INFECTION AND IMMUNITY, Feb. 1975, p. 337-342 Copyright 0 1975 American Society for Microbiology
Vol. 11, No. 2 Printed in U.S.A.
In Vitro Interaction of Bacteroides fragilis with Polymorphonuclear Leukocytes and Serum Factors DENNIS A. CASCIATO, JON E. ROSENBLATT, LEONARD S. GOLDBERG, AND RODNEY BLUESTONE* Medical Service, Wadsworth Hospital Center, Veterans Administration, Los Angeles, California 90073, * and Department of Medicine, University of California at Los Angeles School of Medicine, Los Angeles, California 90024
Received for publication 15 August 1974
The sera and polymorphonuclear leukocytes (PMNs) of healthy human subjects were tested against several isolates of Bacteroides fragilis. All sera killed most of the fecal isolates, but were active against only one of the clinical isolates. The degree of killing was directly related to the serum concentration, but was independent of the bacterial inoculum within the range of bacterial concentrations studied. The serum bactericidal activity was heat-labile. Engulfment and killing of B. fragilis by PMNs were demonstrated consistently. The opsonin involved in phagocytosis was also heat labile. Calculations involving log-transformed data permitted the quantitative studv of the separate and combined effects of serum and PMNs on bacteria which were killed by serum alone. Whereas serum alone usually killed 0.5 log of serum-sensitive bacteria, the addition of PMNs was usually associated with one log further killing. The studies reported here demonstrate the presence of heat-labile serum factors in normal human sera which kill B. fragilis directly and which promote its phagocytosis and killing by PMNs. These observations provide a foundation for investigations into host defense mechanisms against anaerobes.
Anaerobic bacteria constitute a major proportion of the normal flora of human respiratory, intestinal, and genitourinary tracts (10, 14, 25). Infections caused by these organisms are increasingly being recognized as major problems in clinical medicine (2, 8, 27). Although certain diseases may predispose patients to infection caused by anaerobes (8, 9), minimal information is available regarding specific host defense mechanisms. Phagocytosis and killing by polymorphonuclear leukocytes (PMNs) have been shown to be of primary importance in host defense against facultative and aerobic bacteria (3, 24, 28, 30, 31). Serum bactericidal activity, especially against gram-negative bacteria, is also well recognized (12, 13, 21, 29, 31). The present investigation was undertaken to study the in vitro interaction of PMNs and serum factors with Bacteroides fragilis, the anaerobe most commonly involved in human infections (2, 8). This report clearly demonstrates bactericidal activity of normal human sera against B. fragilis and engulfment and killing of this organism by PMNs. MATERIALS AND METHODS Glassware. Glassware was siliconized by immersion in 1% Siliclad (Clay Adams, Parsippany, N.J.).
Incubation mixture tubes were soaked overnight in acid dichromate and resiliconized before each use. Preparation of bacteria. B. fragilis subsp. thetaiotaomicron Wadsworth Anaerobic Bacteriology Laboratory (WAL) 2030. a human fecal isolate, was used as the basic test organism. This stable organism is used as a reference strain in antimicrobial susceptibility testing and in current studies of anaerobe identification and subspeciation at the Wadsworth Anaerobic Laboratory. Nine other strains were also studied, including four stool and five blood culture isolates. Frozen stock cultures were thawed, transferred to tubes of chopped meat glucose (Scott Laboratories, Fiskeville, R.I.) under flowing oxygen-free carbon dioxide, and incubated at 37 C for 24 to 72 h. The organisms were then subcultured on Brucella blood agar plates containing 10 ,g of vitamin K1 per ml and incubated anaerobically for 24 h in a GasPak (BBL) jar. Preliminary experiments revealed that bacterial survival was good under assay conditions in distilled water with 0.1% gelatin (water-gel) and minimal essential medium with Eagle salts (GIBCO, Grand Island, N.Y.) containing 0.1% gelatin (MEM-gel). Accordingly, these diluents were used for bactericidal assays. The bacteria harvested from the Brucella blood agar plates containing 10 Ag of vitamin K1 per ml were washed and suspended to an 0.60 optical density at 620 nm (approximately 109 bacteria per ml) using a Gilford spectrophotometer. Inocula for the assay were 1:100 or 1:1,000 dilutions of the suspension in MEM-
337
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CASCIATO ET AL.
