Arthrobacter gandavensis strains

Abstract

An Arthrobacter gandavensis strain having an activity against Clostridium perfringens selected from the strains AP1 filed with DSMZ on Feb. 19, 2014 under the number DSM 28444, AP2 filed with DSMZ on Feb. 19, 2014 under the number DSM 28445, AP3 filed with DSMZ on Feb. 19, 2014 under the number DSM 28446 or AP4 filed with DSMZ on Feb. 19, 2014 under the number DSM 28447.

Claims

1. A method of providing antibacterial activity against Clostridium perfringens, comprising administering a composition including a bacterial strain of Arthrobacter gandavensis strain selected from the group consisting of strain AP1 (DSMZ DSM 28444), strain AP2 (DSMZ DSM 28445), strain AP3 (DSMZ DSM 28446), and strain AP4 (DSMZ DSM 28447) to a non-human animal or human, wherein the bacterial strain produces at least one peptide selected from the group consisting of SEQ ID NOS: 1-16.

2. The method of claim 1, wherein the composition is a liquid or powder.

3. The method of claim 1, wherein the composition is a nutritional additive and is administered to a non-human animal.

4. The method of claim 1, wherein the composition is an animal feed that comprises a nutritional base and a nutritional additive comprising the peptide or bacterial strain, and the animal feed is administered to a non-human animal.

5. The method of claim 1, wherein the composition is a food or drug, and is administered to a human.

6. The method of claim 1, wherein the composition is administered to a non-human animal to treat or reduce the likelihood of developing intestinal dysbacteriosis.

7. The method of claim 1, wherein the composition is administered to a monogastric animal to treat or reduce the likelihood of developing necrotic enteritis.

8. The method of claim 1, wherein the composition is administered to a breeding animal to improve growth performance.

9. The method of claim 1, wherein the composition is administered to a breeding animal to improve zootechnical performance.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Detection of the gene rumC1 by PCR

(2) FIG. 2: Chromosomal profile by pulsed-field gel electrophoresis of the strains AP1, AP2, AP3 and AP4.

(3) FIG. 3: Percentage of survival in the food of the strains AP1, AP2, AP3 and AP4

(4) FIG. 4: Assay of interleukins IL8 by ELISA in the culture supernatant of the cells Caco-2 after contact with the bacteria in the presence or absence of IL1

(5) FIG. 5: The activity tests with the bacterial culture supernatants against the strain Clostridium perfringens

(6) FIG. 6: an activity anti-C. perfringens after a first pre-purification step has been carried out: purification of supernatants on Sep-Pak column with an elution at 40% of acetonitrile (ACN, see WO2008/152252).

(7) FIG. 7: An illustration of the data set forth in Table 10.

(8) The present invention will be illustrated by the following examples

EXAMPLE 1: ISOLATION OF THE BACTERIAL STRAINS RUMC+

(9) The search of cultivable strains harboring the rumC-like genes was undertaken from the cecal and ileal microbiota of pigs.

(10) In a first stage, the bacteria are cultured in the following media: M17: promoting the lactococci LB: allowing the growth of Bacillus sp. and enterococci BEA: medium counter-selecting the Bacteroides group

(11) The clones are then selected for their ability to inhibit the growth of Clostridium perfringens. The presence of the genes rumC is therefore highlighted by PCR (FIG. 1).

(12) TABLE-US-00001 TABLE 1 List of primers used to amplify the different target DNA fragments Hybridization Size of the Temperature amplified Target pair of primers (T.sub.opt) fragment gene(s) FC1 (SEQ ID No. 33)- 55 C. 700 pb rumC1 RC1(SEQ ID No. 34) FC2C3 (SEQ ID No. 35)- 60 C. 800 pb rumC2- RC2C3(SEQ ID No. 36) rumC3 FC4C5 (SEQ ID No. 37)- 55 C. 400 pb rumC4- RC4C5(SEQ ID No. 38) rumC5

(13) Four strains were retained: AP1, AP2, AP3 and AP4.

