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LAMVAB—A new selective medium for the isolation of lactobacilli from faeces
Journal of Microbiological Methods 29 (1997) 77–84
Journal of Microbiological Methods
LAMVAB—A new selective medium for the isolation of lactobacilli from faeces R. Hartemink*, V.R. Domenech, F.M. Rombouts Section Food Chemistry and Microbiology, Department of Food Science, Wageningen Agricultural University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands Received 28 November 1996; revised 4 March 1997; accepted 15 April 1997
Abstract The development of a new selective medium, LAMVAB, for the isolation of Lactobacillus species from faeces is described. The medium is highly selective due to a low pH and the presence of vancomycin (20 mg l 21 ). In addition, the medium contains cysteine–HCl to enhance anaerobic conditions, and bromocresol green as a pH indicator. Competing faecal flora is inhibited by the low pH (enterobacteria, bacteroides and other Gram-negative anaerobes, clostridia) and the vancomycin (enterococci, bifidobacteria, clostridia). Characterization of the strains isolated on LAMVAB from human faeces included all common faecal lactobacilli and no other lactic acid bacteria. In one sample, vancomycin-resistant cocci were present and in another sample, yeasts were isolated. It can be concluded that the new medium is highly selective and permits the growth of all common faecal lactobacilli. The new medium can also be applied as a selective medium for lactobacilli in probiotics, which contain mixed populations of lactobacilli, bifidobacteria and enterococci. 1997 Elsevier Science B.V. Keywords: Lactobacillus; Faecal flora; Vancomycin
1. Introduction Lactobacilli are Gram-positive anaerobic or facultative aerobic rods, which can be isolated from a large number of sources. Lactobacilli can be isolated from the human and animal body (oral cavity, stomach, intestines, vagina), plants and material of plant origin, sewage and fermented products. Lactobacilli are strictly fermentative and acid-resistant. Growth is enhanced by acidic conditions. The final pH in broth cultures is generally below 4.5 [1]. Lactobacilli are considered to have beneficial effects on human and animal health. Therefore, *Corresponding author. Tel.: 131 317 482241; fax: 131 317 484893; e-mail: [email protected]
several species of lactobacilli are used as probiotics. Worldwide, many products with probiotic lactobacilli are marketed [2,3]. To have a beneficial effect on the health of the host, it is necessary that the probiotic strain is able to survive in the gastro-intestinal tract [2]. It is therefore necessary to determine the number of lactobacilli in faeces and, if possible, to identify the different species directly. This can be done using specific selective media, DNA probes or immunological techniques. Lactobacilli are generally isolated on rich, slightly acidic media, such as MRS, LBS and Rogosa agars [4–6]. For most (fermented) food products, these media allow selective isolation of lactobacilli, as they are generally the most abundant group of bacteria present.
0167-7012 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved PII S0167-7012( 97 )00025-0
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Table 1 Species of lactobacilli isolated from human or animal faeces [1] Species
Isolated from
Lactobacillus acidophilus Lact. amylovorus Lact. aviarius subsp. araffinosus Lact. aviarius subsp. aviarius Lact. brevis Lact. casei Lact. coryniformis Lact. crispatus Lact. curvatus Lact. fermentum Lact. gallinarium Lact. gasseri Lact. hamsteri Lact. intestinalis Lact. johnsonii Lact. murinus Lact. paracasei subsp. paracasei Lact. paracasei subsp. rhamnosus Lact. plantarum Lact. reuteri Lact. salivarius Lact. vaccinostercus
Human, animals Pig Chicken Chicken Human, cow, rat Human Cow Human Cow Human Chicken Human Hamster Mouse, rat Chicken, calf, mouse, pig Mouse, rat Human Human Human Human, animals Human, hamster, chicken Cow
In faeces, however, lactobacilli are present in relatively low numbers [7,8]. In faecal samples, several lactobacilli have been isolated (Table 1), in humans, the main lactobacilli are Lact. acidophilus, Lact. casei and Lact. paracasei [1]. The counts of lactobacilli in faeces are highly variable between species and persons, even within a single person [4,7,8]. The media mentioned above are not selective, as growth of other acid-resistant bacteria is not restricted and, in particular, bifidobacteria, streptococci and enterococci are able to grow on these media [9,10]. Growth of other intestinal bacteria is restricted, but not negligible. It is therefore necessary to have a highly selective medium, which allows the isolation of low numbers of lactobacilli in the presence of high numbers of bifidobacteria, streptococci and / or enterococci. In this report, we describe the development of a new selective medium for the isolation of faecal lactobacilli. Lactobacilli are highly acid-resistant, with growth being possible at an initial pH of 5.0 [1]. Most other intestinal bacteria are not able to grow at this acidic pH. Bifidobacteria, enterococci and streptococci are
not inhibited [11–13]. Contrary to other Gram-positive bacteria, most lactobacilli are resistant to the antibiotic vancomycin, while Gram-negative bacteria are generally resistant [14–17]. Vancomycin has already been applied previously for the selective isolation of lactobacilli from brewery products [18]. It is not useful as a selective agent in dairy products containing Leuconostoc or Pediococcus species, as these are vancomycin resistant [14,19,20]. Enterococci, streptococci and bifidobacteria are susceptible to vancomycin [21–23]. Furthermore, resistance to vancomycin is not inducible and only a few vancomycin-resistant enterococci have been isolated [24]. Vancomycin-resistant bifidobacteria have not been reported. Although lactobacilli are aerotolerant, anaerobic conditions favour their isolation and growth. It was decided to add cysteine as a reducing agent to improve the isolation of lactobacilli. The final medium consisted of MRS agar, with vancomycin, low pH (5.0) and cysteine as selective and elective agents, and was named Lactobacillus Anaerobic MRS with Vancomycin and Bromocresol green, or LAMVAB.
2. Materials and methods
2.1. LAMVAB The medium consists of three different components: Solution A: MRS broth (Merck 1.10661), 104.4 g / l, with cysteine–HCl (0.5 g / l) and bromocresol green (0.05 g / l). The pH of this solution was adjusted to 5.060.1 using 4 M HCl before autoclaving. Solution B: Agar, 40 g / l. Solution C: Vancomycin hydrochloride (local pharmacy, .95% purity), 2 mg / ml in water. Solution C should be kept at 0–48C and was found to be stable for at least three months. Solutions A and B are sterilized at 1218C for 15 min. Solution C is sterilized by filtration using a 0.2-mm filter. Preparation of the medium involves sterilization of equal volumes of solutions A and B. Solution B is cooled down to 508C in a water bath. Solution A is cooled down to room temperature. To 500 ml of
R. Hartemink et al. / Journal of Microbiological Methods 29 (1997) 77 – 84
solution A, 10 ml of solution C are added aseptically. Finally, solution B is added to the MRS–vancomycin (A1C) mixture. Plates have to be poured immediately after mixing. This procedure results in a final vancomycin concentration of 20 mg / l.
