Detection of Lactobacillus acidophilus in Feces of Humans, Pigs, and Chickens1

Transcription

1 AFPPED MICROBIOOGY, Oct. 1975, p Copyright 1975 American Society for Microiology Vol. 30, No. 4 Printed in U.SA. Detection of Lactoacillus acidophilus in Feces of Humans, Pigs, and Chickens1 S. E. GILLILAND,* M. L. SPECK, AND C. G. MORGAN Department offood Science, North Carolina State University, Raleigh, North Carolina Received for pulication 4 June 1975 Lactoacilli in fecal material from humans, pigs, and chickens were enumerated on lactoacillus selective agar (LBS). In all samples, higher numers of lactoacilli were detected when plates were incuated in a system flushed with C02 rather than in air. Much higher numers of acteria from human feces were detected when the LBS agar plates were incuated anaeroically in a hydrogencaron dioxide atmosphere (GasPak) than when incuated in C02. The acteria from human feces isolated on LBS agar incuated anaeroically were predominately ifidoacteria. Cultures from all three sources isolated on LBS agar incuated under C02 were lactoacilli, including Lactoacillus acidophilus. Differences were oserved in iochemical characteristics of some of the L. acidophilus isolated from all three sources. Guanine plus cytosine ase ratios of deoxyrionucleic acid isolated from L. acidophilus cultures from humans were lower, in most cases, than those from pigs and chickens. Many reports have een pulished regarding the importance of lactoacilli in maintaining alanced intestinal flora necessary for the health of humans (for a review, see reference 18). Therapy involving the consumption of viale Lactoacillus acidophilus has een eneficial in treating certain gastrointestinal disorders (18). There are indications that the iotypes of lactoacilli present in the intestinal tract vary among different host animals (11). A possile "host specificity" for intestinal microorganisms has een suggested (12). For these and other reasons there is a need for more selectivity in the quantitation of lactoacilli in the intestinal tract; also more detailed information is needed on lactoacilli found in the intestines of different hosts. In the present study, lactoacillus selection (LBS) agar incuated under different conditions was used in efforts to differentiate various groups of lactoacilli. Characteristics of cultures ofl. acidophilus isolated from the feces of humans, pigs, and chickens also were oserved. MATERIALS AND METHODS Source and maintenance of cultures. L. acidophilus NCFM (of human origin) was from the culture collection in the Food Science Department of North Carolina State University. L. acidophilus 4962 was from the American Type Culture Collection, Rockville, Md. L. acidophilus strains CNRZ 216 and CNRZ 218 were from the Centre National de I Paper no of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, N.C. 541 Recherches Zootechnique (Jouy-en-Josas, France). Other lactoacilli were isolated from fecal material of either humans, pigs, or chickens and are so designated. All L. acidophilus strains were routinely propagated in sterile lactoacillus MRS roth (Difco Laoratories, Detroit, Mich.) y using a 1% inoculum and incuation at 37 C for 18 h. At least three successive transfers were made prior to characterization experiments. The cultures were stored at 5 C etween transfers. The Bifidoacterium cultures were propagated in a similar manner except that the tues were incuated and stored etween transfers at 5 C in a GasPak system (BBL, Cockeysville, Md.). Enumeration procedures. The fecal samples were diluted with sterile 1% peptone, and duplicate plates were prepared for each dilution. The plates were poured with LBS agar (BBL) prepared from individual ingredients, and an overlay of the same medium was added. Some plates were placed in a plastic ag, flushed for 1 min with caron dioxide, and sealed (LBS-CO2 counts). For incuation in an atmosphere of hydrogen and caron dioxide, plates were placed in a GasPak system (LBS-GP counts). This atmosphere contained approximately 7% CO2 (BBL). In addition, some plates were incuated aeroically. All plates were incuated for 48 h at 37 C. Isolation procedures. Colonies were picked from countale plates of LBS-CO2 and LBS-GP and inoculated into tues containing 10 ml of sterile MRS roth. The tues containing isolates from the LBS- CO2 plates were incuated aeroically, and those from the LBS-GP plates were incuated anaeroically in a GasPak system. Cultures that grew within 24 to 48 h were diluted and plated y the pour plate method with MRS agar (MRS roth plus 1.5% agar). Isolated colonies from these plates were picked and maintained in MRS roth for characterization test.

