Old Herborn University Seminar Monograph 3: Consequences of antimicrobial therapy for the composition of the microflora of the digestive tract. Editors: Carl Erik Nord, Peter J. Heidt, Volker Rusch, and Dirk van der Waaij. Institute for Microbiology and Biochemistry, Herborn-Dill, Germany: 47-54 (1993).

SPECIFIC INACTIVATION OF ANTIMICROBIAL AGENTS AND ITS INTERINDIVIDUAL DIFFERENCES

GJALT W. WELLING, and GERDA GROEN Laboratory for Medical Microbiology, University of Groningen, Groningen, The Netherlands.

INTRODUCTION ß-Lactamases are enzymes of bacte­ tivity and a broad spectrum (Heymes et rial origin which hydrolyse the C-N al., 1977; Drasar et al., 1978; Neu et bond in the ß-lactam ring of a penicillin al., 1979). Its spectrum of activity in­ or a cephalosporin. The effect of these cludes, in addition to Haemophilus in­ enzymes was first observed by Abra­ fluenzae and the Enterobacteriaceae, ham and Chain (1940), immediately af­ some Pseudomonas aeruginosa and ter the first report on the clinical use of Bacteroides fragilis strains. benzylpenicillin (Chain et al., 1940). Aztreonam (SQ 26,776) is a syn­ For an adequate antibiotic therapy it is thetic monobactam that is minimally important to know the resistance of a (<1%) absorbed after oral administra­ bacterial species against ß-lactam antibi­ tion (Swabb et al., 1983). Its poor in­ otics. Similarly, the resistance of the testinal absorption and the selective ac­ bacterial flora of an individual may be of tivity against Gram-negative bacilli importance in this respect. At present (Sykes et al., 1982) render this drug many ß-lactamases from a large number particularly suitable for selective decon­ of different strains of bacteria have been tamination (de Vries-Hospers et al., purified and characterized (Bush, 1984) of immunocompromised patients. 1989). The gastrointestinal tract is the Carumonam (Ro 17-2301; AMA­ largest source of bacteria in humans. 1080) is also a synthetic monobactam. Some 400 distinct species of bacteria In vitro it has been shown to be active can normally be isolated from the faeces against many, predominantly Gram- (Holdeman et al.,1976). Some of these negative, aerobic rods, primarily mem­ bacteria may produce ß-lactamases and bers of the families Enterobacteriaceae, release them into the intestinal contents, Neisseriaceae; Haemophilus spp., and thereby interfering with the activity of Pseudomonas aeruginosa. It is not ac­ antibiotics that reach the lower intestinal tive or only weakly active against Gram- tract. In the present study, the effect of positive and anaerobic bacteria. It origi­ faecal enzyme preparations on 4 antibi­ nates from sulfazecin, an N-sulfonated otics was studied i.e. benzylpenicillin monocyclic ß-lactam antibiotic first dis­ (penicillin G), cefotaxime, aztreonam covered in the culture broth of and carumonam. Pseudomonas acidophila sp. nov. Benzylpenicillin, the first discovered (Imada et al., 1985). Modification of penicillin, is effective in vitro against this compound resulted in carumonam, Gram-positive cocci and some Gram- an antibiotic with a high antibacterial ac­ negative bacteria. It is also active against tivity (Kishimoto et al., 1983). a number of anaerobic microorganisms. These 4 antibiotics were incubated Cefotaxime is a third-generation with faecal enzyme preparations from cephalosporin with a high intrinsic ac­

47 12 healthy human volunteers and the formance liquid chromatography remaining amount of antibiotic was (HPLC) . quantitated by reversed-phase high-per-