2.2. Growth of different intestinal bacteria on LAMVAB Selected intestinal bacteria from stock cultures were pre-cultured anaerobically in thioglycolate broth for 24–48 h at 378C. From these cultures, a streak was made, using a sterile cotton swab, on LAMVAB and RCA (reinforced clostridial agar, Oxoid) plates. RCA was used as a control medium. The following strains were used: Lactobacilli (150 strains, .10 species), bacteroides (10 strains, 5 species), clostridia (15 strains, 13 species), bifidobacteria (25 strains, .12 species), eubacteria (2 strains, 2 species), streptococci, lactococci and enterococci (50 strains, .10 species), anaerobic cocci (5 strains, 3 species), fusobacteria (5 strains, 3 species), enterobacteria (15 strains, 7 species), actinomyces (4 strains, 3 species) and two strains of Megasphaera elsdenii.
2.3. Inhibition of intestinal bacteria by LAMVAB To determine which component of the LAMVAB medium inhibits different groups of intestinal bacteria, a selection of isolates (Table 2) was tested for growth on LAMVAB, LAMVAB with a pH value of 6.5 (L65) and on LAMVAB without vancomycin (L2). The inoculation was carried out as described above.
2.4. Comparison of LAMVAB with MRS To compare the growth of intestinal lactobacilli and possible competing flora on MRS and LAMVAB, 71 strains of lactobacilli, bifidobacteria, Actinomyces species, streptococci and lactococci (Table 3) were grown in MRS broth. From these broths, 10-fold dilutions were prepared as described above, and inoculated on MRS and LAMVAB agars. The plates were incubated anaerobically at 378C for 72 h.
79
Table 2 Growth of selected bacteria on LAMVAB, LAMVAB at pH 6.5 (L65) or LAMVAB without vancomycin (L2) Species
L65
L2
LAMVAB
Actinomyces naeslundii Aeromonas hydrophila Bacillus cereus Bacillus megatherium Bacteroides fragilis Bacteroides ovatus Bifidobacterium adolescentis Bifidobacterium dentium Bifidobacterium infantis Bifidobacterium longum Enterobacter cloacae Enterococcus faecalis Enterococcus species Escherichia coli Lactococcus lactis Listeria innocua Proteus mirabilis Proteus vulgaris Pseudomonas fragi Pseudomonas sp. Salmonella infantis Salmonella montevideo Staphylococcus aureus Streptococcus mutans Streptococcus oralis Yersinia enterocolitica
No Yes Yes Yes Yes Yes No No No No Yes Yes No Yes No Yes Yes Yes Yes Yes Yes Yes No No No Yes
Yes No No No No No Yes Yes Yes Yes No No Yes No Yes No No No No No No No Yes Yes Yes No
No No No No No No No No No No No No No No No No No No No No No No No No No No
Growth was considered positive if colonies were visible. This included pinpoint colonies.
2.5. Isolation of lactobacilli from faeces To determine the selectivity of LAMVAB as a medium for the isolation of lactobacilli, faecal samples (from humans and animals) were taken and dilutions were plated on MRS and LAMVAB agars. Faecal samples were taken as fresh as possible and an aliquot was transferred into a pre-weighed bottle with buffered medium. This medium consisted of buffered peptone water (Oxoid) with cysteine–HCl (0.5 g / l) and Tween 80 (1 ml / l). The pH was adjusted to 6.560.1 before autoclaving at 1218C for 15 min. After sampling, the bottles were imported into an anaerobic chamber, with an atmosphere of 80% nitrogen, 10% hydrogen and 10% carbon dioxide (HoekLoos SHK 050H, HoekLoos, Rotterdam, The Netherlands). The samples were mixed thoroughly using an Ultra Turrax mixer. A 10-fold series of dilutions was prepared of the homogenates
80 R. Hartemink et al. / Journal of Microbiological Methods 29 (1997) 77 – 84 Table 3 Counts of selected lactobacilli and possible competing flora on MRS (control medium) and LAMVAB Strain
Lactobacillus acidophilus Lact. acidophilus Lact. acidophilus Lact. acidophilus Lact. bulgaricus Lact. casei Lact. casei Lact. fermentum Lact. fermentum Lact. helveticus Lact. plantarum Lact. reuteri Lact. sake Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Actinomyces israelii Ac. naeslundii Ac. naeslundii Ac. viscosus Bifidobacterium adolescentis B. animalis B. boum B. choerinum B. dentium B. globosum B. infantis B. longum B. longum B. pseudocatenulatum B. pseudolongum
Our number
CH rp 128 126 Lb-1 casCH casY 124 123 Lb-4 Lb-9 Lb-10 Lb. sake 626 824 199 168 856 818 812 711 819 844 832 TR-3 SST-1 SST-3 SST-8 SST-11 TST-1 TR-6 624 622 613 620 604 204 SST-7 Ac-1 41 Ac-2 40 88 Bi-35 Bi-37 Bi-42 Bi-39 Bi-41 223 Bi-17 219 Bi-40 Bi-33
Origin a
Dairy starter Dairy starter Swine faeces Swine faeces IM Dairy starter Dairy starter Swine faeces Swine faeces Type Type Type ATO Human faeces Swine faeces Human faeces Human faeces Swine faeces Swine faeces Swine faeces Human faeces Swine faeces Swine faeces Swine faeces Oral cavity Oral cavity Oral cavity Oral cavity Oral cavity Oral cavity Oral cavity Human faeces Human faeces Human faeces Human faeces Human faeces Human faeces Oral cavity MA Oral cavity, Nijm MA Oral cavity, Nijm Type Type Type Type Type Type Type Human faeces Type Type Type