EXAMPLE 2: IDENTIFICATION OF THE RETAINED STRAINS

(14) 2.1 rDNA 16S

(15) A fragment (about 1550 bp) of the gene encoding for rRNA 16S (corresponding to the positions 8-1541 in the numbering system of Escherichia coli) was amplified by PCR using conserved primers (16F8: 5-AGAGTTTGATCCTGGCTGAG-3 (SEQ ID No. 39) and 16R1541: 5-AAGGAGGTGATCCAGCCGCA-3) (SEQ ID No. 40) and then sequenced.

(16) The sequences obtained were subjected to a comparison in the databanks using a research program of sequence homology of the type BLAST

(17) Strain AP1 (SEQ ID No.)=>99.45% of identity with Arthrobacter gandavensis R 5812

(18) Strain AP2 (SEQ ID No.)=>99.37% of identity with Arthrobacter gandavensis R 5812

(19) Strain AP3 (SEQ ID No.)=>99.44% of identity with Arthrobacter gandavensis R 5812

(20) Strain AP4 (SEQ ID No.)=>99.31% identity with Arthrobacter gandavensis R 5812

(21) 2.2. PFGE Identification

(22) The strains AP1, AP2, AP3 and AP4 belonging to the genus Arthrobacter must be genetically differentiated at the genetic level. The reference technique for identifying at the intra-specific level of the bacterial strains consists in establishing their chromosomal profile by pulsed-field gel electrophoresis (FIG. 2) (Analysis Report No: LR251012Biocane).

(23) The strains AP3 and AP4 appear identical. However, the sequencing of the rumC genes, as well as their reduced peptide sequences seems, for its part, to indicate that AP3 and AP4 are two distinct strains (see Example 4, point 4.2).

EXAMPLE 3: CHARACTERIZATION OF THE STRAINS

(24) 3.1 Resistance to pH and to Bile Salts

(25) The strains are subjected to two treatments to determine their resistance to the acidity and to the bile salts. Acidity: buffer NaCl 0.85%, pH2, containing pepsin (1 mg/mL). Isotonic buffer bile salts (K.sub.2HPO.sub.4 1.24%, H.sub.2PO.sub.4 0.76%, trisodium citrate 0.1%, [NH.sub.4].sub.2SO.sub.4 0.6%, pH6.7) containing 0.2% of bile salts (50% sodium cholate, 50% sodium deoxycholate)

(26) TABLE-US-00002 TABLE 2 Percentage of survival of the different strains AP1 AP2 AP3 AP4 Acidity 0% 15% 12% 90% BS 0% 10% 8% ND BS: Bile salts; ND: Not determined

(27) The strain AP4 resists better the conditions imitating the gastric medium. Generally, all the strains are more sensitive to the bile salts but the survival is however sufficient, except for the strain AP1.

(28) 3.2 Fermentation Parameters

(29) The analysis of their fermentation parameters was performed on a culture supernatant obtained after growth in BHI-YH in semi-anaerobiosis (In vivo analysis Labs).

(30) TABLE-US-00003 TABLE 3 Assay of the fermentation parameters BHI- AP1 AP2 AP3 AP4 YH Lactic acid % 0.01 0.01 0.01 0.02 0.04 Ammonia nitrogen g/L <0.05 <0.05 <0.05 <0.05 <0.05 Fumaric acid % <0.01 <0.01 <0.01 <0.01 <0.01 Acetic acid g/L 0.16 0.14 0.13 0.15 0.15 Propionic acid g/L <0.1 <0.1 <0.1 <0.1 <0.1 Isobutyric acid g/L <0.1 <0.1 <0.1 <0.1 <0.1 Butyric acid g/L <0.1 <0.1 <0.1 <0.1 <0.1 Isovaleric acid g/L <0.1 <0.1 <0.1 <0.1 <0.1 Valeric acid g/L <0.1 <0.1 <0.1 <0.1 <0.1

(31) As expected, it is difficult to evaluate the fermentation parameters for the strains Arthrobacter. The culture conditions do not allow highlighting the production of any metabolite produced by the fermentation. The presence of propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid has also been checked. However none of the four strains appear to be productive in our culture conditions.

