Influence of intensive and extensive breeding on lactic acid bacteria isolated from Gallus gallus domesticus ceca

Veterinary Microbiology 120 (2007) 142–150 www.elsevier.com/locate/vetmic

Influence of intensive and extensive breeding on lactic acid bacteria isolated from Gallus gallus domesticus ceca Marcelo R. Souza a, Joa˜o L. Moreira b, Fla´vio H.F. Barbosa c, ´ lvaro C. Nunes b, Jacques R. Nicoli c,* Moˆnica M.O.P. Cerqueira a, A a

Departamento de Tecnologia e Inspec¸a˜o de Produtos de Origem Animal, Escola Veterina´ria, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil b Departamento de Biologia Geral, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil c Departamento de Microbiologia, Instituto de Cieˆncias Biolo´gicas, 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

1. Introduction * Corresponding author. Tel.: +55 31 3499 27 57; fax: +55 31 3499 27 30. E-mail address: [email protected] (J.R. Nicoli).

The gastrointestinal microbiota plays an important role in nutrition, detoxification of certain compounds, growth performance, and protection against infection

0378-1135/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2006.10.019

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

(McCracken and Lorenz, 2001). Lactic acid bacteria (LAB) colonize in high population levels (higher than 107 viable cells per gram of contents) 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 important 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 controlling the gastrointestinal microbial ecosystem, enhancing the growth of indı´genous 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. Sensitivity 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 Brazilian ‘‘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

143

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 domesticus), 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, microaerophilic and anaerobic conditions were also performed using MRS agar (Difco, Sparks, USA) incubated during 48 h at 37 8C. Finally, the isolated microorganisms 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.

144

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

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 8C and 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 mixture was 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 (GAATCGCTAGTAATCG) that anneals to a conserved region of the 16S rRNA gene and primer 23-1B (GGGTTCCCCCATTCGGA) 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 deoxyribonucleotide triphosphate, reaction buffer, 5 U of Taq DNA polymerase (Phoneutria Biotecnologia & Servic¸os, 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 enzymes chosen 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.lundberg.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 Lactobacillus casei group and in the 23S gene for Lactobacillus acidophilus group. For several lactobacilli species no spacer nucleotide sequences were deposited at the present time and only fragments of 16S and/or 23S genes 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), chloramphenicol (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, concentrations 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.

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

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 Pe´rez 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

145

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

Table 1 Number and frequency (%) of Lactobacillus spp., Enterococcus spp. and other LAB isolated from cecum of commercial broilers and free-range chickens Microorganism

Free-range chickens 14 days

Lactobacillus. acidophilus Lactobacillus agilis Lactobacillus crispatus Lactobacillus johnsonii Lactobacillus reuteri Lactobacillus salivarius Lactobacillus vaginalis LAB Enterococcus spp. Total

3 0 0 1 2 2 0 1 2

(27.2) (0) (0) (9.1) (18.2) (18.2) (0) (9.1) (18.2)

11 (100)

Commercial broilers

180 days 5 1 0 0 2 3 2 2 4

(26.4) (5.3) (0) (0) (10.5) (15.8) (10.5) (10.5) (21.0)

19 (100)

Total 8 1 0 1 4 5 2 3 6

(26.7) (3.3) (0) (3.3) (13.3) (16.7) (6.7) (10.0) (20.0)

30 (100)

4 days 0 0 1 4 3 2 0 1 12

(0) (0) (4.3) (17.3) (13.0) (8.9) (0) (4.3) (52.2)

23 (100)

45 days 1 0 3 1 4 3 0 3 2

(5.9) (0) (17.6) (5.9) (23.6) (17.6) (0) (17.6) (11.8)

17 (100)

Total 1 0 4 5 7 5 0 4 14

(2.5) (0) (10.0) (12.5) (17.5) (12.5) (0) (10.0) (35.0)

40 (100)

146

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

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 (Ko´lar et al., 2002; Edens, 2003).

