259
DIAGN MICROBIOLINFECTDIS 1991;14:259-264
In vitro Antibacterial Activity of Trospectomycin (U-63,366F) Against Anaerobic Bacteria and Aerobic GramPositive Cocci in Chile F. Montiel, G. Kaltwasser, M.E. Pinto, and M. Lam
The in vitro activity of trospectomycin sulfate was compared with those of several antimicrobials, against 301 anaerobic bacteria and 613 aerobic Gram-positive cocci. Trospectomycin was about 4- to 32-fold more active than was spectinomycin. Trospectomycin exhibited consistently good activity against all Bacteroides fragilis group isolates, except Bacteroides vul-
gatus, and against all other anaerobes comparable or higher to that of clindamycin. The trospectomycin"s activity was most similar to that of vancomycin, even against methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus epidermidis.
INTRODUCTION
al., 1986; Thomas and Fritzem, 1985). In a preliminary report (Jacobus and Tally, 1988) it has been demonstrated as having excellent activity against the Bacteroides fragilis group, some other Bacteroides spp., and Bacteroides melaninogenicus. To evaluate the potential of the drug, we studied the in vitro activity of trospectomycin against 301 strains of anaerobes and 613 strains of Gram-positive cocci, and compared its activity with that of other antimicrobial agents.
Trospectomycin (U-63366; 6'-n-propyl spectinomycin pentahydrate sulfate) is a new parenteral aminocyclitol antibiotic similar in structure to spectinomycin (White et al., 1983). Spectinomycin and trospectomycin act by binding to the 30-S ribosome subunit and inhibits protein synthesis (Laborde and Mourey, 1987). The in vitro activity of trospectomycin has been reported to be superior to that of spectinomycin, with good activity against Mycoplasma and Ureaplasma species (Peeters et al., 1984; Yancey and Klein, 1988; Zurenko et al., 1988). It has excellent activity against many Gram-positive (Rolston et al., 1988) and Gram-negative bacteria such as Neisseria gonorrhoeae and Haemophilus ducreyi (Barry et al., 1989; Peeters et al., 1984; Sanson-Le Pors et From the Laboratoriode MicrobiologiaCllnica, UDA Laboratorios Clinicos, Centro de Diagn6stico. Escuelade Medicina PontificiaUniversidad Cat61icade Chile (F.M., G.K., M.L); and Laboratoriode Bacteriologia,(M.E.P.), Hospital San Juan de Dios, Santiago, Chile. Address reprint requests to Dr. F. Montiel, Laboratoriode Microbiologia Clinica, Escuela de Medicina, PontificiaUniversidad Cat61ica de Chile, PO Box 114-D, Santiago, Chile. Received 10 May, revised and accepted 24 July 1990. © 1991 Elsevier Science PublishingCo., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/91/$3.50
MATERIALS A N D METHODS Organisms Gram-negative and Gram-positive anaerobic bacteria used in this study were isolated from clinical samples of hospitalized patients at the Clinical Hospital of the Catholic University of Chile, and at the Hospital San Juan de Dios. Included were 126 B. fragilis group isolates (12 B. distasonis, 88 B. fragilis, 8 B. thetaiotaomicron, and 18 B. vulgatus), 8 Bacteroides spp., 12 B. melaninogenicus, 9 Fusobacterium spp., 92 Peptostreptococcus spp., 30 Clostridium perfringens, 6 Clostridium spp., 12 Gram-positive non-sporulating anaerobic bacilli~ and 6 Propionibacterium acnes. Grampositive cocci used in this study were also isolated
260
F. Montiel et al.
from clinical samples of hospitalized patients at the Clinical Hospital of the Catholic University of Chile. Included were 299 Staphylococcus aureus, 150 S. epidermidis, 22 Streptococcus spp. group A, 44 Streptococcus spp. group B, and 98 Enterococcus faecalis.
Antibiotics Laboratory-grade standard antimicrobials were provided as follows: clindamycin, spectinomycin, and trospectomycin by the Upjohn Company (Kalamazoo, MI); metronidazole, chloramphenicol, ampicillin and gentamicin by Laboratorio Chile in Chile. All the antibiotics were weighed, dissolved in appropriate solvents and produced a stock solution, which was freshly prepared every week as recommended by standard laboratory procedures (Anhalt and Washington, 1985; McGowan, 1988).