(32) 3.3 Tests of Survival to Temperature

(33) The strains are sensitive to the high temperatures. Indeed, none survives beyond 70 C.

(34) According to these results, it is hardly possible to provide for an addition of these strains during the granulation.

(35) 3.4 Tests of Survival in Water and Food

(36) The strain Arthrobacter sp. AP4 survives very well in water (Table 4 hereinafter). It even seems that it is able to grow in these conditions. Indeed, after seven days in water, the bacterial population has doubled.

(37) TABLE-US-00004 TABLE 4 Percentage of survival in water D1 D2 D3 D4 D7 AP4 134% 144% 130% 166% 206%

(38) In food, the population remains relatively stable even after 21 days (FIG. 3). According to these results, the addition of the strains in the drinking water as well as in food is possible.

(39) 3.5 Anti-Inflammatory Potential

(40) The modulation of the inflammatory profile is estimated by assay of interleukins IL8 by ELISA in the culture supernatant of the Caco-2 cells (intestinal cell line) after contact with the bacteria in the presence or absence of IL1 (induction molecule of the inflammation).

(41) These results were obtained on the supernatants of cells cultured in well. The bacteria present no pro-inflammatory activity (low secretion of IL8 in the absence of IL1) nor anti-inflammatory activity (amount of IL8 in the presence of IL1 identical to the control).

(42) The experiment was repeated with the strain AP4 but with Caco-2 cells cultured on filter. In this case, this strain has a pro-inflammatory activity.

(43) 3.6 Enzymatic Profiles

(44) The API ZYM system is a semi-quantitative method of research of enzymatic activities. The enzymatic tests are inoculated with a dense bacterial suspension.

(45) TABLE-US-00005 TABLE 5 Results of the readings of the strips Api Zym AP1 AP2 AP3 AP4 I II I II I II I II 1. Control without a substrate 2. Alkaline phosphatase 3. Esterase (C4) 5 5 5 4 5 4 5 5 4. Esterase lipase (C8) 1 3 4 4 3 4 3 5 5. Lipase (C14) 6. Leucine arylamidase 5 5 5 5 5 5 5 5 7. Valine arylamidase 3 1 4 1 4 1 4 1 8. Cystine arylamidase 3 4 4 4 9. Trypsin 10. Alpha-Chymotrypsine 11. Acid Phosphatase 3 3 4 3 4 2 4 3 12. Naphtol phosphohydrolase 1 1 1 1 13. Alpha-galactosidase (melibiase) 14. Beta-galactosidase (lactase) 15. Beta-glucuronidase (hyaluronidase) 16. Alpha-glucosidase (maltase) 2 1 17. Beta-glucosidase (cellulase) 18. N-acetyl-beta-glucosaminidase (chitinase) 19. Alpha-mannosidase 20. Alpha-fucosidase 1 I: first assay; II: second assay

(46) Some activities are indisputably found regardless of the used strain. This is the case of the esterase, esterase lipase, leucine arylamidase, valine arylamidase and acid phosphatase. Some activities do not seem very stable, such as for the cystine arylamidase.

(47) The strain AP1 presents a profile different from the other Arthrobacter in the use of the maltose and fucose for example.

(48) 3.7 Resistance to Antibiotics

(49) A first assay was conducted in the laboratory. The antimicrobial susceptibility testing was performed using antibiotic diffusion discs (BBL Sensi-Disc Susceptibility Test Discs).

(50) The tested antibiotics were used in the following quantities: Bacitracin 10 g, erythromycin 15 g, penicillin G 10 g, ampicillin 10 g, vancomycin 30 g, streptomycin 300 g, chloramphenicol 30 g, ciprofloxacin 5 g, fosfomycin 200 g, rifamycin 25 g, and trimethoprim/sulfamethoxazole 1.25 g/23.75 g. The results of the antimicrobial susceptibility testing are subjected to a reading abacus in order to determine the level of sensitivity of the strain relative to the measured inhibition diameter.