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 considered 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 chickens, 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,

3.2. Antimicrobial susceptibility All the isolates were sensitive to penicillin and ampicillin (Table 2). Ko´lar 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

Table 2 Frequency of resistance to antimicrobial drugs (%) of Lactobacillus and Enterococcus isolated from ceca of commercial broilers and free-range chickens Fowls

Antimicrobials CTX

E14 L E E180 L E TE L E I4 L E I45 L E TI L E TG L E

AMO

ANL

TET

AMP

VAN

OXA

GNT

CLO

ERI

AMK

PEN

75 100

0 0

50 50

67.5 100

100 100

12.5 0

0 0

87.5 100

0 0

0 0

0 0

100 100

0 0

0 0

100 75

53.8 0

0 0

38.5 0

61.5 0

84.6 25

7.7 0

7.6 0

79.9 25

0 0

0 0

0 0

100 83.3

71.4 33.3

0 0

42.9 16.7

71.4 33.33

90.5 50

9.5 0

4.7 0

80.9 50

0 0

0 16.7

100 100

90 58.3

0 0

50 0

70 66.7

100 83.3

0 16.7

40 16.7

90 91.7

0 0

0 0

100 100

0 0

58.3 100

66.7 100

91.7 100

0 0

16.7 0

83.3 100

0 0

0 7.14

0 14.3

100 100

81.8 64.3

0 0

54.5 14.3

68.2 71.4

95.4 85.7

0 14.3

27.3 28.6

86.4 92.9

0 0

0 5

0 10

100 45

72.0 55

0 0

48.8 15

69.8 60

93.0 75

4.6 10

16.3 10

83.7 80

0 0

0 8.3 0 0

75 100

E14: 14 days old, free-range chickens; E180: 180 days old, commercial broilers; TE: total free-range chickens; I4: 4 days old, commercial broilers; I45: 45 days old, commercial broilers; TI: total commercial broilers; TG: total; L: Lactobacillus spp.; E: Enterococcus spp. CTX (ceftriaxone), AMO (amoxicillin), ANL (nalidixic acid), TET (tetracycline), AMP (ampicillin), VAN (vancomycin), OXA (oxacillin), GNT (gentamicin), CLO (chloramphenicol), ERI (erythromycin), AMK (amikacina) and PEN (penicillin).

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

results. Resistance to ceftriaxone and amoxicillin was only observed in three Enterococci isolated from 4-dayold commercial broiler chicks. Concerning susceptibility 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 freerange chickens and from all the commercial broilers were resistant to nalidixic acid. Ko´lar 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 Lactobacillus isolates were resistant to vancomycin. This is quite interesting, since vancomycin resistance is considered as intrinsic to Lactobacillus (Ko´lar 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

147

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 relationship 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 breeding 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, independently 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-dayold 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 Lactobacillus 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

Table 3 Mean frequency (%) of resistance to all antimicrobial drugs of Lactobacillus and Enterococcus isolated from chicken’s ceca, according to the breeding and age Free-range chickens

Commercial broilers

Total

14 days

180 days

Total

4 days

45 days

Total

Lactobacillus Enterococcus

41.0 a 45.8 a

36.1 a 10.4 b

39.3 a 22.2 a,b

45.0 a 38.2 a

41.0 a 50.0 a

42.8 a 41.1 a

40.7 30.4

Total

42.4 a

28.9 b

33.9 b

42.0 a

37.8 a

41.0 a

39.4

Different letters (a, b) indicate significant statistical difference between the data in the same line (P < 0.05).

55/64 (86%) 28/37 bAB (76%) 8/15 bA (53%) 20/22 aA (91%) 27/27 aA (100%) 17/17 aA (100%) Total

Enterococcus spp.

10/10 aA (100%)

6/12 bA (50%), 22.85 aA 2/3 aA (67%), 18.67aA 8/10 abA (80%), 30.91 aA 12/12 aA (100%), 15.53 aB

14 days

Different letters (a, b, A and B) indicate significant statistical difference between the data in the same line and in the same column (P < 0.05).

35/43 (81%), 24.65 20/21 (95%), 17.48 45 days 4 days

21/21 aA (100%), 22.42 aA 6/6 aA (100%), 17.86 aA 13/13 aA (100%), 22.41 aA 4/4 aA (100%), 17.37 aA Lactobacillus spp.

8/8 aA (100%), 22.43 aA 2/2 aA (100%), 18.34 aA

180 days

14/22 26.88 14/15 17.10

Total intensive Commercial broilers Total extensive Free-range chickens Microorganism

Table 4 In vitro antagonism frequency and mean inhibition zone diameter (mm) according to the breeding and age

bA (64%), aA aB (93%), aB

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

Total

148

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 microorganisms 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 Table 5 In vitro adhesion test (%) to apolar (xylene), acid (chloroform) and basic (ethyl acetate) solvents of the LAB isolated from ceca of commercial broilers and free-range chickens Xylene

Chloroform

Ethyl acetate

Free-range chickens 14 days 33.65 aA 180 days 6.77 aB

47.99 bA 29.46 bB

31.47 aA 10.89 aB

Total extensive

20.21 aA

38.73 bA

21.18 aA

Commercial broilers 4 days 21.52 aA 45 days 4.18 aB

29.78 aB 14.53 aC

25.35 aA 6.13 aC

Total intensive

22.16 aB

15.74 aBC

12.85 aB

Different letters (a, b, A, and B) indicate significant statistical difference between the data in the same line and in the same column (P < 0.05).

