Vol. 23, No. 3
INFECTION AND IMMUNITY, Mar. 1979, p. 559-563 0019-9567/79/03-0559/05$02.00/0
Volatile Fatty Acids and Aerobic Flora in the Gastrointestinal Tract of Mice Under Various Conditions B. M. BYRNE AND J. DANKERT* Laboratory for Medical Microbiology and Department of Hospital Epidemiology, University Hospital, Groningen, The Netherlands Received for publication December 1978
Volatile fatty acids are reported to exert a repressive effect upon Enterobacteriaceae and Pseudomonas species in vitro and in vivo in young mice. The mean total volatile fatty acid concentration in the cecal samples of conventional mice fed ad libitum was 81.7 pmol/g (wet weight), which is antibacterial in vitro, and in the rectal samples it was 41.1 umol/g (wet weight). The mean count of Enterobacteriaceae in the cecum was only 102/g, whereas in the rectum it was 10'/g. Volatile fatty acid levels were influenced by food intake and increased to peak levels approximately 6 to 10 h after eating and then declined. In mice fasted for 17 h, the butyric acid concentration was considerably lower and the number of cecal samples positive for Enterobacteriaceae increased. When fasted for 4 days, mice had extremely low cecal and rectal volatile fatty acid concentrations and the Enterobacteriaceae and enterococci counts increased to mean of 2 x 106/ g and 3 x 106/g, respectively, in the cecum and to means of 107 and 5 X 106/g in the rectum. We conclude that volatile fatty acids are probably one of the many interference mechanisms which are involved with control of the levels of Enterobacteriaceae (and enterococci) in the large intestine of mice.
The gastrointestinal flora is regulated by both direct and indirect interference mechanisms which have been reviewed recently by Savage (14). One of the former mechanisms includes the production of toxic microbial metabolites such as volatile fatty acids (VFA) of which the undissociated state (7, 8) is antagonistic for Enterobacteriaceae species and Pseudomonas aeruginosa. The inhibitory activity of these acids has been thoroughly investigated in vitro (1, 2, 3, 7, 8, 9). The VFA are metabolic end products of the anaerobes in particular (10) and are produced mainly in the cecum. In vivo experiments in mice show that the decrease in VFA levels after antibiotic treatment is associated with a lowered resistance to infection by Salmonella species (2, 3, 8) and by Staphylococcus aureus (9) and with a strong increase in the numbers of coliform bacilli (6). The latter authors have also observed that in young mice the increase in VFA results in a decrease in coliform numbers. However, Freter and Abrams (5) find that VFA levels in the gastrointestinal tract of mice do not always correlate with the Escherichia coli population levels. VFA levels are constantly changing due to variations in the bacterial metabolic activity on the one hand and due to absorption and peristalsis on the other hand. With this in mind, we have tried to investigate the relationship between VFA concentration and the intes-
tinal aerobic flora in mice fed ad libitum and under restricted feeding conditions.
MATERIALS AND METHODS Mice. Female Swiss random mice of strain Cpb.SE (TNO, Zeist, The Netherlands), 8 to 12 weeks old, were maintained individually in Macrolan cages containing autoclaved wood shavings. The mice were fed autoclaved food pellets (Hope Farms, Woerden, The Netherlands) and received sterilized water ad libiturm, except where otherwise stated. For collection of fresh fecal samples, mice were placed in cages containing wired grids. Samples were collected immediately after they were passed to prevent VFA loss. VFA and aerobic bacterial levels in the cecal and fecal samples of conventional mice. A total of 40 mice were screened daily for 7 days for fecal VFA to determine VFA levels and the day-to-day variation. Bacteriological culturing was also performed. A total of 21 mice were sacrificed to determine VFA concentrations and levels of aerobic bacteria in the large intestine (group A). Contents of cecum and rectum were removed and divided for VFA analysis and bacteriological culturing. Experimental procedures. To assess the influence of food intake upon VFA levels in the feces, four mice were fed at fixed times, i.e., at 8.00 to 9.00, 12.00 to 13.00, and 16.00 to 17.00 h for 5 days. On the fifth day, every fecal stool passed was collected individually from 11.00 to 23.00 h and analyzed for VFA. To study the influence of food restriction upon VFA and bac-
559
560
INFECT. IMMUN.