(51) The 4 strains of Arthrobacter are sensitive to all the tested antibiotics.

(52) TABLE-US-00006 TABLE 6 sensitivity to antibiotics AP1 AP2 AP3 AP4 Ampicillin S S S S Erythromycin S S S S Gentamicin S S S S Kanamycin S S S S Streptomycin R R R R Tetracyclin R S S S Chloramphenicol R/S R/S S S Vancomycin S S S S Ciprofloxacin ND* ND* ND* ND* Linezolid ND* ND* ND* ND* Clindamycin R S S S Tylosin ND* ND* ND* ND* *ND: not determined because of the absence of break-point

(53) According to these tests, all the strains are resistant to the Streptomycin. The strain AP1 is the one which is the most resistant.

(54) 3.8 Adhesion Tests

(55) The bacterial adhesion is estimated on the Caco-2 cells (epithelial cell line).

(56) TABLE-US-00007 TABLE 7 Bacterial count and adhesion rates Strains CFU.sub.I CFU.sub.A AP1 4.90 .Math. 10.sup.8 1.50 .Math. 10.sup.3 AP2 1.50 .Math. 10.sup.9 3.00 .Math. 10.sup.3 AP3 1.75 .Math. 10.sup.8 6.00 .Math. 10.sup.3 AP4 1.42 .Math. 10.sup.9 3.00 .Math. 10.sup.4 CFU.sub.I: initial enumeration CFU.sub.A: enumeration after adhesion

(57) Although low, all our strains have adhesiveness to the intestinal cells. The strains AP3 and AP4 seem to adhere more effectively.

EXAMPLE 4 VALIDATION OF THE CONCEPT

(58) 4.1 Harmlessness of the Strains

(59) In a first step, the bacteria were observed by electron microscopy in order to check the absence of morphological character associated with the pathogenicity.

(60) The morphology of the cells is variable (coccoid sticks), which is consistent with the characterization of the genus Arthrobacter. The cells are devoid of flagella and pili.

(61) In a second step, an in vivo test was established. 10.sup.7 bacteria were intragastrically administered to germfree mice (3 animals per strain). Daily and for 5 days, a sampling of faeces is performed. An analysis of these saddles by optical microscopy allowed to confirm the presence of the bacteria during at least 4 days, highlighting their survival in the digestive tract. An absence of mortality, intestinal lesions and clinical signs (prostration, diarrhea . . . ) should be noted. These results support the experimentation on the cell line Caco-2 (Absence of lysis or cell detachment).

(62) 4.2 Sequencing of the Genes rumC

(63) The sequences of the different rumC-like genes present in our strains were compared to the sequences of the strain R. gnavus E1 (See Appendices).

(64) TABLE-US-00008 TABLE 8 Percentage of identity of the rumC-like genes relative to the R. gnavus E1 genes (the identifiers of sequences in the table hereinafter correspond to the sequences identified in the strains AP1 to AP4). AP1 AP2 AP3 AP4 rumC1 78.12 99.48 93.23 98.44 (SEQ ID (SEQ ID (SEQ ID (SEQ ID No 17) No 22) No 27) No 30) rumC2 97.92 55.73 86.46 84.9 (SEQ ID (SEQ ID (SEQ ID (SEQ ID No 18) No 23) No 28) No 31) rumC3 100 44.79 89.06 89.06 (SEQ ID (SEQ ID (SEQ ID (SEQ ID No 19) No 24) No 29) No 32) rumC4 60.98 75.14 / / (SEQ ID (SEQ ID No 20) No 25) rumC5 52.2 66.88 / / (SEQ ID (SEQ ID No 21) No 26)

(65) The conservation of the genes is different depending on the strains. The rumC1 gene is the one that is the more conserved. Generally, the rumC4 and rumC5 genes are very divergent, even too divergent, to be sequenced for the strains AP3 and AP4.

(66) The same analysis has been performed by comparing the deduced peptide sequences (See appendices).

