ARS VETERINARIA, Jaboticabal, SP, Vol. 19, nº 2, 115-118, 2003.
ISSN 0102-6380
POTENTIAL USE OF LACTOBACILLI AS PROBIOTICS IN
POULTRY VERIFIED BY ITS RESISTANCE TO BILE SALTS,
HYDROCHLORIC ACID AND ANTIBIOTICS
(USO POTENCIAL DE LACTOBACILOS COMO PROBIÓTICO EM AVICULTURA
VERIFICADO POR SUA RESISTÊNCIA A SAIS BILIARES,
ÁCIDO CLORÍDRICO E ANTIBIÓTICOS)
A.A. MEDEIROS1, R. P. SCHOCKEN-ITURRINO2, J. O. SORBARA3,
A. C. PAULILLO2, A. BERCHIERI2, B. P. H. RONCHI4, A. P.M. CARNEIRO5
SUMMARY
In the present study Lactobacillus acidophilus and L. crispatus strains were isolated from the intestinal tract of
chickens and subjected to in vitro tests in order to verify its tolerance to digestive fluids (bile salts and hydrochloride acid)
and resistance to antibiotics commonly used as growth promoters in poultry. All lactobacilli strains suspensions presented
absorbance of 0.3 in less than 8 hours of incubation, which confirmed its tolerance to bile salts. L. acidophilus strain A5,
and L. crispatus A2, A4 and A6 had survival rates of 74.05%, 83.90%, 84.41% and 78.7% after 1 hour of incubation in pH
2.0, respectively. All Lactobacillus sp. strains were resistant to the antibiotics avilamicin and avoparcin and sensitive to
zinc bacitracin. Based on the results the Lactobacillus sp. strains isolated can be considered suitable to be used as probiotics
in poultry due to its tolerance to bile salts and hydrochloride acid, and resistance to growth promoters.
KEY-WORDS: Lactobacillus acidophilus. Lactobacillus crispatus. Growth promoters. Digestive fluids
RESUMO
No presente estudo, cepas de Lactobacillus acidophilus e L. crispatus foram isoladas no trato intestinal de galinhas
e submetidas a testes “in vitro” para verificar sua tolerância aos fluidos digestivos (sais biliares e ácido clorídrico) e
resistência aos antibióticos comumente usados como promotores de crescimento em avicultura. Todas as culturas de
lactobacilos apresentaram absorbância de 0,3 em menos de 8 horas de incubação, o que confirmou sua tolerância aos sais
biliares. As cepas A5 de L. acidophilus e A2, A4 e A6 de L. crispatus demostraram respectivamente, taxas de sobrevivência
de 74,05%, 83,90%, 84,41% e 78,7% após uma hora de incubação em pH 2,0. Todas as cepas de Lactobacillus sp. foram
resistentes aos antibióticos avilamicina e avoparcina e sensíveis à bacitracina de zinco. Com base nos resultados obtidos,
cepas de Lactobacillus sp. podem ser consideradas para uso como probiótico em avicultura devido a sua tolerância aos
sais biliares e ao ácido clorídrico e resistência a promotores de crescimento.
PALAVRAS-CHAVE: Lactobacillus acidophilus. Lactobacillus crispatus. Promotores de crescimento. Fluidos digestivos.
1 Aluno do programa de pós-graduação em medicina veterinária da FCAV, Unesp, Campus de Jaboticabal.
End. Eletrôn.: [email protected]
2 Docentes do Departamento de Patologia Animal da FCAV, Unesp, Campus de Jaboticabal.
3 Aluno do programa de pós-graduação em nutrição animal - ESALQ.
4 Médico veterinário - Granja Planalto Ltda
5 Aluno do programa de pós-graduação em Cirurgia Veterinária da FCAV, Unesp, Campus de Jaboticabal.
115
A.A. MEDEIROS, R. P. SCHOCKEN-ITURRINO, J. O. SORBARA, A. C. PAULILLO, A. BERCHIERI, B.P.H. RONCHI, A. P. M. CARNEIRO. Potential
use of lactobacilli as probiotics in poultry verified by its resistance to bile salts, hydrochloric acid and antibiotics./ Uso potencial de lactobacilos como probiótico
em avicultura verificado por sua resistência aos sais biliares, ácido clorídrico e antibióticos. Ars Veterinaria, Jaboticabal, SP, Vol. 19, nº 2, 115-118, 2003.
INTRODUCTION
Probiotics are considered to be a natural and
efficient alternative to growth promoters, since the
continuous and sometimes indiscriminate use of antibiotics
has been leading to development of resistance in bacterial
species. The increasing concern about the control and
safety in food production makes the use of such growth
promoters in animals a great concern worldwide.
