52a Reunião Anual da Sociedade Brasileira de
Zootecnia
Zootecnia: Otimizando Recursos e Potencialidades
Belo Horizonte – MG, 19 a 23 de Julho de 2015
Isolation, identification and characterization in vitro of lactic acid bacteria as probiotics candidates of
mastitis in cattle 1
Raphael Steinberg da Silva2, Lílian Cristina da Silva e Silva3, Jacques Robert Nicoli4, Elisabeth Neumann5, Quinto
Álvaro Cantini Nunes6
1
Parte de da tese de doutorado do Raphael Steinberg da Silva, financiada pela FAPEMIG
Doutorando em genética, Departamento de Biologia Geral - UFMG, BRA. Bolsista do CNPq/Capes. e-mail: [email protected]
3
Graduanda em Ciências Biológicas UFMG, Belo Horizonte, Brasil, Bolsista da FAPEMIG. e-mail:[email protected]
4
Doutor, Departamento de Microbiologia - UFMG, BRA. e-mail: [email protected]
5
Doutora, Departamento de Microbiologia - UFMG, BRA. e-mail: [email protected]
6
Doutor, Departamento de Biologia Geral - UFMG, BRA. e-mail: [email protected]
2
Resumo: A mastite, a inflamação da glândula mamária, é uma das doenças mais caras e complexas da indústria de
lacticínios. As consequências econômicas da mastite bovina estão relacionados ao tratamento, as perdas de
produção, abate e as mudanças na qualidade do leite. Causada principalmente por bactérias é, muitas vezes, tratada
com antimicrobianos o que gerou uma busca por métodos alternativos como os probióticos. Para a obtenção de
possíveis probióticos, 192 bactérias foram isoladas e após as análises genéticas moleculares 82 linhagens diferentes
obtidas de vacas saudáveis passaram pelos seguintes testes in vitro: ensaios de autoagregação, hidrofobicidade e
coagregação, produção de EPS, biofilme e H2O2, ensaio de sensibilidade a antimicrobianos e atividade antagonista
contra patógenos. De forma geral, os isolados apresentaram baixa autoagregação e hidrofobicidade da superfície
celular, sendo que Lactococcus lactis GIR39S2, Weissella confusa GIR48L1* e W. confusa GIRO21L1*
apresentaram as melhores características para a colonização do epitélio mamário. Essas linhagens potencialmente
probióticas isoladas nesse trabalho podem futuro serem usadas para produzir produtos probióticos para rebanhos.
Palavras–chave: glândula mamária, inflamação, micro-organismos vivos
Abstract: Mastitis, inflammation of the mammary gland is one of the more expensive and complex diseases dairy
industry. The economic consequences of bovine mastitis are related to treatment, culling, production losses and
changes in milk quality. Caused mainly by bacteria is often treated with antibiotics which resulted in a search for
alternative methods such as probiotics. To obtain potential probiotic, 192 bacteria were isolated and after molecular
genetic analysis 82 different strains obtained from healthy cows have passed the following tests in vitro: testing
self aggregation, hydrophobicity and coaggregation, EPS production, biofilm and H2O2, the antimicrobial
sensitivity test and antagonistic activity against pathogens. In general, the isolates showed low autoagregação and
hydrophobicity of the cell surface, and Lactococcus lactis GIR39S2, Weissella confusa GIR48L1 * and W. confusa
GIRO21L1 * presented the best characteristics for the colonization of mammary epithelium. These potentially
probiotic strains isolated in this work can be used to produce future probiotic products for livestock.
Keywords: inflamation, live microorganisms, mammary gland
Introduction
Mastitis is a disease in high impact milk production in cattle. Due important dairy cattle strategy in food
production, disease control is crucial. To deal with international pressure to reduce the use of antimicrobials in
animal production, development of alternative methods of treatment and prevention must be implemented to replace
traditional therapy of antibiotics. Because of the increased use of probiotic bacteria in the control of many
infectious diseases, bacterioterapia offers a possible strategy in controlling mastitis. Probiotic strains must be
isolated from the same ecosystem to which will be applied. Therefore, the isolation, identification and
characterization in vitro and in vivo bacterial inmate mammary gland microbiota can permit the formation of a
select group of bacteria that can be used in the formulation of new treatment in cattle improving the health status of
the mammary gland. The lactic acid bacteria are part of the natural microflora of the bovine mammary gland and
have a long history of safe use being strong candidates for selection of probiotics to the mammary gland. The
objective was to isolate, identify and characterize in in vitro lactic acid bacteria milk and swab intrammary of
healthy cows and subclinical mastitis.
