola Superior de Biotecnologia, Rua Diogo Botelho, 1327, 4169-005, Porto, Portugal.  *Corresponding author: jbarbosa@porto.ucp.pt   Clostridioides difficile infections have been associated with the antibiotics use (altering the intestinal microbiota, enabling the proliferation and toxins segregation by C. difficile).[1,2] Moreover, the increase of antibiotic-resistance of hypervirulent strains turns imperative finding alternative therapeutics with preventive and/or prophylactic effects, as the use of probiotics. Lactic acid bacteria are considered as a major group of probiotic bacteria and their ingestion has been pointed out to confer a range of health benefits.[3] There are several mechanisms by which probiotic strains may exert their inhibitory activity towards undesired enteropathogens: secretion of different metabolites as lactic acid, hydrogen peroxide, short-chain fatty acids, bacteriocins, proteases, etc.[4] Limited information is available on the ability of different probiotic bacteria used in food products to inhibit Clostridium difficile. overnight incubation at 11 C The percentage of lactic acid bacteria with anti-clostridial activity is presented in figures 1A and 1B.  - Of the 450 lactic acid bacteria, 15.1% (68) inhibited the isolate C. difficile H63866, 16% (72) C. difficile I805452, 17.1% (77) C. difficile I805937 and V315639 and 15.3% (69) C. difficile DSMZ1296, as a result of competition between cells (culture against culture). - When the cells were centrifuged and the untreated cell-free supernatant was used, 26 LAB maintained their anticlostridial activity. - Aft r treatment of cell-free supernatant (CFS) of the 26 selected LAB it was proposed that C. difficile was inhibited:  - by the action of lactic acid (12 isolates); - by the production of hydrogen peroxide (3 isolates);  - by the action of other nature compounds, other than bacteriocins since, their activity was not lost after the digestion with the proteolytic enzyme proteinase K (6 isolates); - by the presence of proteinaceous compounds, since their activity was lost after the digestion with the proteolytic enzyme proteinase K (5 isolates). This suggests that their inhibitory mechanism may be bacteriocinogenic.  [1] Bartlett JG (2006) Narrative review: the new epidemic of Clostridium difficile-associated enteric disease. Ann Intern Med 145: 758-764. [2] Gerding DN, Johnson S, Peterson LR, Mulligan ME, Silva J Jr. (1995) Clostridium difficile-associated diarrhea and colitis. Infect Control Hosp Epidemiol 16: 459-477. [3] Sathyabama S, Vijayabharathi R, Devi PB, Kumar MR, Priyadarisini VB (2012) Screening for probiotic properties of strains isolated from feces of various human groups. J Microbiol 50: 603-612. [4] Zhang YC, Zhang LW, Ma W, Yi HX, Yang X, Du M, Shan YJ, Han X, Zhang LL (2012) Screening of probiotic lactobacilli for inhibition of Shigella sonnei and the macromolecules involved in inhibition. Anaerobe 18: 498-503. References Acknowledgements This work was supported by funding from the National Funds from the Fundação para a Ciência e a Tecnologia (FCT) through project UID/Multi/50016/2019. Financial support for author J. Barbosa was provided by a post-doctoral fellowship SFRH/BPD/113303/2015 (FCT). Table 1. Inhibitory activity of cell-free supernatants and neutralized cell-free supernatants of lactic acid bacteria against C. difficile cultures Conclusion and Relevance In conclusion, we have identified food LAB isolates displaying a clear inhibitory activity against five C. difficile strains. Although further in vitro tests are still needed, such as the ability of selected LAB to inhibit the invasion of C. difficile into intestinal Caco-2 cells, these LAB isolates may be used as part of potential anti-C. difficile strategies as alternative to antibiotics or as preventive of C. difficile infections. The objective of this study was to test the anti-clostridial activity of 450 lactic acid bacteria (isolated from various food products) against 5 C. difficile strains. For those lactic acid bacteria demonstrating inhibitory activity, the mechanisms of inhibition was investigated. Introduction Objective overnight incubation at 11 ºC Anticlostridial activity of 450 lactic acid bacteria (LAB) isolated from various food products (fermented meat products, smoked salmon, cheese, seafood, etc) were tested against five C. difficile strains (four gently provided by Hospital de S. Marcos, Braga: C. difficile H63866, C. difficile I805452, C. difficile I805937, C. difficile V315638 and one from German collection of microorganisms and cell cultures: C. difficile DSMZ 1296). Each LAB was cultivated in de Man, Rogosa and Sharpe agar plates (MRS agar) at 30 ºC for 24h and, subsequently, one colony of each isolate was transferred to 10 ml of MRS broth. Each C. difficile strain was cultivated twice in Brain Heart Infusion broth (BHI broth) at 37 ºC for 24 h under anaerobic conditions.  Methods Clostridioides difficile and lactic acid bacteria strains and preparation of inocula Anti-clostridial activity of lactic acid bacteria strains BHI (0.7% w/v agar)  +  0.1% sodium thaurocolate C. difficile suspension 10 µl drops LAB suspension LAB        cell-free supernatant (7000 rpm, 15 min, 4 ºC) Incorporated 1:10 37 ºC for 24 to 48h, anaerobiosis LAB        cell-free supernatant (CFS) Anti-clostridial activity BHI (0.7% w/v agar)  +  0.1% sodium thaurocolate 37 ºC for 24 to 48h, anaerobiosis C. difficile suspension Incorporated 1:10 10 µl drops CFS adjusted to pH 6.5 CFS treated 0.1 mg/ml catalase CFS treated 0.1 mg/ml proteinase K Results Discussion Anti-clostridial activity of lactic acid bacteria strains Figure 1. Percentage of LAB with anticlostridial activity against each  C. difficile strain: (A) culture of LAB and (B) LAB cell-free supernatant against C. difficile culture. The results obtained for the antimicrobial activity of 26 lactic acid bacteria against five C. difficile strains are shown in Table 1. In figure 2 is shown an image of inhibition halo zones obtained. Figure 2. Translucent halo zones obtained for inhibitions of supernatant of several LAB against C. difficile culture I805452. Legend:  (+) – presence of translucent halo zone (-) – absence of translucent halo zone and LAB Cd H63866 Cd I805452 Cd I805937 Cd V315639 Cd DSMZ1296 Cd H63866 Cd I805452 Cd I805937 Cd V315639 Cd DSMZ1296 Cd H63866 Cd I805452 Cd I805937 Cd V315639 Cd DSMZ12961 - - - - - - - - - - - - - - -2 - - - - - - - - - - - - - - -3 - - - - - - - - - - - - - - -4 - + - + + - + - + + - - - - +5 + + + + + - - - + + - - - + +6 - - - - + - - - - + - - - - +7 - - - + + - - - - - - - - - -8 - + - + + - + - + + - - - + +9 + + + + + - + - + + - - - + +10 + + + + + + + + + + + + + + +11 + + + + + + + + + + + + - + +12 + + + + + + + + + + - - + + +13 + + + + + + + + + + - + - + +14 - - - - - - - - - - - - - - -15 - - - - - - - - - - - - - - -16 - - - - - - - - - - - - - - -17 - - - - - - - - - - - - - - -18 - - - - - - - - - - - - - - -19 + + + + + + + + + + + + + + +20 + + + + + + + + + + + + + + +21 + + + + + + + + + + + + + + +22 - - - - - - - - - - - - - - -23 - - - - - - - - - - - - - - -24 - - - - - - - - - - - - - - -25 + + + + + + + + + + + + + + +26 - - - - - - - - - - - - - - -Neutralized supernatant against culture Neutralized supernatant treated with catalase against culture Neutralized supernatant treated with proteinase K against culture

