ANTIMICROBAL AGENTS AND CHEMOTHERAPY, May 1975, p. 698-703 Copyright 0 1975 American Society for Microbiology
Vol. 7, No. 5 Printed in U.S.A.
In Vivo Protection of Fusobacterium necrophorum from Penicillin by Bacteroides fragilis A. S. HACKMAN AND T. D. WILKINS* Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 Received for publication 26 December 1974
A mixed infection of Bacteroides fragilis and Fusobacterium necrophorum was resistant to treatment with penicillin even though a pure F. necrophorum infection could be successfully treated with penicillin. Since B. fragilis alone did not produce infection, these results indicate that B. fragilis can protect F. necrophorum from penicillin in vivo. The extent of protection afforded by a strain of B. fragilis was related to its level of resistance to penicillin. Only a few cells of B. fragilis were required in the initial bacterial injection. Moreover, protection was demonstrated when B. fragilis cells were injected as late as 24 h after the F. necrophorum cells. Protection of F. necrophorum from penicillin by B. fragilis was also demonstrated in vitro.
Bacteroides fragilis is a nonsporing gram-negative anaerobic rod which is frequently isolated from human clinical infections. Most B. fragilis strains are relatively resistant to penicillin. This resistance may be due, at least in part, to production of£-lactamase or other penicillindegrading enzymes. Attempts to detect f,-lactamase production by B. fragilis cells have been successful in some cases but not in others. Holt and Stewart (10) found no ,B-lactamase or amidase activity in eight strains of Bacteroides. However, Garrod (7), Pinkus et al. (12), and Anderson and Sykes (1) have reported 3-lactamase activity in strains of B. fragilis that were resistant to penicillin. Initially, Del Bene and Farrar (4) found cephalosporinase but no penicillinase activity in 10 strains of B. fragilis. Recently, with a more sensitive assay, penicillinase has been detected in these strains (V. Del Bene, personal communication). The difficulty of demonstrating penicillinase activity by B. fragilis cells might be due to the small amount of penicillinase produced by them. The ability of B. fragilis to inactivate penicillin is a matter of considerable clinical interest because B. fragilis is often found as a component of mixed infections in humans. If B. fragilis could inactivate the penicillin in its environment, it might also be able to protect penicillin-susceptible organisms present with it in a mixed infection. To determine whether B. fragilis could pro-
necrophorum infection, reported by Wilkins and Smith (17), which was fatal to mice and which could be treated successfully with penicillin. In this paper, we report evidence that B. fragilis was able to protect F. necrophorum from penicillin in vivo. Not only was the mixed infection much more resistant to penicillin than the pure F. necrophorum infection, but the extent of protection depended on the level of penicillin resistance which characterized the strain of B. fragilis in the mixed infection. We have also demonstrated the ability of B. fragilis to protect F. necrophorum from penicillin in vitro.
MATERIALS AND METHODS Source of organisms. F. necrophorum VPI 6054A (ATCC 27852), 10 strains of B. fragilis subsp. fragilis, three strains of B. fragilis subsp. vulgatus, one strain of B. fragilis subsp. distasonis, and two strains of B. fragilis subsp. thetaiotaomicron were obtained from the V. P. I. Anaerobe Laboratory culture collection. All ten strains of B. fragilis subsp. fragilis, two of the three strains of B. fragilis subsp. vulgatus (VPI 4245 and VPI 5710), and one of the two strains of B. fragilis subsp. thetaiotaomicron (VPI 9511) were isolates from human clinical infections. The remaining strains were isolates from stool specimens. Identifications were done by L. V. Holdeman and W. E. C. Moore according to procedures in the V. P. I. Anaerobe Laboratory Manual (8). Anaerobic glove box. A chamber (Coy Manufacturing Co., Ann Arbor, Mich.) similar to that described by Aranki and Freter (2) was used. tect a penicillin-susceptible pathogen in vivo, Preparation and injection of bacteria. Growth of we have studied a mixed infection of B. fragilfs cultures, preparation of cells, and- injection proceand Fusobacterium necrophorum in mice. This dures followed the methods of Wilkins and Smith mixed infection was based on a oure F. (17), except that B. fragilis cells were not washed and 698
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CHEMOTHERAPY OF A MIXED ANAEROBIC INFECTION
were mixed with the F. necrophorum cells under CO2 prior to injection. Bacterial injections, in volumes of 0.1 ml, were made subcutaneously beneath the loose skin of the groin on the animal's left side. Unless otherwise specified, approximately 2 x 107 cells of F. necrophorum and 108 cells of B. fragilis were given in each bacterial injection. To determine whether B. fragilis could produce an infection in the absence of F. necrophorum, B. fragilis cells were injected subcutaneously in 0.1-ml volumes (10w to 109 cells per injection). Forty representative strains of B. fragilis were tested. The mice were inspected daily for external signs of abscess formation and were autopsied at 14 days. To determine the effect of injecting B. fragilis cells after the F. necrophorum cells, an initial injection of 2 x 107 cells of F. necrophorum was made, followed by a separate injection of B. fragilis cells in the same location at 4, 8, 12 or 24 h. Two strains of B. fragilis subsp. fragilis were tested (3625 and 4147). To determine the minimum number of B. fragilis cells required to protect F. necrophorum from penicillin, the number of B. fragilis cells in the injection mixture was varied by 10-fold dilutions with the number of F. necrophorum cells kept constant at 2 x 107 cells per injection. Two strains of B. fragilis (3625 and 4147) were tested. To determine whether heat-killed B. fragilis cells could protect F. necrophorum, heat-killed cells were prepared by heating a 16-h chopped meat carbohydrate broth (8) culture of B. fragilis under CO2 at 80 C for 20 min. The heat-killed cells were mixed with F. necrophorum cells prior to injection. Injection of antibiotics. Benzylpenicillin, potassium salt (1,595 U per mg; Sigma, St. Louis, Mo.) was dissolved in distilled water and injected in 0.1-ml volumes into the intraperitoneal cavity on the animal's right side. All procedures involved in antibiotic injections were aerobic. Unless otherwise stated, penicillin therapy consisted of injections of 2,500 Ag (125 mg/kg) at 4, 20, 28, 44 and 52 h after bacterial challenge (schedule A). This injection schedule was chosen primarily for convenience. The protection phenomenon could also be demonstrated when penicillin was injected at 24, 48, 72 and 96 h after bacterial