The word “probiotic” was first reported in 1974,
and in agreement with its definition comprises microorganisms or substances that contribute to the intestinal microbial balance (FULLER, 1986). Microorganisms used
as probiotic must possess basic characteristics such as capacity of adapting to the intestine of the host, and surviving during its passage though the gastrointestinal system,
i.e. resistance to digestive fluids – hydrochloric acid and
bile salts. Additionally, it must not deteriorate the feeds
which serves as a vehicle for the microorganism (FULLER,
1992).
Lactobacilli are among the most useful bacterial
species in the gastrointestinal tract in poultry, and
Lactoabacillus acidophilus is considered to be the most
effective in protecting the gastrointestinal system against
pathogens. Besides its protective effect, the increase in
production of eggs and alimentary conversion in laying
hens fed with ration supplemented with L. acidophilus was
already described (HADDADIN et al., 1996).
The aim of the present study was to test in vitro
Lactobacillus sp. strains in order to assess its tolerance to
digestive fluids and evaluate its resistance to antibiotics
commonly used in poultry ration as growth promoters.
MATERIAL AND METHODS
The six strains used in the study comprised: L. acidophilus A1, L. acidophilus A3, L. acidophilus A5, L.
crispatus A2, L. crispatus A4 and L. crispatus A6, which
were isolated from healthy chicken raised extensively.
After bacterial isolation, the strains were maintained at 70°C in no fat milk (NFM) reconstituted to 10%.
The tolerance to bile salts was evaluated according
to the methodology described by GILLILAND et al. (1985)
as follows: 1% of the culture was inoculated into Man
Rogosa and Sharpe broth (MRS) (Merck Laboratories,
Darmstadt, Germany), containing bile salts at a concentration of 0.3% (Oxgall™, Difco Laboratories, Detroit,
USA). MRS broth with no bile salts was used as a negative control. Bacterial cultures were incubated at 37°C,
and the cell growth was monitored with a spectrophotom-
eter (Shimadzu UV 140-02) at a wavelength of 600nm until
it reached absorbance of 0.3. In addition, for evaluating
the tolerance of the lactobacilli strains to hydrochloric acid,
a 0.16 M HCl solution was used in order to reduce the pH
of 100 mL 10% NFM to 2.0. Those strains were inoculated
in milk at a final concentration of 1% with pH 2.0 or pH 6.4
(control). One, 2 and 3 hours after exposition to hydrochloric acid, a 0.1 N NaOH solution was added to neutralize the test samples, which were plated onto MRS agar by
the pour plate method (YAKULT, 1980). The number of
colony forming units (CFU/mL) was quantified after incubation at 37°C overnight. Both tests were performed in
duplicate.
Sensitivity to antibiotics was determined by the
diffusion method on Mueller Hinton agar, using impregnated disks with the following antibiotics: zinc bacitracin
(10U), virginiamicin (15ppm), avoparcin (10ppm) and
avilamicin (10ppm).
RESULTS AND DISCUSSION
Tolerance to bile salts is an important property of
microorganisms intended to be used as probiotics, since
an inhibitory effect on the growth of intestinal pathogens
might be achieved because of the possible colonization of
the host intestine by those microorganisms.
According to GILLILAND et al. (1985), a microorganism is considered tolerant to bile salts when its bacterial suspension reaches an absorbance value of 0.3 at a
wavelength of 600nm in up to 8 hours of incubation. All
lactobacilli strains suspensions presented absorbance of
0.3 in less than 8 hours of incubation. In the control group,
the mean values of the number of hours for the bacterial
suspension of strains A1, A5 and A6 to reach that value
differed from each other (P<0.05) (Table 1). No significant differences were observed among strains A2, A3 and
A4 (P>0.05), even though their mean values also differed
from those presented by the former strains. As for the
bacterial suspensions supplemented with 0.3% bile salts,
all results were similar to those mean values showed by
the control group, with the exception of strain A3, whose
mean value was also similar to the one presented by strain
A5.
Table 2 shows the results obtained in the tolerance
test to hydrochloric acid, according to the period of incubation (T0 to T4). Lactobacillus crispatus strain A6 presented the best performance among all lactobacilli strains,
since the presence of viable cells was observed after 3
hours of incubation in pH 2.0. L. acidophilus strain A5,
and L. crispatus A2, A4 and A6 had survival rates of 74.05%,
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A.A. MEDEIROS, R. P. SCHOCKEN-ITURRINO, J. O. SORBARA, A. C. PAULILLO, A. BERCHIERI, B.P.H. RONCHI, A. P. M. CARNEIRO. Potential
use of lactobacilli as probiotics in poultry verified by its resistance to bile salts, hydrochloric acid and antibiotics./ Uso potencial de lactobacilos como probiótico
em avicultura verificado por sua resistência aos sais biliares, ácido clorídrico e antibióticos. Ars Veterinaria, Jaboticabal, SP, Vol. 19, nº 2, 115-118, 2003.
