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Mice delivered by Caesarian section were used to develop a new mouse colony which has been maintained in an environment protected from contact with common mouse pathogens, but not in the germ-free state. These mice, designated as NCS, were compared with animals of the same sex and age coming from the parent colony maintained under ordinary conditions. The NCS mice grew more rapidly than ordinary mice on complete diets; moreover, they continued to gain weight—although at a slower rate—when fed deficient diets which caused ordinary mice to stop growing, or to lose weight. The NCS mice proved much more susceptible than ordinary mice to certain experimental bacterial infections. In contrast, they were much more resistant than ordinary mice to the lethal effect of large doses of endotoxins. However, they responded to injection of minute amounts of these endotoxins by a marked increase in susceptibility to staphylococcal infection. Bacteriological studies revealed striking qualitative differences between the intestinal flora of NCS and ordinary mice. When NCS mice were contaminated—either by contact or by feeding—with a strain of Escherichia coli recovered from the intestine of ordinary mice, they acquired the characteristics of the latter animals with regard to weight gain on various diets, and to response to bacterial pathogens and endotoxins. NCS mice have been found well suited to the study of several nutritional, bacteriological, and immunological problems and it appears that their production on a large scale will not present unsurmountable difficulties.

Adult mice from seven different colonies were studied with regard to (a) the numbers and types of bacteria that could be cultivated from their stools; (b) their resistance to the lethal effect of endotoxins prepared from three strains of Gram-negative bacilli. See PDF for Structure In six of the seven colonies, the stools yielded large numbers of various types of lactobacilli, enterococci, and Gram-negative bacilli. Most animals in these colonies died within 48 hours following injection of endotoxin. The other mouse colony (NCS) has been maintained for the past three years at the Rockefeller Institute under exacting sanitary conditions; it is free of many types of common mouse pathogens. The stool flora of NCS mice yielded very large numbers of viable lactobacilli (109 per gm), representing at least three different morphological types. In contrast, it contained only few enterococci and Gram-negative bacilli (less than 106 per gm). Moreover, E. coli, Proteus sp., and Pseudomonas sp. could not be recovered from the stools under normal conditions. NCS mice proved resistant to the lethal effect of endotoxins. These characteristics of the NCS colony prevailed whether the animals were housed continuously in individual cages on wire grids, or grouped continuously in large cages with wood shavings as litter. However, the composition of the bacterial flora could be rapidly and profoundly altered by a variety of unrelated disturbances such as sudden changes in environmental temperature, crowding in cages, handling of the animals, administration of antibacterial drugs, etc. The first effect of the change was a marked decrease in the numbers of lactobacilli and commonly an increase in the numbers of Gram-negative bacilli and enterococci. When tested 3 weeks after these disturbances some NCS animals were found to have become susceptible to the lethal effects of endotoxin.

Cultures of tubercle bacilli (typical bovine and human strains) known to differ in the severity of the lesions they induce in experimental animals, were injected in various doses into the cerebrum, peritoneal cavity, or blood stream of mice. Quantitative determinations of the numbers of living bacilli present in the tissues at different intervals of time after infection led to the following classification of the cultures tested:— (a) Certain well known variant forms of tubercle bacilli were found to be unable to multiply in vivo, although they could survive for many weeks in the tissues of mice. These organisms proved to be truly avirulent. (b) Other variant forms underwent multiplication in vivo, even when extremely small infective doses were used, but could not give rise to progressive disease. It is proposed to designate these strains, which produce only abortive infections, as "attenuated." Different levels of attenuation could be detected. The maximum numbers of living bacilli that were recovered from the tissues corresponded directly to the severity and duration of the abortive lesions that could be produced by the strain in guinea pigs or in mice and were characteristic for each strain tested. The two BCG substrains tested were found to differ markedly in their level of attenuation. (c) The cultures virulent for guinea pigs were also capable of establishing a progressive infection in mice even when small infective doses were used. In the case of the attenuated and virulent strains, the population of living bacilli present in the lungs was at first much lower than that in the spleen, but it continued to increase in the former organs throughout the period of observation. This was notably true in the case of the virulent cultures. In contrast, the numbers of living bacilli in the spleen rapidly reached a maximum in the case of all cultures and then decreased progressively. For a given infective dose, and a given interval of time after inoculation, the maximum levels of living bacterial population attained in the spleen and in the lungs proved to be a direct expression of the virulence of the strain.