challenge. To determine the effect of increasing the frequency of penicillin injections, injections were given at 4, 12, 20, 28, 36, 44 and 52 h after bacterial challenge (schedule B). To determine the effect of increasing the duration of penicillin therapy, injections were given at 4, 20, 28, 44, 52, 68, 76, 92 and 100 h after bacterial challenge (schedule C). Source of mice. Male white Swiss mice, 18 to 21 g, were obtained from Flow Laboratories, Dublin, Va. Determination of mortality. The mortality rate for each experiment was determined by counting the number of mice dead by 21 days after bacterial challenge. Each mortality rate reported in the results was based on at least two separate experiments involving 10 mice each. Statistical analysis. Determination of the statistical significance of differences in average mortality rates was made by using a two-sided t test. Mortality
699
rates from 20 control groups of 10 mice each, inoculated with F. necrophorum alone, were averaged. This
control average was compared with the average of the mortality rates from the mixed infection groups of 10 mice each. A difference was considered statistically significant if the P value was less than 0.05. Determination of minimal inhibitory concentrations. Minimal inhibitory concentration (MIC) values of penicillin for the strains tested were determined using the agar dilution technique. An inoculum of 105 cells per spot from a Steer's replicator (15) was used with supplemented brain heart infusion agar (BHI-S) plates (8) and an incubation time of 48 h. Viable cell counts. Viable cell counts of F. necrophorum and B. fragilis in the inoculation mixture were made separately using the roll tube procedure in BHI-S agar medium (8). Cultural examination of abscesses. All manipulations were carried out in an anaerobic chamber. Abscess material was removed with sterile forceps and homogenized in 1.0 ml of dilution fluid (8) in a ground glass tissue homogenizer. Tenfold dilutions of the homogenate were made with dilution fluid and 0.1 ml of each dilution was spread on triplicate BHI-S agar plates containing 5% sheep blood and 0.05% cysteine. Colonies were counted after the plates had been incubated 48 h in an anaerobic chamber at 37 C. F. necrophorum colony types were tentatively identified by the narrow zone of hemolysis produced on the blood agar plates. B. fragilis colony types were nonhemolytic. Characteristic colonies of both F. necrophorum and B. fragilis were picked, and identification was verified by Gram stain and by the biochemical tests outlined in the V. P. I. Anaerobe Laboratory Manual (8). To determine whether F. necrophorum and B. fragilis were growing in the abscesses in the absence of penicillin, abscess material was streaked on BHI-S plates with 5% sheep blood and 0.05% cysteine, and the ratio of F. necrophorum to B. fragilis was estimated after 48 h of incubation of the plates in an anaerobic chamber. Determination of penicillin-degrading activity. For our experiments, the in vitro plate assay technique of Holt and Stewart (9) was modified by using BHI-S medium and membrane filters (0.45-Atm pore size; Millipore Corp.). All operations were performed in an anaerobic chamber. The media contained 0, 0.25, 2.0, or 8.0 U of penicillin per ml. The B. fragilis strains were inoculated (0.1 ml per filter) on the surface of filters placed on the plates. The plates were incubated at 37 C for 24 h. After the filters were aspetically removed, the plates were swabbed with F. necrophorum (107 cells per ml). Growth of F. necrophorum on the plates after 48 h of incubation was interpreted as indicating penicillin-degrading activity in the B. fragilis strain. Assays of cell-free preparations were done similarly except that 0.1 ml of the cell-free preparation was applied directly to the agar medium and, after drying, the plates were swabbed with F. necrophorum. Cell-free preparations. Maximally turbid cultures (10 ml) of B. fragilis grown in chopped meat carbohydrate broth (8) were centrifuged at 6620 x g for 15
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ANTIMICROB. AGENTS CHEMOTHER.
HACKMAN AND WILKINS
min. The supernatant fluid was filter sterilized (membrane-filter, Millipore Corp.; 0.45-,um pore size) and assayed for penicillin-degrading activity. The cell pellet was resuspended in 5 ml of 0.1 M phosphate buffer (pH 7) and the cells were lysed by sonic treatment. The cell lysate was centrifuged at 35,000 x g for 15 min and the supernatant fluid was filter sterilized and tested for penicillin-degrading activity. Part of this supernatant fluid was also heated at 80 C for 15 min prior to assay.
penicillin therapy was statistically significant (P < 0.001). Substituting 108 heat-killed B. fragilis cells for the same number of live cells in the mixed infection resulted in a mortality which was not significantly different from that due to the pure F. necrophorum infection. Mice injected with B. fragilis as well as F. necrophorum and treated with penicillin developed the same symptoms as mice injected with F. necrophorum alone and not treated with penicillin, but in the case of the mixed infection both symptoms and death were delayed. In Fig. 1, the number of deaths per day due to the pure F. necrophorum infection without penicillin therapy is compared with the number of deaths per day, due to the mixed infection treated with penicillin. Values for the mixed infection represent pooled data from all 10 strains of B. fragilis for a total of 100 mice. The distribution of deaths dtu to the mixed infection without penicillin therapv was the same as the distribution due to the pure P. necrophorum infection without penicillin therapy. B. fragilis cells alone were not capable of initiating an infection; none of the 40 strains of B. fragilis injected without F. necrophorum produced subcutaneous ab-
RESULTS In vivo penicillin protection. Without penicillin therapy, the pure F. necrophorum infection was fatal for 97% of the mice injected. An abscess formed beneath the skin at the point of injection and remained localized in the subcutaneous tissue. Death occurred within 14 days. With penicillin therapy (schedule A), the mortality due to the pure F. necrophorum infection was reduced to 13%. If, however, B. fragilis cells were included with F. necrophorum in the inoculation mixture, penicillin therapy was no longer effective. Ten strains of B. fragilis subsp. fragilis with penicillin MICs ranging from 16 to 64 U per ml were tested in the mixed infection with penicillin therapy. Results are shown in Table 1. The difference between the average scesses. To determine the number of B. fragilis cells mortality due to the pure F. necrophorum to protect the F. necrophorum from required infection with penicillin therapy and the average mortality due to the mixed infection with penicillin, we varied the number of B. fragilis cells in the inoculation mixture, keeping the number of F. necrophorum cells constant. Two TABLE 1. Ability of B. fragilis strains with penicillin strains of B. fragilis were tested. In the case of MICs ranging from 16 to above 64 U/ml to protect F. one strain (3625) only about 10 cells of B. necrophorum from penicillin in vivo and in vitro Subspecies and strain of B. fragilis in the mixed infection
Penicillin
No. of 20 mice dead
In vitro
50
penicillin-
(U/mi) duringactivity
21dy00.25
VPI 2556-1 VPI 2633 VPI 2758A VPI 3390 VPI 3625 VPI 4147 VPI 5708 VPI 6805
64 32
16 32 32 64 32
18 12 16 19 20 17 17 18 17 20
+ + + + + + + +
+ -
+ + + + + +
+
-
+
+
cron VPI 9511
30 -
C,) 2520LLI
CD 15
-
10-
5
-
o
4
> 64
17
+
+
a Mean percentage of mortality (±+ standard deviation) = 87% (±+ 11%).