2 542 GILLILAND, SPECK, AND MORGAN Classification of isolates. Only those isolates that were gram-positive rods and catalase negative were considered for further identification. Tests for catalase were made y adding 5 ml of 3% hydrogen peroxide to the cell pellet otained y centrifuging (12,000 x g for 10 min) 10 ml of an MRS roth culture. Cultures were considered catalase negative if no visile gassing was oserved. Cultures were examined for growth at 15 and 45 C in MRS roth and for gas production y the methods of Rogosa et al. (17); for the latter, 5 ml of 1.5% sterile agar was used as the overlay. MRS roth with glucose and eef extract omitted (19) was the asal medium used for determination of ammonia production from arginine, esculin hydrolysis, and carohydrate fermentations. Esculin hydrolysis and ammonia production were determined as descried y Davis (4). Aility of cultures to ferment various carohydrates was evaluated as descried y Rogosa and Sharpe (16), except that amygdalin, meliiose, and raffinose were not employed. Cultures of anaeroic organisms were incuated in a GasPak system. To check for ranched cellular forms ofthe anaeroic isolates, the cultures were grown in the lowcalcium medium of Kojima et al. (7) in which yeast extract (0.5%) was sustituted for the eef liver infusion. Cellular morphology was determined y examining, with a microscope, methylene lue stains of the cultures. Determination of DNA ase composition. Deoxyrionucleic acid (DNA) was isolated from representative lactoacilli and ifidoacteria y the method of Marmur (9). The percentage of guanine plus cytosine (G+C) in the DNA samples was determined from the thermal melting point of the DNA y using procedures descried y Marmur and Doty (10). The change in asorance of the heated DNA was measured automatically y using a Beckman DU spectrophotometer equipped with a Gilford model 2000 automatic recording photometer (Gilford Laoratories, Inc., Oerlin, Ohio). The cuvette chamer was heated y a Haake model FE constanttemperature circulation ath (Gilford Laoratories, Inc., Oerlin, Ohio) filled with ethylene glycol. RESULTS Enumeration of lactoacilli. L. acidophilus NCFM did not form colonies on LBS agar incuated under aeroic conditions (Tgle 1). How- TABLE 1. Enumeration of lactoacilli y using LBS agara L. acidophi- Chicken Pig Type of count lus NCFM feces feces (CFU/swa) (CFU/g) (CFU/g) LBS-aeroic NG 2.0 x x 108 LBS-CO2 5.5 x x x 108 LBS-GP 5.5 x x x 108 a CFU, Colony-forming units; NG, no growth. Concentrated culture. APPL. MICROBIOL. ever, the culture grew equally well on LBS-CO2 and LBS-GP. Colonies developed from chicken and pig feces plated on LBS agar and incuated aeroically. Few differences were oserved etween the colony counts on LBS-CO2 and those on LBS-GP. The LBS-CO2 and LBS-GP counts were approximately 2.5 and 5.5 times greater than the LBS-aeroic counts for chickens, and pigs, respectively. Marked differences in counts were otained when human feces was plated on LBS-CO2 and LBS-GP (Tale 2). In all sujects studied, much higher numers were oserved when the fecal material was plated on LBS-GP. Aeroic conditions were not evaluated ecause of the poor growth of L. acidophilus incuated aeroically on LBS agar. Furthermore, the lactoacilli from pig and chicken feces grew less well on LBS agar incuated aeroically than on LBS- CO2 and LBS-GP. Isolation of lactoacilli. Cultures isolated from the highest dilutions of human fecal material on LBS-GP were anaeroic and had characteristics of Bifidoacterium species (Tale 3). All of the isolates, with the exception of BA1, TABLE 2. Sample Enumeration of lactoacilli in human feces y using LBS agar LBS-CO2 CFUO/g LBS-GP A 2.5 x x 108 M 2.5 x x 108 S 5.8 x x 108 W 1.5 X x 108 a Colony-forming units. TABLE 3. Characterization of isolates from human feces plated on LBS agar and incuated in a GasPak system Biochemical Isolate Branching reactions ofbifidoacterium mol% G+C Sp. BA1 a + BA2 BA3 BS BS2 + + BS3 +? 60.9 BW1 + + BW BW BW BW5 + + a Curved rods, no definite ranching. Unale to suculture after initial isolation.