Log N counts on MRS
LAMVAB
8.40 8.67 7.05 6.88 8.10 8.45 8.09 8.21 7.64 8.14 7.40 7.94 8.20 8.25 8.15 6.95 7.50 7.30 7.15 7.27 8.43 7.03 6.12 8.62 7.45 7.77 8.26 7.28 6.78 7.21 8.00 7.74 8.43 8.58 8.12 7.60 7.13 6.88 8.09 7.95 8.92 8.03 6.00 7.48 8.92 8.71 8.80 8.27 8.33 8.50 8.78 7.43 8.12
8.62 8.74 6.99 6.90 8.05 8.40 7.99 8.11 7.73 8.01 7.58 7.95 8.17 8.20 8.19 7.13 7.48 7.43 7.18 7.35 8.40 7.02 6.07 8.80 7.18 ,2.0 ,2.0 6.91 6.52 7.17 7.74 7.99 8.48 8.71 8.26 7.43 7.09 6.88 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0
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Table 3. Continued Strain
B. pullorum B. species B. species B. species B. species B. species B. species B. suis Enterococcus faecalis Lactococcus lactis L. lactis L. lactis Streptococcus gordonii Strep. mitis Strep. mutans Strep. mutans Strep. oralis Strep. species
Our number
Bi-38 683 636 383 628 665 380 211 Ec-1 Lc-4 Lc-3 Lc-1 42 46 44 36 45 TR-11
Origin a
Type Human faeces Human faeces Human faeces Human faeces Human faeces Human faeces Type ATO NIZO NIZO NIZO Oral cavity, Nijm Oral cavity, Nijm Oral cavity, Nijm Type Oral cavity, Nijm Oral cavity, Nijm
Log N counts on MRS
LAMVAB
7.33 8.17 8.02 8.62 8.50 8.85 6.78 8.67 8.09 7.88 7.92 7.86 8.32 8.23 8.21 8.01 7.65 7.65
,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 7.07 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0
Counts are expressed as logarithmic numbers. a These strains were obtained from: Type: type strains, obtained from DSM (Braunsweig, Germany); MA5Maastricht University, The Netherlands; NIZO5Netherlands Institute for Dairy Research, Ede, The Netherlands; ATO5AgroTechnological Institute, Wageningen, The Netherlands; IM5Dept. Industrial Microbiology, Wageningen Agricultural University, The Netherlands; Nijm5Dept. Oral Microbiology, Nijmegen University, The Netherlands.
using RPS. RPS consisted of neutralized bacterial peptone (Oxoid; 0.5 g / l), NaCl (Merck; 8.0 g / l) and cysteine–HCl (0.5 g / l). The pH was adjusted to 6.760.1 before autoclaving at 1218C for 15 min. The tubes were kept under anaerobic conditions for at least 16 h before use. Finally, 30 ml of the 3rd, 5th and 7th dilutions was pipetted on LAMVAB or MRS plates. The plates were incubated under anaerobic conditions at 378C for 72 h.
blue, due to the uptake of bromocresol green into the cell. As not all lactobacilli showed this color and remained white, the color of the colonies was not considered to be characteristic. Several isolates from fish failed to grow on LAMVAB. Further identification showed that these isolates were carnobacteria and not lactobacilli (M. Dapkevicius, personal communication). Carnobacteria fail to grow at pH 5.0.
3.2. Inhibition of intestinal bacteria by LAMVAB 3. Results
3.1. Growth of different intestinal bacteria on LAMVAB None of the intestinal strains, except for the lactobacilli and one strain of Streptococcus gordonii were able to grow on LAMVAB. All lactobacilli from all origins were able to grow on LAMVAB. Growth was determined as colonies over 1 mm in diameter and a yellow discoloration of the medium, due to acid production. Most colonies were green or
Comparing the growth on LAMVAB with that on LAMVAB at pH 6.5 (L65) and on LAMVAB without vancomycin (L2) showed that Gram-negative bacteria were mainly inhibited by the low pH (Table 2) and Gram-positive bacteria were inhibited by the vancomycin.
3.3. Comparison of LAMVAB with MRS Comparing the new LAMVAB medium with MRS as a reference medium showed that only 2 out of 38 lactobacilli failed to grow on LAMVAB (Table 3).
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These two strains were both isolated from the oral cavity. All other lactobacilli showed no reduction in counts on LAMVAB. None of the bifidobacteria, Actinomyces species and cocci grew on LAMVAB, with the exception of Streptococcus gordonii, a strain isolated from the oral cavity.
3.4. Isolation of lactobacilli from faeces Lactobacilli could be selectively isolated from samples of human faeces. Out of 20 samples of human faeces, lactobacilli could be detected in 15 samples. The counts ranged from (log N) 2.5 to 9.0. With the exception of enterococci in one sample and yeasts in another sample, no other bacteria were observed. Faecal samples from 19 different animals were plated on MRS and LAMVAB (Table 4). Growth on MRS was observed in all samples. Bifidobacteria, cocci and spore-forming organisms were observed in 17 samples. In only 2 samples, only lactobacilli were detected on MRS. In 13 of the samples growth on LAMVAB was observed. Lactobacilli were present in all 13 samples. Some cocci were observed in only one of the samples. Bifidobacteria, yeasts or sporeforming organisms were not observed. In only two of the samples were presumptive lactobacilli observed on MRS, but no growth was observed on LAMVAB.