3
I
n inann
1
I3 I
Subsp. thetaiotaomi-
Mixed infection with penicillin therapy
a 350~ Cr
32 64 16
F. necrophorum infection
I7-
-
40-
8.0
U/ml U/ml
Subsp. fragilis VPI 1522 VPI 2553
45
degrading
5
7
9
It
13
15
17
19
21
DAYS AFTER BACTERIAL CHALLENGE FIG. 1. Comparison of the number of mice dying per day from F. necrophorum without penicillin therapy and the number dying per day from the mixed infection with penicillin therapy.
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fragilis were required to cause the deaths of at least half of the animals injected. In the case of the second strain (4147) at least 104 cells of B. fragilis were required. Cultural examination of abscesses due to the mixed infection (with B. fragilis 3625) with penicillin therapy revealed that, regardless of the initial number of B. fragilis cells administered, the resulting concentration of B. fragilis cells in the abscess rose to 109 to 10's cells per g of abscess within 48 h and remained at this level until death. Without penicillin therapy approximately the same concentration of B. fragilis cells was reached and maintained. The concentration of F. necrophorum cells in the mixed infection abscesses in mice treated with penicillin dropped below 106 cells per g of abscess (the limit of detection) during the period of penicillin therapy. After penicillin therapy ceased, the concentration of F. necrophorum rose to 1015 cells per g and remained at that level. Without penicillin, the concentration of F. necrophorum in abscesses due to both the pure F. necrophorum infection and the mixed infection reached io01 cells per g of abscess within 24 h of bacterial challenge and did not decrease significantly during the course of the infection. B. fragilis cells did not have to be injected at the same time as the F. necrophorum to result in some protection from penicillin. Regardless of whether B. fragilis cells were injected at 4, 8, 12 or 24 h after the F. necrophorum, in the same location, approximately half of the mice died. This mortality was lower (P < 0.01) than the 97% mortality obtained when B. fragilis cells were injected at the same time as F. necrophorum, but was higher (P < 0.01) than the 13% mortality due to F. necrophorum alone. Cultural examination of the abscesses at 7 days after inoculation revealed that both B. fragilis and F. necrophorum were present in approximately equal numbers. Both strains of B. fragilis tested (3625 and 4147) yielded similar results. The extent of penicillin resistance conferred by the B. fragilis was tested by increasing the number of injections and the amount of penicillin given at each injection. Neither increasing the frequency of penicillin injections to once every 8 h for 2 days (schedule B) nor increasing the duration of therapy to two injections daily for 4 days (schedule C) lowered the 97% mortality due to the mixed infection, even when the amount of penicillin per injection was doubled (to 250 mg/kg). Using the basic injection schedule (schedule A), the amount of penicillin per injection was increased from 125 mg/kg to 2,000
701
mg/kg without significantly affecting the mortality due to the mixed infection. To study the relationship between the penicillin resistance of the B. fragilis strain used in the mixed infection and its ability to protect F. necrophorum from penicillin, we tested five strains of B. fragilis with MICs ranging from 1 to 4 U per ml and compared these results with results obtained by using strains with higher MICs. Since we could not find any strains of B. fragilis subsp. fragilis with MICs lower than 8 U per ml, we used five strains with MIC values from 1 to 4 U per ml representing other subspecies of B. fragilis. Cultural examination of abscesses resulting from combining these strains with F. necrophorum without penicillin showed that all five strains were able to grow in the abscess with F: necrophorum equally as well as strains of B. fragilis subsp. fragilis. The effect of combining each of these strains having intermediate MICs with F. necrophorum in the mixed infection and treating with penicillin is shown in Table 2. With penicillin therapy, the average mortality due to the mixed infection with these strains of B. fragilis was significantly lower (P < 0.001) than the average mortality obtained with the strains with higher MIC values but significantly higher (P < 0.004) than the mortality obtained for the penicillin-treated F. necrophorum controls. As a further test of the comparability of different B. fragilis subspecies, a strain of B. fragilis subsp. thetaiotaomicron (VPI 9511), which had an MIC greater than 64 U of penicillin per ml, was also tested in the TABLE 2. Ability of B. fragilis strains with penicillin MICs ranging from 1 to 4 U per ml to protect F. necrophorum from penicillin in vivo and in vitro vitro ~~~~~In
4 Subspecies and strain of B. fragilis in the mixed infection
ofpeilin Pn-No. 20 mic Penipenricillin-
dead c (U/ml) (Um)21during daysa
activity
(0.25 U/mi)
Subsp. vulgatus VPI 0959-1 VPI 4245 VPI 5710
4 2 2
5 7 15
_ _ _
1
10
+
Subsp. thetaiotaomicron VPI 0061-1
Subsp. distasonis VPI 4243
4
4
a Mean percentage of mortality ( i standard deviation) = 41% (020%).
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HACKMAN AND WILKINS
ANTIMICROB. AGENTS CHEMOTHER.
mixed infection (Table 1). The mortality rate number of F. necrophorum cells rose rapidly for this strain was comparable to that for strains until it equaled the number of B. fragilis cells, of B. fragilis subsp. fragilis having MICs in the and the ratio of F. necrophorum to B. fragilis same range. remained constant until death. The final conIn vitro protection. All strains of B. fragilis centration of F. necrophorum cells in the mixed used in the mixed infection in vivo were also infection treated with penicillin was the same as assayed in vitro for penicillin-degrading activity that resulting from a pure F. necrophorum with F. necrophorum as the indicator organism. infection not treated with penicillin. Thus, no Results are given in Tables 1 and 2. All of the antagonism between F. necrophorum and B. strains with MICs of 16 U per ml or above fragilis was observed in the mixed infection and (Table 1) were able to protect F. necrophorum B. fragilis seemed to function primarily to from 0.25 U of penicillin per ml and eight of the protect F. necrophorum from penicillin. Moreten gave protection at 8 U per ml. Only one of over, this protection phenomenon could be the five strains with MICs from 1 to 4 U per ml demonstrated even when the initial concentra(Table 2) was able to protect F. necrophorum in tion of B. fragilis was very low or when B. fragilis cells were injected up to 24 h after the F. vitro. The penicillin-degrading activity was found necrophorum. If this protection of F. necrophorum from to be intracellular and heat labile. Activity was detected in the soluble portion of the cell lysate penicillin by B. fragilis is due to penicillinase but not in the culture supernatant. Heat treat- production by B. fragilis, it would be expected that the less penicillin-resistant strains of B. ment of the cell lysate destroyed its activity. fragilis would be less capable of protection than more resistant strains. We found that strains of DISCUSSION B. fragilis having penicillin MICs between 16 Studies of mixed infections in rabbits and and 64 U per ml were, in fact, more effective as mice have shown that