(67) TABLE-US-00009 TABLE 9 Percentage of identity of the peptide sequences deduced relative to R. gnavus E1 (the identifiers of sequences shown in the table hereinafter correspond to the sequences identified in the strains AP1 to AP4). AP1 AP2 AP3 AP4 RumC1 55.56 98.41 62.3 75.81 (SEQ ID (SEQ ID (SEQ ID (SEQ ID No 1) No 6) No 11) No 14) RumC2 79.37 25.4 77.78 76.19 (SEQ ID (SEQ ID (SEQ ID (SEQ ID No 2) No 7) No 12) No 15) RumC3 100 3.17 92.06 92.06 (SEQ ID (SEQ ID (SEQ ID (SEQ ID No 3) No 8) No 13) No 16) RumC4 7.69 14.29 (SEQ ID (SEQ ID No 4) No 9) Rumc5 15.38 33.96 (SEQ ID (SEQ ID No 5) No 10)

(68) Generally the same conclusions can be drawn. The identities of the deduced sequences are lower than the identities of the genes. In some cases, the peptide sequences are too distant (or truncated; see appendices) to affirm an activity (RumC2_AP3 and RumC4_AP1 for example).

(69) 4.3 Activity Test

(70) The activity tests are carried out with the bacterial culture supernatants against the strain Clostridium perfringens.

(71) The four strains have an anti-C. perfringens activity (FIG. 5). In order to limit the action of an organic acid, the supernatants are neutralized prior to the test. Two tests were carried out in order to confirm the idea that the inhibition of C. perfringens is due to a RumC-like activity. In a first step, supernatants were heated for 10 min at 95 C. before the test. The activity is conserved as is the case for RumC. In a second step, the first pre-purification step was carried out (see WO 2008/152252): purification of the supernatants on column Sep-Pak with an elution with 40% of acetonitrile (ACN, FIG. 6). In this case, the activity is also identified.

(72) 4.4 Evaluation of the Probiotic Properties of the Strains of Arthrobacter in Vivo

(73) The evaluation of the effect of the strains AP3 and AP4 on the growth performances of the broil chickens (weight gain, consumption and consumption index) was carried out under conditions called challenging diet. This diet is a corn-based diet with a standard protein content (23%) for 14 days followed by a high protein (26% of protein), wheat- and barley-based diet, therefore rich in fibers, from 14 to 35 days. This challenging diet corresponds to the control diet of table 10).

(74) The strains AP3 and AP4 were sprayed onto the feed at a concentration allowing the ingestion of 10.sup.8 CFU per day and per animal, from the first day, and this, throughout the duration of the assay, namely 35 days. These diets correspond respectively to the mentions AP3 and AP4 in the table 10.

(75) The diet Lincomycin (8.8%) corresponds to a challenging diet which is added to the lycomycin up to 8.8% (i.e. 5.25 g per ton of feed).

(76) These four treatments were performed on batches of 15 chickens were repeated 12 times (namely 720 chickens in total).

(77) TABLE-US-00010 TABLE 10 in vivo Results. Weight Consumption gain (g) ET % (g) ET % IC ET % Mortality Control 2425 21.4 3974 28.0 1.639 0.009 3.3% AP3 2527 28.2 4.2% 3815 42.6 0.4% 1.51 0.012 7.9% 3.3% AP4 2460 21.5 1.4% 3733 43.3 6.1% 1.518 0.015 7.4% 1.1% Lincomycin 2577 22.4 6.3% 4057 37.7 2.1% 1.575 0.013 3.9% 1.7% (8.8%) ET: Standard Deviation; IC: Consumption Index (consumption of feed required for the increase of the weight gain by 1 kg, IC = Consumption/Weight Gain); %: Percentage of improvement compared to the control; Consumption: feed consumption by the animals over the entire assay

(78) The two tested strains, AP3 and AP4, have similar and positive effects on the growth performances of broil chickens. They allow a decrease of the consumption index by 7 to 8% due to both an increase in the weight gain and a decrease in the consumption.