4. Discussion The isolation of lactobacilli from faeces is generally difficult, due to the presence of competing microorganisms, like bifidobacteria, streptococci and enterococci, which may be present in higher numbers than lactobacilli. Most media used for the isolation of lactobacilli also support the growth of these competing micro-organisms [9,10]. The results presented above indicate that LAMVAB does not support the growth of these bacteria, whereas growth of lactobacilli is unaffected. LAMVAB is easy to prepare, even though the three different components have to be sterilised separately. Due to the low pH of the medium, it is impossible to add the agar to the MRS broth directly. The medium does not require expensive chemicals or
antibiotics. Vancomycin is widely used, relatively inexpensive and is easy to obtain from pharmacies. The medium is selective, due to the low pH, which inhibits the most common Gram-negative bacteria in faeces, and the presence of vancomycin, which inhibits the competing Gram-positive flora (Table 2). This is in accordance with the literature. Vancomycin inhibits Gram-positive bacteria, except lactobacilli. Several studies have shown that faecal lactobacilli are not inhibited by vancomycin [14–18]. Only one study indicated susceptibility of faecal lactobacilli to vancomycin [25]. Susceptibility was reported for Lact. acidophilus and Lact. bulgaricus towards very low concentrations of vancomycin (1 and 4 mg / l). Our results show that most lactobacilli are not affected by the vancomycin, as there is no significant reduction in logarithmic counts between MRS and LAMVAB. Only two unidentified oral isolates failed to grow on LAMVAB. These results are in accordance with the literature [14–18]. On only a few occasions could other bacteria be isolated on LAMVAB. These were cocci, probably enterococci or streptococci and it is known that a small percentage of enterococci are vancomycin resistant [21–23]. Also, we observed growth of Streptococcus gordonii, an oral species, which may be present in small numbers in faeces. Comparison of LAMVAB with MRS indicated a low selectivity of MRS, whereas LAMVAB was highly selective for lactobacilli. Even though we did not compare LAMVAB with other common media, such as Rogosa and LBS agar, it is known that the growth of the bacteria tested on these media is similar to that on MRS [9,10]. LAMVAB was successfully applied to the isolation of lactobacilli from faeces from humans and a number of animals. Lactobacilli were present in most of the animals tested (Table 4), with the exception of the giant bat, giraffe, llama, yak, cheetah and watussi. In those samples, other bacteria were isolated on MRS medium, indicating lower selectivity for this medium. There were only two samples (from the giraffe and watussi) of presumptive lactobacilli that could be isolated on MRS, but not on LAMVAB. It is not known whether these isolates were true lactobacilli or other non-spore-forming non-branched rods. LAMVAB may also be a suitable medium for the
R. Hartemink et al. / Journal of Microbiological Methods 29 (1997) 77 – 84
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Table 4 Morpholgy of bacteria isolated from animal faeces Animal
Medium
Colonies on plate a
Colonies tested b
Possible lactobacilli c
Cocci d
Bifid e
Other f
Grevy’s zebra (Equus grevyi) Capybara (Hydrochaeris hydrochaeris) Giant bat (Pteropus vampyrus) Bongo (Boocercus euryceros) White rhinoceros (Cerathoterium simum) Gorilla (Gorilla gorilla) Chimpanzee (Pan troglodytes) Chimpanzee (Pan troglodytes) Chimpanzee (Pan troglodytes) Three-toed sloth (Bradipus tridactylus) Celebes Hornbill (Aceros cassidix) Giraffe (Giraffa camelopardalis) Mouse deer (Tragulus javanicus) Llama (Lama glama) Elephant (Loxodonta africana) Yak (Bos grunniens) Cheetah (Acinonyx jubatus) Watussi (Bos taurus) Bactrian camel (Camelus bactrianus)
MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB
9 5 8 8 8 n.g. 36 12 52 33 14 3 9 6 17 4 15 50 9 6 61 22 31 n.g. 8 4 8 n.g. 214 25 9 n.g. 12 n.g. 19 n.g. 37 3
8 5 8 4 8
7 5 4 4 0
1 0 4 0 8
0 0 0 0 0
0 0 0 0 0
8 6 8 8 8 3 8 6 8 4 8 8 8 5 8 4 8
3 6 8 8 5 3 6 6 3 4 5 8 8 5 3 4 2
5 0 0 0 1 0 1 0 0 0 0 0 0 0 4 0 6
0 0 0 0 2 0 1 0 5 0 3 0 0 0 1 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
8 3 8
0 3 0
1 0 8
1 0 0
6 (spores) 0 0
8 8 8
1 6 0
1 2 6
6 0 2
0 0 0
8
0
0
0
8 (spores)
8
1
6
0
1 (spores)
8 2
3 2
3 0
2 0
0 0
a
Total number of colonies on the plate tested. The number of colonies tested, preferably of the same dilution for both media, the colonies taken were mainly selected on differences in colony morphology, not incidence. c Possible lactobacilli were rod-shaped, straight or slightly curved, not branched bacteria and showed no spores. d Cocci were all coccal-shaped bacteria, mono-, duplo- or streptococci. e Bifid were rod-shaped bacteria with branches, this included possible Bifidobacterium, Actinomyces and Propionibacterium species. f Others included spore-forming rods, yeasts and moulds. n.g.5no visible growth. b
isolation of lactobacilli from probiotics. These products often contain lactobacilli and bifidobacteria. As bifidobacteria fail to grow on LAMVAB, selective isolation of the lactobacilli is possible. We have
succesfully used LAMVAB to isolate different species of lactobacilli from dairy products in the presence of streptococci and bifidobacteria (data not shown).
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It can be concluded that LAMVAB is highly selective for lactobacilli. It allows growth of all faecal lactobacilli tested and suppresses all other bacteria encountered in faecal material, with an occasional exception of cocci and yeasts. The medium is more selective than the media commonly used for the isolation of lactobacilli from faeces and probiotics.
[11]
[12]
[13]
Acknowledgements The authors gratefully acknowledge the financial support for this study, which is part of a multi-donor funded research programme on the role of nondigestible oligosaccharides in food and feed. Also, we would like to thank the staff of Burger’s Zoo, Arnhem, for their help with sampling the animal faeces.