penicillin-resistant facul- protectors of F. necrophorum than strains havtative and aerobic organisms are capable of ing MICs between 1 and 4 U per ml. Although protecting penicillin-susceptible pathogens the strains with lower MICs belonged to differfrom penicillin in vivo (13, 16). Since this ent subspecies of B. fragilis than most of the protection phenomenon was also demonstrated strains with higher MICs, they were found to be using penicillinase extracted from penicillin- equally capable of growing with F. necrophorum resistant organisms (13), it is likely that the in the abscess. Moreover, a strain of B. fragilis protective ability of these organisms was due subsp. thetaiotaomicron with an MIC greater largely to penicillinase production. Penicillin- than 64 U per ml protected F. necrophorum ase production by penicillin-resistant strains of from penicillin as effectively as strains of B. B. fragilis has been shown to occur in vitro, fragilis subsp. fragilis with comparable MICs. although at a very low level (1, 7, 12). It has not Thus subspecies differences in ability to survive been possible, however, to determine whether and grow in vivo probably did not contribute penicillinase production by B. fragilis might be significantly to the lower mortality observed significant in vivo due to the lack of a suitable with the strains having lower MIC values. Protection of F. necrophorum from penicillin animal infection. Using the F. necrophorum infection described by B. fragilis was also demonstrated in vitro. A previously (17) as a starting point, we have plate assay was used because of the high sensideveloped a mixed infection model including B. tivity of this type of assay (3). Penicillinfragilis and F. necrophorum. Since B. fragilis degrading activity was found in all of the strains cells grow very well with F. necrophorum in the which produced high mortality in mice and in infection, this model may prove useful for only one of the strains which produced lower testing the in vivo susceptibility of B. fragilis to mortality. Most of the strains which protected F. necrophorum in vitro could do so when the antibiotics. The growth of B. fragilis in the mixed infec- penicillin concentration was as high as 8 U per tion did not appear to be affected by penicillin. ml. The ability of these strains to effect such a B. fragilis cells grew rapidly to a concentration sizeable reduction in the concentration of peniof 1010 cells per g of abscess regardless of cillin (from 8 U per ml to below 0.125 U per ml) whether penicillin therapy was given. In con- suggests that over a period of 24 h dividing B. trast, the number of F. necrophorum cells fragilis cells produce sizeable amounts of penidropped several logs during penicillin therapy. cillin-degrading activity. The penicillin-degrading activity of B. fragilis With the cessation of therapy, however, the
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appeared to be intracellular. This finding is consistent with the observation by Pinkus et al. (12) that conditions which promoted lysis of B. fragilis cells seemed to enhance their penicillindegrading activity. Also, the penicillinases of many gram-negative aerobic and facultative bacteria have been found to be intracellular (11). Thus the ability of B. fragilis to protect F. necrophorum from penicillin may involve lysis of some of the B. fragilis cells. In most human clinical studies, penicillin has been found to be relatively ineffective in treating B. fragilis infections (6). Recently, however, it has been reported that massive doses of penicillin have been used successfully to treat some B. fragilis infections (E. J. Benner, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 17th, San Francisco, Calif., Abstr. 396, 1974). In the case of our mixed infection in mice, penicillin, even at very high levels, was not effective against B. fragilis in a soft tissue abscess. The protection phenomenon appears to be due mainly to production of penicillinase by B. fragilis. However, other factors cannot be ruled out completely. F. necrophorum can exist as an L-form in the presence of penicillin if properly stabilized (5, 14), and B. fragilis might act in some manner to stabilize these L-forms. Alternatively, B. fragilis cells might alter the permeability of the abscess to penicillin or interfere with elimination of F. necrophorum by the mouse's immune system. Whatever the mechanism, the fact that penicillin-resistant strains of B. fragilis were capable of protecting F. necrophorum from penicillin indicates that protection of other penicillin-susceptible pathogens might also occur in vivo. Our results strongly suggest that all components of a mixed infection should be considered in determining treatment. ACKNOWLEDGMENTS We would like to acknowledge the excellent technical assistance of Sarah Chalgren and Jane Wong. This research was supported by Public Health Service
703
grant no. GM 14604 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Anderson, J. D., and R. B. Sykes. 1973. Characterization of a ,B-lactamase obtained from a strain of Bacteroides fragilis resistant to #-lactamase antibiotics. J. Med. Microbiol. 6:201-206. 2. Aranki, A., and Freter, R. 1972. Use of anaerobic glove boxes for the cultivation of strictly anaerobic bacteria. Am. J. Clin. Nutr. 25:1329-1334. 3. Citri, N., and R. Pollock. 1966. The biochemistry and function of ,8-lactamase (penicillinase). Adv. Enzymol. 28:237-323. 4. Del Bene, V. E., and W. E. Farrar. 1973. Cephalosporinase activity in Bacteroides fragilis. Antimicrob. Agents Chemother. 3:369-372. 5. Dienes, L. 1948. Isolation of L-type cultures from Bacteroides with the aid of penicillin and their reversion into the usual bacilli. J. Bacteriol. 56:445-446. 6. Felner, J. M., and V. R. Dowell. 1971. "Bacteroides" bacteremia. Am. J. Med. 50:787-795. 7. Garrod, L. P. 1955. Sensitivity of four species of Bacteroides to antibiotics. Br. Med. J. 2:1529-1531. 8. Holdeman, L. V., and W. E. C. Moore (ed.). 1973. Anaerobe laboratory manual, 2nd ed. Virginia Polytechnic Institute and State University, Blacksburg. 9. Holt, R. J., and G. T. Stewart. 1963. Techniques for the rapid and sensitive detection of penicillinase. J. Clin. Pathol. 16:263-267. 10. Holt, R. J., and G. T. Stewart. 1964. Production of amidase and ,t-lactamase by bacteria. J. Gen. Micro-. biol. 36:203-213. 11. Neu, H. C. 1974. The role of ,8-lactamases in the resistance of gram negative bacteria to penicillin and cephalosporin derivatives. Infect. Dis. Rev. 3:133-149. 12. Pinkus, G., G. Veto, and A. I. Braude. 1968. Bacteroides penicillinase. J. Bacteriol. 96:1437-1438. 13. Shilo, M., and N. Citri. 1964. The role of penicillinase in staphylococcal infections. Br. J. Exp. Pathol. 45:192197. 14. Smith, W. E., S. Mudd, and J. Miller. 1948. L-type variation and bacterial reproduction by large bodies as seen in electron micrograph studies of Bacteroides funduliformis. J. Bacteriol. 56:603-619. 15. Steers, E., E. L. Foltz, and B. S. Graves. 1959. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311. 16. Tacking, R. 1954. Penicillinase producing bacteria in mixed infections in rabbits treated with penicillin. Acta Pathol. Microbiol. Scand. 35:445-454. 17. Wilkins, T. D., and L. DS. Smith. 1974. Chemotherapy of an experimental Fusobacterium (Sphaerophorus) necrophorum infection in* mice. Antimicrob. Agents Chemother. 5:658-662.
Vol. 7, No. 5 Printed in U.S.A.