MATERIALS AND METHODS Faecal samples Enzymatic inactivation Faecal samples were collected from An amount of 25 µl of antibiotic (1 12 healthy adult volunteers (5 females, mg/ml phosphate-buffered saline, pH 7 males). During a period of 24 months 7.2 [PBS]) was incubated for 20 h at samples were collected at months 0, 6, 37°C with 200 µl faecal enzyme prepa­ 14 and 24. The volunteers did not re­ ration. As controls were incubated, 25 ceive antimicrobial drugs, at least not µl PBS and 200 µl enzyme preparation, two weeks prior to the collection of the to account for the background from the faecal samples. From volunteer 9, faecal faecal enzyme preparation and 25 µl an­ samples were also collected during a tibiotic solution (1 mg/ml PBS) and 200 shorter period of time (2 weeks). Faecal µl PBS to account for any nonenzymatic samples were stored at -20°C. degradation of the antibiotic. The incu­ bation was terminated by putting the Faecal enzyme preparations samples on ice. Aliquots of 50 µl of the Faecal enzyme preparations were incubation mixture were analyzed in prepared as described by Welling et al. duplicate by HPLC. Peak heights were (1987). 0.5 g of faeces was suspended proportional to concentration and the and homogenized in 1.5 ml of dem­ percentage inactivation was calculated ineralized water containing 0.1% (w/v) by considering the peak height obtained Triton X-100. The suspensions were after HPLC of the incubation mixture centrifuged for 10 min (9000 x g). The with antibiotic and without faecal en­ supernatants were centrifuged for 60 zyme preparation as 0% enzymatic inac­ min at 100,000 x g (50Ti rotor, tivation. Beckman L5-65 ultracentrifuge). The supernatants were dialyzed against High-performance liquid chro- phosphate-buffered saline, pH 7.2. The matography retentate was used as enzyme prepara­ The chromatography system con­ tion. sisted of a Waters M 6000A pump, a Rheodyne 7125 injector and a Pye- Antimicrobial agents Unicam LC-UV detector. The reversed­ Benzylpenicillin (sodium salt) was phase (C18) column used for all HPLC from Gist-Brocades NV, Delft, The assays was a Nucleosil 10 C-18 column Netherlands. Cefotaxime (sodium salt) (250 x 4.6 mm) from Nacherey-Nagel, was from Roussel B.V., Hoevelaken, Düren, Germany) equipped with a The Netherlands. Aztreonam (SQ guard column containing the same ma­ 26,776) and its inactive open ring form terial. (SQ 26,992) were a gift from the Elution conditions were as follows: Squibb Institute for Medical Research, the column was eluted at a flow-rate of Princeton, NJ, USA. Carumonam (Ro 1.5 ml/min for benzylpenicillin with 15 17-2301/010) was a gift from mM sodium phosphate pH 7.2 and Hoffmann-La Roche BV, Mijdrecht, methanol in a ratio of 70 : 30 (v/v). The The Netherlands. absorbance was monitored at 214 nm;

48 Figure 1: Percentage inactivation of cefotaxime (O), benzylpenicillin (V), aztreonam (P), carumonam (Ì) after 20 h incubation at 37°C by faecal enzyme preparations from l2 healthy human volunteers at month 0, 6, 14, 24. cefotaxime with 7 mM phosphoric acid phate (adjusted to pH 3.0 with 1 M and methanol in a ratio 60 : 40 (v/v). K2HPO4) and methanol in a ratio of 70 The absorbance was monitored at 254 : 30 (v/v). The absorbance was moni­ nm; aztreonam and carumonam with 5 tored at 293 nm. mM tetrabutylammoniumhydrogensul­

RESULTS Inactivation of benzylpenicillin, cefo­ value 30%), aztreonam for 28% taxime, aztreonam and carumonam was (median value 19%) and carumonam for investigated with an HPLC-assay. The 13% (median value 11%). In Figure 1 principle of this assay is that the antibi­ the average values of inactivation at otic is incubated with a faecal enzyme month 0, 6, 14 and 24 are shown. This preparation. In addition appropriate figure also shows that in this group of control samples are similarly treated and volunteers the extent of inactivation of a the disappearance of the antibiotic can particular antibiotic is relatively be monitored by reversed-phase HPLC. constant. This may be entirely different In order to obtain information on the within an individual (see Figure 2 as an variability of the inactivation of the anti­ example). Cefotaxime inactivation biotics by faecal enzyme preparations, it remains at a high relatively constant was studied over a period of 24 months level. Carumonam is hardly inactivated, at month 0, 6, 14 and 24. The average but differences in inactivation from 37% percentage inactivation after 20 h of in­ (month 0) to 4% (month 14) may occur. cubation at 37°C determined with faecal The variability in inactivation of enzyme preparations from l2 volunteers, benzylpenicillin and aztreonam is larger. ranged from 97 to 13%. Cefotaxime The faecal enzyme preparation of this was inactivated for 97% (median value volunteer inactivated benzylpenicillin for 98%), benzylpenicillin for 44% (median 100% at month 0 and 14, but