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by growth on a semi-selective medium and GLC assay of acetic acid production, Microbiol. Ecol. Health Dis. 2 (1989) 227–230. J.M. Hardie, R.A. Whiley, The genus Streptococcus, in: B.J.B. Wood, W.H. Holzapfel (Eds.), The Genera of Lactic Acid Bacteria, Blackie Academic Press, London, 1995, pp. 54–125. B. Sgorbati, B. Biavati, D. Palenzona, The genus Bifidobacterium, in: B.J.B. Wood, W.H. Holzapfel (Eds.), The Genera of Lactic Acid Bacteria, Blackie Academic Press, London, 1995, pp. 279–306. L.A. Devriese, B. Pot, The genus Enterococcus, in: B.J.B. Wood, W.H. Holzapfel (Eds.), The Genera of Lactic Acid Bacteria, Blackie Academic Press, London, 1995, pp. 327– 367. J.M. Swenson, R.R. Facklam, C. Thornsberry, Antimicrobial susceptibility of vancomycin-resistant Leuconostoc, Pediococcus, and Lactobacillus species., Antimicrob. Agents Chemother. 34 (1990) 543–549. R.E. Holliman, G.P. Bone, Vancomycin resistance of clinical isolates of lactobacilli, J. Infect. 16 (1988) 279–283. ´ Tyuri, K.P. Lentsner, M.E. ´ Mikel’saar, A.A. Lentsner, M.E. V.M. Shilov, N.N. Liz’ko, Antibiotic sensitivity as an additional characteristic for refining the species classification of lactobacilli, Appl. Biochem. Microbiol. 16 (1981) 535– 538. A.W. Chow, N. Cheng, In vitro activities of daptomycin (LY146032) and paldimycin (U-70,138F) against anaerobic gram-positive bacteria, Antimicrob. Agents Chemother. 32 (1988) 788–790. W.J. Simpson, J.R.M. Hammond, R.B. Miller, Avoparcin and vancomycin: useful antibiotics for the isolation of brewery lactic acid bacteria, J. Appl. Microbiol. 64 (1988) 299–309. P.G. Bille, W.E. Espie, W.M.A. Mullan, Evaluation of media for the isolation of leuconostocs from fermented dairy products, Milchwissenschaft 47 (1992) 637–640. A. Tsakri, A. Maniatis, Leuconostoc, Pediococcus and Lactobacillus sp.: Gram-positive vancomycin-resistant bacteria, Acta Microbiol. Hell. (in Greek with English summary) 36 (1991) 312–320.. E. Potgieter, M. Carmichael, H.J. Koornhof, L.J. Chalkley, In vitro antimicrobial susceptibility of viridans streptococci isolated from blood cultures, Eur. J. Clin. Microbiol. Infect. Dis. 11 (1992) 543–546. K.S. Lim, C.S. Huh, Y.J. Baek, Antimicrobial susceptibility of bifidobacteria, J. Dairy Sci. 76 (1993) 2168–2174. M. Green, K. Barbadora, M. Micaelis, Recovery of vancomycin-resistant Gram-positive cocci from pediatric liver transplant recipients, J. Clin. Microbiol. 29 (1991) 2503– 2506. F. Huygens, Vancomycin resistance is not inducible in Leuconostoc, Lactobacillus, Pediococcus and Enterococcus faecalis A256, S. Afr. J. Sci. 91 (1995) 94–98. B. Pacher, W. Kneifel, Development of a culture medium for the detection and enumeration of bifidobacteria in fermented milk products, Int. Dairy J. 6 (1996) 43–64.
Journal of Microbiological Methods
LAMVAB—A new selective medium for the isolation of lactobacilli from faeces R. Hartemink*, V.R. Domenech, F.M. Rombouts Section Food Chemistry and Microbiology, Department of Food Science, Wageningen Agricultural University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands Received 28 November 1996; revised 4 March 1997; accepted 15 April 1997
Abstract The development of a new selective medium, LAMVAB, for the isolation of Lactobacillus species from faeces is described. The medium is highly selective due to a low pH and the presence of vancomycin (20 mg l 21 ). In addition, the medium contains cysteine–HCl to enhance anaerobic conditions, and bromocresol green as a pH indicator. Competing faecal flora is inhibited by the low pH (enterobacteria, bacteroides and other Gram-negative anaerobes, clostridia) and the vancomycin (enterococci, bifidobacteria, clostridia). Characterization of the strains isolated on LAMVAB from human faeces included all common faecal lactobacilli and no other lactic acid bacteria. In one sample, vancomycin-resistant cocci were present and in another sample, yeasts were isolated. It can be concluded that the new medium is highly selective and permits the growth of all common faecal lactobacilli. The new medium can also be applied as a selective medium for lactobacilli in probiotics, which contain mixed populations of lactobacilli, bifidobacteria and enterococci. 1997 Elsevier Science B.V. Keywords: Lactobacillus; Faecal flora; Vancomycin
1. Introduction Lactobacilli are Gram-positive anaerobic or facultative aerobic rods, which can be isolated from a large number of sources. Lactobacilli can be isolated from the human and animal body (oral cavity, stomach, intestines, vagina), plants and material of plant origin, sewage and fermented products. Lactobacilli are strictly fermentative and acid-resistant. Growth is enhanced by acidic conditions. The final pH in broth cultures is generally below 4.5 [1]. Lactobacilli are considered to have beneficial effects on human and animal health. Therefore, *Corresponding author. Tel.: 131 317 482241; fax: 131 317 484893; e-mail: [email protected]
several species of lactobacilli are used as probiotics. Worldwide, many products with probiotic lactobacilli are marketed [2,3]. To have a beneficial effect on the health of the host, it is necessary that the probiotic strain is able to survive in the gastro-intestinal tract [2]. It is therefore necessary to determine the number of lactobacilli in faeces and, if possible, to identify the different species directly. This can be done using specific selective media, DNA probes or immunological techniques. Lactobacilli are generally isolated on rich, slightly acidic media, such as MRS, LBS and Rogosa agars [4–6]. For most (fermented) food products, these media allow selective isolation of lactobacilli, as they are generally the most abundant group of bacteria present.
0167-7012 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved PII S0167-7012( 97 )00025-0
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R. Hartemink et al. / Journal of Microbiological Methods 29 (1997) 77 – 84
Table 1 Species of lactobacilli isolated from human or animal faeces [1] Species
Isolated from
Lactobacillus acidophilus Lact. amylovorus Lact. aviarius subsp. araffinosus Lact. aviarius subsp. aviarius Lact. brevis Lact. casei Lact. coryniformis Lact. crispatus Lact. curvatus Lact. fermentum Lact. gallinarium Lact. gasseri Lact. hamsteri Lact. intestinalis Lact. johnsonii Lact. murinus Lact. paracasei subsp. paracasei Lact. paracasei subsp. rhamnosus Lact. plantarum Lact. reuteri Lact. salivarius Lact. vaccinostercus
Human, animals Pig Chicken Chicken Human, cow, rat Human Cow Human Cow Human Chicken Human Hamster Mouse, rat Chicken, calf, mouse, pig Mouse, rat Human Human Human Human, animals Human, hamster, chicken Cow
In faeces, however, lactobacilli are present in relatively low numbers [7,8]. In faecal samples, several lactobacilli have been isolated (Table 1), in humans, the main lactobacilli are Lact. acidophilus, Lact. casei and Lact. paracasei [1]. The counts of lactobacilli in faeces are highly variable between species and persons, even within a single person [4,7,8]. The media mentioned above are not selective, as growth of other acid-resistant bacteria is not restricted and, in particular, bifidobacteria, streptococci and enterococci are able to grow on these media [9,10]. Growth of other intestinal bacteria is restricted, but not negligible. It is therefore necessary to have a highly selective medium, which allows the isolation of low numbers of lactobacilli in the presence of high numbers of bifidobacteria, streptococci and / or enterococci. In this report, we describe the development of a new selective medium for the isolation of faecal lactobacilli. Lactobacilli are highly acid-resistant, with growth being possible at an initial pH of 5.0 [1]. Most other intestinal bacteria are not able to grow at this acidic pH. Bifidobacteria, enterococci and streptococci are
not inhibited [11–13]. Contrary to other Gram-positive bacteria, most lactobacilli are resistant to the antibiotic vancomycin, while Gram-negative bacteria are generally resistant [14–17]. Vancomycin has already been applied previously for the selective isolation of lactobacilli from brewery products [18]. It is not useful as a selective agent in dairy products containing Leuconostoc or Pediococcus species, as these are vancomycin resistant [14,19,20]. Enterococci, streptococci and bifidobacteria are susceptible to vancomycin [21–23]. Furthermore, resistance to vancomycin is not inducible and only a few vancomycin-resistant enterococci have been isolated [24]. Vancomycin-resistant bifidobacteria have not been reported. Although lactobacilli are aerotolerant, anaerobic conditions favour their isolation and growth. It was decided to add cysteine as a reducing agent to improve the isolation of lactobacilli. The final medium consisted of MRS agar, with vancomycin, low pH (5.0) and cysteine as selective and elective agents, and was named Lactobacillus Anaerobic MRS with Vancomycin and Bromocresol green, or LAMVAB.