Susceptibility Testing Mean inhibitory concentrations (MICs) for all isolates were determined by an agar dilution method (NCCLS, 1985a and b; Washington, 1985), using Wilkins-Chalgren agar (Oxoid Limited, Basingstoke, Hampshire, England, UK) for anaerobes and Mueller-Hinton for Gram-positive cocci. The inoculum was prepared in thioglycolate broth (BD Microbiology Systems, Cockeysville, MD) for anaerobes, with five or more colonies of the original culture, incubated for a period of 6-24 hr or until visible turbidity was observed, at 36°C. The cultures thus prepared were adjusted to give a 0.5 MacFarland opacity. For Gram-positive aerobic cocci, the suspension adjusted to 0.5 MacFarland turbidity was then diluted 1:10 in sterile broth to obtain the desired inculum concentration.
The inoculum was transferred over the dry surface of the plates containing the antibiotics with a I-ram-thick prong of a 37-multipoint inoculator (Cathra International, St. Paul, MN). The inoculated plates were incubated at 36°C in an aerobic or in anaerobic atmosphere created with disposable hydrogen/carbon dioxide generators and palladium-coated catalyst pellets. MICs were determined by visual inspection after 24 hr for aerobes and after 48 hr for anaerobes. MICs were defined as the last concentration yielding no growth. The various antibiotics were incorporated into the agar. Antibiotics concentrations were as follows: trospectomycin, 0.625-20 ~g/ml; spectinomycin, 20-320 ~g/ml, chloramphenicol, 0.5-32 ~g/ml; metronidazole, clindamycin 0.25-16 ~g/ml; for anaerobes; and trospectomycin, 2.5-80 ~g/ml; spectinomycin, 5-320 p,g/ml; ampicillin, 0.5-32 p,g/ml; clindamycin, 0.25-16 ~g/ml; and gentamicin, 0.5-16 ~,g/ml; for Gram-positive cocci. Quality control of the MICs was accomplished using B. fragilis ATCC 28285, B. thetaiotaomicron ATCC 29741, C. perfringens ATCC 13124, S. aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and Escherichia coli ATCC 25922 according to the conditions established (Jones et al., 1987; NCCLS, 1985a and b).
RESULTS AND DISCUSSION The in vitro antibacterial activities of trospectomycin, spectinomycin, and reference antibiotics are summarized in Table 1. Comparison of the MICs for 90% of the strains tested shows that trospectomycin was more active than spectinomycin against all aerobic and anaerobic species.
TABLE 1 Comparative In Vitro Activities of Trospectomycin and Reference Antibiotics Tested Against 914 Bacterial Isolates MIC (~g/ml) a Microorganism Gram-positive aerobic cocci Staphylococcus aureus
Staphylococcus epidermidis
No. of Isolates
Antibiotic
Range
50%
90%
299b Trospectomycin Spectinomycin Clindamycin Ampicillin Gentamicin
42.5-40 20-30 40.25->16 40.5->32 40.5->16
10 20 80 > 160 40.25 0.5 4 >32 40.5 >16
15(F Trospectomycin Spectinomycin Clindamycin Ampicillin Gentamicin
42.5-40 45->320 40.25->16 40.5->32 40.5->16
5 10 80 80 40.25 >16 4 >32 2 >16
261
T r o s p e c t o m y c i n in vitro Activity
TABLE 1
Continued MIC (l~g/ml)"
Microorganism
No. of Isolates
Antibiotic
Range
50%
90%
Streptococcus spp. group A
22
Trospectomycin Spectinomycin Clindamycin Ampicillin Gentamicin
42.5-5 45--40 ~0.25-0.5 40.5-4 ~16
~2.5 20 40.25 ~0.5 2
42.5 40 0.5 4 16
Enterococcus faecalis
98
Trospectomycin Spectinomycin Clindamycin Ampicillin Gentamicin
~0.25->80 10->320 ~0.25->16 ~0.5-->32 40.5->16
5 80 16 2 16
10 80 >16 4 >16
Anaerobes
Bacteroides fragilis group
126
Trospectomyan 40.625->20 5 20 Spectinomycln 420->320 160 >320 Clindamycin ~0.25->16 0.5 2 Chloramphenicol 40.5->32 2 8 Metronidazole ~0.25->16 40.25 1
Bacteroides distasonis
12
Trospectomycm 40.625-20 2.5 Spectinomycln 420->320 320 Clindamycin ~0.25--4 1 Chloramphenicol 1-4 2 Metronidazole ~0.25->16 0.5
Bacteroides fragilis group
88
Trospectomycm 40.625->20 5 10 Spectinomycin 420->320 320 >320 Clindamycm 16 32 2 8 Metronidazole ~16 40.25 0.5
Bacteroides thetaiotaomicron
Bacteroides vulgatus
Bacteroides spp.
