Enteric Pathogen-Normal Flora Interactions1

Transcription

1 THE AMERICAN JOURNAL OF CLINICAL NUTRITION Vol. 23, No. 11, November, 1970, pp S6 Printed in U.S.A. Enteric Pathogen-Normal Flora Interactions1 S TUDIES ON enteric pathogen-normal floral interactions have dealt primarily with the function of the normal intestinal flora as a protective mechanism in preventing establishment of enteric infections. Interest in the phenomenon of antagonism against pathogenic intestinal bacteria has existed for many years. As long ago as 1916, Nissle (1) discussed the possibility that normal intestinal flora may be a significant factor in the natural resistance of human beings and animals to enteric infections. Subsequent reports described antagonistic interactions between coliform organisms (2) and the production of colicines by certain intestinal bacteria that interfere with the multiplication of other bacteria (3). More direct evidence for a possible protective role of the normal intestinal flora was presented by Freter (4, 5) who found that oral administration of antibiotics, to eliminate components of the intestinal flora of mice and guinea pigs, rendered the animals susceptible to infection with Shigella flexneri and Vibrio cholerae. Introduction of an Escherichia coli strain into the intestinal tract of the experimental animals resulted in elimination of the pathogens. At about the same time, Bohnhoff, Drake and Miller (6) reported that oral administration of streptomycin to mice greatly increased their susceptibility to Salmonella enteriditis infection. Elimination of the normal flora 1 Studies by the author, cited in this paper, supported in part by the Army Medical Research and Development Command Contract DA DA C-8l62. Associate Professor of Microbiology, University of Missouri School of Medicine, Columbia, Missouri. DAVID J. HENTGES,2 PH.D. lowered the ID50 of Salmonella from one million viable bacteria per mouse to fewer than 10 bacteria. All this information suggested that the normal intestinal flora represents a major factor that effectively interferes with the establishment of pathogenic bacteria in the intestine. When it was demonstrated that mice and guinea pigs could be infected with Salmonella, Shigella, and Vibrio c/zolerae after elimination of the enteric flora with antibiotics, a convenient experimental animal became available for in vivo studies of en teric pathogen-normal flora in teractions. Having established that the intestinal microflora interferes with multiplication of pathogens in the animals, it seemed important to determine which components of the flora are responsible for the antagonism. Miller and Bohnhoff (7) examined the composition of the intestinal flora of susceptible mice aftei- streptornycin treatment and determined that the gram-negative bacilli were virtually eliminated from the gut. They concluded that disappearance of Bacteroides from the enteric microflora seemed most directly correlated with enhanced susceptibility to Salmonella infection and showed that reestablishment of certain strains of Bacteroides in the intestinal tract, after antibiotic treatment, partially restored resistance to infection with Salmonella enteritidis. Hentges and Freter (8), utilizing a different approach, examined individual components of the intestinal flora for their antagonistic activities against Shigella flexneri. The ability of 22 test strains, including Esche;ichia 1451

2 1452 Hentges Aerobacler aerog Proteus enes vulgaris TABLE Antagonism of different test strains against Escherichla Pseudornonas aetlit iflosa Test Shigella flexneri in vivo organism Species Strain number coli A icaligenes faecalis 2, ,718 2, ,681 2,689 2,803 2,820 2,120 2,258 2,445 In vivo antagonism A, strain was antagonistic; N, strain was not antagonistic; 0, strain did not grow in the mouse intestine, a This strain was able to eliminate Shigella almost completely in quantitative experifllefl ts. coli, A erobacter aerogenes, Protews vulgaris, Pseudom onas aeru gin osa, and A icaligenes fecalis, to suppress the growth of Shigella flexneri in the mouse intestine was determined. Groups of 10 mice, pretreated with antibiotics, were orally infected with mixtures of streptomycin resistant Shigella flexneri and one test strain. A test strain was considered to be antagonistic for Shigella when stool cultures from at least 5 of the 10 mice produced no Shigella colonies 2 clays and 5 days after infection. Results of these studies, repeated several I times, are shown in Table I. Antagonistic behavior appeared to be related to the species of the test strain. Coliform organisms, i.e., E. coli and Aerobacter aerogenes strains, were the most antagonistic, whereas Pseudomonas aeruginosa strains did not inhibit Shigella multiplication. The Alcali- \a genes fecalis strains failed to multiply in \a the mouse intestine. \a An in vitro culture method was then : sought that would reproduce in vivo re- :. stilts. Good correlation between in vivo and in Vitro results was obtained when mixtures A of Shigella and test strains were inoculated, A either into static broth cultures that were A frequently transferred, or into continuous- A flow cultures. Utilizing both of these techniques, Freter (9) determined the 0 mechanisms by which the antagonistic test A strains inhibit Shigella in infusion broth cultures. Filtrates from continuous-flow - cultures of E. coli failed to support multi- N plication of Shigella, provided the filtrates N were kept in a nitrogen atmosphere. Broth N that was in contact through a cellophane N membrane with either static or continuous flow cultures of antagonistic test strains O inhibited the growth of Shigella in a O nitrogen atmosphere. The antagonistic test strains strongly reduced both static and continuous-flow broth cultures, whereas nonantagonistic strains did so to a lesser degree. The Shigella inhibition could be reversed either by aeration of the cultures or by the addition of glucose to the medium. From these results, Freter concluded that antagonism in broth cultures occurs as a result of a competition for carbon sources utilizable in a highly reduced medium. Based on his observation that the cecum of mice, the main site of in vivo multiplication of the microorganisms tested, is in a highly reduced state (Eh my) (see footnote 3), Freter suggested that antagonism against Shigella in the intestines of = observed difference in electromotive force between material being measured and normal hydrogen electrode.