Inhibition of clostridioides difficile by lactic acid bacteria

CONGRESS OF MICROBIOLOGY AND BIOTECHNOLOGY 2019December 5th-7th, 2019University of Coimbra (Pólo II)BOOK OF ABSTRACTS354 III6. Health Microbiology and Biotechnology P289. Inhibition of Clostridioides difficile by lactic acid bacteria Joana Barbosa, Paula Teixeira Escola Superior de Biotecnologia - Universidade Católica Portuguesa E-mail: joanabastos@sapo.ptClostridioides difficile infections have been associated with the antibiotics use (altering the intestinal microbiota, enabling the proliferation and toxins segregation by C. difficile). Moreover, the increase of antibiotic-resistance of hypervirulent strains turns imperative finding alternative therapeutics with preventive and/or prophylactic effects, as the use of probiotics. Lactic acid bacteria are considered as a major group of probiotic bacteria and their ingestion has been pointed out to confer a range of health benefits. There are several mechanisms by which probiotic strains may exert their inhibitory activity towards undesired enteropathogens: secretion of different metabolites as lactic acid, hydrogen peroxide, short-chain fatty acids, bacteriocins, proteases, etc. The objective of this study was to test the anticlostridial activity of 450 lactic acid bacteria (isolated from various food products) against 5 C. difficile strains. For those lactic acid bacteria demonstrating inhibitory activity, the mechanisms of inhibition was investigated. Suspensions of each C. difficile strain were incorporated onto Brain Heart Infusion soft agar with 0.1% sodium thaurocolate and 10 μl drops of each lactic acid bacteria and their supernatants were spotted on the agar plate with the target organism. Clear supernatants of anticlostridial-LAB were sterilized, the pH adjusted, treated with catalase and proteinase K and the procedure was repeated for each treated supernatant. Of the 450 lactic acid bacteria tested, only 77 were able to inhibit at least one C. difficile strain as a result of competition between cells. When the cells were centrifuged and the supernatant was used, 26 maintained their anticlostridial activity apparently: by the action of lactic acid (15), by the production of hydrogen peroxide (3), by the presence of proteinaceous compounds (6) and by the action of other nature compounds (5). Although further in vitro tests are still needed, such as the ability of selected LAB to inhibit the invasion of C. difficile into intestinal Caco-2 cells, these LAB isolates may be potential anti-C. difficile strategies as alternative to antibiotics or as preventive of C. difficile infections. 

Inhibition of Clostridioides difficile by lactic acid bacteria

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Inhibition of Clostridioides difficile by lactic acid bacteria

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Repositório Institucional da Universidade Católica Portuguesa