2. Materials and methods
2.1. LAMVAB The medium consists of three different components: Solution A: MRS broth (Merck 1.10661), 104.4 g / l, with cysteine–HCl (0.5 g / l) and bromocresol green (0.05 g / l). The pH of this solution was adjusted to 5.060.1 using 4 M HCl before autoclaving. Solution B: Agar, 40 g / l. Solution C: Vancomycin hydrochloride (local pharmacy, .95% purity), 2 mg / ml in water. Solution C should be kept at 0–48C and was found to be stable for at least three months. Solutions A and B are sterilized at 1218C for 15 min. Solution C is sterilized by filtration using a 0.2-mm filter. Preparation of the medium involves sterilization of equal volumes of solutions A and B. Solution B is cooled down to 508C in a water bath. Solution A is cooled down to room temperature. To 500 ml of
R. Hartemink et al. / Journal of Microbiological Methods 29 (1997) 77 – 84
solution A, 10 ml of solution C are added aseptically. Finally, solution B is added to the MRS–vancomycin (A1C) mixture. Plates have to be poured immediately after mixing. This procedure results in a final vancomycin concentration of 20 mg / l.
2.2. Growth of different intestinal bacteria on LAMVAB Selected intestinal bacteria from stock cultures were pre-cultured anaerobically in thioglycolate broth for 24–48 h at 378C. From these cultures, a streak was made, using a sterile cotton swab, on LAMVAB and RCA (reinforced clostridial agar, Oxoid) plates. RCA was used as a control medium. The following strains were used: Lactobacilli (150 strains, .10 species), bacteroides (10 strains, 5 species), clostridia (15 strains, 13 species), bifidobacteria (25 strains, .12 species), eubacteria (2 strains, 2 species), streptococci, lactococci and enterococci (50 strains, .10 species), anaerobic cocci (5 strains, 3 species), fusobacteria (5 strains, 3 species), enterobacteria (15 strains, 7 species), actinomyces (4 strains, 3 species) and two strains of Megasphaera elsdenii.
2.3. Inhibition of intestinal bacteria by LAMVAB To determine which component of the LAMVAB medium inhibits different groups of intestinal bacteria, a selection of isolates (Table 2) was tested for growth on LAMVAB, LAMVAB with a pH value of 6.5 (L65) and on LAMVAB without vancomycin (L2). The inoculation was carried out as described above.
2.4. Comparison of LAMVAB with MRS To compare the growth of intestinal lactobacilli and possible competing flora on MRS and LAMVAB, 71 strains of lactobacilli, bifidobacteria, Actinomyces species, streptococci and lactococci (Table 3) were grown in MRS broth. From these broths, 10-fold dilutions were prepared as described above, and inoculated on MRS and LAMVAB agars. The plates were incubated anaerobically at 378C for 72 h.
79
Table 2 Growth of selected bacteria on LAMVAB, LAMVAB at pH 6.5 (L65) or LAMVAB without vancomycin (L2) Species
L65
L2
LAMVAB
Actinomyces naeslundii Aeromonas hydrophila Bacillus cereus Bacillus megatherium Bacteroides fragilis Bacteroides ovatus Bifidobacterium adolescentis Bifidobacterium dentium Bifidobacterium infantis Bifidobacterium longum Enterobacter cloacae Enterococcus faecalis Enterococcus species Escherichia coli Lactococcus lactis Listeria innocua Proteus mirabilis Proteus vulgaris Pseudomonas fragi Pseudomonas sp. Salmonella infantis Salmonella montevideo Staphylococcus aureus Streptococcus mutans Streptococcus oralis Yersinia enterocolitica
No Yes Yes Yes Yes Yes No No No No Yes Yes No Yes No Yes Yes Yes Yes Yes Yes Yes No No No Yes
Yes No No No No No Yes Yes Yes Yes No No Yes No Yes No No No No No No No Yes Yes Yes No
No No No No No No No No No No No No No No No No No No No No No No No No No No
Growth was considered positive if colonies were visible. This included pinpoint colonies.
