Bacteroides fragilis Group*

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1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 18, No. 4 Copyright 1988, Institute for Clinical Science, Inc. Post-Antibiotic Effect in Bacteroides fragilis Group* DAVID J. SIVERHUS, B.S.,t CHARLES E. EDM ISTON, Jr., PH.D.,f JOHN C. CLAUSZ, Ph.D.,1 and MICHAEL P. GOHEEN M.S.* tsurgical Microbiology Research Laboratory, Department of Surgery, Medical College of Wisconsin, Milwaukee, WI and tdepartment o f Pathology, Indiana University School of Medicine, Indianapolis, IN ABSTRACT Post-antibiotic effect (PAE) is the transient suppression of bacterial growth after brief antimicrobial exposure. While numerous reports have described PAE with aerobic and facultative microorganisms, virtually no studies have been conducted with anaerobic isolates. Intraabdominal isolates of the Bacteroides fragilis group were exposed for one hour to antibiotic (cefoxitin, cefotetan, and imipenem) concentrations two to four times the minimal inhibitory concentration (MIC). Post-antibiotic effect was described as the difference betw een the time required for microbial growth in the test versus the control to increase one Log10 above the quantitation observed immediately after drug removal. Bacteroides fra gilis, B. ovatus, B. thetaiotaomicron and B. vulgatus exhibit PAEs for all test compounds. The time intervals for the PAEs were strain variable and ranged from six to 50 hours. No PAE was demonstrated with B. distasonis strains by the broth dilution technique. The results suggest that brief high dose exposure of some members of the B. fragilis group to anaerobe active beta-lactams produces a prolonged suppression in growth. In theory, a prolonged PAE could influence the dosage regimentation of selective antibiotics. as 1944, it has only recently been recognized as having potential clinical relev an ce.2 M any in vitro stu dies have described PAE s for a wide range of gram p o sitive and gram n egative aerobic microorganisms. T he a e ro b ic gram p o sitiv e cocci exhibit PAE s varying from 0.5 to three $01.80 Institute for Clinical Science, Inc. Introduction While the phenomena of post-antibiotic effect (PAE) was described as early *Address reprint requests to: Charles E. Edmiston, Jr., P h.d., D epartm ent of Surgery, Medical College of Wisconsin, 8700 W. Wisconsin Ave., Milwaukee, WI

2 hours following brief antimicrobial exposu re.3,10 The PAE s w hich have been reported for a number of different antimicrobials against the Enterobacteriaceae appear to be strain variable and dependent upon drug classification. In general, drugs which inhibit protein synthesis produce PAE s of one to five hours in duration in the gram negatives, while the beta-lactams antibiotics appear to be poor inducers of PAE.714 However, imipenem has been dem onstrated to produce a significant PAE with strains of Pseudomonas aeruginosa,9 The post-antibiotic effect has also been dem onstrated in a limited num ber of in vivo animal investigations.8 To date, little data have been presented which docum ent PAE for the anaerobic microbial populations.5 The Bacteroides fragilis group are significant pathogens recove re d from su rg ic a l in fe c tio n s, and together with the Enterobacteriaceae, comprise the predom inant flora associated w ith intraabdom inal sepsis.1 Cefoxitin, cefotetan, and imipenem are b ro ad sp ectru m beta-lactam s w hich dem onstrate good to excellent in vitro anaerobe activity and are often used in the surgical practice. In the presen t study, these th re e antim icrobials are tested against clinical isolates of the Bacteroides fragilis group to assess the presence or absence of a post-an tib io tic effect. Materials and Methods O r g a n is m s Organism s w ere obtained from the Surgical Microbiology Research Laboratory intraabdominal isolate library. Four strains each of Bacteroides fragilis group w ere re c o v e re d from fro zen stock ( 70 C) and streaked to Rrucella blood agar supplem ented with Vitamin K and hemin to check for purity. The following strains w ere used: Bacteroides fragilis POST-ANTIBIOTIC EFFECT IN BACTEROIDES (A203-11, G35-8, A285-28, A400-18); B acteroides vulgatu s (A4, A262-13, A269-11, A301-7); Bacteroides ovatus, (A203-3, A280-15, A324-1, A400-16); Bacteroides thetaiotaomicron (A224-4, A237-17, B276-15, A318-4); and Bacteroides distasonis (A5, A221-6, A248-21, A256-4). None of the test strains was encapsulated and all strains tested positive for beta-lactamase activity by the nitrocefin disk method. M in im a l I n h ib it o r y C o n c e n t r a t io n ( M IC ) The anaerobic isolates were tested for antimicrobial susceptibility by the broth dilution technique.11 Standard antimicrobial pow ders (cefoxitin and im i penem * and cefotetanf) w ere kindly provided by pharmaceutical companies. The anaerobic susceptibilities were perfo rm ed in W ilk in s-c h alg ren b ro th. Briefly, following 48 hours incubation on supplem ented Brucella blood agar, five colonies from each strain w ere inoculated to W ilkins-c halgren bro th and incubated 24 hours at 35 C in an anaerobic chamber. The inoculum was adjusted to a 0.5 McFarland Standard with Wilkins-Chalgren broth. The inoculum density in each tube was approximately 6.0 Log10 colony forming units (cfu) per ml. The tubes were incubated anaerobically at 35 C for 48 hours. The M IC was defined as the lowest concentration of antimicrobial agent that inhibited visual growth of the organism. Bacteroides fra gilis ATCC was included as a control organism to verify drug potency and procedure adequacy. D e t e r m in a t io n o f P o s t -A n t ib io t ic E f f e c t (P A E ) In vitro PAE was determ ined by the broth centrifugation m ethod (11). Bac- *Merck Sharp & Dohme, W est Point, PA. tstuart Pharmaceuticals Inc., Wilmington, DE.

