Influence of Intensive and Extensive Breeding on Lactic Acid Bacteria Isolated From Gallus Gallus Domesticus Ceca

Page 1

www.elsevier.com/locate/vetmic

Veterinary Microbiology 120 (2007) 142–150

Influence of intensive and extensive breeding on lactic acid

bacteria isolated from Gallus gallus domesticus ceca

Marcelo R. Souza a, Joao L. Moreira b, Flavio H.F. Barbosa c,Monica M.O.P. Cerqueira a, Alvaro C. Nunes b, Jacques R. Nicoli c,*

a Departamento de Tecnologia e Inspecao de Produtos de Origem Animal, Escola Veterinaria,

Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazilb Departamento de Biologia Geral, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais,

Belo Horizonte, MG, Brazilc Departamento de Microbiologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais,

C.P. 486, 30161-970 Belo Horizonte, MG, Brazil

Received 31 January 2006; received in revised form 10 October 2006; accepted 17 October 2006

Abstract

In the present study, lactic acid bacteria (LAB) from the cecum of chickens bred either under intensive (commercial broilers) or

extensive (free-range) conditions were isolated, identified and some of their probiotic characteristics determined. The LAB

identified by 16S–23S rRNA PCR-ARDRAwere mainly of Lactobacillus species and to a lesser extent of Enterococcus spp. for all

animals. Free-range chickens showed a higher presence of Lactobacillus acidophilus while Lactobacillus reuteri and Lactobacillus

johnsonii were more frequently recovered from commercial broilers. Lactobacillus crispatus was found only in commercial

broilers, Lactobacillus vaginalis and Lactobacillus agilis only in free-range chickens and Lactobacillus salivarius in both types.

Enterococcus isolates from ceca of commercial broilers showed a higher resistance to antimicrobial drugs. Lactobacillus isolates

from free-range chickens presented a higher frequency of in vitro antagonistic activity against selected pathogens than from

commercial broilers. All LAB isolates had predominantly non-hydrophobic surfaces, but with variations depending on age of the

chickens and breeding conditions. Animal breeding caused variation on composition, antimicrobial susceptibility, antagonistic

activity and surface hydrophobicity of LAB from chicken cecum. LAB isolates from ceca of free-range chickens have potential as

probiotic agents, which may be used in the future as replacing the use of antimicrobials as growth promoters.

# 2006 Elsevier B.V. All rights reserved.

Keywords: Lactic acid bacteria; Microbiota; Ceca; Free-range chickens; Broiler chickens; Probiotics

* Corresponding author. Tel.: +55 31 3499 27 57;

fax: +55 31 3499 27 30.

E-mail address: [email protected] (J.R. Nicoli).

0378-1135/$ – see front matter # 2006 Elsevier B.V. All rights reserved

doi:10.1016/j.vetmic.2006.10.019

1. Introduction

The gastrointestinal microbiota plays an important

role in nutrition, detoxification of certain compounds,

growth performance, and protection against infection

.

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M.R. Souza et al. / Veterinary Microbiology 120 (2007) 142–150 143

(McCracken and Lorenz, 2001). Lactic acid bacteria

(LAB) colonize in high population levels (higher than

107 viablecellspergramofcontents) thegastrointestinal

tract of broiler chickens, but LAB species differ

depending on the anatomical site (Zhu and Joerger,

2003). The lactobacilli are predominant populations

in association with other bacterial genera in the

cecum (Zhu et al., 2002), but alone in the crop (Guan

et al., 2003) and the ileum (Knarreborg et al., 2002).

In Brazil, commercial broiler chickens are reared in

large-scale farms and fed with growth promoters.

Free-range chickens (‘‘caipira’’) are not raised

according to modern breeding technologies and eat

only what they find on the ground and corn grains.

The breeding environment and feeding are impor-

tant factors in determining the intestinal microbial

communities (Knarreborg et al., 2002). Growth

promoters in the feed alter the intestinal microbiota

and induce a dissemination of antimicrobial resistance

(WHO/FAO/OIE, 2003). Hence, there is an increasing

interest in developing alternative methods of control-

ling the gastrointestinal microbial ecosystem, enhan-

cing the growth of indıgenous beneficial bacteria (i.e.,

prebiotics) or introducing viable bacteria (i.e.,

probiotics) that benefit the host (Nashashon et al.,

1996). Probiotics are preferentially isolated from the

gastrointestinal microbiota of the animal species of

interest and frequently selected among LAB. Sensi-

tivity to antimicrobials (to avoid resistance transfer),

production of inhibitory substances (to antagonize

pathogenic microorganisms) and hydrophobic cell

wall (to facilitate adhesion to intestinal epithelium) are

other desirable properties for probiotic use.