We have observed that a number of synthetic unsaturated ring-containing acids endowed with auxin (plant hormone) activity can exert a bacteriostatic effect on the growth of certain bacterial species. The following substances were selected as representatives of different chemical types of auxins: indole-3-acetic acid, β-(indole-3)-propionic acid, -(indole-3)-n-butyrie acid, α-naphthalene-acetic acid, β-naphthoxyacetic acid, 2,4-dichlorophenoxyacetic acid, 2,3,5-triiodoben-zoic acid.1The order of activity of these different substances on a few bacterial species is illustrated in Table I in which are given the concentrations of auxins required to cause a 50% inhibition of growth in a medium containing by weight 0.1% enzymatic hydrolysate of casein and 0.02% yeast extract. It should be emphasized that the inhibitory concentrations recorded in Table I have only a relative value since, as will be indicated later, tryptophane and peptone can antagonize the bacteriostatic action of auxins. It is also worth record...

Avery and Cullen1 obtained from both rough and smooth pneumococci a ferment capable of lysing heat-killed pneumococci. In unpublished experiments (Dubos) it was found that the same ferment preparation renders ineffective in rabbits the capsular type specific antigen of killed cells of virulent pneumococci.A hydrolytic action of the ferment preparation of pneumococcus on two tissue polysaccharides is described here. This action seems to be identical with that of the same ferment on the pneumococci.From bovine vitreous humor and from human umbilical cord two polysaccharides have been obtained in pure form, seemingly identical in composition, rotation, and general physical behavior.2 They are composed of acetylglucosamine and glucuronic acid, the components having been shown to be present in equimolar ratio by isolation or quantitative analysis.The 2 polysaccharide acids yielded reducing sugar following hydrolysis by the ferment obtained from pneumococcus autolysates. The hydrolysis is optimal between pH 5 a...

Mice maintained on various types of diets were found to become more susceptible to tuberculosis when deprived of food for periods of 30 hours shortly after infection. In contrast, the susceptibility of the animals to the disease was unaffected by undernutrition resulting from limitation of food intake to a low but constant daily level. The resistance of mice to tuberculosis appeared to be independent—within wide limits—of the protein content of the diet. It is true that mice fed a diet very low in protein and high in carbohydrate proved highly susceptible, but resistance was normal if part of the carbohydrate was replaced by fat (peanut oil)—without any change in the protein content of the food. Resistance to tuberculosis could be consistently and markedly decreased by adding sodium citrate (or glutarate) to a variety of diets. The survival time following infection was greatly shortened if dinitrophenol or thyroxine were administered per os in amounts sufficient to limit the weight gains of non-infected controls. There was usually a lag period of several days before the infection-enhancing effect of these metabolic stimulants became manifest. The procedures which increased the susceptibility of mice to infection with virulent tubercle bacilli also made it possible to establish in these animals a fatal infection with BCG. There was no constant relation between weight gains of uninfected mice on the various regimens, and the effect of the latter on susceptibility to tuberculosis. These findings appear compatible with, but do not prove, the hypothesis that a decrease in resistance to infection can be brought about by metabolic disturbances which cause either a depletion of the glycogen reserves of the body, or a reduction in the glycolytic activity of inflammatory cells, or an increase in the concentration of certain polycarboxylic acids and ketones in the tissues.

At the 22nd Annual Meeting of The Society for Investigative Dermatology, in New York, June 27-29, 1961, Dr. Rene J. Dubos, Member and Professor, The Rockefeller Institute, New York, will deliver the first annual Herman Beerman Lecture. Dr. Dubos's topic will be “Molecules, Social Systems and Dermatology/' and the lecture will be given on Wednesday, June 28th, at 2:00 P. M., at the Barbizon-Plaza Hotel, New York.