3 3 2 8 SIVERHUS, EDMISTON, CLAUSZ, AND GOHEEN teroides fragilis group test strains were grown overnight (24 hours) in Wilkins- Chalgren broth. The broth cultures were diluted to an optical density of 0.3 at 580 nm and one ml of the adjusted inoculum was added to nine ml of Wilkins-Chalgren broth containing the test antimicrobial and a tube containing 9-ml of antibio tic fre e b ro th (control). T he final microbial concentration in each tube was approximately 7.5 Log10 cfu per ml. The antimicrobials w ere added in test concentrations which varied from two to four times the MIC of the isolate. Test and control cu ltu res w ere incubated anaerobically at 35 C on a rotating rack. At one hour post-antibiotic exposure, the broth cultures were centrifuged (1200 X g for 10 minutes) and washed (3 X ) with buffered diluent to remove the test drug. The control cultures were also washed (3 x ). D rug elim ination was confirmed by the removal of a 200 xl aliquot from the final wash solution for drug quantitation by biological assay (E. coli indicator strain #1346). Following the final wash, antibiotic and control cultures were returned to the rotating rack and incubated anaerobically at 35 C. Samples (100 xl) were removed for quantitative plate counting at 0, 1 (pre and post-wash), 4, 12, 24, 36, and 48 hours (additional sample taken at 96 hours for B. thetaiotaomicron). Dilutions were carried out in buffered gelatin diluent, plated to supplem ented Wilkins-Chalgren agar and incubated anaerobically for 48 hours. Microbial quantitation was expressed as the Log10 cfu per ml. The post-antibiotic effect was defined to be the difference betw een the time required for the colony forming units in the test (T = times in hours) versus contro l (C = tim es in hours) tu b es to in crease 1.0 Log10 above the count observed im m ediately following drug rem oval.4 Therefore, the PAE = T-C: time interval during which normal bacterial grow th was in h ib ited after drug removal. S c a n n in g E l e c t r o n M ic r o s c o p y (S E M ) Bacteroides fragilis strain A and Bacteroides distasonis strain A5 w ere inoculated to Wilkins-Chalgren broth as in the previous PAE procedure. B. fra gilis was tested against cefoxitin and cefotetan while B. distasonis was inoculated to a tube containing im ipenem. The antibiotic concentrations were two times the MIC for cefoxitin and cefotetan (32 xg per ml) and four times the MIC for im ipenem (20 xg per ml). Samples (200 xl) for SEM were removed at initial inoculation and one, 8, 12, 24, 36 and 48 hours post-d rug rem oval. The bro th samples were passed through a 0.45 xm m illipore filter and prefixed for two hours in Karnovsky s fixative at ph 7.2 followed by post-fixation in one percent osm ium tetro x id e.6 Specim ens w ere washed (2 X ) pre and post-fixation in 0.15M sodium cacodylate buffer. Follow ing serial dehydration the filters w ere critical point dried from liquid C 0 2 coated with goldpalladium and observed in a Phillips 500 scanning electron microscope at 25 kv and a spot size of eight nm. Results The post-antibiotic effects for cefoxitin, cefotetan, and imipenem against 20 Bacteroides fragilis group strains are reported in tables I through III. Figures 1 through 3 demonstrate the microbial growth kinetics for selected strains following one hour antimicrobial exposure. Cefoxitin (table I) exhibited a PAE for all B. fragilis strains tested (mean 11.3 hours). Three B. vulgatus isolates demonstrated a PAE with cefoxitin while all strains of B. ovatus exhibited a PAE (Mean 9.4 hours). Two B. thetaiotaomicron strains produced PAE s of 6.5 and 7