In Vivo Protection of Fusobacterium necrophorum from Penicillin by Bacteroides fragilis A. S. HACKMAN AND T. D. WILKINS* Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 Received for publication 26 December 1974
A mixed infection of Bacteroides fragilis and Fusobacterium necrophorum was resistant to treatment with penicillin even though a pure F. necrophorum infection could be successfully treated with penicillin. Since B. fragilis alone did not produce infection, these results indicate that B. fragilis can protect F. necrophorum from penicillin in vivo. The extent of protection afforded by a strain of B. fragilis was related to its level of resistance to penicillin. Only a few cells of B. fragilis were required in the initial bacterial injection. Moreover, protection was demonstrated when B. fragilis cells were injected as late as 24 h after the F. necrophorum cells. Protection of F. necrophorum from penicillin by B. fragilis was also demonstrated in vitro.
Bacteroides fragilis is a nonsporing gram-negative anaerobic rod which is frequently isolated from human clinical infections. Most B. fragilis strains are relatively resistant to penicillin. This resistance may be due, at least in part, to production of£-lactamase or other penicillindegrading enzymes. Attempts to detect f,-lactamase production by B. fragilis cells have been successful in some cases but not in others. Holt and Stewart (10) found no ,B-lactamase or amidase activity in eight strains of Bacteroides. However, Garrod (7), Pinkus et al. (12), and Anderson and Sykes (1) have reported 3-lactamase activity in strains of B. fragilis that were resistant to penicillin. Initially, Del Bene and Farrar (4) found cephalosporinase but no penicillinase activity in 10 strains of B. fragilis. Recently, with a more sensitive assay, penicillinase has been detected in these strains (V. Del Bene, personal communication). The difficulty of demonstrating penicillinase activity by B. fragilis cells might be due to the small amount of penicillinase produced by them. The ability of B. fragilis to inactivate penicillin is a matter of considerable clinical interest because B. fragilis is often found as a component of mixed infections in humans. If B. fragilis could inactivate the penicillin in its environment, it might also be able to protect penicillin-susceptible organisms present with it in a mixed infection. To determine whether B. fragilis could pro-
necrophorum infection, reported by Wilkins and Smith (17), which was fatal to mice and which could be treated successfully with penicillin. In this paper, we report evidence that B. fragilis was able to protect F. necrophorum from penicillin in vivo. Not only was the mixed infection much more resistant to penicillin than the pure F. necrophorum infection, but the extent of protection depended on the level of penicillin resistance which characterized the strain of B. fragilis in the mixed infection. We have also demonstrated the ability of B. fragilis to protect F. necrophorum from penicillin in vitro.
MATERIALS AND METHODS Source of organisms. F. necrophorum VPI 6054A (ATCC 27852), 10 strains of B. fragilis subsp. fragilis, three strains of B. fragilis subsp. vulgatus, one strain of B. fragilis subsp. distasonis, and two strains of B. fragilis subsp. thetaiotaomicron were obtained from the V. P. I. Anaerobe Laboratory culture collection. All ten strains of B. fragilis subsp. fragilis, two of the three strains of B. fragilis subsp. vulgatus (VPI 4245 and VPI 5710), and one of the two strains of B. fragilis subsp. thetaiotaomicron (VPI 9511) were isolates from human clinical infections. The remaining strains were isolates from stool specimens. Identifications were done by L. V. Holdeman and W. E. C. Moore according to procedures in the V. P. I. Anaerobe Laboratory Manual (8). Anaerobic glove box. A chamber (Coy Manufacturing Co., Ann Arbor, Mich.) similar to that described by Aranki and Freter (2) was used. tect a penicillin-susceptible pathogen in vivo, Preparation and injection of bacteria. Growth of we have studied a mixed infection of B. fragilfs cultures, preparation of cells, and- injection proceand Fusobacterium necrophorum in mice. This dures followed the methods of Wilkins and Smith mixed infection was based on a oure F. (17), except that B. fragilis cells were not washed and 698
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CHEMOTHERAPY OF A MIXED ANAEROBIC INFECTION
were mixed with the F. necrophorum cells under CO2 prior to injection. Bacterial injections, in volumes of 0.1 ml, were made subcutaneously beneath the loose skin of the groin on the animal's left side. Unless otherwise specified, approximately 2 x 107 cells of F. necrophorum and 108 cells of B. fragilis were given in each bacterial injection. To determine whether B. fragilis could produce an infection in the absence of F. necrophorum, B. fragilis cells were injected subcutaneously in 0.1-ml volumes (10w to 109 cells per injection). Forty representative strains of B. fragilis were tested. The mice were inspected daily for external signs of abscess formation and were autopsied at 14 days. To determine the effect of injecting B. fragilis cells after the F. necrophorum cells, an initial injection of 2 x 107 cells of F. necrophorum was made, followed by a separate injection of B. fragilis cells in the same location at 4, 8, 12 or 24 h. Two strains of B. fragilis subsp. fragilis were tested (3625 and 4147). To determine the minimum number of B. fragilis cells required to protect F. necrophorum from penicillin, the number of B. fragilis cells in the injection mixture was varied by 10-fold dilutions with the number of F. necrophorum cells kept constant at 2 x 107 cells per injection. Two strains of B. fragilis (3625 and 4147) were tested. To determine whether heat-killed B. fragilis cells could protect F. necrophorum, heat-killed cells were prepared by heating a 16-h chopped meat carbohydrate broth (8) culture of B. fragilis under CO2 at 80 C for 20 min. The heat-killed cells were mixed with F. necrophorum cells prior to injection. Injection of antibiotics. Benzylpenicillin, potassium salt (1,595 U per mg; Sigma, St. Louis, Mo.) was dissolved in distilled water and injected in 0.1-ml volumes into the intraperitoneal cavity on the animal's right side. All procedures involved in antibiotic injections were aerobic. Unless otherwise stated, penicillin therapy consisted of injections of 2,500 Ag (125 mg/kg) at 4, 20, 28, 44 and 52 h after bacterial challenge (schedule A). This injection schedule was chosen primarily for convenience. The protection phenomenon could also be demonstrated when penicillin was injected at 24, 48, 72 and 96 h after bacterial
challenge. To determine the effect of increasing the frequency of penicillin injections, injections were given at 4, 12, 20, 28, 36, 44 and 52 h after bacterial challenge (schedule B). To determine the effect of increasing the duration of penicillin therapy, injections were given at 4, 20, 28, 44, 52, 68, 76, 92 and 100 h after bacterial challenge (schedule C). Source of mice. Male white Swiss mice, 18 to 21 g, were obtained from Flow Laboratories, Dublin, Va. Determination of mortality. The mortality rate for each experiment was determined by counting the number of mice dead by 21 days after bacterial challenge. Each mortality rate reported in the results was based on at least two separate experiments involving 10 mice each. Statistical analysis. Determination of the statistical significance of differences in average mortality rates was made by using a two-sided t test. Mortality
