In Vitro Antimicrobial Activity and Probiotic Potential of Bifidobacterium and Lactobacillus against Species of Clostridium

doi:10.3390/nu11020448

. 2019 Feb 21;11(2):448.

doi: 10.3390/nu11020448.

In Vitro Antimicrobial Activity and Probiotic Potential of Bifidobacterium and Lactobacillus against Species of Clostridium

Cinara R A V Monteiro^{1

2}, Monique S do Carmo^{3

4}, Bruna O Melo⁵, Matheus S Alves⁶, Camilla I Dos Santos⁷, Sílvio G Monteiro^{8

9}, Maria Rosa Q Bomfim¹⁰, Elizabeth S Fernandes¹¹, Valério Monteiro-Neto^{12

13}

Affiliations

¹ Programa de Pós-graduação em Ciências da Saúde, Universidade Federal do Maranhão, Av. dos Portugueses, campus do Bacanga, São Luís 65065545, MA, Brazil. [email protected].
² Programa de Pós-graduação, Universidade Ceuma, Rua dos Castanheiros No. 1, jardim Renascença II, São Luís 65075120, MA, Brazil. [email protected].
³ Programa de Pós-graduação em Ciências da Saúde, Universidade Federal do Maranhão, Av. dos Portugueses, campus do Bacanga, São Luís 65065545, MA, Brazil. [email protected].
⁴ Programa de Pós-graduação, Universidade Ceuma, Rua dos Castanheiros No. 1, jardim Renascença II, São Luís 65075120, MA, Brazil. [email protected].
⁵ Programa de Pós-graduação, Universidade Ceuma, Rua dos Castanheiros No. 1, jardim Renascença II, São Luís 65075120, MA, Brazil. [email protected].
⁶ Programa de Pós-graduação, Universidade Ceuma, Rua dos Castanheiros No. 1, jardim Renascença II, São Luís 65075120, MA, Brazil. [email protected].
⁷ Programa de Pós-graduação, Universidade Ceuma, Rua dos Castanheiros No. 1, jardim Renascença II, São Luís 65075120, MA, Brazil. [email protected].
⁸ Programa de Pós-graduação, Universidade Ceuma, Rua dos Castanheiros No. 1, jardim Renascença II, São Luís 65075120, MA, Brazil. [email protected].
⁹ Departamento de Biologia, Universidade Federal do Maranhão, Av. dos Portugueses, campus do Bacanga, São Luís 65065545, MA, Brazil. [email protected].
¹⁰ Programa de Pós-graduação, Universidade Ceuma, Rua dos Castanheiros No. 1, jardim Renascença II, São Luís 65075120, MA, Brazil. [email protected].
¹¹ Programa de Pós-graduação, Universidade Ceuma, Rua dos Castanheiros No. 1, jardim Renascença II, São Luís 65075120, MA, Brazil. [email protected].
¹² Programa de Pós-graduação em Ciências da Saúde, Universidade Federal do Maranhão, Av. dos Portugueses, campus do Bacanga, São Luís 65065545, MA, Brazil. [email protected].
¹³ Programa de Pós-graduação, Universidade Ceuma, Rua dos Castanheiros No. 1, jardim Renascença II, São Luís 65075120, MA, Brazil. [email protected].

PMID: 30795551
PMCID: PMC6412307
DOI: 10.3390/nu11020448

In Vitro Antimicrobial Activity and Probiotic Potential of Bifidobacterium and Lactobacillus against Species of Clostridium

Cinara R A V Monteiro et al. Nutrients. 2019.

. 2019 Feb 21;11(2):448.

doi: 10.3390/nu11020448.