Bacteroides melaninogenicus
8
18
8
12
Trospectomycm Spectinomycin Clindamycm Chloramphenicol Metronidazole
2.5-10 2.5 160->320 320 0.5-1 1 2-8 2 40.25-0.5 ~0.25
10 >320 1 4 1
a ----
Trospectomycm ~0.625->20 5 >20 Spectinomycin 420->320 160 >320 Clindamycin 40.25->16 0.5 >16 Chloramphenicol 40.5-8 2 4 Metronidazole 40.25->16 40.25 >16 Trospectomycm Spectinomycin Clindamycin Chloramphenicol Metronidazole
4 0 . 6 2 5 - > 2 0
2.5
420->320 160 40.25->16 ~0.25 40.5-16 2 40.25->16 ~0.25
Trospectomycm ~0.625-20 Spectinomycm 40->320 Clindamycin ~0.25-2 Chloramphenicol 40.5-16 Metronidazole 40.25->16
--
----
2.5 20 80 >320 40.25 2 2 4 0.5 >16
262
F. Montiel et al.
TABLE 1
Continued MIC (~g/ml) a
Microorganism
No. of Isolates
Fusobacterium spp.
Antibiotic
Range
50%
90%
Trospectomycm 40.625-10 Spectinomycm 420-320 Clindamycin 40.25->16 Chloramphenicol 40.5-32 Metronidazole 40.25->16
5 160 0.5 2 40.25
------
5 20 80 >320 40.25 8 2 4 0.5 >16
Peptostreptococcus spp.
92
Trospectomycm 40.625->20 Spectinomycm 420->320 Clindamycln 40.25->16 Chloramphenicol 40.5->32 Metronidazole 40.25->16
Clostridium perfringens
30
Trospectomycm 40.625->20 10 20 Spectinomycm 420->320 320 >320 Clindamycin 40.25~ 0.5 2 Chloramphenicol 1->32 2 4 Metronidazole ~16 0.5 1
Clostridium spp.
Gram-positive nonsporulating anaerobic bacilli
Propionibacterium aches
Trospectomycm 40.625-10 Spectinomycm 420--160 40.25-2 Clindamycm Chloramphenicol 40.5-4 40.25-1 Metronidazole 12
5 160 40.25 2 40.25
------
Trospectomycm 40.625-20 5 20 Spectinomycm 420->320 160 >320 Clindamycln 40.25->16 40.25 8 40.5-2 1 2 Chloramphenicol Metronidazole 40.25->16 40.25 >16 Trospectomycm 40.625-2.5 Spectinomycm 420->320 Clindamycm 40.25-40.25 40.5-1 Chloramphenicol 40.25->16 Metronidazole
1.25 160 40.25 40.5 >16
----
a50%, 90% MIC for 50% and 90% of isolates tested. blncludes 54 methicillin-resistant strains. qncludes 41 methicfllin-resistant strains. d__, Not calculated; 20 ~g/ml). The activity of trospectomycin for the Grampositive anaerobes was good, a n d the MICgo was 20 ~g/ml for C. perfringens, Peptostreptococcus spp., and for the Gram-positive n o n s p o r u l a t i n g anaerobic bacilli. O u r results s h o w e d that clindamycin s h o w e d a good activity against all the isolates; with an MIC9o of 2 ~g/ml for Bacteroides fragilis g r o u p isolates, comparable to previous multicenter studies (Rosenblatt, 1984; Tally et al., 1985). An analysis of susceptibilities by species revealed that clindamycin h a d excellent activity against B. distasonis and B. fragilis (MIC90,
Trospectomycin in vitro Activity
1-2 ~g/ml, respectively) and poor activity against B. vulgatus (MICg0, > 16 ~g/ml). The activity against B. melaninogenicus and Clostridium perfringens was excellent with an MIC9o of 2 ~g/ml. The MICgo for Peptostreptococcus spp., and gram-positive nonsporulating anaerobic bacilli was of 8 p,g/ml. Our results also showed that chloramphenicol presented a good activity against all the isolates; with a MIC9o of 8 ~,g/ml for Bacteroides fragilis group isolates. Metronidazole presented an excellent activity against the B. fragilis group (MIC9o, 1 ~g/ml) comparable to previous multicenter studies (Rosenblatt, 1984; Tally et al., 1985). Trospectomycin has better activity than spectinomycin for every strain of aerobic Gram-positive cocci tested. The MICg0 for trospectomycin was 20 p.g/ml for S. aureus, 10 p,g/ml for S. epidermidis and Enterococcus faecalis, and ~2.5 ~g/ml for Streptococcus spp. groups A and B. Zurenko et al. (1988) have reported similar results. Trospectomycin was equally active as ampicillin against the enterococci (MIC9o, 10 }zg/ml). When we set aside the methicillin-resistant (MR) Staphylococcus strains, we found that the trospectomycin's MIC9o for S. aureus and S. epidermidis was 20 ~g/ml. All the staphylococci tested were susceptible to ~2 p~g/ml of vancomycin and the MR Staphylococcus aureus, and MR Staphylococcus epidermidis were resistant to >4 p,g/ml of oxacillin. In summary, our results indicate excellent in vitro activity of trospectomycin against B. fragilis group (except Bacteroides vulgatus ) and Peptostreptococcus spp., Clostridium perfringens, and Gram-positive nonspo-
263