4 TABLE I Post-Antibiotic Effect of Cefoxitin Against B a c t e r o i d e s Fragilis Group O r g a n i s m (MIC)* POST-ANTIBIOTIC EFFECT IN BACTEROIDES 32 9 Concent r a t i o nf (ligml) P A E (hrs) B. f r a g i l i s A (16) G35-8 (16) A (16) A (32) B. v u l g a t u s A (8) 16 None A4 (16) A (16) A301-7 (32) B. ovatus A (8) A203-3 (16) A324-1 (16) A (32) B. thetaiotaomicron A224-4 (16) 32 None A (16) B (32) A318-4 (32) 64 None B. d i s t a s o n i s A221-6(8) 16 None A5 (16) 16 None A (16) 32 None A256-4 (16) 64 None *MIC = minimal inhibitory concentration, pg per ml fpost-antibiotic effect (PAE) test concentration and test antimicrobials. The results with B. distasonis were similar to the other two drugs with the exception that strain A221-6 exhibited a PAE of 4.5 hours. Overall, 85 percent of the test isolates exhibited a post-antibiotic effect to imipenem. Figures 1 A through D dem onstrate the PAE kinetics of four B. fragilis group strains following exposure to cefoxitin. W ith the exception of B. distasonis (A5), the growth of the other three isolates exhibited a prolonged growth suppression following drug rem oval. Sim ilar re su lts w ere observed w ith th e te st strains following brief exposure to cefotetan and imipenem (Figures 2 and 3). W hile a significant drop in m icrobial recovery was observed with most strains following drug removal, microbial recovery approximated control values at study end. In general, the shortest PAEs were produced by cefoxitin while the PAEs for hours, respectively. None of the four test strains of B. distasonis exhibited a PAE with cefoxitin. Overall, 65 percent of the B. fragilis group strains exhibited a PAE following brief exposure to cefoxitin. Extended PAE s were documented for cefotetan (table II) with B. fragilis at two times the MIC. Two B. vulgatus strains exhibit a post-antibiotic effect at four times the M IC. Four of the B. ovatus and three of the B. thetaiotaomicron isolates dem onstrated extended PAE s in excess of 18 hours. No PAE was demonstrated with the B. distasonis strains. All strains of the Bacteroides fragilis group w ere tested against im ipenem, (table III) at four times the individual MIC. B. fragilis, B. vulgatus, B. ovatus, and B. thetaiotaom icron all exhibited PAE s w ith im ipenem in excess of 15 hours. The B. thetaiotaomicron strains exhibited the highest PAE s (Range: 24.5 to 50 hours) relative to the other strains TABLE II Post-Antibiotic Effect of Cefotetan Against B a c t e r o i d e s F r a g i l i s Group O r g a n i s m (MIC)* C o n c e n - tration"\ (vgml) P A E (hrs) B. fragilis G35-8 (8) A (8) A (16) A (16) B. vulgatus A4 (4) A (8) A (8) A301-7 (16) B. ovatus A203-3 (16) A (32) A324-1 (32) A (64) B. thetaiotaomicron A224-4 (32) A (32) B (64) A318-4 (64) 128 None B. d i s t a s o n i s A221-6 (16) 32 None A (16) 32 None A5 (32) 64 None A256-4 (32) 64 None *MIC = minimal inhibitory concentration, pg per ml fpost-antibiotic effect (PAE) test concentration