Authors

Affiliations

¹ Programa de Pós-graduação em Ciências da Saúde, Universidade Federal do Maranhão, Av. dos Portugueses, campus do Bacanga, São Luís 65065545, MA, Brazil. [email protected].
² Programa de Pós-graduação, Universidade Ceuma, Rua dos Castanheiros No. 1, jardim Renascença II, São Luís 65075120, MA, Brazil. [email protected].
³ Programa de Pós-graduação em Ciências da Saúde, Universidade Federal do Maranhão, Av. dos Portugueses, campus do Bacanga, São Luís 65065545, MA, Brazil. [email protected].
⁴ Programa de Pós-graduação, Universidade Ceuma, Rua dos Castanheiros No. 1, jardim Renascença II, São Luís 65075120, MA, Brazil. [email protected].
⁵ Programa de Pós-graduação, Universidade Ceuma, Rua dos Castanheiros No. 1, jardim Renascença II, São Luís 65075120, MA, Brazil. [email protected].
⁶ Programa de Pós-graduação, Universidade Ceuma, Rua dos Castanheiros No. 1, jardim Renascença II, São Luís 65075120, MA, Brazil. [email protected].
⁷ Programa de Pós-graduação, Universidade Ceuma, Rua dos Castanheiros No. 1, jardim Renascença II, São Luís 65075120, MA, Brazil. [email protected].
⁸ Programa de Pós-graduação, Universidade Ceuma, Rua dos Castanheiros No. 1, jardim Renascença II, São Luís 65075120, MA, Brazil. [email protected].
⁹ Departamento de Biologia, Universidade Federal do Maranhão, Av. dos Portugueses, campus do Bacanga, São Luís 65065545, MA, Brazil. [email protected].
¹⁰ Programa de Pós-graduação, Universidade Ceuma, Rua dos Castanheiros No. 1, jardim Renascença II, São Luís 65075120, MA, Brazil. [email protected].
¹¹ Programa de Pós-graduação, Universidade Ceuma, Rua dos Castanheiros No. 1, jardim Renascença II, São Luís 65075120, MA, Brazil. [email protected].
¹² Programa de Pós-graduação em Ciências da Saúde, Universidade Federal do Maranhão, Av. dos Portugueses, campus do Bacanga, São Luís 65065545, MA, Brazil. [email protected].
¹³ Programa de Pós-graduação, Universidade Ceuma, Rua dos Castanheiros No. 1, jardim Renascença II, São Luís 65075120, MA, Brazil. [email protected].

PMID: 30795551
PMCID: PMC6412307
DOI: 10.3390/nu11020448

Abstract

Many Clostridium species are found as commensal members of the intestinal microbiota. However, imbalances of the microbiota may lead to certain infections caused by these microorganisms, mainly Clostridium butyricum, Clostridium difficile, and Clostridium perfringens. In many cases, infection recurrence can occur after antibiotics, indicating the need for novel therapeutic options that act on the pathogens and also restore the microbiota. Herein, the in vitro antimicrobial activity and probiotic potential of clinical and reference strains of Bifidobacterium and Lactobacillus were investigated against Clostridium species. Antimicrobial activity was evaluated by the agar spot test and inhibition of gas production. Then, the probiotic potential of selected strains was assessed by analyzing their coaggregation ability, adhesive properties to host cells and mucin, tolerance to acidic pH and bile salts, and antimicrobial susceptibility profiles. Lactobacillus plantarum ATCC 8014 was the most promising strain based on its inhibitory activity against Clostridium spp. Also, this strain met criteria to be considered a probiotic based on its coaggregation ability, adhesive properties, and tolerance to harsh pH and bile acid salt conditions. The results indicate that among the studied strains, L. plantarum ATCC 8014 presents probiotic potential for controlling infections induced by the studied Clostridium species and should be further evaluated in in vivo animal models.

Keywords: Clostridium; Lactobacillus plantarum; antimicrobial activity; dysbiosis; probiotics.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Figures

**Figure 1**
Inhibition of gas production assay. The lower layer corresponds to the RCM agar inoculated with *Clostridium butyricum* ATCC 860 and the upper layer is MRS medium with 0.7% agar and 100 mM phosphate buffer inoculated with *Bifidobacterium* and *Lactobacillus* strains; cultures were incubated at 37 °C for 24 h under anaerobic conditions. (a) Gas production by *C. butyricum* in buffered MRS medium, indicating the absence of inhibitory activity of *Lactobacillus* and *Bifidobacterium* strains. (b) Inhibitory activity of five *Lactobacillus* and two *Bifidobacterium* strains on gas production by *Clostridium butyricum*.