rulating anaerobic bacilli isolates. At the same time, these results enable us to conclude that the majority of the staphylococcal strains, even the methicillin resistant, will be inhibited by trospectomycin. Trospectomycin then appears to be a potentially useful antistaphylococcal drug, because 13-1actamase production by staphylococci did not influence the activity of aminocyclitols. The MICs9o for these staphylococci fall in concentrations capable of being reached in serum. The results of preliminary pharmacokinetic studies in humans (Novak et al., 1987) following a single 20-min infusion of a 1000-mg dose showed that the average peak concentration in serum was 81.2 p~g/ml and that the half-life in serum was 2.18 hr. We can conclude then that trospectomycin looks promising for anaerobes and aerobic Gram-positive cocci, compared to other reference antibiotics. These data, together with the data obtained from other investigators for Neisseria (Barry et al., 1989), Mycoplasma and Ureaplasma (Klein et al., 1988; Yancey and Klein, 1988), and Chlamydia (Gordon and Bross, 1988), make trospectomycin a potential antibiotic for use in pelvic inflamatory disease. Its activity, better tissue penetration, and very long tissue half-life of - 3 days (Burrows et al., 1987) gives trospectomycin the best characteristics for this application.
We thank the Upjohn Co. Kalamazoo, Michigan, and the following people for manuscript suggestions and technical support: Dr. D. Batts, Dr. D. Griffith, Dr. D. Zambrano, and G. Zurenko.
REFERENCES Anhalt J, Washington II JA (1985) Preparation and storage of antimicrobial solutions. In Manual of Clinical Microbiology. Eds, EH Lennette, A Balows, JW Hausler, and HJ Shadomy Jr. Washington DC: American Society for Microbiology, pp 1019-1020. Barry AL, Jones RN, Thornsberry C (1989) Antibacterial activity of trospectomycin (U-63366F) and initial eva[uations of disk diffusion susceptibility tests. Antimicrob Agents Chemother 33:569-572. Burrows M, Osgood TA, Dring LG (1987) Tissue distribution of 3H-trospectomycin in rats dosed intravenously [abstr 273]. In 27th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), New York. Gordon G, Bross JE (1988) Trospectomycin activity against anaerobic gram negative bacilli [abstr 538]. In 28th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), Los Angeles. Klein LK, Case CA, Yancey RJ Jr (1988) In vitro activity of trospectomycin sulfate against ureaplasmas of human and veterinary importance [abstr 537]. In 28th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), Los Angeles.
Jacobus NV, Tally FP (1988) Activity of trospectomycin against Bacteroides fragilis and other Bacteroides species. Antimicrobial Agents Chemother 32:584-586. Jones RN, Barry AL, Fuchs PC, Allen SD (1987) Ceftizoxime and cefoxitin susceptibility testing against anaerobic bacteria: comparison of results from three NCCLS methods and quality control recommendations for the reference agar dilution procedure. Diagn Microbiol Infect Dis 8:87-94. Laborde AL, Mourey RJ (1987) Mechanism of action of trospectomycin, a novel spectinomycin analog [abstr 266]. In 27th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), New York. McGowan JE Jr (1988) Current concepts and approaches to antimicrobial agent susceptibility testing. In CUMITECH 25. Washington DC: American Society for Microbiology. National Committee for Clinical Laboratory Standards (NCCLS) (1985a) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 2nd ed. Approved standard M7-A. Villanova PA: NCCLS.
264
National Committee for Clinical Laboratory Standards (NCCLS) (1985b) Reference agar dilution procedure for antimicrobial susceptibility testing of anaerobic bacteria. Approved standard Mll-A. Villanova PA: NCCLS. Novak E, Zurenko GE, Paxton LM, Francom SF, Oliver LK (1987) Safety and preliminary pharmacokinetics of intravenously administered trospectomycin sulfate (U63,366F) in man: a novel spectinomycin analogue [abstr 271]. In 27th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), New York. Peeters ME, Van Dyck E, Plot P (1984) In vitro activities of the spectinomycin analog U63,366 and four quinolone derivatives against Neisseriagonorrhoeae. Antimicrob Agents Chemother 26:608-609. Rolston K, Ho DH, Bodey GP (1988) Gram-positive activity of trospectomycin [abstr 536]. In 28th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), Los Angeles. Rosenblatt JE (1984) Antimicrobial susceptibility testing of anaerobic bacteria. Rev Infect Dis 6S:242-248. Sanson-Le Pors MJ, Casin IM, Thebault MC, Arlet G, Perol Y (1986) In vitro activities of U-63366, a spectinomycin analog; roxithromycin (RU 28965), a new macrolide an-