3 Interactions of Enteric Pathogen and Normal Flora 1453 mice is also the result of a competition for fermentable carbon sources. Interactions between components of the normal intestinal flora and Shigella flexneri were also studied in vitro by Hentges (10, 11). A total of 15 strains of coliform organisms, isolated from human feces, were examined for their inhibitory activities against Shigella in a chemically defined medium. Included in the study were strains of Escherichia coli, Kiebsiella aerogenes, Enterobacter aerogenes, and slow lactose-fermenting strains of both E. coli and K. aerogenes. The organisms were grown in a liquid medium composed of 1% glutamic acid, 1% glucose, 0.4% NH4C1, 2% Na2HPO4, 0.5% KH2PO4, and 0.01% niacin. Experiments showed that the Shigella strain required glucose as a carbon energy source, glutamic acid as a nitrogen source, and niacin as an accessory growth factor. Fourteen of the 15 strains tested inhibited Shigella growth in mixed culture. The exception, a slow lactose-fermenting E. coli strain, interestingly, had a stimulatory effect on Shigella growth. Typical results obtained with Shigella and antagonistic test strains in pure and mixed cultures are illustrated in Fig. 1. The graph on the left in the figure represents Shigella and Klebsiella growth curves and on the right, Shigella and E. coli growth curves. Results show that pure and mixed culture growth curves were identical for both E. coli and Kiebsiella. Multiplication rates for Shigella were the same in pure and mixed culture during the early stages of growth, but in the mixture, exponential growth was interrupted and Shigella entered into either a stationary phase or a death phase. Coliform organisms exerted an effect on Shigella in mixture that ranged from bacteriostatic to bactericidal. None of the coliform strains produced colicines active against Shigella. An analysis of mixed culture environments at the time Shigella inhibition occurred revealed that inhibition was not due to depletion BACTERtOSTATIC EFFECT BACTERIC:DAL EFFECT Ho,ts Incubation Hours Incubo an Fic. 1. Inhibition of Shigella by coliform organisms. of glucose, glutamic acid, or niacin. Essential nutrients remaining at the time inhibition occurred were considerably in excess of concentrations required to support Shigella growth. Antagonistic coliform organisms produced, however, formic and acetic acids in the culture medium, presumably as metabolic end products, in concentrations that inhibited Shigella growth. The organisms also greatly i-educed the culture medium to an E5 of approximately -350 my. The influence of volatile fatty acids and anaerobiosis on Shigella growth are illustrated in Fig. 2. The lines in the figure are not intended to repiesent growth curves but simply to illustrate differences in viable Shigella populations after 24 hr incubation. The figure shows that anaerobiosis and fatty acids in themselves had some effect in decreasing the viable Shigella populations, but a combination of the two factors produced a bactericidal effect on Shi gel/a similar to the effect observed in mixed cultures. Under the same conditions, the acids were only moderately toxic for the coliform strains. These data indicate that, in the presence of adequate concentrations of nutrients, Shigella antagonism by the coliform organisms is the result of volatile acid production and concomitant reduction of the medium. Although coliform organisms constitute an important segment of the indigenous