A study has been made of the genetic aspects of the difference between two inbred strains of mice (C57B1/6 and Swiss) in response to experimental infection with mammalian tubercle bacilli. Males and females, 4 to 6 weeks of age were inoculated intravenously with 0.2 ml of a 1/50 culture dilution of Mycobacterium tuberculosis var. bovis (Vallée strain) grown in tween albumin medium. Mean survival time for C57B1 animals was 28.1 ± 0.6 days and for Swiss, 55.3 ± 0.6 days postinfection. The characteristic survival time of the two strains was reversed in mice receiving a smaller infective dose. The age of mice at the time of inoculation also affected the results of infection: both C57B1 and Swiss, inoculated at 12 months of age, died at the same rate, but when inoculated at older ages, C57B1 survived slightly longer. Bacteriologic studies demonstrated that there was no significant difference between the two mouse strains with regard to the numbers of viable units of tubercle bacilli recovered from various organs during the 2 week period following infection with a 10–3 culture dilution of Vallée. Moreover, the standard infective inoculum (1/50 culture dilution) did not activate corynebacterial pseudotuberculosis in C57B1 mice, a strain known to be latently infected with Corynebacterium kutscheri, rapid multiplication of tubercle bacilli occurred, but no corynebacteria were recovered. When C57B1 and Swiss strains were crossed, survival tests after infection with the standard inoculum demonstrated that mice of the F1 generation were more resistant than either parent. Whether the overdominance was due to a new combination of parental genes for resistance or to heterosis was not determined. The increased litter size of the F1 mice, an evidence of increased vigor, supports the view that heterosis was involved. In backcrosses to the resistant strain (Swiss), survival time gradually became stabilized at approximately the parental level. In the 1st backcross to the susceptible strain (C57B1), survival times fell into two classes indicating segregation of genes, with perhaps dominance of genes from the Swiss. After repeated backcrosses to C57B1, mice of the 4th backcross generation had a survival time essentially the same as that of the original parental strain. On the basis of having obtained progeny characterized by the original parental susceptibilities after genetic tendencies had been intermingled by crossbreeding, it was concluded that hereditary factors influenced the response of mice to experimental infection with M. tuberculosis. The number of genes was not determined.

Certain strains of yeast were cultured frequently from the feces of adult CFW mice and Long-Evans and Sprague-Dawley rats, but not from infants of those murine strains, or from adults or infants of NCS or NCS-D mice. When the yeasts could be cultured from the feces, they could also be grown from all areas of the digestive tracts of the animals, but especially from the stomachs, where they formed layers on the epithelium of the glandular mucosa. Three of the yeast isolates, one each from the three murine colonies, were provisionally classified in the genus Torulopsis of the asporogenous yeasts. These yeast strains failed to colonize the digestive tubes of suckling infant mice of either the CFW or NCS-D colonies. In contrast, they colonized the guts of adult NCS-D mice and formed layers in their stomachs; tests with the yeast from CFW mice revealed that this strain colonized the guts and formed layers in the stomachs of germ-free CFW mice. When established in NCS-D mice, the yeast strains did not affect qualitatively or quantitatively the growth of the animals or the composition of the bacterial flora in the gastrointestinal tracts. Moreover, they did not elicit an unusual inflammatory response in the digestive tracts; nor were they pathogenic for NCS mice when injected by the intraperitoneal or intravenous routes. The yeasts thus appear to be harmless saprophytes that are able to flourish in the environment of the surface of the secreting epithelium of the murine stomach. The findings conform with the view that some types of microorganisms of the gastrointestinal tract are not just mixed randomly but rather occupy microenvironments in almost pure culture. This concept is important to the understanding of the ecology of the gut microflora.