Material e Methods
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52a Reunião Anual da Sociedade Brasileira de
Zootecnia
Zootecnia: Otimizando Recursos e Potencialidades
Belo Horizonte – MG, 19 a 23 de Julho de 2015
Samples of 123 cows (healthy and subclinical mastitis) were collected, serial dilutions were made, the
samples are plating on MRS agar and the plates were incubated in anaerobic conditions. The gram-positive colonies
and negative catalase were collected, the total were obtained 314 isolated. The bacteria were identified by rep-PCR
for differentiation of strains and subjected to molecular identification by DNA sequencing of the 16S rRNA gene.
After the final identification, 82 different strains isolated from healthy animals were selected for in vitro tests. The
self aggregation was determined after 4 h of incubation at ambient temperature and absorbance measurements were
done on a spectrophotometer at 600 nm. The hydrophobicity of the cell surface of the isolates was assessed by
Microbial Adhesion method Solvent using xylene and measuring the absorbance at 600nm. To evaluate the
production of exopolysaccharides (EPS), isolates were striated in modified MRS agar and analyzed the presence of
viscosity in the colonies. To test for biofilm production absorbance was measured at OD 570nm after incubation of
the bacteria with crystal violet, followed by washing and addition of acetic acid. For screening of hydrogen
peroxide production (H2O2) cultures were striated in MRS Agar containing 1 mM 3,3 ', 5,5'-tetramethylbenzidine
and 2 U / mL horseradish peroxidase type II. The production of H2O2 was measured after exposure of the colonies
O2 for 10 min. The antimicrobial susceptibility was determined by the drug's diffusion in agar using discs
impregnated with relevant antibiotics for treatment of mastitis. The test to detect antagonistic BAL isolated from
bovine mammary gland was performed by diffusion technique in double layer agar, using as revealing pathogens
bacteria related to the etiology of mastitis.
Results and Discussion
Among the 82 isolates selected for evaluation of the potential probiotic in vitro, 6 belonged to the genus
Pediococcus, Lactococcus 14, 12 Weissella, 10 Lactobacillus, Enterococcus 23 and 17 Streptococcus. These
isolates obtained from samples of raw milk and intramammary swab were evaluated in vitro tests.
The autoagregação capacity and hydrophobicity were evaluated to assess cell surface characteristics. Only
18.29% of the isolates were considered autoagregação high and 53.66% of the isolates showed low autoagregação.
The same trend was observed for the hydrophobicity. Most isolates (85.37%) showed low cell surface
hydrophobicity and only 9.76% of the isolates showed high hydrophobicity.
Biofilm production capacity on mucosal isolates of BAL was assessed using EPS production tests on
modified MRS and biofilm production in polystyrene plates. Of the 82 isolates tested, 17 were able to produce EPS
in culture medium modified and approximately half of the tested isolates (52.44%) showed no ability to produce
biofilms. Among the BAL evaluated 35.37% were weak producing biofilm, 4.88% producing moderate biofilm
and 7.32% were strong biofilm producers.
Among the producers of H2O2 31.71% were classified as poor producers of H2O2, 34.15% were considered
moderate producers of H2O2 and 12.20% were classified as strong producers of H2O2. The strengths of H2O2
producers were found only representatives of the genus Weissella (10 Weissella of 12 tested), W. confusa
GIR48L1* and W. confusa GIRO21L1* in addition to producing strong H2O2 were also EPS producing and
producing strong biofilm.
The sensitivity of 82 isolates from BAL to important antimicrobials in veterinary clinical practice related
to mastitis was evaluated. Only 4 isolates were susceptible to vancomycin, the others were resistant. Only P.
pentosaceus GIR50S1 * was resistant to ampicillin, and the other were sensitive to this drug. Three isolates were
sensitive to gentamicin and the other were resistant. Only P. pentosaceus GIR50S1 was sensitive and W.
paramesenteroides GIR46L4 * was moderately sensitive to amikacin, the remaining were resistant. E. faecalis
GIRO33L2 * was the only isolated resistant to erythromycin, another 14 isolates were moderately sensitive and
remaining were sensitive. P. pentosaceus GIR50S1 was the only isolated resistant to ceftriaxone, other eight
isolates were moderately susceptible and others were resistant to this antibiotic. All isolates were resistant to
streptomycin. E. faecalis GIRO33L2 * was the only isolated moderately sensitive to oxacillin, P. pentosaceus
GIR44S3 * and Lactococcus lactis GUZ3S2 * were sensitive and the rest were resistant to this antimicrobial. None
isolate was resistant to penicillin G. and 8 isolates were resistant to amoxicillin.