3 VOL. 30, 1975 formed ranched rods in the medium of Kojima et al. (7); this is typical for this species. Furthermore, all of the isolates from human feces plated on LBS-GP (with the exception of BS3) had iochemical characteristics of Bifidoacterium species (15). The G+C contents of five strains (including BS3) tested were within the range reported for Bifidoacterium (1). Cultures isolated from human, pig, and chicken fecal material plated on LBS-CO2 were all classified as lactoacilli. Of the 20 isolated from human feces, 6 possessed characteristics closely resemling L. acidophilus. Three additional isolates did not closely fit the fermentation pattern descried y Rogosa and Sharpe (16) for L. acidophilus; however, their characteristics were closer to those of L. acidophilus than to any other organism of the thermoacterium group. Of 12 lactoacilli isolated from pigs, none was identical tol. acidophilus. However, eight of the isolates had characteristics more nearly similar to L. acidophilus than to any other lactoacilli. Of 12 isolates otained from chickens, 3 had characteristics closely resemling L. acidophilus and 2 additional isolates more closely resemled L. acidophilus than any other lactoacilli in the thermoacterium group. Complete fermentation patterns for five representative isolates characterized as L. acidophilus from humans, pigs, and chickens are presented in Tale 4. Four laoratory strains of L. acidophilus were included for comparison. The fermentation patterns for these strains conformed closely with the characteristics of L. acidophilus (15) with the exception of CNRZ 216. Isolates HA3 and HM2 (human origin) possessed the same characteristics as L. acidophilus. Isolate HA1 was similar except for an apparent variation in the aility to ferment maltose and galactose. Isolate HA2 differed from L. acidophilus in that it failed to ferment trehalose. Isolate HM6 was variale with respect to the fermentation of celloiose and salicin, and failed to ferment trehalose or hydrolyze esculin. All of the isolates of pig origin differed from the fermentation pattern of L. acidophilus ecause of negative or variale actions on celloiose, salicin, and trehalose. All except PA3 were also variale with respect to the hydrolysis of esculin. Of the five representative isolates from chickens, isolates C1, C2, and C3 possessed characteristics identical to those of L. acidophilus. Isolate C7, however, was variale on mannitol, and isolate Cl1 fermented mannitol ut did not attack esculin, celloiose, or salicin. G+C content of DNA. The G+C content for isolates identified as L. acidophilus ranged DETECTION OF L. ACIDOPHILUS IN FECES 543 from 36.7 to 43.7 mol%, except for isolate C11, which was 29.8 mol% (Tale 4), and therefore could not e considered as L. acidophilus. The G+C contents for two of the three isolates of human origin (HA3 and HM2) were closer to the percentages oserved for the laoratory strains than were those for the isolates otained from chicken and pig fecal material. Isolate PA 19 (of pig origin) also appeared to have a G+C content close to that oserved for the laoratory strains. In general, G+C contents for the isolates (except PA19 and C11) otained from the chicken and pig fecal material were higher than those oserved for the laoratory strains. Isolate HA1 (of human origin) also possessed a higher G+C content than those oserved for the laoratory strains. DISCUSSION In evaluating the effect(s) offeeding L. acidophilus on the microial flora of the intestinal tract, the selection of the proper medium for detection of the organism eing fed is of great importance. In addition, it appears that selection of an L. acidophilus strain compatile with the host is also important. LBS agar has highly selective qualities for lactoacilli. The conditions under which LBS agar is incuated have een shown in this study to yield different counts for the same sample of fecal material, indicating a possile means of more selectively enumerating the lactoacilli. Although colonies from chicken and pig feces were oserved on LBS agar when incuated aeroically, L. acidophilus NCFM failed to show growth under these conditions. Rogosa and Sharpe (16) indicated that high concentrations of acetate caused y evaporation during aeroic incuation may e inhiitory to some lactoacilli. L. acidophilus NCFM and other strains may e sensitive to these higher levels of acetate caused y evaporation. Thus, this medium should not e used aeroically for the enumeration ofl. acidophilus. On the other hand, incuation under strictly anaeroic conditions (GasPak system) enales anaeroic Bifidoacterium species to grow and completely prevents the detection of L. acidophilus from some sources (i.e., human feces). Anaeroic incuation of LBS plates would provide a method of enumerating ifidoacteria. Not all organisms detected on LBS-CO2 were classified as typical L. acidophilus; therefore, this method of incuation cannot e used to enumerate L. acidophilus selectively. Such results also indicate that lactoacilli other than L. acidophilus are present in the intestinal flora of humans, pigs, and chickens. Comparison of the fermentation patterns of