**Figure 2**
Gram staining of bacterial isolates before (a–d) and after (e–g) the coaggregation assays of *L. plantarum* ATCC 8014 with the three species of *Clostridium.* (a) *L. plantarum*, (b) *C. butyricum*, (c) *C. difficile,* and (d) *C. perfringens* controls. Coaggregation of *L. plantarum* and (e) *C. butyricum*, (f) *C. difficile,* or (g) *C. perfringens*. Formation of bacterial aggregates were observed after mixing *L. plantarum* with each *Clostridium* strain. Gram stained slides were visualized by light microscopy under a 100× oil immersion objective.

**Figure 3**
Microscopic visualization of adhesion assays of (a) *L. plantarum* ATCC 8014 and (b) *L. fermentum* ATCC 23271 to HeLa cells after inoculation of approximately 10⁷ CFU of bacterial suspensions. Cell monolayers grown on coverslips were stained by the Gram’s method and examined by light microscopy under a 100× oil immersion objective. Note the presence of numerous gram-positive bacilli adhered to HeLa cells.

See this image and copyright information in PMC

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References

1. Perez-Lopez A., Behnsen J., Nuccio S.P., Raffatellu M. Mucosal immunity to pathogenic intestinal bacteria. Nat. Rev. Immunol. 2016;16:135–148. doi: 10.1038/nri.2015.17. - DOI - PubMed
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1. Sonnenburg J.L., Bäckhed F. Diet–microbiota interactions as moderators of human metabolism. Nature. 2016;535:56. doi: 10.1038/nature18846. - DOI - PMC - PubMed
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[1] Perez-Lopez A., Behnsen J., Nuccio S.P., Raffatellu M. Mucosal immunity to pathogenic intestinal bacteria. Nat. Rev. Immunol. 2016;16:135–148. doi: 10.1038/nri.2015.17. - DOI - PubMed

[2] Perez-Lopez A., Behnsen J., Nuccio S.P., Raffatellu M. Mucosal immunity to pathogenic intestinal bacteria. Nat. Rev. Immunol. 2016;16:135–148. doi: 10.1038/nri.2015.17. - DOI - PubMed

[3] Reinoso Webb C., Koboziev I., Furr K.L., Grisham M.B. Protective and pro-inflammatory roles of intestinal bacteria. Pathophysiology. 2016;23:67–80. doi: 10.1016/j.pathophys.2016.02.002. - DOI - PMC - PubMed

[4] Reinoso Webb C., Koboziev I., Furr K.L., Grisham M.B. Protective and pro-inflammatory roles of intestinal bacteria. Pathophysiology. 2016;23:67–80. doi: 10.1016/j.pathophys.2016.02.002. - DOI - PMC - PubMed

[5] Sonnenburg J.L., Bäckhed F. Diet–microbiota interactions as moderators of human metabolism. Nature. 2016;535:56. doi: 10.1038/nature18846. - DOI - PMC - PubMed

[6] Sonnenburg J.L., Bäckhed F. Diet–microbiota interactions as moderators of human metabolism. Nature. 2016;535:56. doi: 10.1038/nature18846. - DOI - PMC - PubMed

[7] Kau A.L., Ahern P.P., Griffin N.W., Goodman A.L., Gordon J.I. Human nutrition, the gut microbiome and the immune system. Nature. 2011;474:327–336. doi: 10.1038/nature10213. - DOI - PMC - PubMed

[8] Kau A.L., Ahern P.P., Griffin N.W., Goodman A.L., Gordon J.I. Human nutrition, the gut microbiome and the immune system. Nature. 2011;474:327–336. doi: 10.1038/nature10213. - DOI - PMC - PubMed

[9] Littman D.R., Pamer E.G. Role of the commensal microbiota in normal and pathogenic host immune responses. Cell Host Microbe. 2011;10:311–323. doi: 10.1016/j.chom.2011.10.004. - DOI - PMC - PubMed

[10] Littman D.R., Pamer E.G. Role of the commensal microbiota in normal and pathogenic host immune responses. Cell Host Microbe. 2011;10:311–323. doi: 10.1016/j.chom.2011.10.004. - DOI - PMC - PubMed

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In Vitro Antimicrobial Activity and Probiotic Potential of Bifidobacterium and Lactobacillus against Species of Clostridium

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In Vitro Antimicrobial Activity and Probiotic Potential of Bifidobacterium and Lactobacillus against Species of Clostridium

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