gel. Suspensions ot B. fragili.s were examined for clumping or chaining and plate counts were related to pipetted volumes and dilution factors to determine if pour plate colony counts accurately reflected the number of viable bacteria. Effect of oxygen exposure. The oxygen exposure for the organisms was substantial during preparation for and performance of' the assay. After the reaction period, the organisms encountered f'urther oxygen exposure because of' the practical necessity of filling each jar with plates before adding the GasPak and the inherent delay in obtaining anaerobiosis with the GasPak envelope. Bacterial survival was compared after preparation of' the organism was carried out in 'he anaerobic glove box (1) and in room air. Survival was also compared when anaerobiosis was established at several intervals atter plate pouring. Serum. The sera from 17 healthy human volunteers were either used t'resh or stored at 70 C. Frozen specimens were thawed only once and tested either individually or as pools of four donors. Heat inactivation was accomplished by incubation in a 56 C water bath for 30 min. Twenty percent serum solutions were prepared in MEM for the assay. Leukocyte suspensions. Leukocyte-rich suspensions from 11 healthy volunteers were prepared by the method of Douglas et al. (5). Blood was collected in plastic syringes and anticoagulated with 5 U of' heparin per ml (Ries Biologicals. Los Angeles, Calif'.), Ervthrocvtes were lvsed by incubating blood twice for 5 min with 0.87%. ammonium chloride, and were washed and suspended in MEM with 5 U of heparin per ml to a final concentration of' 10( PMNs per ml. The viability of the PMNs was determined by trvpan blue dye exclusion; survival of' the phagocytes at the end of the assays was always greater than 98%,. Morphological assessment of phagocytosis. PMN suspensions were allowed to adhere to glass cover slips placed in humidified petri dishes at 37 C; after 30 min a suspension containing 10'-. serum and 107 bacteria per ml was applied to the adherent cells. After incubation for 1 h at 37 C. the excess bacteria were rinsed off the cover slips with MEM-gel. The dried slides were stained by the Giemsa method and examined by light microscopy. Bactericidal tests. All studies were performed in duplicate in sterile siliconized glass screw-cap test tubes (13 by 100 mm) in room air. The serum bactericidal test system of 3.0-ml volume contained final concentrations of' 8%7c serum and 105 or 106 bacteria per ml. The effect of serum concentration was measured by performing bactericidal assays with final serum concentrations ranging from 2 to 40%. Modifications of the methods of Hirsch and Strauss (15) were used for the PMN bactericidal tests. The final 3.0-ml volume contained 5 x 10' PMNs per ml. 8% serum, and 106 to 2 x 106 bacteria per ml. Parallel control mixtures were included to study killing by serum alone, bacterial survival in heat-inactivated serum, killing by leukocytes without opsonin, and heat stability of the opsonin. The mixtures were incubated at 37 C with endover-end agitation and portions were taken at timed intervals. PMN lysis and bacterial dilutions were
INFECT. IMMUN.
accomplished in distilled water. Pour plates were made and incubated anaerobically in GasPak jars for 48 to 72 h. Colonies were counted, duplicates were averaged, and data were expressed as percentages of the initial (zero time) number of bacteria surviving at each interval. To determine the number of bacteria which were engulfed but not killed by PMNs. the incubation mixture remaining af'ter the 2-h assay was centrifuged at 150 x g. The PMN button was washed, freezethawed with dry ice (to lyse cells and liberate surviving bacteria), and resuspended to 1.0 ml. Pour plates were made and the bacterial survival was compared with that in the supernant and in the routine 2-h portions. These PMN-associated bacteria were taken to represent the bacteria which were engulf'ed but not killed. Expression of data. Bacterial killing curves have been shown usually to be exponential functions and to follow log-normal distributions (16). Thus, numerical expressions of bacterial survival should involve logarithmic rather than arithmetic values of surviving bacteria. Our data were converted to percentage of bacteria surv iving and the log,. of the percentage alive was derived. The amount of killing in logs from the start of the experiment to a given time interval was calculated by subtracting the log1o percentage alive at that time from 2.00 (log 100% at time zero). Quantative differences in the degrees of killing expressed in logs by the components of the test systems could thereby be compared. The killing by PMNs of serum-sensitive bacteria was calculated by subtracting the logs killed by serum alone from the logs killed by the combination of serum and PMNs.
RESULTS Assessment of clumping. Clumping was not observed by microscope studies of bacterial suspensions 100-f'old more concentrated than usual test conditions. Furthermore, plate counts were linearly related to the volume of bacterial suspension pipetted, and bacterial concentrations were directly related to the dilution f'actors over a four-log range. One colony-forming unit was consequently interpreted to reflect one surviving bacterium within the limits of sampling precision. Effect of oxygen exposure. The studies on the effect of' oxygen exposure showed that the duration of' air exposure before, during, and after the assay did not affect the survival of these strains of' B. fragilis in diluents or active serum. Furthermore, the bacteria survived well in inactive solutions (such as heated serum) during the assay under conditions of maximal aeration. These observations permitted the perf'ormance of' the entire assay on the laboratory bench in a manner similar to previously reported bactericidal tests with facultative or aerobic organisms without special precautions to minimize oxygen exposure.