F. Montiel et al.
tibiotic; and five quinolone derivatives against Haemophilus ducreyi. Antimicrob Agents Chemother 30:512-513. Tally FP, Cuchural GJ, Jacobus NV, et al. (1985) Nationwide study of the susceptibility of the Bacteroidesfragilis group in the United States. Antimicrob Agents Chemother 28:675-677. Thomas RC, Fritzem EL (1985) Spectinomycin modification. III. Spectinomycin analogs with C-3'-branched chain sugar. J Antibiot 28:208-219. Washington II JA (1985) Susceptibility tests: agar dilution. In Manual of Clinical Microbiology. Eds, EH Lennette, A Balows, JW Hausler Jr, and HJ Shadomy. Washington DC: American Society for Microbiology, pp 967-971. White DR, Maring CJ, Cain GA (1983) Synthesis and in vitro properties of alkylspectinomycin analogs. J Antibiot 36:339-342. Yancey RJ, Klein LK (1988) In vitro activity of trospectomycin sulfate against Mycoplasma and Ureaplasma species from humans. J Antimicrob Chemother 21:731-736. Zurenko GE, Yagi BH, Vavra JJ, Wentworth BB (1988) In vitro antibacterial activity of trospectomycin (U-63366F): a novel spectinomycin analog. Antimicrob Agents Chemother 32:216-223.
DIAGN MICROBIOLINFECTDIS 1991;14:259-264
In vitro Antibacterial Activity of Trospectomycin (U-63,366F) Against Anaerobic Bacteria and Aerobic GramPositive Cocci in Chile F. Montiel, G. Kaltwasser, M.E. Pinto, and M. Lam
The in vitro activity of trospectomycin sulfate was compared with those of several antimicrobials, against 301 anaerobic bacteria and 613 aerobic Gram-positive cocci. Trospectomycin was about 4- to 32-fold more active than was spectinomycin. Trospectomycin exhibited consistently good activity against all Bacteroides fragilis group isolates, except Bacteroides vul-
gatus, and against all other anaerobes comparable or higher to that of clindamycin. The trospectomycin"s activity was most similar to that of vancomycin, even against methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus epidermidis.
INTRODUCTION
al., 1986; Thomas and Fritzem, 1985). In a preliminary report (Jacobus and Tally, 1988) it has been demonstrated as having excellent activity against the Bacteroides fragilis group, some other Bacteroides spp., and Bacteroides melaninogenicus. To evaluate the potential of the drug, we studied the in vitro activity of trospectomycin against 301 strains of anaerobes and 613 strains of Gram-positive cocci, and compared its activity with that of other antimicrobial agents.
Trospectomycin (U-63366; 6'-n-propyl spectinomycin pentahydrate sulfate) is a new parenteral aminocyclitol antibiotic similar in structure to spectinomycin (White et al., 1983). Spectinomycin and trospectomycin act by binding to the 30-S ribosome subunit and inhibits protein synthesis (Laborde and Mourey, 1987). The in vitro activity of trospectomycin has been reported to be superior to that of spectinomycin, with good activity against Mycoplasma and Ureaplasma species (Peeters et al., 1984; Yancey and Klein, 1988; Zurenko et al., 1988). It has excellent activity against many Gram-positive (Rolston et al., 1988) and Gram-negative bacteria such as Neisseria gonorrhoeae and Haemophilus ducreyi (Barry et al., 1989; Peeters et al., 1984; Sanson-Le Pors et From the Laboratoriode MicrobiologiaCllnica, UDA Laboratorios Clinicos, Centro de Diagn6stico. Escuelade Medicina PontificiaUniversidad Cat61icade Chile (F.M., G.K., M.L); and Laboratoriode Bacteriologia,(M.E.P.), Hospital San Juan de Dios, Santiago, Chile. Address reprint requests to Dr. F. Montiel, Laboratoriode Microbiologia Clinica, Escuela de Medicina, PontificiaUniversidad Cat61ica de Chile, PO Box 114-D, Santiago, Chile. Received 10 May, revised and accepted 24 July 1990. © 1991 Elsevier Science PublishingCo., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/91/$3.50
MATERIALS A N D METHODS Organisms Gram-negative and Gram-positive anaerobic bacteria used in this study were isolated from clinical samples of hospitalized patients at the Clinical Hospital of the Catholic University of Chile, and at the Hospital San Juan de Dios. Included were 126 B. fragilis group isolates (12 B. distasonis, 88 B. fragilis, 8 B. thetaiotaomicron, and 18 B. vulgatus), 8 Bacteroides spp., 12 B. melaninogenicus, 9 Fusobacterium spp., 92 Peptostreptococcus spp., 30 Clostridium perfringens, 6 Clostridium spp., 12 Gram-positive non-sporulating anaerobic bacilli~ and 6 Propionibacterium acnes. Grampositive cocci used in this study were also isolated
