Studies of Intestinal Microflora

Transcription

1 JOURNAL OF BACTERIOLOGY, Aug. 1967, p Vol. 94, No American Society for Microbiology Printe'd in U.S.A. Studies of Intestinal Microflora VI. Effect of X Irradiation on the Fecal Microflora of the Rat ALBERT S. KLAINER, SHERWOOD GORBACH, AND LOUIS WEINSTEIN Infectious Disease Service, New England Medical Center Hospitals, and the Department of Medicine, Tufts University School ofmedicine, Boston, Massachusetts Received for publication 24 May 1967 The effect of sublethal, midlethal, and lethal doses of total-body X irradiation on the fecal microbial population of the rat was studied. No changes were observed in animals receiving sublethal doses of X ray. Midlethal and lethal doses produced an increase in the numbers of fecal coliforms, staphylococci, streptococci, and fungi; these changes were transient in rats that survived, but were more marked and persisted during life in those that died. The possible role of these alterations in intestinal microflora in post-irradiation infection and in death is discussed. Midlethal doses of whole-body X irradiation for 1 month prior to irradiation. They were fed a diet frequently produce premortem bloodstream invasion by the microorganisms normally resident animals were 2 to 3 months old and weighed approxi- of Purina laboratory chow pellets and water. The in the intestinal tract (1-3, 8, 12, 14, 19, 20). This mately 140 g at the time they were exposed to X ray. probably results from (i) radiation injury to the Four groups of 10 animals each were given a single intestinal mucosa, (ii) depression of host defense total-body exposure of 550, 1,050, 1,550, or 2,150 R of X ray. A Picker mechanisms, and (iii) changes in Deep Therapy Machine (260 kv, 15 the intestinal ma, distance 53 cm, with filters of 1-mm Al, 0.25-mm microflora (5, 7, 9, 10, 15, 16, 18, 19). That infection by intestinal bacteria plays a major role in of 0.25-mm Cu for the higher doses) was used; this Cu, and 0.25-mm Sn for the 550 R dose and a filter radiation death appears to be demonstrated by delivered 47 R/min for the 550 R dose and 145 R/min the fact that these organisms can be recovered for the other doses. from the blood and organs of animals dying after Fresh stool samples were obtained twice during exposure to lethal doses of total-body irradiation the week prior to irradiation and at various times after and by the reduction of death rates by treatment treatment with X ray. The feces were collected in sterile with antibiotics (4, 6, 11, 13, 15, 17). containers and were either cultured immediately or were stored at 4 C Most for 2 hr or studies of the effects of X irradiation less before culture. on Random samples were studied in duplicate to check the intestinal microflora of animals at levels of the accuracy of the microbiological assay methods, the exposure significantly different from the LD5o reliability of which had been documented by the results of other studies in this laboratory. range have not been carried out under carefully controlled, standardized, and comparable conditions. For this reason, the investigations reported tainer and thoroughly comminuted in sterile saline (9 Each stool specimen was weighed in a sterile con- in this paper were undertaken. The purpose of ml for samples weighing 750 to 1,000 mg, 9.5 ml for these experiments was to determine whether or those 250 to 750 mg, and 10 ml for those less than 250 not treatment with sublethal, midlethal, and mg). Serial 10-fold dilutions were made in sterile lethal doses of total-body X physiological saline to a irradiation dilution of produced 109; a separate pipette was used for each transfer. Samples of 1 ml significant qualitative or quantitative changes in of appropriate dilutions were incorporated into liquefied agar media and pour plates were made; three the microbial population of the feces of rats. All the bacteriological techniques employed were dilutions of each specimen were cultured in each carefully standardized, and the results were subjected to statistical analysis. 1. After varying periods of incubation at 37 C or room medium. The types of media used are shown in Table temperature (Table 1), the number of organisms per MATERIALS AND METHODS gram of feces was determined by direct plate counting. The organisms isolated were identified on the basis of One-month-old white female Sprague-Dawley rats colonial and cellular morphology and Gram-staining weighing approximately 120 g were kept in separate characteristics. Selected colonies were isolated in subculture for specific identification. cages in an animal room under standard conditions 378