Because the gastrointestinal microbiota in Brazi-

lian ‘‘caipira’’ chickens is not known, the objectives of

the present work were to determine the influence of

intensive or extensive breeding conditions on LAB

composition in ceca of chickens (Gallus gallus

domesticus) and to evaluate probiotic properties of

these bacteria.

2. Materials and methods

2.1. Animals

Ten Cobb commercial broilers (Gallus gallus

domesticus) intensively raised in a large-scale chicken

farm were used. Five of them were 4 days old and

other five 45 days old. The feed varied according to

age (pre-initial 1–7 days; initial 8–21 days; growth 22–

40 days and finishing 41–45 days old) and contained

corn, soy meal, degommed soy meal, bone and meat

meal, salt and vitamin premixes. The first three age

stages also received coccidiostatics. The manufacturer

did not identify the antimicrobials present in the feed.

Ten free-range chickens (Gallus gallus domesti-

cus), five 14-day-old and other five 180-day-old, bred

under extensive conditions were also used. They did

not have a specific breed, being the result of several

crossings among indigenous farm chickens. Feeding

was based on corn grain, grass, vegetable wastes,

insects, ticks and earthworms. No antimicrobial drug

was used.

2.2. Isolation and physiological characterization

of LAB

The animals were transported to the laboratory and

immediately sacrificed by cervical dislocation. Cecum

was removed under aseptic conditions inside a laminar

flow hood (VECO, Campinas, Brazil). Luminal content

and mucous scraping of each fowl were recovered,

weighed and submitted to serial decimal dilution in

buffered saline (5.61 g NaCl; 1 g KH2PO4; 2 g

Na2HPO4 and 0.11 g KCl in 1000 ml distillated water)

up to 10�5. Materials were introduced immediately

into an anaerobic chamber (Forma Scientific Company,

Marietta, USA, containing an atmosphere of N2 85%,

H2 10% and CO2 5%) and 0.1 ml of each dilution was

spread onto plates containing Man, Rogosa and Sharp

(MRS) agar (Merck, Darmstadt, Germany). The plates

were incubated in the anaerobic chamber for 48 h at

37 8C. Each colony presenting distinct morphology

was isolated, stained by Gram and tested for

catalase. Respiratory tests under aerobic, microaer-

ophilic and anaerobic conditions were also performed

using MRS agar (Difco, Sparks, USA) incubated

during 48 h at 37 8C. Finally, the isolated microorgan-

isms were inoculated into 5 ml MRS broth (Difco) and

anaerobically incubated for 48 h at 37 8C. After

growth, 500 ml of the broth were transferred to

Eppendorf tubes containing 50 ml of sterile glycerol

before freezing at �18 8C. The remaining broth was

used for molecular identification of the isolated

bacteria.

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M.R. Souza et al. / Veterinary Microbiology 120 (2007) 142–150144

2.3. DNA extraction

Chromosomal DNA was isolated from overnight

cultures of all bacterial isolates in 10 ml MRS broth

(Difco). After washing the cells with deionised water,

the pellet was suspended in 1 ml of 5 M LiCl and

incubated for 1 h with constant shaking. After a second

washing with 1 ml of deionised water, the pellet was

suspended in 1 ml protoplasting buffer (50 mM Tris–

HCl pH 8.0, 10 mM EDTA, 10 mg/ml of lysozyme,

100 mg/ml of RNAse). After incubation for 1 h at 37 8Cand centrifugation for 5 min, the pellet was suspended

in 500 ml of protoplasting buffer without lysozyme and

100 ml of 10% sodium dodecyl sulfate were added to

allow cells to lyse. After lysis, the mixturewas extracted

once with phenol, phenol–chloroform–isoamyl alcohol

(25:24:1) and with chloroform–isoamyl alcohol (24:1).

After isopropanol precipitation, the DNAwas dissolved

in 100 ml of TE buffer.