A study has been made of the fate of staphylococci and tubercle bacilli resuspended in aqueous media at slightly acid reactions. The tests were carried out at several acid reactions in balanced ionic media containing 0.5 per cent serum albumin. These experimental conditions were selected in order to approximate those which are probably encountered by pathogenic agents in inflammatory areas and in the intracellular environment of the leucocytes after phagocytosis. The viability of the microorganisms at a given pH was markedly influenced by the composition of the medium, being decreased by addition to the latter of lactic, acetic, propionic, and butyric acids, and increased by the addition of certain ketone bodies such as dihydroxyacetone and pyruvic, ß-hydroxybutyric, α-ketoglutaric, and oxalacetic acids. The presence of ketone bodies in the medium afforded to the microorganisms some protection against the bactericidal effect of lactic and acetic acids at acid reactions. The minimum and the optimum pH for growth were found to be dependent on the composition of the medium. Both were higher in the presence of lactic, acetic, propionic, and butyric acids than in the media without organic acids added. In contrast, the addition of ketone bodies to the medium allowed microbial multiplication even in acid media (approximately at pH 5.3 or even lower). The fact that lactic acid antagonizes, whereas ketone bodies favor, the survival and multiplication of staphylococci and tubercle bacilli at acid reactions, is discussed in relation to the high susceptibility to infection which is often associated with ketosis of various etiology.

A crystalline substance capable of suppressing the growth of a variety of mycobacteria in vitro has been isolated from extracts of tissue in acidified dilute ethanol. This inhibitory material was found to be equally active against virulent, attenuated, and avirulent variants of human and bovine tubercle bacilli, but had little or no effect on saprophytic mycobacteria and on several non-acid-fast microorganisms under the conditions of the test. Its inhibitory activity on the growth of tubercle bacilli was essentially independent of the size of the inoculum within the limits studied. The crystalline material appeared to exert a bactericidal action on the susceptible organisms. Tubercle bacilli maintained in the presence of the agent for 4 days failed to grow when transferred to inhibitor-free media. The findings were not appreciably altered by minor variations in the composition of the medium or by shift in its reaction. When certain preparations of whole serum were used in the medium in place of albumin, no antimycobacterial activity was observed; however, this activity was restored by adding bovine albumin (fraction V) to the media containing whole serum. By chemical purification and analysis, the inhibitory material was identified as spermine, an organic base widely distributed in animal tissues.

A stable, water-soluble substance which possesses potent antimycobacterial activity under certain conditions in vitro has been prepared from calf thymus. This substance has been tentatively named thymus peptide. In final concentrations of 1 to 10 µg. per ml. of an albumin medium it inhibits the growth of various strains of mammalian mycobacteria, but manifests only little or no inhibitory activity against a variety of other microbial species. The ability of thymus peptide to inhibit the multiplication of tubercle bacilli diminishes when the inoculum is large, or when the medium is acidic. It is also markedly antagonized by addition of enzymatic hydrolysate of casein or beef heart infusion broth to the culture medium. Thymus peptide does not exert a rapid bactericidal action on tubercle bacilli, but organisms exposed to this compound for longer than 2 weeks could not be made to multiply in ordinary culture media. Substances similar or identical to the thymus peptide preparation could be extracted from calf spleen, sheep thymus, beef lymph nodes, and calf pancreas, but not from calf lung or calf liver.

THE gastrointestinal microflora of specific pathogen free mice usually includes several species of anaerobic bacteria. In a healthy state these organisms greatly outnumber the coliform bacilli and enterococci that are commonly but erroneously assumed to be the most characteristic inhabitants of the intestinal tract1–3. Earlier publications from this laboratory have presented the hypothesis that several species of anaerobic bacteria have evolved with the mouse, live symbiotically with it and play essential parts in its anatomical development as well as in physiological functions. Such organisms are intimately associated with gastrointestinal anatomy and can be regarded as constituting its autochthonous microflora. This hypothesis, if valid, applies equally well to other omnivorous animals, including man1–3.

The bacterial species that are most abundant in the gastrointestinal tract under normal conditions are anaerobic and have exacting growth requirements. As they display only a narrow range of biochemical activities, it is probable that the chemical transformation of metabolites in the gastrointestinal tract is less pronounced when these organisms predominate than when others gain the upper hand under pathologic conditions. In other words, the composition of the gastrointestinal flora determines the nature of the bioactive substances that are produced through metabolic transformation of amino acids, bile acids and other metabolites. Such biochemical activities may be as important as the orthodox pathologic lesions caused by pathogens.