2.5. Isolation of lactobacilli from faeces To determine the selectivity of LAMVAB as a medium for the isolation of lactobacilli, faecal samples (from humans and animals) were taken and dilutions were plated on MRS and LAMVAB agars. Faecal samples were taken as fresh as possible and an aliquot was transferred into a pre-weighed bottle with buffered medium. This medium consisted of buffered peptone water (Oxoid) with cysteine–HCl (0.5 g / l) and Tween 80 (1 ml / l). The pH was adjusted to 6.560.1 before autoclaving at 1218C for 15 min. After sampling, the bottles were imported into an anaerobic chamber, with an atmosphere of 80% nitrogen, 10% hydrogen and 10% carbon dioxide (HoekLoos SHK 050H, HoekLoos, Rotterdam, The Netherlands). The samples were mixed thoroughly using an Ultra Turrax mixer. A 10-fold series of dilutions was prepared of the homogenates
80 R. Hartemink et al. / Journal of Microbiological Methods 29 (1997) 77 – 84 Table 3 Counts of selected lactobacilli and possible competing flora on MRS (control medium) and LAMVAB Strain
Lactobacillus acidophilus Lact. acidophilus Lact. acidophilus Lact. acidophilus Lact. bulgaricus Lact. casei Lact. casei Lact. fermentum Lact. fermentum Lact. helveticus Lact. plantarum Lact. reuteri Lact. sake Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Lact. species Actinomyces israelii Ac. naeslundii Ac. naeslundii Ac. viscosus Bifidobacterium adolescentis B. animalis B. boum B. choerinum B. dentium B. globosum B. infantis B. longum B. longum B. pseudocatenulatum B. pseudolongum
Our number
CH rp 128 126 Lb-1 casCH casY 124 123 Lb-4 Lb-9 Lb-10 Lb. sake 626 824 199 168 856 818 812 711 819 844 832 TR-3 SST-1 SST-3 SST-8 SST-11 TST-1 TR-6 624 622 613 620 604 204 SST-7 Ac-1 41 Ac-2 40 88 Bi-35 Bi-37 Bi-42 Bi-39 Bi-41 223 Bi-17 219 Bi-40 Bi-33
Origin a
Dairy starter Dairy starter Swine faeces Swine faeces IM Dairy starter Dairy starter Swine faeces Swine faeces Type Type Type ATO Human faeces Swine faeces Human faeces Human faeces Swine faeces Swine faeces Swine faeces Human faeces Swine faeces Swine faeces Swine faeces Oral cavity Oral cavity Oral cavity Oral cavity Oral cavity Oral cavity Oral cavity Human faeces Human faeces Human faeces Human faeces Human faeces Human faeces Oral cavity MA Oral cavity, Nijm MA Oral cavity, Nijm Type Type Type Type Type Type Type Human faeces Type Type Type
Log N counts on MRS
LAMVAB
8.40 8.67 7.05 6.88 8.10 8.45 8.09 8.21 7.64 8.14 7.40 7.94 8.20 8.25 8.15 6.95 7.50 7.30 7.15 7.27 8.43 7.03 6.12 8.62 7.45 7.77 8.26 7.28 6.78 7.21 8.00 7.74 8.43 8.58 8.12 7.60 7.13 6.88 8.09 7.95 8.92 8.03 6.00 7.48 8.92 8.71 8.80 8.27 8.33 8.50 8.78 7.43 8.12
8.62 8.74 6.99 6.90 8.05 8.40 7.99 8.11 7.73 8.01 7.58 7.95 8.17 8.20 8.19 7.13 7.48 7.43 7.18 7.35 8.40 7.02 6.07 8.80 7.18 ,2.0 ,2.0 6.91 6.52 7.17 7.74 7.99 8.48 8.71 8.26 7.43 7.09 6.88 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0
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81
Table 3. Continued Strain
B. pullorum B. species B. species B. species B. species B. species B. species B. suis Enterococcus faecalis Lactococcus lactis L. lactis L. lactis Streptococcus gordonii Strep. mitis Strep. mutans Strep. mutans Strep. oralis Strep. species
Our number
Bi-38 683 636 383 628 665 380 211 Ec-1 Lc-4 Lc-3 Lc-1 42 46 44 36 45 TR-11
Origin a
Type Human faeces Human faeces Human faeces Human faeces Human faeces Human faeces Type ATO NIZO NIZO NIZO Oral cavity, Nijm Oral cavity, Nijm Oral cavity, Nijm Type Oral cavity, Nijm Oral cavity, Nijm
Log N counts on MRS
LAMVAB
7.33 8.17 8.02 8.62 8.50 8.85 6.78 8.67 8.09 7.88 7.92 7.86 8.32 8.23 8.21 8.01 7.65 7.65
,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0 7.07 ,2.0 ,2.0 ,2.0 ,2.0 ,2.0
Counts are expressed as logarithmic numbers. a These strains were obtained from: Type: type strains, obtained from DSM (Braunsweig, Germany); MA5Maastricht University, The Netherlands; NIZO5Netherlands Institute for Dairy Research, Ede, The Netherlands; ATO5AgroTechnological Institute, Wageningen, The Netherlands; IM5Dept. Industrial Microbiology, Wageningen Agricultural University, The Netherlands; Nijm5Dept. Oral Microbiology, Nijmegen University, The Netherlands.
using RPS. RPS consisted of neutralized bacterial peptone (Oxoid; 0.5 g / l), NaCl (Merck; 8.0 g / l) and cysteine–HCl (0.5 g / l). The pH was adjusted to 6.760.1 before autoclaving at 1218C for 15 min. The tubes were kept under anaerobic conditions for at least 16 h before use. Finally, 30 ml of the 3rd, 5th and 7th dilutions was pipetted on LAMVAB or MRS plates. The plates were incubated under anaerobic conditions at 378C for 72 h.
blue, due to the uptake of bromocresol green into the cell. As not all lactobacilli showed this color and remained white, the color of the colonies was not considered to be characteristic. Several isolates from fish failed to grow on LAMVAB. Further identification showed that these isolates were carnobacteria and not lactobacilli (M. Dapkevicius, personal communication). Carnobacteria fail to grow at pH 5.0.
3.2. Inhibition of intestinal bacteria by LAMVAB 3. Results
3.1. Growth of different intestinal bacteria on LAMVAB None of the intestinal strains, except for the lactobacilli and one strain of Streptococcus gordonii were able to grow on LAMVAB. All lactobacilli from all origins were able to grow on LAMVAB. Growth was determined as colonies over 1 mm in diameter and a yellow discoloration of the medium, due to acid production. Most colonies were green or
Comparing the growth on LAMVAB with that on LAMVAB at pH 6.5 (L65) and on LAMVAB without vancomycin (L2) showed that Gram-negative bacteria were mainly inhibited by the low pH (Table 2) and Gram-positive bacteria were inhibited by the vancomycin.
3.3. Comparison of LAMVAB with MRS Comparing the new LAMVAB medium with MRS as a reference medium showed that only 2 out of 38 lactobacilli failed to grow on LAMVAB (Table 3).
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These two strains were both isolated from the oral cavity. All other lactobacilli showed no reduction in counts on LAMVAB. None of the bifidobacteria, Actinomyces species and cocci grew on LAMVAB, with the exception of Streptococcus gordonii, a strain isolated from the oral cavity.
3.4. Isolation of lactobacilli from faeces Lactobacilli could be selectively isolated from samples of human faeces. Out of 20 samples of human faeces, lactobacilli could be detected in 15 samples. The counts ranged from (log N) 2.5 to 9.0. With the exception of enterococci in one sample and yeasts in another sample, no other bacteria were observed. Faecal samples from 19 different animals were plated on MRS and LAMVAB (Table 4). Growth on MRS was observed in all samples. Bifidobacteria, cocci and spore-forming organisms were observed in 17 samples. In only 2 samples, only lactobacilli were detected on MRS. In 13 of the samples growth on LAMVAB was observed. Lactobacilli were present in all 13 samples. Some cocci were observed in only one of the samples. Bifidobacteria, yeasts or sporeforming organisms were not observed. In only two of the samples were presumptive lactobacilli observed on MRS, but no growth was observed on LAMVAB.