2.4. PCR amplification of 16S–23S rDNA

intergenic spacer

The 16S–23S intergenic spacer region amplification

was carried out according to Tilsala and Alatossava

(1997) by using the primer 16-1A (GAATCGCTAG-

TAATCG) that anneals to a conserved region of the 16S

rRNA gene and primer 23-1B (GGGTTCCCCCA-

TTCGGA) that anneals to a conserved region of the 23S

rRNA gene using a PTC-1001 Thermal cycler (MJ

Research). The reaction mixture (50 ml) contained

10 pM of each primer, 0.2 mM of each deoxyribonu-

cleotide triphosphate, reaction buffer, 5 U of Taq DNA

polymerase (Phoneutria Biotecnologia & Servicos,

Belo Horizonte, Brazil) and 5 ml of template DNA

solution. The amplification program was 95 8C for

2 min, 35 cycles of 95 8C for 30 s, 55 8C for 1 min,

72 8C for 1 min and finally 72 8C for 10 min. PCR

products were electrophoresed in a 1.4% agarose gel

and visualized by UV transillumination after staining

with an ethidium bromide solution (5 mg/ml).

2.5. Amplified ribosomal DNA restriction analysis

(16S–23S rRNA)

PCR-ARDRA (amplified ribosomal DNA restriction

analysis) of the 16S–23S rRNA intergenic regions was

performed according to Moreira et al. (2005). Briefly,

the 16S–23S rRNA intergenic spacer regions of LAB

were amplified by PCR and submitted to restriction

analysis by a set of 12 enzymeschosen after compilation

of nucleotide sequences already deposited at the

GenBank and in silico restriction digestion using the

Webcutter 2.0 tool (Max Heiman 1997; http://rna.lund-

berg.gu.se/cutter2/). SphI, NcoI and NheI enzymes cut

inside 16S gene, SspI, SfuI, DraI, VspI, HincII and

EcoRI enzymes cut inside the intergenic region, and

AvrII and HindIII enzymes cut inside 23S gene.

EcoRV enzyme cut inside spacer region for Lactoba-

cillus casei group and in the 23S gene for Lactobacillus

acidophilus group. For several lactobacilli species no

spacer nucleotide sequences were deposited at the

present timeandonly fragmentsof16Sand/or23Sgenes

were found. All restriction enzymes were purchased

from Promega Corporation (Madison, USA).

2.6. Susceptibility of the microorganisms to

antimicrobial drugs

The susceptibility test to antimicrobials ceftriaxone

(30 mg), amoxicillin (10 mg), nalidixic acid (30 mg),

tetracycline (30 mg), ampicillin (45 mg), vancomycin

(30 mg), oxacillin (1 mg), gentamicin (10 mg), chlor-

amphenicol (30 mg), erythromycin (15 mg), amikacin

(30 mg) and penicillin (10 U) were carried out

according to specific assays for LAB as described by

Charteris et al. (1998). The isolated microorganisms

were grown on MRS agar (Difco), under anaerobiosis,

for 24–48 h at 37 8C. From their colonies, concentra-

tions of 108 viable cells (McFarland scale) were

prepared using 3.5 ml of 0.85% buffered saline. Swabs

from those dilutions were spread onto the surface of

14 cm diameter plates containing MRS agar (Difco).

The drug disks (Oxoid, Basingstoke, England) were

distributed on the surface of the plates, which were

incubated under anaerobiosis, for 24–48 h at 37 8C.

Then, the diameters of the inhibition zones were

determined using a digital pachymeter. Quality control

of discs containing the antimicrobials was performed

using E. coli ATCC 25922.

2.7. Determination of the antagonistic activity by

‘‘in vitro’’ assay

The isolated bacteria were cultured in MRS broth

(Difco) for 24 h at 37 8C in the anaerobic chamber.

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M.R. Souza et al. / Veterinary Microbiology 120 (2007) 142–150 145

After growth, an aliquot (5 ml) of the culture was

spotted onto MRS agar (Difco). After incubation at

37 8C for 48 h under anaerobic conditions, the cells

were killed by exposure to chloroform during 20 min.

Residual chloroform was allowed to evaporate and

Petri dishes were overlaid with 3.5 ml of BHI or MRS

soft (0.7%) agar (Difco) which had been inoculated

with 0.2 ml of a 24 h culture of Salmonella enterica

serotype Typhimurium 864, Escherichia coli ATCC

25723, L. acidophilus ATCC 4356, Enterococcus

faecalis ATCC 19433, Listeria monocytogenes ATCC

15313 or Staphylococcus aureus ATCC 29213.