699
rates from 20 control groups of 10 mice each, inoculated with F. necrophorum alone, were averaged. This
control average was compared with the average of the mortality rates from the mixed infection groups of 10 mice each. A difference was considered statistically significant if the P value was less than 0.05. Determination of minimal inhibitory concentrations. Minimal inhibitory concentration (MIC) values of penicillin for the strains tested were determined using the agar dilution technique. An inoculum of 105 cells per spot from a Steer's replicator (15) was used with supplemented brain heart infusion agar (BHI-S) plates (8) and an incubation time of 48 h. Viable cell counts. Viable cell counts of F. necrophorum and B. fragilis in the inoculation mixture were made separately using the roll tube procedure in BHI-S agar medium (8). Cultural examination of abscesses. All manipulations were carried out in an anaerobic chamber. Abscess material was removed with sterile forceps and homogenized in 1.0 ml of dilution fluid (8) in a ground glass tissue homogenizer. Tenfold dilutions of the homogenate were made with dilution fluid and 0.1 ml of each dilution was spread on triplicate BHI-S agar plates containing 5% sheep blood and 0.05% cysteine. Colonies were counted after the plates had been incubated 48 h in an anaerobic chamber at 37 C. F. necrophorum colony types were tentatively identified by the narrow zone of hemolysis produced on the blood agar plates. B. fragilis colony types were nonhemolytic. Characteristic colonies of both F. necrophorum and B. fragilis were picked, and identification was verified by Gram stain and by the biochemical tests outlined in the V. P. I. Anaerobe Laboratory Manual (8). To determine whether F. necrophorum and B. fragilis were growing in the abscesses in the absence of penicillin, abscess material was streaked on BHI-S plates with 5% sheep blood and 0.05% cysteine, and the ratio of F. necrophorum to B. fragilis was estimated after 48 h of incubation of the plates in an anaerobic chamber. Determination of penicillin-degrading activity. For our experiments, the in vitro plate assay technique of Holt and Stewart (9) was modified by using BHI-S medium and membrane filters (0.45-Atm pore size; Millipore Corp.). All operations were performed in an anaerobic chamber. The media contained 0, 0.25, 2.0, or 8.0 U of penicillin per ml. The B. fragilis strains were inoculated (0.1 ml per filter) on the surface of filters placed on the plates. The plates were incubated at 37 C for 24 h. After the filters were aspetically removed, the plates were swabbed with F. necrophorum (107 cells per ml). Growth of F. necrophorum on the plates after 48 h of incubation was interpreted as indicating penicillin-degrading activity in the B. fragilis strain. Assays of cell-free preparations were done similarly except that 0.1 ml of the cell-free preparation was applied directly to the agar medium and, after drying, the plates were swabbed with F. necrophorum. Cell-free preparations. Maximally turbid cultures (10 ml) of B. fragilis grown in chopped meat carbohydrate broth (8) were centrifuged at 6620 x g for 15
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ANTIMICROB. AGENTS CHEMOTHER.
HACKMAN AND WILKINS
min. The supernatant fluid was filter sterilized (membrane-filter, Millipore Corp.; 0.45-,um pore size) and assayed for penicillin-degrading activity. The cell pellet was resuspended in 5 ml of 0.1 M phosphate buffer (pH 7) and the cells were lysed by sonic treatment. The cell lysate was centrifuged at 35,000 x g for 15 min and the supernatant fluid was filter sterilized and tested for penicillin-degrading activity. Part of this supernatant fluid was also heated at 80 C for 15 min prior to assay.
penicillin therapy was statistically significant (P < 0.001). Substituting 108 heat-killed B. fragilis cells for the same number of live cells in the mixed infection resulted in a mortality which was not significantly different from that due to the pure F. necrophorum infection. Mice injected with B. fragilis as well as F. necrophorum and treated with penicillin developed the same symptoms as mice injected with F. necrophorum alone and not treated with penicillin, but in the case of the mixed infection both symptoms and death were delayed. In Fig. 1, the number of deaths per day due to the pure F. necrophorum infection without penicillin therapy is compared with the number of deaths per day, due to the mixed infection treated with penicillin. Values for the mixed infection represent pooled data from all 10 strains of B. fragilis for a total of 100 mice. The distribution of deaths dtu to the mixed infection without penicillin therapv was the same as the distribution due to the pure P. necrophorum infection without penicillin therapy. B. fragilis cells alone were not capable of initiating an infection; none of the 40 strains of B. fragilis injected without F. necrophorum produced subcutaneous ab-
RESULTS In vivo penicillin protection. Without penicillin therapy, the pure F. necrophorum infection was fatal for 97% of the mice injected. An abscess formed beneath the skin at the point of injection and remained localized in the subcutaneous tissue. Death occurred within 14 days. With penicillin therapy (schedule A), the mortality due to the pure F. necrophorum infection was reduced to 13%. If, however, B. fragilis cells were included with F. necrophorum in the inoculation mixture, penicillin therapy was no longer effective. Ten strains of B. fragilis subsp. fragilis with penicillin MICs ranging from 16 to 64 U per ml were tested in the mixed infection with penicillin therapy. Results are shown in Table 1. The difference between the average scesses. To determine the number of B. fragilis cells mortality due to the pure F. necrophorum to protect the F. necrophorum from required infection with penicillin therapy and the average mortality due to the mixed infection with penicillin, we varied the number of B. fragilis cells in the inoculation mixture, keeping the number of F. necrophorum cells constant. Two TABLE 1. Ability of B. fragilis strains with penicillin strains of B. fragilis were tested. In the case of MICs ranging from 16 to above 64 U/ml to protect F. one strain (3625) only about 10 cells of B. necrophorum from penicillin in vivo and in vitro Subspecies and strain of B. fragilis in the mixed infection
Penicillin
No. of 20 mice dead
In vitro
50
penicillin-
(U/mi) duringactivity
21dy00.25
VPI 2556-1 VPI 2633 VPI 2758A VPI 3390 VPI 3625 VPI 4147 VPI 5708 VPI 6805
64 32
16 32 32 64 32
18 12 16 19 20 17 17 18 17 20
+ + + + + + + +
+ -
+ + + + + +
+
-
+
+
cron VPI 9511
30 -
C,) 2520LLI
CD 15
-
10-
5
-
o
4
> 64
17
+
+
a Mean percentage of mortality (±+ standard deviation) = 87% (±+ 11%).
3
I
n inann
1
I3 I
Subsp. thetaiotaomi-
Mixed infection with penicillin therapy
a 350~ Cr
32 64 16
F. necrophorum infection
I7-
-
40-
8.0
U/ml U/ml
Subsp. fragilis VPI 1522 VPI 2553
45
degrading
5
7
9
It
13
15
17
19
21
DAYS AFTER BACTERIAL CHALLENGE FIG. 1. Comparison of the number of mice dying per day from F. necrophorum without penicillin therapy and the number dying per day from the mixed infection with penicillin therapy.