260
F. Montiel et al.
from clinical samples of hospitalized patients at the Clinical Hospital of the Catholic University of Chile. Included were 299 Staphylococcus aureus, 150 S. epidermidis, 22 Streptococcus spp. group A, 44 Streptococcus spp. group B, and 98 Enterococcus faecalis.
Antibiotics Laboratory-grade standard antimicrobials were provided as follows: clindamycin, spectinomycin, and trospectomycin by the Upjohn Company (Kalamazoo, MI); metronidazole, chloramphenicol, ampicillin and gentamicin by Laboratorio Chile in Chile. All the antibiotics were weighed, dissolved in appropriate solvents and produced a stock solution, which was freshly prepared every week as recommended by standard laboratory procedures (Anhalt and Washington, 1985; McGowan, 1988).
Susceptibility Testing Mean inhibitory concentrations (MICs) for all isolates were determined by an agar dilution method (NCCLS, 1985a and b; Washington, 1985), using Wilkins-Chalgren agar (Oxoid Limited, Basingstoke, Hampshire, England, UK) for anaerobes and Mueller-Hinton for Gram-positive cocci. The inoculum was prepared in thioglycolate broth (BD Microbiology Systems, Cockeysville, MD) for anaerobes, with five or more colonies of the original culture, incubated for a period of 6-24 hr or until visible turbidity was observed, at 36°C. The cultures thus prepared were adjusted to give a 0.5 MacFarland opacity. For Gram-positive aerobic cocci, the suspension adjusted to 0.5 MacFarland turbidity was then diluted 1:10 in sterile broth to obtain the desired inculum concentration.
The inoculum was transferred over the dry surface of the plates containing the antibiotics with a I-ram-thick prong of a 37-multipoint inoculator (Cathra International, St. Paul, MN). The inoculated plates were incubated at 36°C in an aerobic or in anaerobic atmosphere created with disposable hydrogen/carbon dioxide generators and palladium-coated catalyst pellets. MICs were determined by visual inspection after 24 hr for aerobes and after 48 hr for anaerobes. MICs were defined as the last concentration yielding no growth. The various antibiotics were incorporated into the agar. Antibiotics concentrations were as follows: trospectomycin, 0.625-20 ~g/ml; spectinomycin, 20-320 ~g/ml, chloramphenicol, 0.5-32 ~g/ml; metronidazole, clindamycin 0.25-16 ~g/ml; for anaerobes; and trospectomycin, 2.5-80 ~g/ml; spectinomycin, 5-320 p,g/ml; ampicillin, 0.5-32 p,g/ml; clindamycin, 0.25-16 ~g/ml; and gentamicin, 0.5-16 ~,g/ml; for Gram-positive cocci. Quality control of the MICs was accomplished using B. fragilis ATCC 28285, B. thetaiotaomicron ATCC 29741, C. perfringens ATCC 13124, S. aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and Escherichia coli ATCC 25922 according to the conditions established (Jones et al., 1987; NCCLS, 1985a and b).
RESULTS AND DISCUSSION The in vitro antibacterial activities of trospectomycin, spectinomycin, and reference antibiotics are summarized in Table 1. Comparison of the MICs for 90% of the strains tested shows that trospectomycin was more active than spectinomycin against all aerobic and anaerobic species.