The milk is biological fluid secreted by mammals with important nutritional functions for the offspring
development. Humans with the habit of continuing to drink milk after weaning started using in feeding.The milk
got other animals, which are now being created for this purpose. The factors mainly associated health and animal
welfare directly affect milk production. The technological relevance of bacteria present in milk, for example BAL,
is widely recognized, as they are critical to the production of cheese and other dairy products. The use of probiotics
in animal husbandry should be initiated by in vitro selection studies for the screening of antimicrobial activity,
characterization studies and cell surface adhesion and the susceptibility to antibiotics. Gaggia et al.( 2010).
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52a Reunião Anual da Sociedade Brasileira de
Zootecnia
Zootecnia: Otimizando Recursos e Potencialidades
Belo Horizonte – MG, 19 a 23 de Julho de 2015
Approximately half of the isolates showed low self aggregation and only 18.29% of the isolates were
considered high aggregation. This trend was also observed for the cell surface hydrophobicity. It was found on
correlation (Pearson correlation coefficient = - 0.1551, P = 0.1642) between the hydrophobicity and the
autoagregação isolated on individual characteristics even when the values are low.
The milk isolates, 89.2% had low value of self aggregation and only 10.8% have medium value of
autoagregação and none had high among the BAL isolated in northern Argentina Espeche et al (2009). Similar to
those isolated in this work, Espeche et al. (2009) reported that the great majority of strains isolated from milk BAL
had a low hydrophobicity, and the minority those with moderate values (6.9%) and high (7.8%) adhesion to
hydrophobic solvents. Espeche et al. (2012) reported, as we found, a low coefficient of Pearson (0.215) obtained
from analysis of the relationship of BAL surface properties (hydrophobicity and autoagregação), showing that these
features do not present statistical correlation. However, BAL strains which have a low degree of hydrophobicity can
still be self-aggregating hydrophilic, they may have glycoproteins or glycosylated peptides on their surface. Some
studies show that isolated BAL from milk do not show EPS production in vitro screening test (Franciosi et al, 2009;
Espeche et al, 2012). Among the 82 isolates, 7 were classified as strong biofilm producers and half of tested isolates
produced no biofilm in the test on polystyrene plates. This is the first work reported in the literature that evaluates
the BAL biofilm production potential isolated from the mammary gland. Moreover, 53% of the isolates did not
produce EPS producing biofilm, and between the BAL are strong or moderate producing biofilm EPS produced
only 24%.
The vast majority of isolates from BAL evaluated in this study were H2O2 producers, of which 34.15%
were moderate and 12.20% strong producers of H2O2. Unlike our results, Espeche et al. (2012) found that 63% of
isolates were major producers of H2O2 belonged to the species P. pentosaceus. According to Espeche et al. (2009),
most milk BAL strains (71.6%) were negative for the production of H2O2 and only 8.8% were classified as high
producers. In this same study, it was reported that most isolated BAL of healthy cows was producing H2O2, which
did not occur with isolated from cows with subclinical mastitis. An important criterion for the selection of probiotic
bacteria for mastitis is the antimicrobial susceptibility due to the risk of lateral transfer of genes conferring
resistance to inmates and potentially pathogenic members of the indigenous microbiota. Plasmids carrying
antibiotic resistance is uncommon in the BAL, but if this occurs the safety of this line should be questioned.
Overall, this intrinsic resistance profile was submitted by BAL obtained in this work, with some exceptions.
Conclusions
The strains to be selected for future studies as potential candidates for probiotics for ecosystem should have
had no quote as pathogens associated with mastitis, be isolated from healthy teats inhibit at least one pathogen
screening antagonism, have high or medium hydrophobicity and autoagregação. Added to this, the production of
EPS or biofilm characteristics are desirable but not obligatory and the evaluation of the susceptibility gene should
avoid lateral transfer resistance. Thus, the bacteria were selected based on those principles and will continue to
evaluate the cell.
References
Gaggia, F.; Mattarelli P.; Biavati, B. Probiotics and prebiotics in animal feeding for safe food production.
Int. J. Food Microbiol, v. 141, p. S15– S28, 2010.
Espeche, M. C.; Otero, M. C.; Sesma, F.; Nader-Macias, M. E. F. Screening of surfaceproperties and
antagonistic substance s production by lactic acid bacteria isolated from themammary gland of healthy and mastitic
cows. Vet Microbiol, v. 135, p. 346–357, 2009.
Espeche, M. C.; Pellegrino, M.; Frola, I.; Larriestra, A.; Bogni, C.;NADER-MACÍAS, M. E. F. Lactic acid
bacteria from raw milk as potentially beneficial strains to prevent bovine mastitis. Anaerobe. v. 18, p. 103 -109,
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Franciosi, E.; Settanni, L.; Cavazza, A.; Poznanski, E. Biodiversity and technological potential of wild
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