Lactobacillus salivarius 35, Lactobacillus johnsonii

51, L. acidophilus, and L. salivarius 67 isolated and

identified in the present work were also used as

indicator strains. After 24 h of incubation at 37 8C,

under aerobic or anaerobic conditions depending on

the indicator strain, the plates were evaluated for the

presence of a growth inhibition zone.

2.8. Hydrophobic cell surface test

The test was performed according to Perez et al.

(1998). The microorganisms were grown in MRS

broth (Difco), under anaerobic conditions, at 37 8C for

24 h. After three activations, they were centrifuged at

1100–1500 � g for 15 min and the cells twice washed

in buffer (KH2PO4–Na2HPO4 50 mM, pH 7.0),

suspended in KNO3 (0.1 M, pH 6.2) and the optical

density (ODA) determined at 600 nm. Then, 4 ml of

the bacterial suspension were added to 1 ml of xylene

(apolar solvent), chloroform (acid solvent) or ethyl

Table 1

Number and frequency (%) of Lactobacillus spp., Enterococcus spp. and ot

chickens

Microorganism Free-range chickens

14 days 180 days

Lactobacillus. acidophilus 3 (27.2) 5 (26.4)

Lactobacillus agilis 0 (0) 1 (5.3)

Lactobacillus crispatus 0 (0) 0 (0)

Lactobacillus johnsonii 1 (9.1) 0 (0)

Lactobacillus reuteri 2 (18.2) 2 (10.5)

Lactobacillus salivarius 2 (18.2) 3 (15.8)

Lactobacillus vaginalis 0 (0) 2 (10.5)

LAB 1 (9.1) 2 (10.5)

Enterococcus spp. 2 (18.2) 4 (21.0)

Total 11 (100) 19 (100)

acetate (basic solvent). After 5 min, the phases were

vortexed for 2 and 60 min later, after the separation of

the phases, the OD600 nm (ODB) was determined in the

aqueous phase. The percentage of bacterial adhesion

to the solvents was obtained according to the

following formula: [(ODA �ODB) � 100)]/ODA.

2.9. Statistical analysis

The data were statistically analyzed using the

Fisher exact and Kruskal–Wallis tests, at a probability

level of 0.05, using the Saeg 8.1 software.

3. Results and discussion

3.1. Cecum microbiota

Table 1 shows the distribution of lactic acid bacteria

isolated from the cecum of the animals. The isolated

that were not identified at the genus level were

considered as LAB. Seven different Lactobacillus

species were identified in the ceca of the animals.

Young and finished broilers, either free or commercially

bred, presented similar number of Lactobacillus

species. Lu et al. (2003) also observed that lactobacilli

diversity in chicken cecum did not increase throughout

the life of the animals. Comparing to Enterococcus spp.,

more Lactobacillus species were observed in all the

animals, except for 4-day-old broiler chickens, in which

the number of Enterococcus species was similar to

that of Lactobacilli. Alterations of the microbiota

her LAB isolated from cecum of commercial broilers and free-range

Commercial broilers

Total 4 days 45 days Total

8 (26.7) 0 (0) 1 (5.9) 1 (2.5)

1 (3.3) 0 (0) 0 (0) 0 (0)

0 (0) 1 (4.3) 3 (17.6) 4 (10.0)

1 (3.3) 4 (17.3) 1 (5.9) 5 (12.5)

4 (13.3) 3 (13.0) 4 (23.6) 7 (17.5)

5 (16.7) 2 (8.9) 3 (17.6) 5 (12.5)

2 (6.7) 0 (0) 0 (0) 0 (0)

3 (10.0) 1 (4.3) 3 (17.6) 4 (10.0)

6 (20.0) 12 (52.2) 2 (11.8) 14 (35.0)

30 (100) 23 (100) 17 (100) 40 (100)

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M.R. Souza et al. / Veterinary Microbiology 120 (2007) 142–150146

composition in the cecum of broiler chickens were also

reported by Sarra et al. (1992), while Jin et al. (1998)