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

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 Tecnolo´gico (CNPq) and Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas Gerais (FAPEMIG). The authors are grateful to Maria Gorete Barbosa Ribas for valuable technical help.

References Alzueta, C., Rodriguez, M.L., Cutuli, M.T., 2003. Effect of whole and demucilaged linseed in broiler chicken diets on digesta viscosity, nutrient utilization and intestinal microflora. Brit. Poultry Sci. 44, 67–74. Andreatti Filho, R.L., Crocci, A.J., 2002. Efeito protetor da microbiota cecal congelada e liofilizada sobre a infecc¸a˜o experimental de frangos de corte por Salmonella enterica sorovar Enteritidis. Arq. Br. Med. Vet. Zootec. 54, 140–145. Axelsson, L.T., Ahrne, S.E.I., Andersson, M.C., Stahl, S.R., 1998. Identification and cloning of a plasmid-encoded erythromycin resistance determinant for Lactobacillus reuteri. Plasmid 20, 171–174. Bedford, M., 2000. Removal of antibiotic growth promoters from poultry diets: implications and strategies to minimize subsequent problems. World Poultry Sci. J. 56, 347–365. Brasil Ministe´rio da Agricultura, Pecua´ria e Abastecimento, 2003. Instruc¸a˜o Normativa no. 9 de 27/06/2003. Proibic¸a˜o de Cloranfenicol e Nitrofuranos para Uso Veterina´rio e Suscetı´veis de Emprego na Alimentac¸a˜o de Todos os Animais e Insetos. Ministe´rio da Agricultura, Brası´lia, p. 12.

149

Charteris, W.P., Kelly, P.M., Morelli, L., Collins, J.K., 1998. Antibiotic susceptibility of potentially probiotic Lactobacillus species. J. Food Protect. 61, 1636–1643. Council of the Europe Union, 2003. Council Regulation on the Authorization of the Additive Avilamycin in Feeding Stuffs. Accessed October 15, 2003. Online: http://register.consilium.eu.int/pdf/em/03/st06/st06120en03.pdf. Danielsen, N., Wind, A., 2003. Susceptibility of Lactobacillus spp. to antimicrobial agents. Intern. J. Food Microbiol. 82, 1–11. Edens, F.W., 2003. An alternative for antibiotic use in poultry: probiotics. Rev. Br. Cieˆnc. Avı´cola 5, 1–40. Fuller, R., 1989. Probiotics in man and animals. J. Appl. Bacteriol. 66, 365–378. Garriga, M., Pascual, M., Monfort, J.M., Hugas, M., 1998. Selection of lactobacilli for chicken probiotics adjuncts. J. Appl. Microbiol. 84, 125–132. Gong, J., Forster, R.J., Yu, H., Chambers, J.R., Wheatcroft, R., Sabour, P.M., Chen, S., 2002. Molecular analysis of bacterial populations in the ileum of broiler chickens and comparison with bacterial in the caecum. FEMS Microbiol. 41, 171– 179. Guan, L.L., Hagen, K.E., Tannock, G.W., Korver, D.R., Fasenko, G.M., Alisson, G.E., 2003. Detection and identification of Lactobacillus species in crops of broilers of different ages by using PCR-denaturing gradient gel electrophoresis and amplified ribosomal DNA restriction analysis. Appl. Environ. Microbiol. 69, 6750–6757. Gusils, C., Gonzalez, S.M., Oliver, G., 1999. Lactobacilli isolated from chicken intestines: potential use as probiotics. J. Food Protect. 62, 252–256. Jin, L.Z., Ho, Y.W., Ali, M.A., Abdullah, N., Ong, K.B., Jaladulin, S., 1996. Effect of adherent Lactobacillus sp. on in vitro adherence of Salmonella to epithelial cells of chicken. J. Appl. Bacteriol. 81, 201–206. Jin, L.Z., Ho, Y.W., Abdullah, N., Ali, M.A., Jalaludin, S., 1998. Effects of adherent Lactobacillus cultures on growth, weight of organs and intestinal microflora and volatile fatty acids in broilers. Anim. Feed Sci. Technol. 70, 197–209. Knarreborg, A., Simon, M.A., Engberg, R.M., Jensen, B.B., Tannock, G.W., 2002. Effect of dietary fat source and subtherapeutic levels of antibiotic on the bacterial community in the ileum of broiler chickens at various ages. Appl. Environ. Microbiol. 68, 5918–5924. Ko´lar, M., Pantu`e`ek, R., Bardoo`, J., Va´gnerova´, I., Typovska´, H., Doskar, J., Va´lka, I., 2002. Occurrence of antibiotic-resistant bacterial strains isolated in poultry. Vet. Med. Czech. 47, 52–59. Kozlova, E.V., Malinovskaia, I.V., Aminov, R.I., Kovalenko, N.K., Voronin, A.M., 1992. Antibiotic resistance of Lactobacillus strains. Antibiot. Khimioter. 37, 5–12. Lu, J., Idris, U., Harmon, B., Hofacre, C., Maurer, J.J., Lee, M.D., 2003. Diversity and succession of the intestinal bacteria community of the maturing broiler chicken. Appl. Environ. Microbiol. 69, 6816–6824. Maiorka, A., Santin, E., Sugeta, S.M., Almeida, J.G., Mari, M., 2001. Utilizac¸a˜o de prebio´ticos, probio´ticos ou simbio´ticos em dietas para frangos. Rev. Br. Cieˆnc. Avı´cola 3, 15–30.