Table 1 – Tolerance of Lactobacillus acidophilus and L. crispatus strains to bile salts in MRS medium
No. of hours α
Strain
M RS broth without Oxgall™
Lactobacillus acidophilus
Lactobacillus crispatus
β
MRS broth with 0.3% Oxgall™
A1
4.74 a
5.34 a
A3
3.99 b
4.04 b/c
A5
3.01 c
3.56 c
A2
3.93 b
4.12 b
A4
4.21 b
4.32 b
A6
1.85 d
2.07 d
Table 2 – Number of colony forming units of Lactobacillus acidophilus and L. crispatus incubated in 10% NFM pH 2.0
and plated onto MRS agar plates
α
α
β
β
m e an values
v a lu e s of
o f the
th e log
n u mnumber
b e r o f hof
o ucolony
rs fo r th
e b a cte rial
sp en sio n toobtained
reach abaccording
so rb an c e to
o f the
0 .3 period
a t 6 0 0 nofm incubation.
. m e a n s n o tmeans
sh arinnot
g a
mean
forming
unitssu(CFU/mL)
co m m o n letter d iffer sig n ific an tly (T u k e y test, P < 0 .0 5 ).
sharing a common letter differ significantly (Tukey test, P<0.05). γ NG: no bacterial growth.
Colony Forming Units (CFU/mL) α
Strain
T0
Lactobacillus acidophilus
Lactobacillus crispatus
β
T1
T2
T3
A1
6.11 a
2.95 f
NG γ
NG
A3
6.13 a
3.69 e
NG
NG
A5
5.82 a
4.31 d
NG
NG
A2
6.52 a
5.47 a
NG
NG
A4
5.84 a
4.93 c
NG
NG
A6
6.73 a
5.27 b
3.07
3.04
83.90%, 84.41% and 78.7% after 1 hour of incubation, respectively. Similar survival rates were reported by BOLIN
et al. (1997). Considering the fact that the expected amount
of time for the feeds to be in contact with gastric fluids is
approximately 30 minutes in poultry, those Lactobacillus
sp. strains might be suitable for using as probiotics and
represent an effective alternative to the use of growth promoters.
It is worth noticing that under “in vivo” conditions,
117
the buffer effect of the gastrointestinal content can contribute to the bacterial survival, and that was described by
FULLER (1992) for both crop (pH 4.0 to 5.0) and gizzard
(pH 1.0 to 2.0).
All lactobacilli strains were resistant to the antibiotics avilamicin and avoparcin and sensitive to zinc bacitracin (Table 3). L. crispatus A1 was the only strain which
showed resistance to virginiamicin. The antibiotic concentrations used for the diffusion test were similar to the con-
A.A. MEDEIROS, R. P. SCHOCKEN-ITURRINO, J. O. SORBARA, A. C. PAULILLO, A. BERCHIERI, B.P.H. RONCHI, A. P. M. CARNEIRO. Potential
use of lactobacilli as probiotics in poultry verified by its resistance to bile salts, hydrochloric acid and antibiotics./ Uso potencial de lactobacilos como probiótico
em avicultura verificado por sua resistência aos sais biliares, ácido clorídrico e antibióticos. Ars Veterinaria, Jaboticabal, SP, Vol. 19, nº 2, 115-118, 2003.
Table 3 – Sensitivity of Lactobacillus acidophilus and L. crispatus to different antibiotics used as growth promoters in
poultry by the disk diffusion method
R: resistant S: sensitive.
centrations of growth promoters used in poultry ration.
Resistance to antibiotics was also described by JOHNSON
et al. (1987). Those authors reported resistance in all L.
acidophilus strains to gentamicin, nalidixic acid, and penicillin.
The antibiogram results have to be taken into consideration when preparing the poultry diet, since the sensitivity to some antibiotics makes it impossible that Lactobacillus sp. strains can be used as probiotics in combination with growth promoters such as avoparcin and
avilamicin.
CONCLUSION
Based on the present results, those Lactobacillus
sp. strains can be considered suitable for using as probiotics
in poultry due to its tolerance to digestive fluids and resistance to avilamicin and avoparcin.
ACKNOWLEDGMENTS
We would like to thank the FAPESP (Fundação de
Amparo à Pesquisa do Estado de São Paulo) for the financial support (Project nº. 97/00275-5) and the Granja
Planalto Ltda for given us the animals used in the present
study.
ARTIGO RECEBIDO: Julho/2002
APROVADO: Maio/2003
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