5 3 3 0 SIVERHUS, EDMISTON, CLAUSZ, AND GOHEEN TABLE III Post-Antibiotic Effect of Imipenem Against Bacteroides Fragilis Group Organism (MIC)* Concentrationf (Mgml) PAE (hrs) B. fragilis A (0.5) G35-8 (0.5) A (1) A (1) B. vulgatus A4 (0.5) A (1) A (1) A301-7 (2) B. ovatus A203-3 (0.5) A (0.5) A324-1 (1) A (2) B. thetaiotaomicron A (0.5) A224-4 (1) B (1) A318-4 (2) B. distasonis A5 (0.5) 2 None A221-6 (1) A (2) 8 None A256-4 (2) 8 None *MIC = minimal inhibitory concentration, ug per ml fpost-antiobiotic effect (PAE) test concentration cefotetan were interm ediate to cefoxitin and imipenem (highest). Figures 4 and 5 A -D document the morphologic changes induced in B. fra gilis (A203-11) following one hour exposure to cefotetan and cefoxitin. Cellular elongation was the most common nonlethal defect seen following brief drug exposure. The cellular changes w ere similar to those observed in gram negative cell populations grown in the presence of subinhibitory beta-lactam antimicrobials. No morphologic alterations were seen in B. distasonis (A5) following imipenem exposure (Figures 5 C and D). Discussion The major importance of the post-antibiotic effect resides w ith its potential impact upon the frequency of antimicrobial administration. Theoretically, a prolonged PAE would allow antimicrobial serum and tissue levels to fall below the MIC and still be therapeutically effective. Because of the persistence of inhibitory activity, the dosage interval could conceivably be w idened. H ow ever, there is at present limited clinical evidence which corroborates the in vitro phenomena. Furtherm ore, the administration of high dose antimicrobials in less frequent intervals, while therapeutically valid, may induce a higher incidence of toxicity. Limited clinical trials, utilizing once a day high dose aminoglycoside therapy, appear to be prom ising for facultative infections. H ow ever, the small study populations prevent complete acceptance.1213 The results of the present study suggest that cefoxitin, cefotetan and im i penem are capable of producing a significant PAE at concentrations two to four times the MIC in some members of the Bacteroides fragilis group. The length of the PAE s were variable for the B. fra gilis group isolates. Furtherm ore, the duration of the PAE s were considerably longer than the reported values for aerobicfacultative isolates. It is interesting to note that a number of the strains exhibited MICs that were near or at the resistant breakpoints for cefoxitin and cefotetan. Regardless of this, brief exposure to high level beta-lactams resulted in significant static growth (inhibition range: 6 to 36 hours). As a rule, the cell wall active drugs pro duce relatively short PAE s w hen tested against facultative gram negative microorganisms. The mechanism for the extended PAE s in the B. fragilis group is at present unknown. However, scanning electron microscopy demonstrates that in the case of a susceptible cell population (Figures 4 and 5) the drug can induce non-lethal damage to the cell wall. These non-lethal changes may be precipitated by the covalent binding of the beta-lactams to the penicillin binding proteins (PBPs saturating the active

6 POST-ANTIBIOTIC EFFECT IN BACTEROIDES Co j> O PAE ot Cefoxitin with Bacteroides fragilis A (MIC = 16ugml) PAE of Cefoxitin with Bacteroides vulgatus A4 (MIC = 16ugml) \ e A B -5 9 ' c> I s if I 8 - t CD Z 1 I T ^ a y u_ \ 6 - \ \ - A o -J S 6.5 \ = 5 - \ r V " V. w 6.0 i i i t i i i r t 1 i i i i i r- i i A J LOG TIME IN HOURS I CONTROL ORGANISM * ANTIBIOTIC TIME IN HOURS I CONTROL ORGANISM * ANTIBIOTIC PAE of Cefoxitin with Bacteroides ovatus A203-3 (MIC = I6ugml) PAE of Cefoxitin with Bacteroides distasonis A5 (MIC = 1 6 u g m l) I CONTROL ORGANISM * ANTIBIOTIC l CONTROL ORGANISM ANTIBIOTIC FIGURE 1. Post-Antibiotic effect for B. fragilis (A203-11), B. vulgatus (A4), B. ovatus (A203-3) and B. distasonis (A5) after one hour exposure to cefoxitin. Left and right vertical lines indicate time at which control (solid line) and test (broken line) growth has increased 1.0 Log10 colony forming units following drug removal: PAE = T-C.