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CHEMOTHERAPY OF A MIXED ANAEROBIC INFECTION
fragilis were required to cause the deaths of at least half of the animals injected. In the case of the second strain (4147) at least 104 cells of B. fragilis were required. Cultural examination of abscesses due to the mixed infection (with B. fragilis 3625) with penicillin therapy revealed that, regardless of the initial number of B. fragilis cells administered, the resulting concentration of B. fragilis cells in the abscess rose to 109 to 10's cells per g of abscess within 48 h and remained at this level until death. Without penicillin therapy approximately the same concentration of B. fragilis cells was reached and maintained. The concentration of F. necrophorum cells in the mixed infection abscesses in mice treated with penicillin dropped below 106 cells per g of abscess (the limit of detection) during the period of penicillin therapy. After penicillin therapy ceased, the concentration of F. necrophorum rose to 1015 cells per g and remained at that level. Without penicillin, the concentration of F. necrophorum in abscesses due to both the pure F. necrophorum infection and the mixed infection reached io01 cells per g of abscess within 24 h of bacterial challenge and did not decrease significantly during the course of the infection. B. fragilis cells did not have to be injected at the same time as the F. necrophorum to result in some protection from penicillin. Regardless of whether B. fragilis cells were injected at 4, 8, 12 or 24 h after the F. necrophorum, in the same location, approximately half of the mice died. This mortality was lower (P < 0.01) than the 97% mortality obtained when B. fragilis cells were injected at the same time as F. necrophorum, but was higher (P < 0.01) than the 13% mortality due to F. necrophorum alone. Cultural examination of the abscesses at 7 days after inoculation revealed that both B. fragilis and F. necrophorum were present in approximately equal numbers. Both strains of B. fragilis tested (3625 and 4147) yielded similar results. The extent of penicillin resistance conferred by the B. fragilis was tested by increasing the number of injections and the amount of penicillin given at each injection. Neither increasing the frequency of penicillin injections to once every 8 h for 2 days (schedule B) nor increasing the duration of therapy to two injections daily for 4 days (schedule C) lowered the 97% mortality due to the mixed infection, even when the amount of penicillin per injection was doubled (to 250 mg/kg). Using the basic injection schedule (schedule A), the amount of penicillin per injection was increased from 125 mg/kg to 2,000
701
mg/kg without significantly affecting the mortality due to the mixed infection. To study the relationship between the penicillin resistance of the B. fragilis strain used in the mixed infection and its ability to protect F. necrophorum from penicillin, we tested five strains of B. fragilis with MICs ranging from 1 to 4 U per ml and compared these results with results obtained by using strains with higher MICs. Since we could not find any strains of B. fragilis subsp. fragilis with MICs lower than 8 U per ml, we used five strains with MIC values from 1 to 4 U per ml representing other subspecies of B. fragilis. Cultural examination of abscesses resulting from combining these strains with F. necrophorum without penicillin showed that all five strains were able to grow in the abscess with F: necrophorum equally as well as strains of B. fragilis subsp. fragilis. The effect of combining each of these strains having intermediate MICs with F. necrophorum in the mixed infection and treating with penicillin is shown in Table 2. With penicillin therapy, the average mortality due to the mixed infection with these strains of B. fragilis was significantly lower (P < 0.001) than the average mortality obtained with the strains with higher MIC values but significantly higher (P < 0.004) than the mortality obtained for the penicillin-treated F. necrophorum controls. As a further test of the comparability of different B. fragilis subspecies, a strain of B. fragilis subsp. thetaiotaomicron (VPI 9511), which had an MIC greater than 64 U of penicillin per ml, was also tested in the TABLE 2. Ability of B. fragilis strains with penicillin MICs ranging from 1 to 4 U per ml to protect F. necrophorum from penicillin in vivo and in vitro vitro ~~~~~In
4 Subspecies and strain of B. fragilis in the mixed infection
ofpeilin Pn-No. 20 mic Penipenricillin-
dead c (U/ml) (Um)21during daysa
activity
(0.25 U/mi)
Subsp. vulgatus VPI 0959-1 VPI 4245 VPI 5710
4 2 2
5 7 15
_ _ _
1
10
+
Subsp. thetaiotaomicron VPI 0061-1
Subsp. distasonis VPI 4243
4
4
a Mean percentage of mortality ( i standard deviation) = 41% (020%).
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HACKMAN AND WILKINS
ANTIMICROB. AGENTS CHEMOTHER.
mixed infection (Table 1). The mortality rate number of F. necrophorum cells rose rapidly for this strain was comparable to that for strains until it equaled the number of B. fragilis cells, of B. fragilis subsp. fragilis having MICs in the and the ratio of F. necrophorum to B. fragilis same range. remained constant until death. The final conIn vitro protection. All strains of B. fragilis centration of F. necrophorum cells in the mixed used in the mixed infection in vivo were also infection treated with penicillin was the same as assayed in vitro for penicillin-degrading activity that resulting from a pure F. necrophorum with F. necrophorum as the indicator organism. infection not treated with penicillin. Thus, no Results are given in Tables 1 and 2. All of the antagonism between F. necrophorum and B. strains with MICs of 16 U per ml or above fragilis was observed in the mixed infection and (Table 1) were able to protect F. necrophorum B. fragilis seemed to function primarily to from 0.25 U of penicillin per ml and eight of the protect F. necrophorum from penicillin. Moreten gave protection at 8 U per ml. Only one of over, this protection phenomenon could be the five strains with MICs from 1 to 4 U per ml demonstrated even when the initial concentra(Table 2) was able to protect F. necrophorum in tion of B. fragilis was very low or when B. fragilis cells were injected up to 24 h after the F. vitro. The penicillin-degrading activity was found necrophorum. If this protection of F. necrophorum from to be intracellular and heat labile. Activity was detected in the soluble portion of the cell lysate penicillin by B. fragilis is due to penicillinase but not in the culture supernatant. Heat treat- production by B. fragilis, it would be expected that the less penicillin-resistant strains of B. ment of the cell lysate destroyed its activity. fragilis would be less capable of protection than more resistant strains. We found that strains of DISCUSSION B. fragilis having penicillin MICs between 16 Studies of mixed infections in rabbits and and 64 U per ml were, in fact, more effective as mice have shown that penicillin-resistant facul- protectors of F. necrophorum than strains havtative and aerobic organisms are capable of ing MICs between 1 and 4 U per ml. Although protecting penicillin-susceptible pathogens the strains with