TABLE 1 Comparative In Vitro Activities of Trospectomycin and Reference Antibiotics Tested Against 914 Bacterial Isolates MIC (~g/ml) a Microorganism Gram-positive aerobic cocci Staphylococcus aureus
Staphylococcus epidermidis
No. of Isolates
Antibiotic
Range
50%
90%
299b Trospectomycin Spectinomycin Clindamycin Ampicillin Gentamicin
42.5-40 20-30 40.25->16 40.5->32 40.5->16
10 20 80 > 160 40.25 0.5 4 >32 40.5 >16
15(F Trospectomycin Spectinomycin Clindamycin Ampicillin Gentamicin
42.5-40 45->320 40.25->16 40.5->32 40.5->16
5 10 80 80 40.25 >16 4 >32 2 >16
261
T r o s p e c t o m y c i n in vitro Activity
TABLE 1
Continued MIC (l~g/ml)"
Microorganism
No. of Isolates
Antibiotic
Range
50%
90%
Streptococcus spp. group A
22
Trospectomycin Spectinomycin Clindamycin Ampicillin Gentamicin
42.5-5 45--40 ~0.25-0.5 40.5-4 ~16
~2.5 20 40.25 ~0.5 2
42.5 40 0.5 4 16
Enterococcus faecalis
98
Trospectomycin Spectinomycin Clindamycin Ampicillin Gentamicin
~0.25->80 10->320 ~0.25->16 ~0.5-->32 40.5->16
5 80 16 2 16
10 80 >16 4 >16
Anaerobes
Bacteroides fragilis group
126
Trospectomyan 40.625->20 5 20 Spectinomycln 420->320 160 >320 Clindamycin ~0.25->16 0.5 2 Chloramphenicol 40.5->32 2 8 Metronidazole ~0.25->16 40.25 1
Bacteroides distasonis
12
Trospectomycm 40.625-20 2.5 Spectinomycln 420->320 320 Clindamycin ~0.25--4 1 Chloramphenicol 1-4 2 Metronidazole ~0.25->16 0.5
Bacteroides fragilis group
88
Trospectomycm 40.625->20 5 10 Spectinomycin 420->320 320 >320 Clindamycm 16 32 2 8 Metronidazole ~16 40.25 0.5
Bacteroides thetaiotaomicron
Bacteroides vulgatus
Bacteroides spp.
Bacteroides melaninogenicus
8
18
8
12
Trospectomycm Spectinomycin Clindamycm Chloramphenicol Metronidazole
2.5-10 2.5 160->320 320 0.5-1 1 2-8 2 40.25-0.5 ~0.25
10 >320 1 4 1
a ----
Trospectomycm ~0.625->20 5 >20 Spectinomycin 420->320 160 >320 Clindamycin 40.25->16 0.5 >16 Chloramphenicol 40.5-8 2 4 Metronidazole 40.25->16 40.25 >16 Trospectomycm Spectinomycin Clindamycin Chloramphenicol Metronidazole
4 0 . 6 2 5 - > 2 0
2.5
420->320 160 40.25->16 ~0.25 40.5-16 2 40.25->16 ~0.25
Trospectomycm ~0.625-20 Spectinomycm 40->320 Clindamycin ~0.25-2 Chloramphenicol 40.5-16 Metronidazole 40.25->16
--
----
2.5 20 80 >320 40.25 2 2 4 0.5 >16
262
F. Montiel et al.
TABLE 1
Continued MIC (~g/ml) a
Microorganism
No. of Isolates
Fusobacterium spp.
Antibiotic
Range
50%
90%
Trospectomycm 40.625-10 Spectinomycm 420-320 Clindamycin 40.25->16 Chloramphenicol 40.5-32 Metronidazole 40.25->16
5 160 0.5 2 40.25
------
5 20 80 >320 40.25 8 2 4 0.5 >16
Peptostreptococcus spp.
92
Trospectomycm 40.625->20 Spectinomycm 420->320 Clindamycln 40.25->16 Chloramphenicol 40.5->32 Metronidazole 40.25->16
Clostridium perfringens
30
Trospectomycm 40.625->20 10 20 Spectinomycm 420->320 320 >320 Clindamycin 40.25~ 0.5 2 Chloramphenicol 1->32 2 4 Metronidazole ~16 0.5 1
Clostridium spp.