4 1454 Hentges..a 0. Hours Incubohon control ocids FIG. 2. Influence of anaerobiosis and fatty acids on Shigella growth in defined medium. 0 SHIGELLA, PURE CULTURE RE / 1tLA BACTEROIDES PURE & MIXED CUL Ui MIXED CULTURE 53 SI HOURS INCUBATION, SHIGELLA HOURS INCUBATION, BACTEROIDES FIG. 3. Multiplication of Shigella in an exponential phase Bacteroides culture. Inhibitory effect. intestinal flora, there is considerable evidence that anaerobic bacteria, such as Bacteroides, are the predominant organisms in the intestine of man and many animals (12-15). We recently reported results of a study of in vitro interactions between Shi ge/la flexneri and Bacteriodes fragilis strains, isolated from human feces (16). Results showed that when Shigella and Bacteroides were simultaneously inoculated into trypticase soy medium, supplemented with glucose, no Shigella inhibition could be detected. inhibition was apparent only when Shigella was inoculated into estabhished Bacteroides cultures. This is illustrated in Fig. 3. The Shigella viable population after 48 hr incubation in exponential phase Bacteroides cultures was approximately I,000-fold smaller than its population, in a similarly inoculated, control medium. A comparison of Shigella pure and mixed culture growth curves reveals that the diminished Shigella population, in mixture, resulted from a decreased growth rate and premature interruption of exponential growth. Shigella failed to grow at all in stationary phase Bacteroides cultures. An analysis of the stationary phase cultures revealed that acetic and propionic acids were present in sufficiently high concentrations and the ph of the culture medium was sufficiently low to account for inhibidon of Shi ge/la growth. Apparently, during growth, Bacteroides strains produce the acids that interfere with Shige/la multiplication at the ph levels present in the culture medium. In glucose-free Bacteroides cultures, on the other hand, Shigella multiplied almost as well as in the control medium despite the presence of high concentrations of volatile acids. During Bacteroides multiplication, the ph of glucose-free medium remained above 7.0. At the higher ph levels, the acids had little or no toxic effect on Shige/la. These results reaffirm the importance of the volatile acid-ph interrelationship in the inhibition of Shigella growth. At ph levels above neutrality, the acids are almost entirely in the dissociated state, whereas at lower levels, the proportion of undissociated acid molecules increases. Previous work demonstrated that the undissociated acid molecules are responsible for Shi ge/la inhibition (17). Only in a medium of low ph, containing a large proportion of undissociated acid molecules, is Shigella growth inhibited. The work of Meynell (18) and Bohnhoff, Miller and Martin (19, 20) indicates that volatile fatty acids, produced by the normal gut flora in mice, interfere with multiplication of Salmonella in the intestine. Their experiments demonstrated that Salmonella multiplication was inhibited in vitro by suspensions of intestinal contents

5 Interactions of Enteric Pathogen and Normal Flora 1455 from noi-mal mice. Most effective was material from the cecum and transverse colon. The inhibitory activity was not impaired by filtration or heat sterilization. Volatile fatty acids were iecoveied from the intestinal contents of the mice in concentrations that inhibited Salmonella growth at the ph level of the intestines (ph 6.1). Oxidation-reduction (OR) potential also influenced the inhibitory activity of the acids. At an E,, of -200 my, the OR potential measured in the cecums of mice, the volatile acids exerted a weakly bactericidal effect on Salmonella. Oral administration of streptomycin eliminated much of the intestinal flora of the mice and produced conditions that favored multiplication of Salmonella. An increase in oxidation-reduction potential and ph of the intestinal contents was noted accompanied by decrease in volatile fatty acid concentration. From these data it was concluded that protection against Salmonella infection in mice is related to volatile fatty acid production by the intact flora with a concomitant decrease in the oxidation-reduction potential and ph level of the intestinal contents. Studies are currently in progress in our laboratory to determine the mechanisms by which the normal intestinal flora provides protection against Shi gel/a infection in germfree mice. The animals, monocontaminated with antagonistic test strains are subsequently challenged with Shi ge/la. Formal and othei-s (21) previously demonstrated that germfree guinea pigs succumbed after oral infection with Shigel/a flexneri 2a. Animals monocontaminated with E. co/i, however, survived challenge with the dysentery bacilli. Experiments with Shigella flexneri and an antagonistic E. co/i strain in germfree mice demonstrated that both organisms survived and multiplied in the cecum. Figure 4 illustrates that the organisms attained populations of between 1010 and 10 viable organisms/g dry wt of cecal material and appeared to et 0 Ui 12. > II. 0LTTh t5 io o 40 o o SHIGELLA HOURS AFTER INFECTION FIG. 4. The E. coli and Shigella populations in the cecums of monocontaminated mice. 2 8 >4 (1 0 COLI 2 SHIGELLA DAYS AFTER CHALLENGE WITH SHIGELLA FIG. 5. The fate of Shigella in the cecums of mice monocontaminated with E. coli. persist at this level. Shigella failed to multiply, on the other hand, in the cecums of mice monocontaminated with E. co/i but declined in numbers until they stabilized at approximately 102 organisms/g cecal material. These results may be visualized in Fig. 5. It is interesting that Shigella could be recovered from the cecums of the animals 8 days after challenge. Additional studies are currently being carried out on the interrelationships between Bacteroides strains and Shigella flex neri in germfree mice. Future experiments will be designed to determine the mechanisms responsible for the antagonism against Shigella in vivo. a