49 Figure 2: Percentage inactivation of cefotaxime (O), benzylpenicillin (V), aztreonam (P), carumonam (Ì) after 20 h incubation at 37°C by faecal enzyme preparations from one volunteer at month 0, 6, 14, 24. for only 5% at month 6 and at month Groen, 1989). Figure 3 shows the inac­ 24, 42% inactivation was measured. tivation of aztreonam by the faecal en­ Aztreonam inactivation ranged from zyme preparations of one volunteer. For 84% at month 6 to 15% at month 24. example at day 9 we found hardly any Similar variability in inactivation was inactivation (7%) and then a rapid in­ observed with the other faecal enzyme crease in inactivation to 69% at day 12. preparations except for those which The inactivation of aztreonam by faecal hardly inactivated a particular antibiotic enzymes from 2 other volunteers at all. This prompted us to investigate showed fluctuations from 0 to 15% (day this inactivation also during a shorter 1 and 3, respectively) and 43 to 22% period of time. Faecal enzyme prepara­ (day 7 and 9, respectively). The results tions from 3 volunteers were used to of the long and the short study period determine the inactivation of aztreonam show that inactivation cannot be pre­ over a period of 14 days (Welling and dicted.

DISCUSSION Our first study on the effect of faecal Gram-negative bacilli persisted, aztreo­ enzymes on antibiotics was initiated by nam was not detectable. Ehret et al. the results of de Vries-Hospers et al. (1987), who conducted a similar study (1984). Aztreonam was orally adminis­ with 8 volunteers, found 64 to 876 mg tered to 10 volunteers in order to elimi­ of aztreonam/kg in the faeces of 6 vol­ nate selectively the potentially patho­ unteers, while the remaining two had genic Gram-negative bacteria. During low levels or no aztreonam at all in the this selective decontamination with az­ faeces after oral administration of 300 treonam faecal counts of Gram-negative mg/day. These results indicated that az­ bacilli decreased in most volunteers. In treonam can be inactivated by faecal the faeces of two volunteers in whom material. We showed that this inactiva­

50 Figure 3: Percentage inactivation of aztreonam (P) after 20 h incubation at 37°C by faecal enzyme preparations from one volunteer over 14 days. tion is most probably due to ß-lactamase all faecal enzyme preparations. This activity and that this enzyme activity could be due to ß-lactamases produced could be inhibited by clavulanic acid by the anaerobic component of the bac­ (Welling et al., 1987) . terial flora. The 4 antibiotics were inac­ Although Swabb et al. (1983) regard tivated to a different extent but when the degradation unlikely, others (Livermore inactivation percentages of the 12 volun­ and Williams, 1981; Phillips et al., teers are averaged, at a fairly constant 1981) have also reported on inactivation level at 0, 6, 14 and 24 months (see of aztreonam by ß-lactamases. In a next Figure 1). This suggests a stability of study (Welling and Groen, 1989) indi­ the bacterial flora which is only mean­ vidual differences in inactivation of az­ ingful when this group of volunteers is treonam were monitored over a longer considered as a population. Examination period of time (24 months, at month 0, of the inactivation of each antibiotic by 6 and 24) and during 14 days. Consid­ individual enzyme preparations at month erable inter- and intra-individual differ­ 0, 6, 14 and 24 provides an entirely dif­ ences were found. The conclusion from ferent picture (see Figure 2). For ex­ that study was that aztreonam inactiva­ ample, benzylpenicillin may be inacti­ tion cannot be predicted and that it may vated for 100% at one particular sam­ be worthwhile to determine prior and pling time (month 0) but not at all at during selective decontamination with another sampling time (month 6). The aztreonam the extent of inactivation. same is true for shorter intervals (Figure This type of inactivation by faecal ß­ 3). At one particular day aztreonam was lactamases will most likely not be lim­ hardly inactivated (day 9) and a few ited to one ß-lactam antibiotic but prob­ days later (day 12) it was inactivated for ably is a widespread phenomenon. This 69% by a faecal enzyme preparation of prompted us to study the effect of faecal the same subject. enzyme preparations on a number of Examination of the individual inacti­ other ß-lactam antibiotics. Surprisingly, vation data also reveals that when caru­ the third generation cephalosporin cefo­ monam is inactivated for more than 20% taxime was most rapidly inactivated by by a particular faecal enzyme prepara­