Gram-positive nonsporulating anaerobic bacilli
Propionibacterium aches
Trospectomycm 40.625-10 Spectinomycm 420--160 40.25-2 Clindamycm Chloramphenicol 40.5-4 40.25-1 Metronidazole 12
5 160 40.25 2 40.25
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Trospectomycm 40.625-20 5 20 Spectinomycm 420->320 160 >320 Clindamycln 40.25->16 40.25 8 40.5-2 1 2 Chloramphenicol Metronidazole 40.25->16 40.25 >16 Trospectomycm 40.625-2.5 Spectinomycm 420->320 Clindamycm 40.25-40.25 40.5-1 Chloramphenicol 40.25->16 Metronidazole
1.25 160 40.25 40.5 >16
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a50%, 90% MIC for 50% and 90% of isolates tested. blncludes 54 methicillin-resistant strains. qncludes 41 methicfllin-resistant strains. d__, Not calculated; 20 ~g/ml). The activity of trospectomycin for the Grampositive anaerobes was good, a n d the MICgo was 20 ~g/ml for C. perfringens, Peptostreptococcus spp., and for the Gram-positive n o n s p o r u l a t i n g anaerobic bacilli. O u r results s h o w e d that clindamycin s h o w e d a good activity against all the isolates; with an MIC9o of 2 ~g/ml for Bacteroides fragilis g r o u p isolates, comparable to previous multicenter studies (Rosenblatt, 1984; Tally et al., 1985). An analysis of susceptibilities by species revealed that clindamycin h a d excellent activity against B. distasonis and B. fragilis (MIC90,
Trospectomycin in vitro Activity
1-2 ~g/ml, respectively) and poor activity against B. vulgatus (MICg0, > 16 ~g/ml). The activity against B. melaninogenicus and Clostridium perfringens was excellent with an MIC9o of 2 ~g/ml. The MICgo for Peptostreptococcus spp., and gram-positive nonsporulating anaerobic bacilli was of 8 p,g/ml. Our results also showed that chloramphenicol presented a good activity against all the isolates; with a MIC9o of 8 ~,g/ml for Bacteroides fragilis group isolates. Metronidazole presented an excellent activity against the B. fragilis group (MIC9o, 1 ~g/ml) comparable to previous multicenter studies (Rosenblatt, 1984; Tally et al., 1985). Trospectomycin has better activity than spectinomycin for every strain of aerobic Gram-positive cocci tested. The MICg0 for trospectomycin was 20 p.g/ml for S. aureus, 10 p,g/ml for S. epidermidis and Enterococcus faecalis, and ~2.5 ~g/ml for Streptococcus spp. groups A and B. Zurenko et al. (1988) have reported similar results. Trospectomycin was equally active as ampicillin against the enterococci (MIC9o, 10 }zg/ml). When we set aside the methicillin-resistant (MR) Staphylococcus strains, we found that the trospectomycin's MIC9o for S. aureus and S. epidermidis was 20 ~g/ml. All the staphylococci tested were susceptible to ~2 p~g/ml of vancomycin and the MR Staphylococcus aureus, and MR Staphylococcus epidermidis were resistant to >4 p,g/ml of oxacillin. In summary, our results indicate excellent in vitro activity of trospectomycin against B. fragilis group (except Bacteroides vulgatus ) and Peptostreptococcus spp., Clostridium perfringens, and Gram-positive nonspo-
263
rulating anaerobic bacilli isolates. At the same time, these results enable us to conclude that the majority of the staphylococcal strains, even the methicillin resistant, will be inhibited by trospectomycin. Trospectomycin then appears to be a potentially useful antistaphylococcal drug, because 13-1actamase production by staphylococci did not influence the activity of aminocyclitols. The MICs9o for these staphylococci fall in concentrations capable of being reached in serum. The results of preliminary pharmacokinetic studies in humans (Novak et al., 1987) following a single 20-min infusion of a 1000-mg dose showed that the average peak concentration in serum was 81.2 p~g/ml and that the half-life in serum was 2.18 hr. We can conclude then that trospectomycin looks promising for anaerobes and aerobic Gram-positive cocci, compared to other reference antibiotics. These data, together with the data obtained from other investigators for Neisseria (Barry et al., 1989), Mycoplasma and Ureaplasma (Klein et al., 1988; Yancey and Klein, 1988), and Chlamydia (Gordon and Bross, 1988), make trospectomycin a potential antibiotic for use in pelvic inflamatory disease. Its activity, better tissue penetration, and very long tissue half-life of - 3 days (Burrows et al., 1987) gives trospectomycin the best characteristics for this application.
We thank the Upjohn Co. Kalamazoo, Michigan, and the following people for manuscript suggestions and technical support: Dr. D. Batts, Dr. D. Griffith, Dr. D. Zambrano, and G. Zurenko.
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National Committee for Clinical Laboratory Standards (NCCLS) (1985b) Reference agar dilution procedure for antimicrobial susceptibility testing of anaerobic bacteria. Approved standard Mll-A. Villanova PA: NCCLS. Novak E, Zurenko GE, Paxton LM, Francom SF, Oliver LK (1987) Safety and preliminary pharmacokinetics of intravenously administered trospectomycin sulfate (U63,366F) in man: a novel spectinomycin analogue [abstr 271]. In 27th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), New York. Peeters ME, Van Dyck E, Plot P (1984) In vitro activities of the spectinomycin analog U63,366 and four quinolone derivatives against Neisseriagonorrhoeae. Antimicrob Agents Chemother 26:608-609. Rolston K, Ho DH, Bodey GP (1988) Gram-positive activity of trospectomycin [abstr 536]. In 28th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), Los Angeles. Rosenblatt JE (1984) Antimicrobial susceptibility testing of anaerobic bacteria. Rev Infect Dis 6S:242-248. Sanson-Le Pors MJ, Casin IM, Thebault MC, Arlet G, Perol Y (1986) In vitro activities of U-63366, a spectinomycin analog; roxithromycin (RU 28965), a new macrolide an-
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