2 VOL. 94, 1967 EFFECT OF X IRRADIATION ON FECAL MICROFLORA 379 TABLE 1. Selective media used for study of fecal microbiolozv Mediuma Culture conditions Predominant organisms recovered Eugon Agar Aerobic, 2 days at 37 C Violet Red Bile Aerobic, 2 days Coliforms Agar at 37 C Mycosel Agar Aerobic, 3 days at 22 C Rogosa SL Aerobic, 3 days Aerobic lacto- Agar at 37 C bacilli Anaerobic, 3 Anaerobic lacdays at 37 C tobacilli Staphylococcus Aerobic, 4 days Medium No. at 37 C 110 S F Medium Aerobic, 4 days Enteric streptoat 37 C cocci Blood agarb Anaerobic, 5 Total anaerat 37 C obes a All media are from Difco, except Mycosel Agar which is from BBL. bconsists of Heart Infusion Agar (Difco) and 0.5 ml of horse blood. Anaerobiosis was achieved by use of sealed airtight containers (Anaerobic Incubator, National Appliance Co., Portland, Ore., or Anaero-Jar, Scientific Products, Evanston, Ill.). Steel wool activated by an acidic copper sulfate solution was employed as the oxygen scavenger. Three cycles of evacuation and replacement with 95% N2 and 5% CO2 were carried out. RESULTS As can be seen in Tables 2 and 6, single exposure of 550 R of total-body X ray, a sublethal dose for the rats used in this study, caused no significant change in the fecal flora of these animals. On the other hand, total body irradiation with 1,050 and 1,550 R caused 20 and 60% mortality, respectively, and produced significant increases in the total numbers of intestinal aerobes only in those animals which later succumbed to radiation effect (Tables 2-4, 6). Increases in the numbers of coliforms were noted in rats receiving either dose of X ray; this change was transient in the animals that survived but persisted in those which died. Exposure to 1,050 R led to a decrease in aerobic and anaerobic lactobacilli in the animals that eventually succumbed; such a change was not observed with a dose of 1,550 R. These midlethal quantities of radiation also produced significant and sustained increases in fecal staphylococci and streptococci in the rats which died; exposure to a higher dose of radiation provoked a transient increase in these organisms in the animals which survived (Table 3). No significant alterations of the numbers of fecal anaerobes or fungi were detected. Total-body exposure to lethal dose of X ray (2,150 R) resulted in significant increases in the numbers of coliform organisms, fungi, staphylococci, and streptococci in the feces (Tables 5 and 6). These changes persisted until death occurred 5 to 12 days after irradiation. The length of survival of the irradiated animals appeared, in general, to be proportional to the dose of X ray used. The rats became lethargic, had a poor appetite, and developed crusting about the eyes, ruffling of the coat, and decreased stool production for 1 to 2 days before death. Twitching, convulsions, and a shock-like picture were apparent just before the animals expired. Black or bloody diarrhea was noted in about 50% of the rats for 24 hr before death. Alterations in the microbial population of the feces were generally first apparent 3 to 5 days after exposure to X ray. These changes persisted until death in the animals that succumbed; in those that survived, the pretreatment fecal flora was reestablished about 11 to 12 days after irradiation. TABLE 2. Effect of total-body X irradiation with 500 R on the fecal flora of rats No. of organismsa/g of feces 4 SE: 3 daysb 7 daysb DISCUsSION The results of these experiments suggest that sublethal doses of total-body X ray produced no significant alterations in the fecal microbial popu : ± 0.09 Coliforms ± Anaerobic lactobacilli ± ± :1: ± l ± t ± ± ± ± 0.09 a Logio organisms = mean value for 10 rats each group. b Number of days after exposure to X ray. c p = d p = e p = f p =