VOL. 11. 1975
Serum bactericidal activity. The results ot' serum bactericidal tests on 10 isolates of B. fragilis are represented in Table 1. All studies were performed with 8% pooled sera and 105 bacteria per ml in the incubation mixtures. Three of the f'ive stool isolates were sensitive to serum, whereas only one ot' the five blood culture isolates was sensitive. Heat inactivation of the serum ablated the bactericidal activity against each of the serum-sensitive strains. Each of the 17 individual and 11 pooled sera at 8% final concentration effectively killed the refterence strain, B. fragilis WAL 20:30. Figure 1 depicts the considerable variation in the degree ot' killing between subjects. At 2 h the percentage of' inoculum surviving ranged trom 20 to 80/c. Generally, killing occurred at a rapid rate during the first hour and at a much slower rate thereafter. Studies extended to 4 h demonstrated a continued slow rate of killing to the end of the assay. Effect of inoculum size. The bactericidal effect of 8% serum mixtures on B. fragilis could be masked by use of a heavy bacterial inoculum. Serum killing was not apparent at incubation mixture bacterial concentrations greater than approximately 3 x 106/ml. However, there was virtually no change in killing at 1 h in more dilute concentrations (106. 5 x 10, 5 -> 104, and 5 x 103 bacteria per ml). Effect of serum concentration. The major factor influencing the magnitude of killing was the serum concentration. Increasing, serum concentrations resulted in progressively greater bacterial killing at all time intervals. There was no killing at any concentration when the serum was heated at 56 C. In Fig. 2 the survival of B. TABLE 1. Serum bactericidal activity against 10 isolates of B. fragilis Stock no.
Subspecies
Cc killed by 8%c pooled serum At 1 h
Stool isolates WAL 1208 WAL 1222 WAL1423 WAL 2030 WAL 2437 Blood culture isolates WAL 256 WAL 1103 WAL 1327 WAL 1329 WAL 2380
339
BA CTEROIDES FRA GILIS
Indeterminate thetaiotaomicron ovatus thetaiotaomicron fragilis
fragilis fragilis fragilis fragilis thetaiotaomicron
At 2 h
Vol. 11, No. 2 Printed in U.S.A.
In Vitro Interaction of Bacteroides fragilis with Polymorphonuclear Leukocytes and Serum Factors DENNIS A. CASCIATO, JON E. ROSENBLATT, LEONARD S. GOLDBERG, AND RODNEY BLUESTONE* Medical Service, Wadsworth Hospital Center, Veterans Administration, Los Angeles, California 90073, * and Department of Medicine, University of California at Los Angeles School of Medicine, Los Angeles, California 90024
Received for publication 15 August 1974
The sera and polymorphonuclear leukocytes (PMNs) of healthy human subjects were tested against several isolates of Bacteroides fragilis. All sera killed most of the fecal isolates, but were active against only one of the clinical isolates. The degree of killing was directly related to the serum concentration, but was independent of the bacterial inoculum within the range of bacterial concentrations studied. The serum bactericidal activity was heat-labile. Engulfment and killing of B. fragilis by PMNs were demonstrated consistently. The opsonin involved in phagocytosis was also heat labile. Calculations involving log-transformed data permitted the quantitative studv of the separate and combined effects of serum and PMNs on bacteria which were killed by serum alone. Whereas serum alone usually killed 0.5 log of serum-sensitive bacteria, the addition of PMNs was usually associated with one log further killing. The studies reported here demonstrate the presence of heat-labile serum factors in normal human sera which kill B. fragilis directly and which promote its phagocytosis and killing by PMNs. These observations provide a foundation for investigations into host defense mechanisms against anaerobes.
Anaerobic bacteria constitute a major proportion of the normal flora of human respiratory, intestinal, and genitourinary tracts (10, 14, 25). Infections caused by these organisms are increasingly being recognized as major problems in clinical medicine (2, 8, 27). Although certain diseases may predispose patients to infection caused by anaerobes (8, 9), minimal information is available regarding specific host defense mechanisms. Phagocytosis and killing by polymorphonuclear leukocytes (PMNs) have been shown to be of primary importance in host defense against facultative and aerobic bacteria (3, 24, 28, 30, 31). Serum bactericidal activity, especially against gram-negative bacteria, is also well recognized (12, 13, 21, 29, 31). The present investigation was undertaken to study the in vitro interaction of PMNs and serum factors with Bacteroides fragilis, the anaerobe most commonly involved in human infections (2, 8). This report clearly demonstrates bactericidal activity of normal human sera against B. fragilis and engulfment and killing of this organism by PMNs. MATERIALS AND METHODS Glassware. Glassware was siliconized by immersion in 1% Siliclad (Clay Adams, Parsippany, N.J.).