4 544 GILLILAND, SPECK, AND MORGAN APPL. MICROBIOL. V1+11 I++++I I++ c a I _ C) Ill+ E X ~+ +l E 1I+ ++I+++ I C" 5 E :;U +,, +l +l _X ; c ef E II+++III+l+IC I +I++ 1,,,,II+I,-+ 04 Ito+,,+++++,++ 1 4c I", N.~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ 5s +I II + aq ~ ~ ~ ~ ~ ~~~~~~' II+I++ 06 C43j] s j; 3( I+ We + ~~~~I + ++I++I I +I + + W I~. + +I E~~~~~V eq~ ~ ~

5 VOL. 30, 1975 DETECTION OF L. ACIDOPHILUS IN FECES 545 isolates identified as L. acidophilus indicate that different iotypes exist. Mitsuoka (11) reported that different iotypes of L. acidophilus existed which were host specific. Morishita et al. (12) descried a similar phenomenon in that a strain of L. acidophilus of human origin would not ecome estalished in the intestines of chickens. The differences oserved in the fermentation patterns and G+C content of the DNA among the isolates of the present study also lends support to the theory that relationships exist etween the host and the strain or iotype ofl. acidophilus that can ecome estalished in the intestine. Thus, it appears that care must e taken in selecting strains of L. a#idophilus for use in dietary preparations intended as a source of lactoacilli for estalishment in the intestinal tract. LITERATURE CITED 1. Buchanan, R. E., and N. E. Gions (ed.) Bergey's manual of determinative acteriology, 8th ed. The Williams & Wilkins Company, Baltimore. 2. Chopra, S. L., A. C. Blackwood, and D. G. Dale Intestinal microflora associated with enteritis of early-weaned pigs. Can. J. Comp. Med. Vet. Sci. 27: Conn, H. O., and M. H. Floch Effects of lactulose and Lactoacillus acidophilus on the fecal flora. Am. J. Clin Nutr. 24: Davis, G. H. G The classification of lactoacilli from the human mouth. J. Gen. Microiol. 13: Gordon, D., J. Macrae, and D. M. Wheater A lactoacillus preparation for use with antiiotics. Lancet 272: Harrison, A. P., Jr., and P. A. Hansen Lactoacilli from turkeys. J. Bacteriol. 60: Kojima, M., S. Suda, S. Hotta, and K. Hamada Induction of pleomorphism in LactoaciUus ifidus. J. Bacteriol. 95: Maceth, W. A. A. G., E. H. Kass, and W. V. Mc- Dermott; Jr Treatment of hepatic encephalopathy y alteration of intestinal flora with Lactoacillws acidophilus. Lancet 1: Marmur, J A procedure for the isolation of deoxyrionucleic acid from microorganisms. J. Mol. Biol. 3: Marmur, J., and P. Doty Determination of the ase composition of deoxyrionucleic acid from its thermal denaturation temperature. J. Mol. Biol. 5: Mitsuoka, T Vergleichende Untersuchungen uer die Laktoazillen aus den Faeces von Menschen, Schweinen und Huhnern. Zentrall. Bakteriol. Parasitenkde. Infektionskr. Hyg. At. 1: Orig. 210: Morishita, Y., T. Mitsuoka, C. Kaneuchi, S. Yamamato, and M. Ogata Specific estalishment of lactoacilli in the digestive tract of germ-free chickens. Jpn. J. Microiol. 15: Muralidhara, K. S., D. C. England, W. E. Sandine, and P. R. Elliker Lactoacillus concentrates effect on coliforms in swine. J. Anim. Sci. 35: Read, A. E., C. F. McCarthy, K. W. Heaton, and J. Laidlaw Lactoacillus acidophilus (Enpac) in treatment of hepatic encephalopathy. Br. Med. J. 1: Reuter, G Designation of type strains for Bifidoacterium species. Int. J. Syst. Bacteriol. 21: Rogosa, M., and M. E. Sharpe An approach to the classification of the lactoacilli. J. Appl. Bacteriol. 22: Rogosa, M., R. F. Wiseman, J. A. Mitchell, and M. N. Disraely Species differentiation of oral lactoacilli from man including descriptions oflactoacillus salivarius nov spec and Lactoacillus celloiosus nov spec. J. Bacteriol. 65: Sandine, W. E., K. S. Muralidhara, P. R. Elliker, and D. C. England Lactic acid acteria in food and health: a review with special reference to enteropathogenic Escherichia coli as well as certain enteric diseases and their treatment with antiiotics and lactoacilli. J. Milk Food Technol. 35: Sharpe, M. E Taxonomy of the lactoacilli. Dairy Sci. Astr. 24: Smith, H. W., and W. E. Cra The faecal acterial flora of animals and man. Its development in the young. J. Pathol. Bacteriol. 82: Tortuero, R Influence of the implanation oflactoacillus acidophilus in chicks on the growth, feed conversion, malasorption of fats syndrome, and intestinal flora. Poultry Sci. 52: