Bacteriófagos líticos como alternativa potencial para o controle de <i>Staphylococcus aureus</i>

O objetivo deste trabalho foi caracterizar bacteriófagos autóctones e determinar sua atividade lítica em Staphylococcus aureus. Seis fagos foram isolados de água de lavagem de pisos de estábulos por meio do enriquecimento de cultura com três estirpes de S. aureus. Todos os fagos foram caracterizados pela digestão do DNA com enzimas de restrição e pelo sequenciamento do fragmento de DNA que codifica a endolisina. Cada fago foi testado contra 100 estirpes de S. aureus isoladas de casos de mastite bovina e de produtos lácteos pelo método de lise em placa. Sequências do gene de endolisina apresentaram alta conservação, com mais de 99% de similaridade a nível do nucleotídeo entre os fagos isolados. Foram identificados três domínios envolvidos no reconhecimento e na lise da parede celular bacteriana. Dois bacteriófagos isolados de estábulos apresentam alta atividade lítica em S. aureus, em ampla gama de estirpes, o que indica seu potencial para estudos de fagoterapia em gado leiteiro ou como agente de controle biológico para produtos lácteos.

Termos para indexação:
Staphylococcus aureus; controle biológico; endolisina; fagos

Autoria

Juliana Almeida Leite

Instituto Oswaldo Cruz, Laboratório de Vírus Respiratório e do Sarampo, Avenida Brasil, no 4.365, Manguinhos, CEP 21040-360 Rio de Janeiro, RJ, Brazil. E-mail: Juliana.leite@ioc.fiocruz.brInstituto Oswaldo CruzBrazilRio de Janeiro, RJ, Brazil Instituto Oswaldo Cruz, Laboratório de Vírus Respiratório e do Sarampo, Avenida Brasil, no 4.365, Manguinhos, CEP 21040-360 Rio de Janeiro, RJ, Brazil. E-mail: Juliana.leite@ioc.fiocruz.br

http://orcid.org/0000-0002-8620-114X

Hyago Passe Pereira

Universidade Federal de Juiz de Fora, Rua José Lourenço Kelmer, s/no, Campus Universitário, São Pedro, CEP 36036-900 Juiz de Fora, MG, Brazil. E-mail: hyago9295@gmail.comUniversidade Federal de Juiz de ForaBrazilJuiz de Fora, MG, Brazil Universidade Federal de Juiz de Fora, Rua José Lourenço Kelmer, s/no, Campus Universitário, São Pedro, CEP 36036-900 Juiz de Fora, MG, Brazil. E-mail: hyago9295@gmail.com

http://orcid.org/0000-0002-8243-4667

Cristiano Amâncio Vieira Borges

Embrapa Gado de Leite, Rua Eugênio do Nascimento, no 610, Dom Bosco, CEP 36038-330 Juiz de Fora, MG, Brazil. E-mail: cristiano.borges@embrapa.br, bruna.alves@embrapa.br, izispinheiro@yahoo.com.br, marta.martins@embrapa.br, edna.arcuri@embrapa.brEmbrapa Gado de LeiteBrazilJuiz de Fora, MG, Brazil Embrapa Gado de Leite, Rua Eugênio do Nascimento, no 610, Dom Bosco, CEP 36038-330 Juiz de Fora, MG, Brazil. E-mail: cristiano.borges@embrapa.br, bruna.alves@embrapa.br, izispinheiro@yahoo.com.br, marta.martins@embrapa.br, edna.arcuri@embrapa.br

http://orcid.org/0000-0001-8120-3288

Bruna Rios Coelho Alves

http://orcid.org/0000-0002-9446-154X

Alessandra Isis Alves Pinheiro Ramos

Marta Fonseca Martins

http://orcid.org/0000-0002-4260-5329

Edna Froeder Arcuri ^**Corresponding author: edna.arcuri@embrapa.br

http://orcid.org/0000-0001-5503-218X

*Corresponding author: edna.arcuri@embrapa.br

SCIMAGO INSTITUTIONS RANKINGS

Figuras | Tabelas

Figuras (6)
Tabelas (1)

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Figure 1.
Lytic activity of the isolated bacteriophages against a Staphylococcus aureus strain isolated from a milk sample collected from a milk cooling tank. 1, phage 1; 2, phage 2; 3, phage 3; 4, phage 4; 5, phage 5; and 6, phage 6.

Thumbnail

Figure 2.
Proportions of Staphylococcus aureus strains isolated from the milk of cows with clinical or subclinical mastitis (n = 64 binomial trials for each phage type), from bulk milk cooling tank (n = 23), and from Minas Frescal cheese (n = 13), showing susceptibility to the isolated bacteriophages, with a confidence interval of 95%.

Thumbnail

Figure 3.
Genomic restriction profiles of the isolated bacteriophages. Viral DNA was extracted and digested with the EcoRI (A), HindIII (B), XhoI (C), and XbaI (D) enzymes. Lanes: 1, molecular weight using the 100bp DNA ladder (Promega Corporation, Madison, WI, USA); 2, phage 1; 3, phage 2; 4, phage 3; 5, phage 4; 6, phage 5; and 7, phage 6.

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Figure 4.
Phylogenetic relationships of the endolysin gene sequences of the isolated bacteriophages. The neighbor-joining tree was generated by the MEGA, version 4, software (Tamura et al., 2007TAMURA, K.; DUDLEY, J.; NEI, M.; KUMAR, S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA): software version 4.0. Molecular Biology and Evolution, v.24, p.1596-1599, 2007. DOI: https://doi.org/10.1093/molbev/msm092.
https://doi.org/10.1093/molbev/msm092... ). The evolutionary distances are expressed in units of the number of nucleotide substitutions per site (scale bar). The analysis was based on 1,000 bootstrap replicates, with a cutoff value of 95%.

Thumbnail

Figure 5.
Bacteriophage phylogeny reconstruction based on the amino acid sequence inferred from the endolysin gene nucleotide sequence. GenBank accession numbers are given after dash (GenBank, 2018GENBANK. Available at: <Available at: http://www.ncbi.nlm.nih.gov/Genbank >. Accessed on: July 9 2018.
http://www.ncbi.nlm.nih.gov/Genbank... ). The analysis was based on 1,000 bootstrap replicates, with a cutoff value of 95%.

Thumbnail

Figure 6.
Amino acid sequence analysis of the endolysins of the isolated bacteriophages, showing the cysteine, histidine-dependent amidohydrolase/peptidases (CHAP), N-acetylmuramyl-L-alanine amidase (amidase 2), and SH3b (involved in the bacterial cell wall recognition) domains, identified by the Pfam, version 24.0, software (Pfam, 2018PFAM. Available at: <Available at: http://pfam.sanger.ac.uk/ >. Accessed on: July 9 2018.
http://pfam.sanger.ac.uk/... ) (A); and amino acid sequence alignment of the CHAP (B), amidase 2 (C), and SH3b (D) domains of the isolated lytic phages and phage phi 11, using the DNASTAR software (DNASTAR, 2018DNASTAR. Available at: <Available at: https://www.dnastar.com/workflows/protein/ >. Accessed on: July 9 2018.
https://www.dnastar.com/workflows/protei... ). Identical amino acids are shaded in gray, conservative substitutions are marked in red and unrelated amino acids are in white.

Thumbnail

Table 1.
Similarity among the endolysin nucleotide sequences of the six isolated bacteriophages (phages 1 to 6) and of the other phages available in the GenBank (2018)GENBANK. Available at: <Available at: http://www.ncbi.nlm.nih.gov/Genbank >. Accessed on: July 9 2018.
http://www.ncbi.nlm.nih.gov/Genbank... .

Figure 1. Lytic activity of the isolated bacteriophages against a Staphylococcus aureus strain isolated from a milk sample collected from a milk cooling tank. 1, phage 1; 2, phage 2; 3, phage 3; 4, phage 4; 5, phage 5; and 6, phage 6.

Figure 2. Proportions of Staphylococcus aureus strains isolated from the milk of cows with clinical or subclinical mastitis (n = 64 binomial trials for each phage type), from bulk milk cooling tank (n = 23), and from Minas Frescal cheese (n = 13), showing susceptibility to the isolated bacteriophages, with a confidence interval of 95%.

Figure 3. Genomic restriction profiles of the isolated bacteriophages. Viral DNA was extracted and digested with the EcoRI (A), HindIII (B), XhoI (C), and XbaI (D) enzymes. Lanes: 1, molecular weight using the 100bp DNA ladder (Promega Corporation, Madison, WI, USA); 2, phage 1; 3, phage 2; 4, phage 3; 5, phage 4; 6, phage 5; and 7, phage 6.