150

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

McCracken, V.J., Lorenz, R.G., 2001. The gastrointestinal ecosystem: a precarious alliance among epithelium, immunity and microbiota. Cell Microbiol. 3, 1–11. Moreira, J.L.S., Mota, R.M., Horta, M.F., Teixeira, S.M.R., Neumann, E., Nicoli, J.R., Nunes, A.C., 2005. Identification to the species level of Lactobacillus isolated in probiotic prospecting studies of human, animal or food origin by 16S-23S rRNA restriction profiling. BMC Microbiol. 5, 15–20. Nashashon, S.N., Nakaue, H.S., Mirosh, L.W., 1996. Performance of single comb white Leghorn layers fed a diet with a live microbial during the growth and egg laying phases. Anim. Feed Sci. Technol. 57, 25–38. Pe´rez, P., Minaard, Y., Disalvo, E., De Antoni, G., 1998. Surface properties of bifidobacterial strains of human origin. Appl. Environ. Microbiol. 64, 21–26. Sanders, M.E., 1999. Probiotics. Food Technol. 53, 67–77. Sarra, P.G., Morelli, L., Bottazzi, F., 1992. The lactic microflora of fowl. The Lactic Acid Bacteria in Health and Disease, vol. 1. Elsevier, New York. Shome, B.R., Senani, S., Padhi, M.K., Saha, S.K., Rai, R.B., Shome, R., 2001. Isolation, identification and characterisation of autochthonous Lactobacillus from chicken intestine. Indian Vet. J. 78, 278–281.

Tilsala, A.T., Alatossava, T., 1997. Development of oligonucleotide primers from the 16S–23S rDNA intergenic sequences for identifying different dairy and probiotic lactic acid bacteria by PCR. Intern. J. Food Microbiol. 35, 49–56. Vandervoorde, L., Christiaens, H., Verstraete, W., 1991. In vitro appraisal of the probiotic value of intestinal lactobacilli. World J. Microbiol. Technol. 7, 587–592. Wage, S.W., 2003. The Role of Enteric Antibiotics in Livestock Production. Avcare Limited, Camberra, p. 338. WHO/FAO/OIE Food Agricultural Organization/World Health Organization Background document for the Joint WHO/FAO/ OIE expert, 2003.In: Workshop on Non-Human Antimicrobials Usage and Antimicrobials Resistance Scientific Assessment, Geneva, Switzerland, December 1–5. Zhu, X.Y., Joerger, R.D., 2003. Composition of microbiota in content and mucus from caeca of broiler chickens as measured by fluorescent in situ hybridization with group-specific, 16S rRNA-targeted oligonucleotides probes. Poultry Sci. 82, 1242– 1249. Zhu, X.Y., Zhong, T., Pandya, Y., Joerger, R.D., 2002. 16S rRNAbased analysis of microbiota from the cecum of broiler chickens. Appl. Environ. Microbiol. 68, 124–137.