also found that Lactobacillus is as a genus of LAB

frequently found in chicken gut microbiota. This is

highly desirable, since Lactobacillus may be consid-

ered beneficial bacteria (Fuller, 1989). Their positive

effects in gut ecosystem include immunomodulation,

bacteriocin production and lactic and acetic acid

production, which decrease the local pH and avoid

undesirable bacteria to develop. L. acidophilus was the

most frequently identified species in free-range chick-

ens, while Enterococcus spp., L. johnsonii and

Lactobacillus reuteri were isolated in larger numbers

in commercial broilers. Lactobacillus crispatus was

found only in commercial broilers, Lactobacillus

vaginalis and Lactobacillus agilis only in free-range

chickens and L. salivarius in both. Several authors

(Shome et al., 2001; Gong et al., 2002; Zhu and Joerger,

2003) also found these bacteria in the ceca of broiler

chickens. These differences may be caused by diet,

Table 2

Frequency of resistance to antimicrobial drugs (%) of Lactobacillus and En

chickens

Fowls Antimicrobials

CTX AMO ANL TET AMP VAN

E14

L 0 0 100 75 0 50

E 0 0 100 100 0 50

E180

L 0 0 100 53.8 0 38.5

E 0 0 75 0 0 0

TE

L 0 0 100 71.4 0 42.9

E 0 0 83.3 33.3 0 16.7

I4

L 0 0 100 90 0 50

E 8.3 16.7 100 58.3 0 0

I45

L 0 0 100 75 0 58.3

E 0 0 100 100 0 100

TI

L 0 0 100 81.8 0 54.5

E 7.14 14.3 100 64.3 0 14.3

TG

L 0 0 100 72.0 0 48.8

E 5 10 45 55 0 15

E14: 14 days old, free-range chickens; E180: 180 days old, commercial

broilers; I45: 45 days old, commercial broilers; TI: total commercial bro

(ceftriaxone), AMO (amoxicillin), ANL (nalidixic acid), TET (tetracyclin

(gentamicin), CLO (chloramphenicol), ERI (erythromycin), AMK (amika

level of stress and the use of growth promoter drugs as

suggested by Alzueta et al. (2003) and Wage (2003).

Enterococcus spp. were also found in cecum microbiota

of broiler chicken (Zhu and Joerger, 2003). Even though

they have been used as probiotics for fowls (Maiorka

et al., 2001), recent publications showed a link between

that genus and the presence and transmission of

antimicrobial resistance genes to other microorganisms

(Kolar et al., 2002; Edens, 2003).

3.2. Antimicrobial susceptibility

All the isolates were sensitive to penicillin and

ampicillin (Table 2). Kolar et al. (2002) also reported

this high sensitivity in bacteria from chicken gut. Even

though penicillin G procaine was once the leading

antimicrobial drug used in farm animals, it was

replaced, especially in large modern scale aviculture

(Edens, 2003). The decrease of its use in commercial

broiler as well as in other livestock may explain these

terococcus isolated from ceca of commercial broilers and free-range

OXA GNT CLO ERI AMK PEN

67.5 100 12.5 0 87.5 0

100 100 0 0 100 0

61.5 84.6 7.7 7.6 79.9 0

0 25 0 0 25 0

71.4 90.5 9.5 4.7 80.9 0

33.33 50 0 0 50 0

70 100 0 40 90 0

66.7 83.3 16.7 16.7 91.7 0

66.7 91.7 0 16.7 83.3 0

100 100 0 0 100 0

68.2 95.4 0 27.3 86.4 0

71.4 85.7 14.3 28.6 92.9 0

69.8 93.0 4.6 16.3 83.7 0

60 75 10 10 80 0

broilers; TE: total free-range chickens; I4: 4 days old, commercial

ilers; TG: total; L: Lactobacillus spp.; E: Enterococcus spp. CTX

e), AMP (ampicillin), VAN (vancomycin), OXA (oxacillin), GNT

cina) and PEN (penicillin).

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M.R. Souza et al. / Veterinary Microbiology 120 (2007) 142–150 147

results. Resistance to ceftriaxone and amoxicillin was

only observed in three Enterococci isolated from 4-day-

old commercial broiler chicks. Concerning suscept-

ibility to amoxicillin, there are reports showing the

resistance of Escherichia and Salmonella isolated from

chicken gut. As the literature does not mention chicken

gut LAB resistant to this drug, a possible resistance

gene transfer between Enterobacteriaceae and LAB

within cecum microbiota could explain the results. All

the microorganisms isolated from 14-day-old free-

range chickens and from all the commercial broilers

were resistant to nalidixic acid. Kolar et al. (2002) also

described this fact in the Czech Republic. The

resistance of Lactobacillus to erythromycin was

observed essentially for L. reuteri. According to

Axelsson et al. (1998), the L. reuteri resistance is

related to a plasmidial gene. The presence of some

strains of Lactobacillus spp. or Enterococcus spp.