BYRNE AND DANKERT
terial levels, 17 mice were sacrificed 15 to 17 h after feeding (group B). Contents of cecum and rectum were removed for VFA analysis and culturing. Lastly, a fasting experiment was performed involving 10 mice (group C). After 4 days of fasting (with sterilized water ad libitum), the contents of the cecum and rectum were collected aseptically and divided for VFA analysis and bacteriological culturing. In some experiments there was insufficient material for both determinations. VFA extraction and analysis. Immediately after collection, samples were stored at -20°C awaiting analysis. The pretreatment and analysis of specimens is similar to that previously described (18) involving vacuum distillation and gas chromatography. To ensure that this method could be applied to minute samples, the detection limit was first determined. Samples were suspended in 1.5 ml of distilled water containing 500 pl of 2-methylvaleric acid as internal standard and 11 ml of H2SO4 per liter and then directly distilled. Quantitative aerobic culturing technique. After collection, samples were stored at 4°C to prevent microbial growth. Culturing was performed, in most cases, within 30 min, but in any case within 20 h. No differences were found when culturing was done immediately or after 20 h. Specimens were weighed, homogenized in nutrient broth (Oxoid Ltd., London) supplemented with 1% (wt/vol) glucose and serially diluted up to 10'9 in the same medium. Tenfold dilutions were prepared in trays (Thovadec, Nieuwkoop, The Netherlands), using calibrated pipettes, and were incubated at 37°C for 18 to 24 h. Subinoculations were prepared on Sheep blood agar, MacConkey agar (Merck, Darmstad, Federal Republic of Germany) selective for gram-negative organisms, Mannitol Salt agar (Oxoid Ltd., London) selective for staphylococci, Aesculine Azide Bile agar (Merck) selective for enterococci, acetamide (111%, wt/vol) agar (Merck) selective for Pseudomonas species, and Sabouraud dextrose agar (pH 5.2, Merck) selective for yeasts. All inoculated media were incubated at 37°C for 24 h, except Sabouraud dextrose agar which was incubated for 72 h at 23°C. The highest dilution from which growth was obtained, in each of the specimens, represented the concentration of microorganisms. Ten to 20 colonies from MacConkey medium, presumed to be gram-negative bacteria, were identified by using the API-system (API 20 Enteric, Analytical Products, Inc., New York, N.Y.). Enterococci cultured on aesculine azide bile agar appear as black colonies. When less than 100 organisms were present, no growth was obtained and this was consequently denoted as
30 20
°
10
0 12356
tolgo
7
1 23567
Enterobacteriocsat
ou
12
35670
t/grom
G. 2. Individual VFA concentrations and Enterteriaceae levels from cecal (0) and rectal (A) les of mice fed ad libitum (a), fasted for 17 h (b), fasted for 4 days (c).