Incubation mixture tubes were soaked overnight in acid dichromate and resiliconized before each use. Preparation of bacteria. B. fragilis subsp. thetaiotaomicron Wadsworth Anaerobic Bacteriology Laboratory (WAL) 2030. a human fecal isolate, was used as the basic test organism. This stable organism is used as a reference strain in antimicrobial susceptibility testing and in current studies of anaerobe identification and subspeciation at the Wadsworth Anaerobic Laboratory. Nine other strains were also studied, including four stool and five blood culture isolates. Frozen stock cultures were thawed, transferred to tubes of chopped meat glucose (Scott Laboratories, Fiskeville, R.I.) under flowing oxygen-free carbon dioxide, and incubated at 37 C for 24 to 72 h. The organisms were then subcultured on Brucella blood agar plates containing 10 ,g of vitamin K1 per ml and incubated anaerobically for 24 h in a GasPak (BBL) jar. Preliminary experiments revealed that bacterial survival was good under assay conditions in distilled water with 0.1% gelatin (water-gel) and minimal essential medium with Eagle salts (GIBCO, Grand Island, N.Y.) containing 0.1% gelatin (MEM-gel). Accordingly, these diluents were used for bactericidal assays. The bacteria harvested from the Brucella blood agar plates containing 10 Ag of vitamin K1 per ml were washed and suspended to an 0.60 optical density at 620 nm (approximately 109 bacteria per ml) using a Gilford spectrophotometer. Inocula for the assay were 1:100 or 1:1,000 dilutions of the suspension in MEM-
337
338
CASCIATO ET AL.
gel. Suspensions ot B. fragili.s were examined for clumping or chaining and plate counts were related to pipetted volumes and dilution factors to determine if pour plate colony counts accurately reflected the number of viable bacteria. Effect of oxygen exposure. The oxygen exposure for the organisms was substantial during preparation for and performance of' the assay. After the reaction period, the organisms encountered f'urther oxygen exposure because of' the practical necessity of filling each jar with plates before adding the GasPak and the inherent delay in obtaining anaerobiosis with the GasPak envelope. Bacterial survival was compared after preparation of' the organism was carried out in 'he anaerobic glove box (1) and in room air. Survival was also compared when anaerobiosis was established at several intervals atter plate pouring. Serum. The sera from 17 healthy human volunteers were either used t'resh or stored at 70 C. Frozen specimens were thawed only once and tested either individually or as pools of four donors. Heat inactivation was accomplished by incubation in a 56 C water bath for 30 min. Twenty percent serum solutions were prepared in MEM for the assay. Leukocyte suspensions. Leukocyte-rich suspensions from 11 healthy volunteers were prepared by the method of Douglas et al. (5). Blood was collected in plastic syringes and anticoagulated with 5 U of' heparin per ml (Ries Biologicals. Los Angeles, Calif'.), Ervthrocvtes were lvsed by incubating blood twice for 5 min with 0.87%. ammonium chloride, and were washed and suspended in MEM with 5 U of heparin per ml to a final concentration of' 10( PMNs per ml. The viability of the PMNs was determined by trvpan blue dye exclusion; survival of' the phagocytes at the end of the assays was always greater than 98%,. Morphological assessment of phagocytosis. PMN suspensions were allowed to adhere to glass cover slips placed in humidified petri dishes at 37 C; after 30 min a suspension containing 10'-. serum and 107 bacteria per ml was applied to the adherent cells. After incubation for 1 h at 37 C. the excess bacteria were rinsed off the cover slips with MEM-gel. The dried slides were stained by the Giemsa method and examined by light microscopy. Bactericidal tests. All studies were performed in duplicate in sterile siliconized glass screw-cap test tubes (13 by 100 mm) in room air. The serum bactericidal test system of 3.0-ml volume contained final concentrations of' 8%7c serum and 105 or 106 bacteria per ml. The effect of serum concentration was measured by performing bactericidal assays with final serum concentrations ranging from 2 to 40%. Modifications of the methods of Hirsch and Strauss (15) were used for the PMN bactericidal tests. The final 3.0-ml volume contained 5 x 10' PMNs per ml. 8% serum, and 106 to 2 x 106 bacteria per ml. Parallel control mixtures were included to study killing by serum alone, bacterial survival in heat-inactivated serum, killing by leukocytes without opsonin, and heat stability of the opsonin. The mixtures were incubated at 37 C with endover-end agitation and portions were taken at timed intervals. PMN lysis and bacterial dilutions were
INFECT. IMMUN.