51 tion, penicillin is also inactivated (for while the metabolic activity measured by 20% or more) by the same enzyme determination of bacterial enzymatic ac­ preparation from 8 out of 8 volunteers tivity may show marked changes. We and aztreonam (for 20% or more) by have found that the enzymatic activity those from 7 out of 8 volunteers. This may be considerably different inter- and type of inactivation could be due to dif­ intra-individually and that it may change ferent enzymes or to one enzyme with with time. This could be due to dietary different substrate affinities since the modulations of the composition of the three antibiotics generally were not in­ bacterial flora. This variable bacterial activated to the same degree. Although population in the colon may produce a this pattern of inactivation was most number of different ß-lactamases or, frequently found, we also observed depending on the composition of the 84% inactivation of aztreonam while flora, variations in the concentration of benzylpenicillin was hardly inactivated one type of enzyme. (5%). The practical consequences of these Several authors have reported on findings are different for different an­ penicillin and cephalosporin resistance tibiotics. When the antibiotic is intended in Bacteroides sp. (Anderson and for selective decontamination of the in­ Sykes, 1973; Britz and Wilkinson, testinal tract (aztreonam and caru­ 1978; Del Bene and Farrar, 1973; monam) it may be worthwhile to know Garrod, 1955; Olsson et al., 1976; to which extent it will presumably be in­ Pinkus et al., 1968; Richmond and activated by the faecal enzymes prior Sykes, 1973). Quantitatively, the and during selective decontamination. Bacteroides fragilis-group is predomi­ When the antibiotic has a broad spec­ nant in the human faecal flora (Meijer- trum and is not intended for selective Severs and van Santen, 1986). They decontamination (benzylpenicillin, cefo­ may represent therefore together with taxime) a fraction of the antibiotic may other anaerobic species in the intestine, reach the lower intestinal tract through a large potential source of antibiotic-in­ the biliary canal after parenteral adminis­ activating enzymes. Meijer-Severs and tration (benzylpenicillin, cefotaxime). It van Santen (1986) found considerable is epidemiologically important when the interindividual differences in Bac­ antibiotic is then inactivated by bacterial teroides cultural counts (between 8.83 enzymes. Then disturbance of the bac­ and 10.24) although the total anaerobic terial flora is prevented and therewith a cultural counts showed only one log dif­ barrier is maintained against coloniza­ ference. According to Simon and tion (colonization resistance, van der Gorbach (1984), the composition of the Waaij, 1982) by potentially pathogenic bacterial flora is relatively stable in sin­ microorganisms from the environment. gle subjects over longer periods of time,