lower MICs belonged to differfrom penicillin in vivo (13, 16). Since this ent subspecies of B. fragilis than most of the protection phenomenon was also demonstrated strains with higher MICs, they were found to be using penicillinase extracted from penicillin- equally capable of growing with F. necrophorum resistant organisms (13), it is likely that the in the abscess. Moreover, a strain of B. fragilis protective ability of these organisms was due subsp. thetaiotaomicron with an MIC greater largely to penicillinase production. Penicillin- than 64 U per ml protected F. necrophorum ase production by penicillin-resistant strains of from penicillin as effectively as strains of B. B. fragilis has been shown to occur in vitro, fragilis subsp. fragilis with comparable MICs. although at a very low level (1, 7, 12). It has not Thus subspecies differences in ability to survive been possible, however, to determine whether and grow in vivo probably did not contribute penicillinase production by B. fragilis might be significantly to the lower mortality observed significant in vivo due to the lack of a suitable with the strains having lower MIC values. Protection of F. necrophorum from penicillin animal infection. Using the F. necrophorum infection described by B. fragilis was also demonstrated in vitro. A previously (17) as a starting point, we have plate assay was used because of the high sensideveloped a mixed infection model including B. tivity of this type of assay (3). Penicillinfragilis and F. necrophorum. Since B. fragilis degrading activity was found in all of the strains cells grow very well with F. necrophorum in the which produced high mortality in mice and in infection, this model may prove useful for only one of the strains which produced lower testing the in vivo susceptibility of B. fragilis to mortality. Most of the strains which protected F. necrophorum in vitro could do so when the antibiotics. The growth of B. fragilis in the mixed infec- penicillin concentration was as high as 8 U per tion did not appear to be affected by penicillin. ml. The ability of these strains to effect such a B. fragilis cells grew rapidly to a concentration sizeable reduction in the concentration of peniof 1010 cells per g of abscess regardless of cillin (from 8 U per ml to below 0.125 U per ml) whether penicillin therapy was given. In con- suggests that over a period of 24 h dividing B. trast, the number of F. necrophorum cells fragilis cells produce sizeable amounts of penidropped several logs during penicillin therapy. cillin-degrading activity. The penicillin-degrading activity of B. fragilis With the cessation of therapy, however, the
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CHEMOTHERAPY OF A MIXED ANAEROBIC INFECTION
appeared to be intracellular. This finding is consistent with the observation by Pinkus et al. (12) that conditions which promoted lysis of B. fragilis cells seemed to enhance their penicillindegrading activity. Also, the penicillinases of many gram-negative aerobic and facultative bacteria have been found to be intracellular (11). Thus the ability of B. fragilis to protect F. necrophorum from penicillin may involve lysis of some of the B. fragilis cells. In most human clinical studies, penicillin has been found to be relatively ineffective in treating B. fragilis infections (6). Recently, however, it has been reported that massive doses of penicillin have been used successfully to treat some B. fragilis infections (E. J. Benner, Prog. Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 17th, San Francisco, Calif., Abstr. 396, 1974). In the case of our mixed infection in mice, penicillin, even at very high levels, was not effective against B. fragilis in a soft tissue abscess. The protection phenomenon appears to be due mainly to production of penicillinase by B. fragilis. However, other factors cannot be ruled out completely. F. necrophorum can exist as an L-form in the presence of penicillin if properly stabilized (5, 14), and B. fragilis might act in some manner to stabilize these L-forms. Alternatively, B. fragilis cells might alter the permeability of the abscess to penicillin or interfere with elimination of F. necrophorum by the mouse's immune system. Whatever the mechanism, the fact that penicillin-resistant strains of B. fragilis were capable of protecting F. necrophorum from penicillin indicates that protection of other penicillin-susceptible pathogens might also occur in vivo. Our results strongly suggest that all components of a mixed infection should be considered in determining treatment. ACKNOWLEDGMENTS We would like to acknowledge the excellent technical assistance of Sarah Chalgren and Jane Wong. This research was supported by Public Health Service
703
grant no. GM 14604 from the National Institute of General Medical Sciences. LITERATURE CITED 1. Anderson, J. D., and R. B. Sykes. 1973. Characterization of a ,B-lactamase obtained from a strain of Bacteroides fragilis resistant to #-lactamase antibiotics. J. Med. Microbiol. 6:201-206. 2. Aranki, A., and Freter, R. 1972. Use of anaerobic glove boxes for the cultivation of strictly anaerobic bacteria. Am. J. Clin. Nutr. 25:1329-1334. 3. Citri, N., and R. Pollock. 1966. The biochemistry and function of ,8-lactamase (penicillinase). Adv. Enzymol. 28:237-323. 4. Del Bene, V. E., and W. E. Farrar. 1973. Cephalosporinase activity in Bacteroides fragilis. Antimicrob. Agents Chemother. 3:369-372. 5. Dienes, L. 1948. Isolation of L-type cultures from Bacteroides with the aid of penicillin and their reversion into the usual bacilli. J. Bacteriol. 56:445-446. 6. Felner, J. M., and V. R. Dowell. 1971. "Bacteroides" bacteremia. Am. J. Med. 50:787-795. 7. Garrod, L. P. 1955. Sensitivity of four species of Bacteroides to antibiotics. Br. Med. J. 2:1529-1531. 8. Holdeman, L. V., and W. E. C. Moore (ed.). 1973. Anaerobe laboratory manual, 2nd ed. Virginia Polytechnic Institute and State University, Blacksburg. 9. Holt, R. J., and G. T. Stewart. 1963. Techniques for the rapid and sensitive detection of penicillinase. J. Clin. Pathol. 16:263-267. 10. Holt, R. J., and G. T. Stewart. 1964. Production of amidase and ,t-lactamase by bacteria. J. Gen. Micro-. biol. 36:203-213. 11. Neu, H. C. 1974. The role of ,8-lactamases in the resistance of gram negative bacteria to penicillin and cephalosporin derivatives. Infect. Dis. Rev. 3:133-149. 12. Pinkus, G., G. Veto, and A. I. Braude. 1968. Bacteroides penicillinase. J. Bacteriol. 96:1437-1438. 13. Shilo, M., and N. Citri. 1964. The role of penicillinase in staphylococcal infections. Br. J. Exp. Pathol. 45:192197. 14. Smith, W. E., S. Mudd, and J. Miller. 1948. L-type variation and bacterial reproduction by large bodies as seen in electron micrograph studies of Bacteroides funduliformis. J. Bacteriol. 56:603-619. 15. Steers, E., E. L. Foltz, and B. S. Graves. 1959. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9:307-311. 16. Tacking, R. 1954. Penicillinase producing bacteria in mixed infections in rabbits treated with penicillin. Acta Pathol. Microbiol. Scand. 35:445-454. 17. Wilkins, T. D., and L. DS. Smith. 1974. Chemotherapy of an experimental Fusobacterium (Sphaerophorus) necrophorum infection in* mice. Antimicrob. Agents Chemother. 5:658-662.