TABLE 2. Mean major VFA concentrations and aerobic bacterial levels in cecal and rectal samples from mice fed ad libitum (group A), fasted for 15 to 17 h (group B), and fasted for 4 days (group C) Bacterial level (counts/g)b Concn (jmol/g, wet wt) of:a Total Propiomc acid Butyric acid Group Specimen Enterobac- Enterococci Acetic acid (C2 teriaceae Eneoci aerobes (C4) (03) 1.0 x 10' 1.0 X 102 1.5 X 106 22.44 (8.92) 4.36 (2.28) 52.30 (22.55) Cecum A (n = 21)
Rectum B (n = 17)
C (n = 10)
32.91 (14.38)
2.36 (1.15)
5.06 (3.10)
(103_106) 2.0 x 107
(104_101s)
(1w-1M~)
(102_104)
(102_1o63)
(103_1o6)
1.0 X 105
Cecum
58.45 (25.99)
4.05 (2.80)
11.92 (5.59)
Rectum
21.59 (10.64)
1.91 (1.46)
1.56 (0.85)
2 x 103 1 X 106 (104_107) (
INFECTION AND IMMUNITY, Mar. 1979, p. 559-563 0019-9567/79/03-0559/05$02.00/0
Volatile Fatty Acids and Aerobic Flora in the Gastrointestinal Tract of Mice Under Various Conditions B. M. BYRNE AND J. DANKERT* Laboratory for Medical Microbiology and Department of Hospital Epidemiology, University Hospital, Groningen, The Netherlands Received for publication December 1978
Volatile fatty acids are reported to exert a repressive effect upon Enterobacteriaceae and Pseudomonas species in vitro and in vivo in young mice. The mean total volatile fatty acid concentration in the cecal samples of conventional mice fed ad libitum was 81.7 pmol/g (wet weight), which is antibacterial in vitro, and in the rectal samples it was 41.1 umol/g (wet weight). The mean count of Enterobacteriaceae in the cecum was only 102/g, whereas in the rectum it was 10'/g. Volatile fatty acid levels were influenced by food intake and increased to peak levels approximately 6 to 10 h after eating and then declined. In mice fasted for 17 h, the butyric acid concentration was considerably lower and the number of cecal samples positive for Enterobacteriaceae increased. When fasted for 4 days, mice had extremely low cecal and rectal volatile fatty acid concentrations and the Enterobacteriaceae and enterococci counts increased to mean of 2 x 106/ g and 3 x 106/g, respectively, in the cecum and to means of 107 and 5 X 106/g in the rectum. We conclude that volatile fatty acids are probably one of the many interference mechanisms which are involved with control of the levels of Enterobacteriaceae (and enterococci) in the large intestine of mice.
The gastrointestinal flora is regulated by both direct and indirect interference mechanisms which have been reviewed recently by Savage (14). One of the former mechanisms includes the production of toxic microbial metabolites such as volatile fatty acids (VFA) of which the undissociated state (7, 8) is antagonistic for Enterobacteriaceae species and Pseudomonas aeruginosa. The inhibitory activity of these acids has been thoroughly investigated in vitro (1, 2, 3, 7, 8, 9). The VFA are metabolic end products of the anaerobes in particular (10) and are produced mainly in the cecum. In vivo experiments in mice show that the decrease in VFA levels after antibiotic treatment is associated with a lowered resistance to infection by Salmonella species (2, 3, 8) and by Staphylococcus aureus (9) and with a strong increase in the numbers of coliform bacilli (6). The latter authors have also observed that in young mice the increase in VFA results in a decrease in coliform numbers. However, Freter and Abrams (5) find that VFA levels in the gastrointestinal tract of mice do not always correlate with the Escherichia coli population levels. VFA levels are constantly changing due to variations in the bacterial metabolic activity on the one hand and due to absorption and peristalsis on the other hand. With this in mind, we have tried to investigate the relationship between VFA concentration and the intes-
tinal aerobic flora in mice fed ad libitum and under restricted feeding conditions.
MATERIALS AND METHODS Mice. Female Swiss random mice of strain Cpb.SE (TNO, Zeist, The Netherlands), 8 to 12 weeks old, were maintained individually in Macrolan cages containing autoclaved wood shavings. The mice were fed autoclaved food pellets (Hope Farms, Woerden, The Netherlands) and received sterilized water ad libiturm, except where otherwise stated. For collection of fresh fecal samples, mice were placed in cages containing wired grids. Samples were collected immediately after they were passed to prevent VFA loss. VFA and aerobic bacterial levels in the cecal and fecal samples of conventional mice. A total of 40 mice were screened daily for 7 days for fecal VFA to determine VFA levels and the day-to-day variation. Bacteriological culturing was also performed. A total of 21 mice were sacrificed to determine VFA concentrations and levels of aerobic bacteria in the large intestine (group A). Contents of cecum and rectum were removed and divided for VFA analysis and bacteriological culturing. Experimental procedures. To assess the influence of food intake upon VFA levels in the feces, four mice were fed at fixed times, i.e., at 8.00 to 9.00, 12.00 to 13.00, and 16.00 to 17.00 h for 5 days. On the fifth day, every fecal stool passed was collected individually from 11.00 to 23.00 h and analyzed for VFA. To study the influence of food restriction upon VFA and bac-
559
560
INFECT. IMMUN.