The resistance of white mice to tuberculous infection could be increased by preliminary vaccination with small amounts of tubercle bacilli killed by contact with 2 per cent phenol. Vaccine prepared from a variant strain of human tubercle bacilli unable to multiply in vivo (H37Ra) proved as active as vaccines prepared from either virulent or attenuated strains. The immunity induced by phenol-killed bacilli persisted for several weeks. Under the conditions of the experiments, however, it was never able to bring about the death of the virulent bacilli used for the challenge infection, even when the infective inoculum was very small. Its protective effect could be detected (a) by the increased survival time of mice infected with a very large dose of virulent bacilli, and particularly (b) by the lower numbers of bacilli present in the organs of mice sacrificed at various periods of time after injection of sublethal infective doses. Under the proper conditions of vaccination the immunity produced in mice by phenol-killed cells of avirulent bacilli was of the same order as that produced by BCG. The protective antigen proved to be susceptible to heat, particularly at acid reactions. It retained its activity when the bacilli were disintegrated and rendered non-acid-fast by grinding with concentrated phenol. It remained in the insoluble cellular debris when the bacilli were extracted with 88 per cent phenol. Reasons are presented to support the view that the antigenic components present in the tubercle bacilli (avirulent as well as virulent) killed with phenol play a significant part in several manifestations of increased resistance to tuberculosis.

When your Institute and Society were founded fifty years ago, the specialists in infectious disease, nutrition, and metabolism were still the scientific heroes of the medical world. Their disciplines had yielded rich harvests of practical results, and had enlarged man's understanding of his relation to the environment. The intellectual atmosphere in medical schools, however, was even then beginning to change. In 1920, the development ofa practical method for the production ofinsulin made it obvious that medicine had entered a new phase; emphasis had shifted from the external to the internal agents of disease. Today the focus of attention is shifting even further away from the preoccupations of the early 1900's. More and more scientific medicine is identified with the esoteric knowledge of molecular biology or biological electronics, and with the spectacular performances of medical engineering in the lungs, the brain, the heart or the kidneys. The ceremony marking your fiftieth anniversary is a salute to the past, but also a dedication to the future. As a representative ofone of the downgraded scientific disciplines, medical microbiology, I should therefore feel embarrassed at speaking for the future. But instead, I feel gently amused because I am convinced that the glamorous achievements of today will appear old fashioned fifty years hence. In fact, I shall take advantage of your invitation to play the prophet, and tell you of some of the topics likely to occupy a prominent place on your programs when you meet to celebrate your hundredth anniversary. As there is no good prospective without retrospective, I shall first try to review briefly the forces which have been at work during the past 2,500 years to prepare the forthcoming change in direction of medical

The fate of hemolytic staphylococci injected intravenousiy into albino mice was followed by determining quantitatively the numbers of living organisms present in the various tissues at different intervals of time after infection. Irrespective of the strain of staphylococcus used, most of the organisms disappeared rapidly from the blood, liver, spleen, and kidneys. This was true even when the infective dose consisted of large numbers of virulent, coagulase-positive staphylococci, capable of producing a fatal disease in a high percentage of the infected mice. The initial rate of removal or destruction of staphylococci was particularly high in the lungs and kidneys. In all cases on the other hand, a few living staphylococci persisted in the various organs for several weeks after infection, even when the organisms were non-virulent and coagulase-negative. Although virulent as well as avirulent staphylococci were eliminated extremely rapidly and efficiently from the kidneys during the initial stage of infection, the microorganisms soon began to multiply in this organ, causing abscesses first detected in the cortex. Death of the animals infected with virulent cultures appeared to be due to the destruction of renal tissue by these abscesses. The abscesses caused by the avirulent strains eventually became sterile, and healed. No convincing difference could be recognized amongst seven strains in their resistance to the bactericidal power of the mouse tissues during the initial phase of the infection. In contrast, marked quantitative differences came to light in their subsequent behavior in the kidneys. The multiplication of the coagulase-negative staphylococci in this organ soon came to an end in all animals and never proceeded far enough to result in fatal disease. The staphylococci of a weakly coagulase-positive strain multiplied somewhat more extensively in the kidneys than did the coagulase-negative, but never sufficiently to cause the death of any animal within the period of observation of 1 month. The three coagulase-positive strains tested yielded the largest bacterial population in the kidneys and caused the death of many of the infected animals. These three virulent strains differed quantitatively amongst themselves with regard to both the rapidity and extent of their multiplication in the kidneys and the lethal power of a given infective dose. Taken together, the findings indicate that the hemolytic strains of staphylococci can be arranged in a continuous spectrum according to their ability to cause disease in albino mice. Although virulence for these animals appeared to be correlated with the production of coagulase, it did not seem to depend upon the ability of this substance to interfere with the bactericidal mechanisms of the mouse organs during the early phase of the infection. Virulence manifested itself chiefly by the production in the kidneys of progressive abscesses originating from the few staphylococci which were not destroyed during the initial bactericidal reaction.