4. Discussion The isolation of lactobacilli from faeces is generally difficult, due to the presence of competing microorganisms, like bifidobacteria, streptococci and enterococci, which may be present in higher numbers than lactobacilli. Most media used for the isolation of lactobacilli also support the growth of these competing micro-organisms [9,10]. The results presented above indicate that LAMVAB does not support the growth of these bacteria, whereas growth of lactobacilli is unaffected. LAMVAB is easy to prepare, even though the three different components have to be sterilised separately. Due to the low pH of the medium, it is impossible to add the agar to the MRS broth directly. The medium does not require expensive chemicals or
antibiotics. Vancomycin is widely used, relatively inexpensive and is easy to obtain from pharmacies. The medium is selective, due to the low pH, which inhibits the most common Gram-negative bacteria in faeces, and the presence of vancomycin, which inhibits the competing Gram-positive flora (Table 2). This is in accordance with the literature. Vancomycin inhibits Gram-positive bacteria, except lactobacilli. Several studies have shown that faecal lactobacilli are not inhibited by vancomycin [14–18]. Only one study indicated susceptibility of faecal lactobacilli to vancomycin [25]. Susceptibility was reported for Lact. acidophilus and Lact. bulgaricus towards very low concentrations of vancomycin (1 and 4 mg / l). Our results show that most lactobacilli are not affected by the vancomycin, as there is no significant reduction in logarithmic counts between MRS and LAMVAB. Only two unidentified oral isolates failed to grow on LAMVAB. These results are in accordance with the literature [14–18]. On only a few occasions could other bacteria be isolated on LAMVAB. These were cocci, probably enterococci or streptococci and it is known that a small percentage of enterococci are vancomycin resistant [21–23]. Also, we observed growth of Streptococcus gordonii, an oral species, which may be present in small numbers in faeces. Comparison of LAMVAB with MRS indicated a low selectivity of MRS, whereas LAMVAB was highly selective for lactobacilli. Even though we did not compare LAMVAB with other common media, such as Rogosa and LBS agar, it is known that the growth of the bacteria tested on these media is similar to that on MRS [9,10]. LAMVAB was successfully applied to the isolation of lactobacilli from faeces from humans and a number of animals. Lactobacilli were present in most of the animals tested (Table 4), with the exception of the giant bat, giraffe, llama, yak, cheetah and watussi. In those samples, other bacteria were isolated on MRS medium, indicating lower selectivity for this medium. There were only two samples (from the giraffe and watussi) of presumptive lactobacilli that could be isolated on MRS, but not on LAMVAB. It is not known whether these isolates were true lactobacilli or other non-spore-forming non-branched rods. LAMVAB may also be a suitable medium for the
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83
Table 4 Morpholgy of bacteria isolated from animal faeces Animal
Medium
Colonies on plate a
Colonies tested b
Possible lactobacilli c
Cocci d
Bifid e
Other f
Grevy’s zebra (Equus grevyi) Capybara (Hydrochaeris hydrochaeris) Giant bat (Pteropus vampyrus) Bongo (Boocercus euryceros) White rhinoceros (Cerathoterium simum) Gorilla (Gorilla gorilla) Chimpanzee (Pan troglodytes) Chimpanzee (Pan troglodytes) Chimpanzee (Pan troglodytes) Three-toed sloth (Bradipus tridactylus) Celebes Hornbill (Aceros cassidix) Giraffe (Giraffa camelopardalis) Mouse deer (Tragulus javanicus) Llama (Lama glama) Elephant (Loxodonta africana) Yak (Bos grunniens) Cheetah (Acinonyx jubatus) Watussi (Bos taurus) Bactrian camel (Camelus bactrianus)
MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB MRS LAMVAB
9 5 8 8 8 n.g. 36 12 52 33 14 3 9 6 17 4 15 50 9 6 61 22 31 n.g. 8 4 8 n.g. 214 25 9 n.g. 12 n.g. 19 n.g. 37 3
8 5 8 4 8
7 5 4 4 0
1 0 4 0 8
0 0 0 0 0
0 0 0 0 0
8 6 8 8 8 3 8 6 8 4 8 8 8 5 8 4 8
3 6 8 8 5 3 6 6 3 4 5 8 8 5 3 4 2
5 0 0 0 1 0 1 0 0 0 0 0 0 0 4 0 6
0 0 0 0 2 0 1 0 5 0 3 0 0 0 1 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
8 3 8
0 3 0
1 0 8
1 0 0
6 (spores) 0 0
8 8 8
1 6 0
1 2 6
6 0 2
0 0 0
8
0
0
0
8 (spores)
8
1
6
0
1 (spores)
8 2
3 2
3 0
2 0
0 0
a
Total number of colonies on the plate tested. The number of colonies tested, preferably of the same dilution for both media, the colonies taken were mainly selected on differences in colony morphology, not incidence. c Possible lactobacilli were rod-shaped, straight or slightly curved, not branched bacteria and showed no spores. d Cocci were all coccal-shaped bacteria, mono-, duplo- or streptococci. e Bifid were rod-shaped bacteria with branches, this included possible Bifidobacterium, Actinomyces and Propionibacterium species. f Others included spore-forming rods, yeasts and moulds. n.g.5no visible growth. b
isolation of lactobacilli from probiotics. These products often contain lactobacilli and bifidobacteria. As bifidobacteria fail to grow on LAMVAB, selective isolation of the lactobacilli is possible. We have
succesfully used LAMVAB to isolate different species of lactobacilli from dairy products in the presence of streptococci and bifidobacteria (data not shown).
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It can be concluded that LAMVAB is highly selective for lactobacilli. It allows growth of all faecal lactobacilli tested and suppresses all other bacteria encountered in faecal material, with an occasional exception of cocci and yeasts. The medium is more selective than the media commonly used for the isolation of lactobacilli from faeces and probiotics.
[11]
[12]
[13]
Acknowledgements The authors gratefully acknowledge the financial support for this study, which is part of a multi-donor funded research programme on the role of nondigestible oligosaccharides in food and feed. Also, we would like to thank the staff of Burger’s Zoo, Arnhem, for their help with sampling the animal faeces.
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