BYRNE AND DANKERT
terial levels, 17 mice were sacrificed 15 to 17 h after feeding (group B). Contents of cecum and rectum were removed for VFA analysis and culturing. Lastly, a fasting experiment was performed involving 10 mice (group C). After 4 days of fasting (with sterilized water ad libitum), the contents of the cecum and rectum were collected aseptically and divided for VFA analysis and bacteriological culturing. In some experiments there was insufficient material for both determinations. VFA extraction and analysis. Immediately after collection, samples were stored at -20°C awaiting analysis. The pretreatment and analysis of specimens is similar to that previously described (18) involving vacuum distillation and gas chromatography. To ensure that this method could be applied to minute samples, the detection limit was first determined. Samples were suspended in 1.5 ml of distilled water containing 500 pl of 2-methylvaleric acid as internal standard and 11 ml of H2SO4 per liter and then directly distilled. Quantitative aerobic culturing technique. After collection, samples were stored at 4°C to prevent microbial growth. Culturing was performed, in most cases, within 30 min, but in any case within 20 h. No differences were found when culturing was done immediately or after 20 h. Specimens were weighed, homogenized in nutrient broth (Oxoid Ltd., London) supplemented with 1% (wt/vol) glucose and serially diluted up to 10'9 in the same medium. Tenfold dilutions were prepared in trays (Thovadec, Nieuwkoop, The Netherlands), using calibrated pipettes, and were incubated at 37°C for 18 to 24 h. Subinoculations were prepared on Sheep blood agar, MacConkey agar (Merck, Darmstad, Federal Republic of Germany) selective for gram-negative organisms, Mannitol Salt agar (Oxoid Ltd., London) selective for staphylococci, Aesculine Azide Bile agar (Merck) selective for enterococci, acetamide (111%, wt/vol) agar (Merck) selective for Pseudomonas species, and Sabouraud dextrose agar (pH 5.2, Merck) selective for yeasts. All inoculated media were incubated at 37°C for 24 h, except Sabouraud dextrose agar which was incubated for 72 h at 23°C. The highest dilution from which growth was obtained, in each of the specimens, represented the concentration of microorganisms. Ten to 20 colonies from MacConkey medium, presumed to be gram-negative bacteria, were identified by using the API-system (API 20 Enteric, Analytical Products, Inc., New York, N.Y.). Enterococci cultured on aesculine azide bile agar appear as black colonies. When less than 100 organisms were present, no growth was obtained and this was consequently denoted as
30 20
°
10
0 12356
tolgo
7
1 23567
Enterobacteriocsat
ou
12
35670
t/grom
G. 2. Individual VFA concentrations and Enterteriaceae levels from cecal (0) and rectal (A) les of mice fed ad libitum (a), fasted for 17 h (b), fasted for 4 days (c).
TABLE 2. Mean major VFA concentrations and aerobic bacterial levels in cecal and rectal samples from mice fed ad libitum (group A), fasted for 15 to 17 h (group B), and fasted for 4 days (group C) Bacterial level (counts/g)b Concn (jmol/g, wet wt) of:a Total Propiomc acid Butyric acid Group Specimen Enterobac- Enterococci Acetic acid (C2 teriaceae Eneoci aerobes (C4) (03) 1.0 x 10' 1.0 X 102 1.5 X 106 22.44 (8.92) 4.36 (2.28) 52.30 (22.55) Cecum A (n = 21)
Rectum B (n = 17)
C (n = 10)
32.91 (14.38)
2.36 (1.15)
5.06 (3.10)
(103_106) 2.0 x 107
(104_101s)
(1w-1M~)
(102_104)
(102_1o63)
(103_1o6)
1.0 X 105
Cecum
58.45 (25.99)
4.05 (2.80)
11.92 (5.59)
Rectum
21.59 (10.64)
1.91 (1.46)
1.56 (0.85)
2 x 103 1 X 106 (104_107) (