resistant to chloramphenicol is highly undesirable,

since the use of that drug in livestock is prohibited in

Brazil (Brasil, 2003). High numbers of LAB isolates

were resistant to gentamicin and amikacin, irrespective

of breeding conditions or age. Kozlova et al. (1992) also

reported the resistance of 136 samples of Lactobacillus

isolated from fowls and 23 from elder human subjects to

several antimicrobials, being most of the bacteria

resistant to aminoglycosids. Only half of the Lactoba-

cillus isolates were resistant to vancomycin. This is

quite interesting, since vancomycin resistance is

considered as intrinsic to Lactobacillus (Kolar et al.,

2002; Danielsen and Wind, 2003; Wage, 2003). Even

though that resistance is genetically determined,

sensitive bacteria might have lost it probably due to

the lack of pressure selection.

Table 3 shows a lower (P < 0.05) total resistance

frequency (10.4%) for Enterococcus spp. isolated

from 180-day-old free-range chickens when compared

Table 3

Mean frequency (%) of resistance to all antimicrobial drugs of Lactobacil

breeding and age

Free-range chickens

14 days 180 days Total

Lactobacillus 41.0 a 36.1 a 39.3 a

Enterococcus 45.8 a 10.4 b 22.2 a,b

Total 42.4 a 28.9 b 33.9 b

Different letters (a, b) indicate significant statistical difference between t

to all other groups. Commercial broilers also

presented a higher percentage (P < 0.05) of isolates

resistant to the tested antimicrobials (41.0%) than

free-range chickens (33.9%). This suggests a relation-

ship between the intensive (commercial) breeding

manner (use of antimicrobial growth promoters) and

the induction of higher presence of bacteria resistant to

antimicrobial drugs in Gallus gallus domesticus gut,

hypothesis formerly suggested by Bedford (2000),

Edens (2003) and Wage (2003). On the other hand,

Lactobacillus species showed similar resistance

frequencies independently of chicken age and breed-

ing conditions. Likewise, the results found in the

present work support the determinations of the

European Parliament, which banned the use of growth

promoters for livestock since January 2006 (Council

of the Europe Union, 2003).

3.3. In vitro antagonistic activities

Table 4 shows that, globally, LAB isolated in the

present work presented a highly frequent antagonistic

capacity (86%). However, this characteristic was more

pronounced (P < 0.05) in free-range chickens, inde-

pendently of bacterial genus and age (100%) when

compared to commercial broilers (76%). This reduced

antagonistic capacity was due to data obtained when

Lactobacillus and Enterococcus isolates from 45-day-

old commercial broilers chickens were tested

(Table 4). Concerning the intensity of antagonistic

phenomenon (determined as the size of inhibitory

zone), higher capacity was observed with Lactoba-

cillus samples from 4-day-old commercial broilers

when compared to Enterococcus (Table 4). The

Lactobacillus species more frequently found in either

breeding groups (L. acidophilus, L. reuteri, L.

johnsonii, L. salivarius) showed a higher frequency

lus and Enterococcus isolated from chicken’s ceca, according to the

Commercial broilers Total

4 days 45 days Total

45.0 a 41.0 a 42.8 a 40.7

38.2 a 50.0 a 41.1 a 30.4

42.0 a 37.8 a 41.0 a 39.4

he data in the same line (P < 0.05).

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M.R. Souza et al. / Veterinary Microbiology 120 (2007) 142–150148

Tab

le4

Invi

tro

anta

gonis

mfr

equen

cyan

dm

ean

inhib

itio

nzo

ne

dia

met

er(m

m)

acco

rdin

gto

the

bre

edin

gan

dag

e

Mic

roorg

anis

mF

ree-

range

chic

ken

sT

ota

lex

tensi

ve

Com

mer

cial

bro

iler

sT

ota

lin

tensi

ve

Tota

l

14

day

s1

80

day

s4

day

s4

5d

ays

La

cto

ba

cill

us

spp

.8

/8aA

(10

0%

),

22

.43

aA

13

/13

aA(1

00

%),

22

.41

aA

21

/21

aA(1

00

%),

22

.42

aA

8/1

0ab

A(8

0%

),

30

.91

aA

6/1

2b

A(5

0%

),

22

.85

aA

14

/22

bA

(64

%),

26

.88

aA

35

/43

(81

%),

24

.65

En

tero

cocc

us

spp

.2

/2aA

(10

0%

),

18

.34

aA

4/4

aA(1

00

%),

17

.37

aA

6/6

aA(1

00

%),

17

.86

aA

12

/12

aA(1

00

%),

15

.53

aB

2/3

aA(6

7%

),

18

.67

aA

14

/15

aB(9

3%

),

17

.10

aB

20

/21

(95

%),

17

.48

To

tal

10

/10

aA(1

00

%)