accomplished in distilled water. Pour plates were made and incubated anaerobically in GasPak jars for 48 to 72 h. Colonies were counted, duplicates were averaged, and data were expressed as percentages of the initial (zero time) number of bacteria surviving at each interval. To determine the number of bacteria which were engulfed but not killed by PMNs. the incubation mixture remaining af'ter the 2-h assay was centrifuged at 150 x g. The PMN button was washed, freezethawed with dry ice (to lyse cells and liberate surviving bacteria), and resuspended to 1.0 ml. Pour plates were made and the bacterial survival was compared with that in the supernant and in the routine 2-h portions. These PMN-associated bacteria were taken to represent the bacteria which were engulf'ed but not killed. Expression of data. Bacterial killing curves have been shown usually to be exponential functions and to follow log-normal distributions (16). Thus, numerical expressions of bacterial survival should involve logarithmic rather than arithmetic values of surviving bacteria. Our data were converted to percentage of bacteria surv iving and the log,. of the percentage alive was derived. The amount of killing in logs from the start of the experiment to a given time interval was calculated by subtracting the log1o percentage alive at that time from 2.00 (log 100% at time zero). Quantative differences in the degrees of killing expressed in logs by the components of the test systems could thereby be compared. The killing by PMNs of serum-sensitive bacteria was calculated by subtracting the logs killed by serum alone from the logs killed by the combination of serum and PMNs.
RESULTS Assessment of clumping. Clumping was not observed by microscope studies of bacterial suspensions 100-f'old more concentrated than usual test conditions. Furthermore, plate counts were linearly related to the volume of bacterial suspension pipetted, and bacterial concentrations were directly related to the dilution f'actors over a four-log range. One colony-forming unit was consequently interpreted to reflect one surviving bacterium within the limits of sampling precision. Effect of oxygen exposure. The studies on the effect of' oxygen exposure showed that the duration of' air exposure before, during, and after the assay did not affect the survival of these strains of' B. fragilis in diluents or active serum. Furthermore, the bacteria survived well in inactive solutions (such as heated serum) during the assay under conditions of maximal aeration. These observations permitted the perf'ormance of' the entire assay on the laboratory bench in a manner similar to previously reported bactericidal tests with facultative or aerobic organisms without special precautions to minimize oxygen exposure.
VOL. 11. 1975
Serum bactericidal activity. The results ot' serum bactericidal tests on 10 isolates of B. fragilis are represented in Table 1. All studies were performed with 8% pooled sera and 105 bacteria per ml in the incubation mixtures. Three of the f'ive stool isolates were sensitive to serum, whereas only one ot' the five blood culture isolates was sensitive. Heat inactivation of the serum ablated the bactericidal activity against each of the serum-sensitive strains. Each of the 17 individual and 11 pooled sera at 8% final concentration effectively killed the refterence strain, B. fragilis WAL 20:30. Figure 1 depicts the considerable variation in the degree ot' killing between subjects. At 2 h the percentage of' inoculum surviving ranged trom 20 to 80/c. Generally, killing occurred at a rapid rate during the first hour and at a much slower rate thereafter. Studies extended to 4 h demonstrated a continued slow rate of killing to the end of the assay. Effect of inoculum size. The bactericidal effect of 8% serum mixtures on B. fragilis could be masked by use of a heavy bacterial inoculum. Serum killing was not apparent at incubation mixture bacterial concentrations greater than approximately 3 x 106/ml. However, there was virtually no change in killing at 1 h in more dilute concentrations (106. 5 x 10, 5 -> 104, and 5 x 103 bacteria per ml). Effect of serum concentration. The major factor influencing the magnitude of killing was the serum concentration. Increasing, serum concentrations resulted in progressively greater bacterial killing at all time intervals. There was no killing at any concentration when the serum was heated at 56 C. In Fig. 2 the survival of B. TABLE 1. Serum bactericidal activity against 10 isolates of B. fragilis Stock no.
Subspecies
Cc killed by 8%c pooled serum At 1 h
Stool isolates WAL 1208 WAL 1222 WAL1423 WAL 2030 WAL 2437 Blood culture isolates WAL 256 WAL 1103 WAL 1327 WAL 1329 WAL 2380
339
BA CTEROIDES FRA GILIS
Indeterminate thetaiotaomicron ovatus thetaiotaomicron fragilis
fragilis fragilis fragilis fragilis thetaiotaomicron
At 2 h