LITERATURE

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52 263 (1989). Koudo, M., and Ochiai, M.: Synthesis of Chain, E.H., Florey, H.W., Gardner, A.D., sulfazecin-type 2-azetidinones with a carbon Heatly, N.G., Jennings, M.A., Orr-Ewing, substituent at the 4-position. J. Antibiot. J., and Sanders, A.G.: Penicillin as 36, 1421-1424 (1983). chemotherapeutic agent. Lancet ii, 226-228 Livermoore, D.M., and Williams, J.D.: In (1940). vitro activity of the monobactam, SQ Del Bene, V.E., and Farrar Jr., W.E.: 26,776, against Gram-negative bacteria and Cephalosporinase activity in Bacteroides its stability to their ß-lactamases, J. fragilis. Antimicrob. Agents Chemother. Antimicrob. Chemother. 8, Suppl. E, 29­ 14, 414-419 (1973) . 37 (1981). de Vries-Hospers, H.G., Welling, G.W., Meijer-Severs, G.J. and van Santen, E.: Swabb, E.A., and van der Waaij, D.: Variations in the anaerobic faecal flora of Selective decontamination of the digestive ten healthy human volunteers with special tract with aztreonam: a study of 10 healthy reference to the Bacteroides fragilis group volunteers. J. Inf. Dis. 150, 636-642 and Clostridium difficile, Zbl. Bakt. Hyg. A (1984). 261, 43-52 (1986). Drasar, F.A., Farrel, W., Howard, A.J., Hince, Neu, H.C., Aswapokee, N., Awapokee, P., and C., Leung, T., and Williams, J.D.: Activity Fu, K.P.: HR-756, a new cephalosporin ac­ of HR756 against Haemophilus influenza, tive against Gram-positive and Gram-nega­ Bacteroides fragilis and Gram-negative rods. tive aerobic and anaerobic bacteria. J. Antimicrob. Chemother. 4, 445-450 Antimicrob. Agents Chemother. 15, 273­ (1978). 281 (1979). Ehret, W., Probst, H., and Ruckdeschel, G.: Olsson, B., Nord, C.E., and Wadström, T.: Determination of aztreonam in faeces of Formation of ß-lactamase in Bacteroides human volunteers: a comparison of re­ fragilis: cell-bound and extracellular activ­ versed-phase high pressure liquid chro­ ity, Antimicrob. Agents Chemother. 9, matography and bioassay. J. Antimicrob. 727-735 (1976). Chemother. 19, 541-549 (1987). Phillips, I., King, A., Shannon, K., and Fu, K.P., and Neu, H.C.: ß-lactamase stability Warren, C.: SQ 26,776: in vitro antibacte­ of HR756, a novel cephalosporin, compared rial activity and susceptibility to ß-lacta­ to that of cefuroxime and cefoxitin. mases. J. Antimicrob. Chemother. 8, Antimicrob. Agents Chemother. 14, 322­ Suppl. E, 103-110 (1981). 326 (1978). Pinkus, G., Veto, G., and Braude, A.I.: Garrod, P.: Sensitivity of four species of Bacteroides penicillinase. J. Bact. 96, 1437­ Bacteroides to antibiotics. Br. Med. J. 2, 1438 (1968). 1529-1531 (1955). Richmond, M.H., and Sykes, R.B.: The ß-lac­ Heymes, R., Lutz, A., and Schrinner, E.: tamases of Gram-negative bacteria and their Experimental evaluation of HR756, a new possible physiological role. Adv. Microb. cephalosporin derivative: pre-clinical study. Physiol. 9, 31-38 (1973). Infection 5, 259-260 (1977). Simon, G.L., and Gorbach, S.L.: Intestinal Holdeman, L.V., Good, I.J., and Moore, flora in health and disease. Gastroenterology W.E.C.: Human faecal flora: variation in 86, 174-193 (1984). bacterial composition within individuals and Swabb, E.A., Sugerman, A.A., and Stern, M.: a possible effect of emotional stress. Appl. Oral bioavailability of the monobactam Environm. Microbiol. 31, 359-375 (1976). aztreonam (SQ 26,776) in healthy subjects. Imada, A., Kondo, M., Okonogi, K., Antimicrob. Agents Chemother. 23, 548­ Yukishige, K., and Kuno, M.: In vitro and 550 (1983). in vivo antibacterial activities of caru­ Sykes, R.B., Bonner, D.P., Bush, K., and monam (AMA-l080), a new N-sulfonated Georgopapadakou, N.H.. Aztreonam (SQ monocyclic ß-lactam antibiotic. 26,776), a synthetic monobactam specifi­ Antimicrob. Agents Chemother. 27, 821­ cally active against aerobic Gram-negative 827 (1985). bacteria. Antimicrob. Agents Chemother. Kishimoto, S., Sendai, M., Hashiguchi, S., 29, 85-92 (1982) . Tomomoto, M., Satoh, Y., Matsuo, T., van der Waaij, D.: Colonization resistance of

53 the digestive tract; clinical consequences and aztreonam by faecal supernatants of healthy implications. J. Antimicrob. Chemother. volunteers as determined by HPLC. J. 10, 263-270 (1982). Antimicrob. Chemother. 24, 805-809 Welling, G.W., Groen, G., Welling-Wester, (1989). S., de Vries-Hospers, H.G., and van der Yotsuji, A.. Minami, S., Inoue, M., and Waaij, D.: Enzymatic inactivation of aztre­ Mitsuhashi, S.: Properties of novel ß-lac­ onam by faecal enzyme preparations from tamase produced by Bacteroides fragilis. healthy volunteers. Infection 15, 188-191 Antimicrob. Agents Chemother. 24, 925­ (1987). 929 (1983). Welling, G.W. and Groen, G.: Inactivation of

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