3 380 KLAINER, GORBACH, AND WEINSTEIN J. BACTERIOL. TABLE 3. Effect of total-body X irradiation with 1,050 R on the fecal flora of rats No. of organismsa g of feces i SE Animals surviving (80%) Animals dying (20%,) 5 daysb 11 days 14 days 5 daysb 11 days Coliforms 8.71 ± ± ± ± 0.18e 0.53 ± i ± i ± i ± i ± ± ±t ± ± ±i ± i: i i ± ± i ± ±: ± ± t ± ± ± O0.1Od lOd 0 ± i ±0.10e 6.70 ± O.lOd 9.5 4± i 0.lod l4f 0 ± i:.o.olf Of 6.00 ± a Logio organisms, mean value for 10 rats in each group. b Number of days after exposure to X ray. c P = d p = e p -=0.o1. f p _ TABLE 4. Effect of total-body X irradiation with 1,550 R on the fecal flora of rats No. of organismsa/g feces of 5sE Rats surviving (40%) Rats dying (60%) 3 days 12 days 3 days Colif Drms 8.55 i i i i4: i t 0.17f i e f 8.60 i t t it == : i it i t 0.18f f ± i :+ 0.14f f 8.92 i 0.31 a Logio organisms, mean value for 10 rats each group. b No. of days after exposure to X ray. cp = d P = e p = f p = lation of rats. On the other hand, exposure to midlethal and lethal doses of radiation appeared to lead to an increase in the numbers of coliform organisms, streptococci, and staphylococci in the feces. Such changes were transient and less marked in animals that survived radiation. These findings, as well as those of Miller and co-workers (6, 12, 13), Vincent et al. (19), Gustafson and Koletsky (4), and Rosoff (15), suggest that these effects are directly related to radiation and do not represent an agonal event. The fact that coliforms and other gram-negative members of the intestinal flora have been found to be the most common organisms causing bacteremia after radiation (12, 13, 15, 19) suggests that qualitative and quantitative alterations in the microbial population of the gut may be as significant as radiation injury to the intestinal mucosa as a cause of the postirradiation syndrome and death. The results of the studies reported in this paper raise the possibility that the fecal staphylococci and streptococci play an independent or supporting role in infection and death after total-body exposure to X ray. Although an increase in intestinal gram-negative organisms and a decrease in lactobacilli after X-ray treatment were noted by Vincent and his

4 VOL. 94, 1967 EFFECT OF X IRRADIATION ON FECAL MICROFLORA 381 coworkers (19), the present investigation indicates that a significant reduction in the numbers of lactobacilli occurred only in animals dying after exposure to 1,050 R and not in those that received doses of X ray smaller or larger than this. This discrepancy is difficult to explain. The decrease in lactobacilli may merely reflect a reciprocal effect of the increase in coliform organisms. It is also possible that reduction in the numbers of lactobacilli causes an increase in coliforms as a result of interference with the activity of various unidentified factors that tend to keep the intestinal microflora relatively stable. The observed increase in the total numbers of aerobic bacteria TABLE 5. Effect of total-body X irradiation with 2,150 R on the fecal flora of rats Coliforms alog,i No. of organismsa/g of feces :4= Rats dying (100%) SE 3 days after X ray 8.84 i i i 0.31e i i i e 7.31 i organisms, mean value for 10 rats each group. b P = c P = d p = e p = and fungi after X irradiation requires further study; that this was not an agonalevent is suggested by the fact that it occurred several days before death. The dose of radiation required to kill the animals (LDso about 1,500 R) appeared to be quite large. It is important to point out, in this regard, that this dose did not differ for the large number of rats studied and that no gross errors in dosimetry were detected. This suggests that the relative resistance to X irradiation was a characteristic of the Sprague-Dawley rats employed in these investigations. As has been the case with other studies, the one reported in this paper was limited by imperfections in the selective culture media used, the inability to recover a large number of microorganisms probably present in the feces, and the lack of information concerning interactions among the heterogeneous collection of organisms populating the intestinal tract. The present investigation confirms the observations of others, who used a wide range of X-ray dosage and standardized culture techniques. ACKNOWLEDGMENTS This investigation was supported by Public Health Service research grant Al 6365, training grant Al 276, and a postdoctoral Fellowship (A. S. Klainer) from the Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. BENNETT, L. R., P. E. RETKERS, M. KRESGE, AND AND J. W. HOWLAND The influence of infection on the hematological effects and mortality following midlethal X-irradiation. U.S. Atomic Energy Comm. UR CHROM, S. A Studies on the effect of roentgen rays upon intestinal epithelium and upon reticuloendothelial cells of liver and spleen. Acta Radiol. 16: TABLE 6. Comparison of numbers offecal bacteria before and after irradiationa 500 R 1,050 R 1,550 R 2,150 R 10 Survivors 8 Survivors 2 Dead 4 Survivors 6 Dead 10 Dead NC NC INCRb NC INCRd NC Coliforms NC INCRb INCRc INCRd INCRd INCRd NC NC NC NC NC INCRd NC NC DECRd NC NC NC NC NC DECRd NC NC NC Staphylocci NC NC INCRb INCRc INCRd INCRd NC NC INCRb INCRd INCRd INCRd NC NC NC NC NC NC a NC = no change in numbers; INCR = increased numbers; and DECR = decreased numbers. b p = c p = f P =

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