17

/17

aA(1

00

%)

27

/27

aA(1

00

%)

20

/22

aA(9

1%

)8

/15

bA

(53

%)

28

/37

bA

B(7

6%

)5

5/6

4(8

6%

)

Dif

fere

nt

lett

ers

(a,

b,

Aan

dB

)in

dic

ate

sig

nifi

can

tst

atis

tica

ld

iffe

ren

ceb

etw

een

the

dat

ain

the

sam

eli

ne

and

inth

esa

me

colu

mn

(P<

0.0

5).

of antagonistic capacity (89–100%) when compared

(25–70%) to species less frequently recovered (L.

crispatus, L. salivarius, L. vaginalis) (data not shown).

According to Sanders (1999) and Edens (2003), L.

acidophilus in vivo antagonistic activity is mediated

by the immune modulation in the gut, leading to a

proper balance in the bacterial populations that share

the same habitat. For L. reuteri the well-known

production of bacteriocin reuterin was probably

responsible for the in vitro inhibitory activity (Edens,

2003). Vandervoorde et al. (1991) also described in

vitro antagonistic activity of Lactobacillus spp. and

Enterococcus spp. recorded from chicken’s crop

against S. Typhimurium. Andreatti Filho and Crocci

(2002) reported that the oral administration of

lyophilized anaerobic cecum microbiota to broiler

chickens decreased the intestinal colonization as well

as the fecal excretion of S. Typhimurium.

3.4. Microbial adhesion test to solvents

The highest values of the adhesion of the micro-

organisms were obtained with chloroform (Table 5).

However, considering all the results, most bacteria

isolated from the ceca of the studied chickens showed

predominantly hydrophilic cellular surface. There was

no difference between results of adhesion tests

comparing Lactobacillus with Enterococcus (data not

shown). Less hydrophilic surfaces (P < 0.05) were

observed for younger animals (14 days old free-range

able 5

n vitro adhesion test (%) to apolar (xylene), acid (chloroform) and

asic (ethyl acetate) solvents of the LAB isolated from ceca of

ommercial broilers and free-range chickens

Xylene Chloroform Ethyl acetate

ree-range chickens

14 days 33.65 aA 47.99 bA 31.47 aA

180 days 6.77 aB 29.46 bB 10.89 aB

otal extensive 20.21 aA 38.73 bA 21.18 aA

ommercial broilers

4 days 21.52 aA 29.78 aB 25.35 aA

45 days 4.18 aB 14.53 aC 6.13 aC

otal intensive 12.85 aB 22.16 aB 15.74 aBC

ifferent letters (a, b, A, and B) indicate significant statistical

ifference between the data in the same line and in the same column

P < 0.05).

T

I

b

c

F

T

C

T

D

d

(

Page 8

M.R. Souza et al. / Veterinary Microbiology 120 (2007) 142–150 149

chickens and 4 days old commercial broilers) when

compared to older ones as well as for all free-range

chickens when compared to commercial broilers. Jin

et al. (1996) described hydrophobic differences

between L. acidophilus and Lactobacillus fermentum

cells isolated from chicken intestines, and in a posterior

report found high levels of in vitro adhesion of L.

acidophilus cells (Jin et al., 1998). Gusils et al. (1999)

showed differences among adhesion values among

bacteria of distinct species. Garriga et al. (1998) related

differences among L. salivarius strains CTC2183 and

2197 in colonizing capacity when inoculated in chicks’

gizzard or ceca.

Concluding, animal breeding and age caused

variation on composition, antimicrobial susceptibility,

antagonistic activity and surface hydrophobicity of

LAB from chicken cecum.

Acknowledgments

This study was supported by grants and fellowships

from Conselho Nacional do Desenvolvimento Cientı-

fico e Tecnologico (CNPq) and Fundacao de Amparo a

Pesquisa do Estado de Minas Gerais (FAPEMIG). The

authors are grateful to Maria Gorete Barbosa Ribas for

valuable technical help.

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