6 1456 Hentges If it is true that the normal flora represents a natural barrier to the establishment of intestinal pathogens as is indicated by these studies, one wonders under what circumstances does the natural defense mechanism break down to predispose an individual to infection? What about the ecology of the intestinal flora during active infection and recovery from infection? These are some of the important questions on enteric flora-pathogen interactions that have not yet been answered. REFERENCES 1. Nissi.u,A. Ueber die Grundlagen einer ursaechlichen Bekaempfung cler pathologischen Darmflora. Deut. Med. lvorc/zschr. 42: 1181, GRATIA, A. Sur un remarquable exemple dantagonisme entre cleux souches de colibacille. Compt. Rend. Soc. Biol. 93: 1040, FREIWRICQ, P., AND M. LEVINE. Antibiotic interrelationships among the enteric group of bacteria. J. Bacteriol. 54: 785, FRETER, R. The fatal enteric cholera infection in the guinea pig, achieved by inhibition of normal enteric flora. J. Infect. Diseases 97: 57, FRETER, R. Experimental enteric Shigella and l7ibrio infections in mice and guinea pigs. I. Exptl. Med. 104: 411, BOHNOFF, M., B. L. DRAKE AND C. P. MILLER. Effect of streptomycin on susceptibility of intestinal tract to Salmonella infection. Proc. Soc. Exptl. Biol. Med. 86: 133, MILLER, C. P., AND M. BOHNHOFF. Changes in the mouse s enteric microflora associated with enhanced susceptibility to Salmonella infection following streptomycin treatment. J. Infect. Diseases 113: 59, HENTGES, D. J., AND R. FRETER. In vivo and in vitro antagonism of intestinal bacteria against Shigella flexneri. I. Correlation between various tests. I. Infect. Diseases 110: 30, FREmIt, R. In vivo and in vitro antagonism of intestinal bacteria against Shigella flexneri. II. The inhibitory mechanism. 1. Infect. Diseases 110: 38, HENTGES, D. J. Inhibition of Shigella flexneri by the normal intestinal flora. I. Mechanisms of inhibition by Klebsiella. J Bacteriol. 93: 1369, HENTGE5, D. J. Inhibition of Shigella flexneri by the normal intestinal flora. II. Mechanisms of inhil)ition by coliform organisms. J. Bacteriol. 97: 513, DRASAR, B. A. Cultivation of anaerobic intestinal bacteria. J. Pathol. Bacteriol. 94: 417, GALL, L. S., AND P. E. RIELY. Determination of aerobic and anaerobic microflora of human feces. Aerospace Med. Res. Lab. Rept. AMRL-TR Republic Aviation Corp., SMITH, H. W., AND W. E. CRABB. The fecal bacterial flora of animals and man: its development in the young. J. Pathol. Bacteriol. 82: 53, ZUBRZYCKI, L., AND E. H. SPAULDING. Studies on the stability of the normal human fecal flora. J. Bacteriol. 83: 968, HENTGES, I). J., AND B. R. MALER. Shigella flexneri, Bacteroides fragilis interactions in vitro. Bacteriol. Proc. p. 75, HENTGES, D. J. Influence of ph on the inhibitory activity of formic and acetic acids for Shigella. J. Bacteriol. 93: 2029, MEYNELL, G. G. Antibacterial mechanisms of the mouse gut. II. The role of E5 and volatile fatty acids in the normal gut. Brit. J. Exptl. Pat/so!. 44: 209, BOHNHOFF, M., C. P. MILLER, AND W. R. MARTIN. Resistance of the mouse s intestinal tract to experimental Salmonella infection. I. Factors which interfere with the initiation of infection by oral inoculation. J. Exptl. Med. 120: 805, BOHNHOFF, M., C. P. MILLER AND W. R. MARTIN. Resistance of the mouse s intestinal tract to experimental Salmonella infection. II. Factors responsible for its loss following streptomycin treatment. I. Exptl. Med. 120: 817, FORMAL, S. B., G. DAMMIN, H. SPRINZ, D. KUNDEL, H. SCHNEIDER, R. E. HoRowrrz AND M. FORBES. Experimental Shigella infections. V. Studies in germ-free guinea pigs. J. Bacteriol. 82: 284, 1961.