7 332 SIVERHUS, EDMISTON, CLAUSZ, AND G O H E E N PAE of Cefotetan with Bacteroides fragilis A (MIC = 16ugml) PAE of Cefotetan with Bacteroides thetaiotaomicron A224-4 (MIC =32ugml) LOG 10 COLONY FORMING UNITS (cfu)ml LOG 10 COLONY FORMING UNITS (cfu)ml CONTROL ORGANISM ANTIBIOTIC PAE of Cefotetan with Bacteroides ovatus A203-3 (MIC = 16ugml) TIME IN HOURS CONTROL * ORGANISM * ANTIBIOTIC PAE of Cefotetan with Bacteroides distasonis A5 (MIC -32ugml) CONTROL ORGANISM ANTIBIOTIC CONTROL * ORGANISM * ANTIBIOTIC F ig u r e 2. Post-antibiotic effect for B. fragilis (A203-11), B. thetaiotaomicron (A224-4), B. ovatus (A203-3) and B. distasonis (A5) after one hour exposure to cefotetan. Left and right vertical lines indicate time at which control (solid line) and test (broken line) growth has increased 1.0 Log10 colony forming units following drug removal: PAE = T-C.

8 I CONTROL «ORGANISM ANTIBIOTIC l CONTROL ORGANISM ANTIBIOTIC POST-ANTIBIOTIC EFFECT IN BACTEROIDES 333 PAE of Imipenem with Bacteroides fragilis G35-8 (MIC = 0.5ugml) PAE of Imipenem with Bacteroides ovatus A203-3 (MIC = 0.5 iflm l) PAE of Imipenem with Bacteroides thetaiotaomicron A224-4 (MIC =1.0 ggml) PAE of Imipenem with Bacteroides distasonis A5 (MIC = O.Sugml) TIME IN HOURS RCONTROL»ORGANISM ANTIBIOTIC I CONTROL ORGANISM * ANTIBIOTIC F ig u r e 3. Post-antibiotic effect for B. fragilis (G35-8), B. ovatus (A203-3), B. thetaiotaomicron (A224-4) and B. distasonis (A5) after one hour exposure to imipenem. Left and right vertical lines indicate time at which control (solid line) and test (broken line) growth has increased 1.0 Log10 colony forming units following drug removal: PAE = T-C.

9 334 SIVERHUS, EDMISTON, CLAUSZ, AND GOHEEN F ig u r e 4. (A) Bacteroides fragilis (A203-11) one hour post-cefotetan removal, cells present on filter exhibit normal morphology (Mag X). (B) Twenty-four hours post-cefotetan removal, numerous filamentous cells present in field (3750 x ). (C) Thirty-six hours post-cefotetan removal, elongated cells demonstrating numer ous (arrows) surface defects (7500 x). (1)) Forty-eight hours post-cefotetan removal, majority of cells have assumed normal morphology. However, a few elongated cells (arrows) remain in culture (3750 X).

10 POST-ANTIBIOTIC EFFECT IN BACTEROIDES 335 F ig u r e 5. (A) Bacteroid.es fragilis (A203-11) culture eight hours post-cefoxitin removal, a few filamentous rods are observed on millipore filter (3750 x). (B) Nearly all B. fragilis cells exhibit normal morphology at 48 hours post-cefoxitin removal (3750x). (C-D ) at 12 and 48 hours post-imipenem removal, Bacteroides distasonis (A5) culture demonstrates normal cellular morphology (7500 x and 1800 x, respectively).