Figure 4. Phylogenetic relationships of the endolysin gene sequences of the isolated bacteriophages. The neighbor-joining tree was generated by the MEGA, version 4, software (Tamura et al., 2007TAMURA, K.; DUDLEY, J.; NEI, M.; KUMAR, S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA): software version 4.0. Molecular Biology and Evolution, v.24, p.1596-1599, 2007. DOI: https://doi.org/10.1093/molbev/msm092.
https://doi.org/10.1093/molbev/msm092... ). The evolutionary distances are expressed in units of the number of nucleotide substitutions per site (scale bar). The analysis was based on 1,000 bootstrap replicates, with a cutoff value of 95%.

Figure 5. Bacteriophage phylogeny reconstruction based on the amino acid sequence inferred from the endolysin gene nucleotide sequence. GenBank accession numbers are given after dash (GenBank, 2018GENBANK. Available at: <Available at: http://www.ncbi.nlm.nih.gov/Genbank >. Accessed on: July 9 2018.
http://www.ncbi.nlm.nih.gov/Genbank... ). The analysis was based on 1,000 bootstrap replicates, with a cutoff value of 95%.

Figure 6. Amino acid sequence analysis of the endolysins of the isolated bacteriophages, showing the cysteine, histidine-dependent amidohydrolase/peptidases (CHAP), N-acetylmuramyl-L-alanine amidase (amidase 2), and SH3b (involved in the bacterial cell wall recognition) domains, identified by the Pfam, version 24.0, software (Pfam, 2018PFAM. Available at: <Available at: http://pfam.sanger.ac.uk/ >. Accessed on: July 9 2018.
http://pfam.sanger.ac.uk/... ) (A); and amino acid sequence alignment of the CHAP (B), amidase 2 (C), and SH3b (D) domains of the isolated lytic phages and phage phi 11, using the DNASTAR software (DNASTAR, 2018DNASTAR. Available at: <Available at: https://www.dnastar.com/workflows/protein/ >. Accessed on: July 9 2018.
https://www.dnastar.com/workflows/protei... ). Identical amino acids are shaded in gray, conservative substitutions are marked in red and unrelated amino acids are in white.

Table 1. Similarity among the endolysin nucleotide sequences of the six isolated bacteriophages (phages 1 to 6) and of the other phages available in the GenBank (2018)GENBANK. Available at: <Available at: http://www.ncbi.nlm.nih.gov/Genbank >. Accessed on: July 9 2018.
http://www.ncbi.nlm.nih.gov/Genbank... .

Phage	Nucleotide identity (%)
Phage	Phage 1	Phage 2	Phage 3	Phage 4	Phage 5	Phage 6
Phage 1	-
Phage 2	99.5%	-
Phage 3	99.7	99.6	-
Phage 4	99.5	99.9	99.6	-
Phage 5	99.8	99.6	99.7	99.6	-
Phage 6	100	99.5	99.8	99.5	99.9	-
phiMR25	98.9	98.7	98.7	98.7	98.8	98.9
92	96.0	95.9	95.8	95.9	95.9	96.0
29	95.7	95.7	95.5	95.7	95.7	95.7
69	95.5	95.6	95.5	95.6	95.5	95.5
phi11	95.2	95.1	95.0	95.1	95.1	95.2
phiMR11	95.1	95.2	95.1	95.2	95.1	95.1
88	94.4	94.5	94.4	94.5	94.4	94.4
phiNM2	94.1	94.0	93.9	94.0	94.0	94.1
52A	93.8	93.9	93.7	93.9	93.7	93.8
80	93.8	93.9	93.7	93.9	93.7	93.8
85	92.7	92.7	92.7	92.7	92.6	92.7
55	92.3	92.4	92.2	92.4	92.2	92.3
phiH5	89.2	89.3	89.2	89.3	89.1	89.2
phiSauS-IPLA88	89.2	89.3	89.3	89.3	89.1	89.2
X2	85.4	85.5	85.4	85.5	85.4	85.4
37	73.8	73.8	73.7	73.8	73.7	73.8
EW	69.5	69.5	69.4	69.5	69.5	69.5

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Bacteriófagos líticos como alternativa potencial para o controle de <i>Staphylococcus aureus</i>

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[1] *Corresponding author: edna.arcuri@embrapa.br