Selective culture media, and equipment for anaerobic incubation of large numbers of specimens, have been developed to facilitate the quantitative enumeration of the various aerobic and anaerobic bacterial species present in the gastrointestinal tract. The evolution of this flora has been followed in young mice from several colonies by cultivating homogenates of the different parts of the gastrointestinal tract at daily intervals from the time of birth to the time of weaning. It has been found that the lactobacilli and anaerobic streptococci become established immediately after birth and persist in large numbers, not only in the large intestine but also in the stomach and in the small intestine. In contrast, the anaerobic bacilli of the bacteroides group become established only after the 16th day; they multiply only in the large intestine but persist in this organ in very large numbers. Other bacterial species become established at different periods of time after birth, exhibit characteristic anatomic localizations, and greatly fluctuate in numbers. In general, the populations of enterobacilli and enterococci decrease precipitously after having reached a maximum level shortly after the beginning of colonization.

Mice of the NCS and NCS-D colonies, bred at the Rockefeller University, harbored in their intestine an endemic strain of slow lactose-fermenting Escherichia coli 081±: :H21 serotype. In addition, NCS mice have recently acquired E. coli 0109±:K48:H14. Both strains persisted during the period of observation, whereas they were not encountered in the feces of mice from two other colonies. Other coliform strains encountered were more transient in their occurrence. Since strains of E. coli 081±: :H21 and 0109±:K48:H14 are extremely uncommon in human beings, it seems probable that they possess specificity for the mouse host.

SummaryAddition to Long's synthetic medium of small amounts of phosphatides and of long chain fatty acids esters of polyhydric alcohols permits submerged and rapid growth of tubercle bacilli; the different groups of mycobacteria appear to exhibit differential optima with reference to these two types of substances.

Gramicidin, an alcohol-soluble, water-insoluble substance extracted from cultures of an aerobic sporulating bacillus, has been found to exert a marked bactericidal effect against Gram positive microorganisms, both in vitro and in vivo.1 When injected by way of the cistern into infected quarters of cows suffering from mastitis caused by Streptococcus agalactiae (Lancefield serological group B), gramicidin apparently cured a number of the infections.2The method originally used for the administration of gramicidin in the treatment of bovine mastitis consisted in diluting 60-240 mg of the bactericidal substance in 1000 cc of aqueous media and injecting this material into the cistern of the infected quarter. As reported earlier, this method of administration caused an intense swelling of the treated quarter, accompanied by a marked elevation of temperature. Although this reaction lasted only a few hours, it was severe enough to prevent repeated treatments on successive days.It has now been found that sterile m...

The nature of the substance in filtrates of whole blood and plasma which gives the color with alkaline picrate (Jaffe's reaction) has been for many years a subject of controversy. One group of investigators believe that this material is true creatinine—others deny that creatinine exists in normal blood. Because of the non-specific methods employed for the identification of creatinine, and the very minute quantities of the chromogenic material available in normal blood, it has been difficult for either group to present convincing evidence.To obtain a definitive answer regarding the nature of the Jaffe-reactive material in blood, and also to develop a specific method for the analysis of creatinine in biological fluids, an attempt was made to obtain a specific enzyme for creatinine. By means of a technique similar to that described by Dubos and Avery1 and Dubos2 it has been possible to isolate 4 different species of soil bacteria with a high degree of adaptability toward a substrate of creatinine. One strain...