11 3 3 6 SIVERHUS, EDMISTON, CLAUSZ, AND GOHEEN sites) which controls cell wall synthesis. F u rth erm o re, the characteristic slow growth of Bacteroides (relative to facultative organisms) would require a longer period for the synthesis of replacement cellular com ponents required for division. Failure of the B. distasonis and o th e r isolates to exhib it a PAE may reflect variation in species PBP affinity for the test beta-lactam s. At present, most of our knowledge concerning the penicillin binding proteins is limited to aerobic and facultative m icrobial data and little is known about the PBPs in anaerobic microorganisms. In conclusion, cefoxitin, cefotetan and im ipenem pro duce a significant, but variable, post-antibiotic effect with some m em bers of the Bacteroides fra gilis group. The clinical significance of this phenomena in the treatm ent of anaerobic infection is at p resent unknown. However, further studies are necessary to evaluate the frequency of post-antibiotic effect among additional Bacteroides strains and the other clinically important anaerobic populations (clostridia, fusobacteria and peptostreptococci). Acknowledgments Thanks are expressed to Ms. Marcey Jaskulski for h e r technical assistance in the preparation of this manuscript. References 1. A h r e n h o l z, D. H. and S i m m o n s, R. L.: Peritonitis and other intraabdominal infections. Surgical Infectious Diseases. Simmons, R. L. and Howard, R. J., ed. New York, Appleton-Century-Croft, 1982, pp B ig g e r, J. W.: The bactericidal action of penicillin on staphylococcus pyogenes. J. Med. Sci. 227: , B u n d t z e n, R. W., G e r b e r, A. U., C o h n, D. L., and C r a ig, W. A.: Post-antibiotic suppression of bacterial growth. Rev. Infect. Dis. 3:28-37, CRAIG, W. A. and G u d m u n d s s o n, S.: The postantibiotic effect. Antibiotics in Laboratory Medicine. Lorian, V., ed. Baltimore, Williams & Wilkins, 1986, pp C r a ig, W. A. and M a t t ie, H.: Postantibiotic effect (PAE) with Bacteroides fragilis. Twenty- Sixth Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society of Microbiology Abstract No. 147, D a w e s, C. J.: Biological Techniques in Electron Microscopy. New York, Barnes & Noble, Inc., 1971, pp G e r b e r, A. U. an d C r a i g, W.A.: G row th kinetics of respiratory pathogens after short exposures to ampicillin and erythrom ycin in vitro. J. Antimicrob. Chem other. 5:581-91, G u d m u n d s s o n, S., T u r n i d g e, J., V o g e l m a n, B., and C r a ig, W.A.: The postantibiotic effect in vitro. Proceedings of 13th International Congress of Chemotherapy. Spitzy, K. H. and Karrer, K., eds. Vienna, Verlag H. Egerm ann, 1983, pp G u d m u n d s s o n, S., V o g e l m a n, B., and C r a ig, W. A.: The in vivo post-antibiotic effect of imipenem and other new antimicrobials. J. Antimicrob. Chemother. 18:67-73, K nzis, M. D., G u t m a n n, L., and A car, J. F.: Recovery period after exposure of Staphylococcus aureus to subinhibitory and bactericidal concentrations of rifampin: clinical implications. J. Antimicrob. Chem other. 13: Suppl C., 1-7, National C om m ittee for Clinical Laboratory Standards. Alternative Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria: P ro p o sed G u id elin e s N C C L S p u b licatio n M17-D, Villanova, PA, N o r d s t r o m, L., C r o n b e r g, S., R i n b e r g, H., T r e n s t r OM, O., and WALDER, M.: Prospective, comparative, randomized clinical study of aminoglycoside given once a day versus three times a day in severe infections. Recent Advances in Chemotherapy. Ishigami, J., ed. Tokyo, University of Tokyo Press, 1985, P P o w e l l, S. H., T h o m p s o n, W. L., L u t h e, M. A., S t e r n, R. C., G r o s s n ik l a u s, D. A., B l o x h a m, D. D., G r o d e n, D. L., J a c o b s, M. R., D i Sc e n n a, A. O., C a s h, H. A., and Klin g e r, J. D. Once-daily vs continuous aminoglycoside dosing: efficacy and toxicity in animal and clinical studies of gentamicin, netilmicin and tobram ycin. J. Infect. Dis 147: , Vo g e l m a n, B. and C r a ig, W. A.: Kinetics of antimicrobial activity. J. Pediatr. 108: , 1986.