Against Bacterial Enteropathogens

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, OCt. 93, p. 509-53 0666-404/3/00509-0S$0.00/0 Copyright C 93, American Society for Microbiology Vol. 4, No. 4 Comparative In Vitro Activities of Ten Antimicrobial Agents Against Bacterial Enteropathogens JAMES R. CARLSON,lt* SCOTT A. THORNTON, HERBERT L. DuPONT, A. HANNA WEST, AND JOHN J. MATHEWSON Department of Pathology and Laboratory Medicine' and Program in Infectious Diseases and Clinical Microbiology, the Medical School, University of Texas Health Science Center, Houston, Texas 7705 Received April 93/Accepted 0 July 93 The in vitro susceptibilities of 50 strains of Salmonella spp., 0 strains of Shigella spp., and 50 enterotoxigenic Escherichia coli, 4 Yersinia enterocolitica, 6 Aeromonas hydrophila, 4 Plesiomonas shigelloides, 9 Vibrio parahaemolyticus, and 30 Campylobacterjejuni strains that were recently isolated from worldwide sources were determined for 0 antimicrobial agents. The antimicrobial agents tested included ampicillin, bicozamycin, doxycycline, enoxacin (CI-99), erythromycin, furazolidone, amdinociilin, norfloxacin, trimethoprim, and trimethoprimsulfamethoxazole. resistance occurred frequently in strains of Salmonella and Shigella spp. and enterotoxigenic E. coli strains. The most active agents against all of the bacteria tested were enoxacin and norfloxacin. and amdinocillin were also highly active against the majority of strains. Trimethoprim and trimethoprim-sulfamethoxazole were inhibitory at low concentrations against all test strains except C. jejuni isolates. The in vitro results of this study confirm the high prevalence of bacterial resistance to ampicillin. However, this work also identifies four antimicrobial agents, enoxacin, furazolidone, norfloxacin, and amdinocilin, that would be appropriate for further testing in clinical trials. Acute diarrhea is a common cause of morbidity and mortality throughout the world. Generally, the most severe, as well as the most frequently occurring, forms of this disease in developing countries are of bacterial etiology (, 3). Antibiotics have been found to shorten the duration of some types of bacterial diarrhea; however, because of the emergence of antimicrobial agent resistance among enteric pathogens, selection of appropriate therapy is often difficult (5). A continual search is required for new agents to which prevalent bacterial enteropathogens are susceptible. Antimicrobial agents have been used to treat diarrhea caused by a variety of bacterial agents (3, -0,, 3). Additionally, antimicrobial agents with activity against prevalent enteropathogens have been successfully employed in preventing diarrhea among travelers to high-risk areas (6, 6). It would be of great benefit to define a single broad-spectrum antimicrobial agent of low toxicity that would be effective for both the prophylaxis and empirical treatment of acute diarrheal disease. The ideal preparation would have limited importance in the treatment of infectious t Present address: Department of Pathology, Clinical Microbiology Laboratories, University of California, Davis, Medical Center, Sacramento, California 957 diseases outside of the intestinal tract and would be taken orally, with high concentrations of the drug achieved intraluminally. The purpose of this study is to compare the in vitro activities of drugs previously or currently used in the prevention and treatment of bacterial diarrhea with those offour newly developed and unlicensed preparations: bicozamycin, an antimicrobial agent with a unique chemical structure that inhibits lipoprotein biosynthesis and assembly to peptidoglycan; amdinocillin, a penicillanic acid derivative; and the nalidixic acid analog, enoxacin (CI-99) and norfloxacin. MATERIALS AND METHODS Antibiotics. Test compounds of known potencies were provided as follows: ampicillin, Bristol Laboratories, Syracuse, N.Y.; bicozamycin, Ciba-Geigy, Ltd., Basel, Switzerland; enoxacin, Warner-Lambert/ Parke-Davis Pharmaceutical Research Division, Ann Arbor, Mich.; doxycycline, Pfizer, Inc., Brooklyn, N.Y.; erythromycin, The Upjohn Co., Kalamazoo, Mich.; furazolidone, Norwich-Eaton Pharmaceuticals, Inc., Norwich, N.Y.; amdinocillin, Hoffmann-La Roche Inc., Nutley, N.J.; norfloxacin, Merck Sharp & Dohme, Rahway, N.J.; and trimethoprim () and -sulfamethoxazole (SMX) Burroughs Wellcome Co. Research Triangle Park, N.C. Bacterial isolates. Bacterial isolates from human stool specimens were obtained from various sources, including the Houston City Health Department, Hous- Downloaded from http://aac.asm.org/ on September 9, 0 by guest 509

50 CARLSON ET AL. ton, Tex.; The University of Texas Medical School, Program in Infectious Diseases and Clinical Microbiology, Houston, Tex., and its field study center in Guadalajara, Mexico; Institut Pasteur, Paris, France; Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; Montreal Children's Hospital, Montreal, Quebec, Canada; and the Central Health Laboratory, Cairo, Egypt. We selected for study 50 strains of Salmonella spp., 0 strains of Shigella spp. and 50 enterotoxigenic (ET) Escherichia coli, 4 Yersinia enterocolitica, 6 Aeromonas hydrophila, 4 Plesiomonas shigelloides, 9 Vibrio parahaemolyticus, and 30 Campylobacter jejuni strains. The ET E. coli strains were shown to produce heat-stable toxin (7 strains), heat-labile toxin (0 strains), or both (33 strains) by previously reported methods (7). All strains were collected after 90 and stored in skim milk at -0 C. Each strain was thawed and subcultured before being tested. Facultative organisms were incubated on 5% sheep blood agar plates at 35C for h. C. jejuni strains were cultured on Campy blood agar (Austin Biological Laboratories, Austin, Tex.) at 4 C for 4 h in Torbal jars (Torsion Balance Co., Clifton, N.J.) in an atmosphere of 5% 0-0%o C0-5% N. Antimicrobial sucepibity testng. Minimal inhibitory concentrations (MICs) were determined for each TABLE. ANTIMICROB. AGENTS CHEMOTHER. 0 antimicrobial agent by broth dilution in microtiter plates (7). Facultative organisms were tested against dilutions of antimicrobial agents in Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.), and Wilkins Chalgren broth (Difco) was used for Campylobacter spp. (4). Control strains of E. coli (ATCC 59) and Staphylococcus aureus (ATCC 593) were tested on each microtiter plate to ensure the potency of each drug. Thymidine phosphorylase (0. U/ml; Burroughs Wellcome Co.) was incorporated into test dilutions of both and to prevent the trailing effect caused by thymidine present in the Mueller-Hinton broth. was tested at a ratio of :9. Microtiter plates were inoculated with 05 cells per well and incubated at 35 C for 4 h, except for C. jejuni strains, which were incubated at 4 C for 4 h. The MIC was defined as the lowest concentration of the drug at which there was complete inhibition of growth. RESULTS MIC ranges and the MIC that inhibited 50 and 90% of the strains (MIC50 and MIC90, respectively) are given in Table. The drugs that showed the greatest activity against Salmonella spp. were enoxacin, furazolidone, norfloxacin,, and (MIC90, sl,ug/ml). These Comparative MICs of selected antimicrobial agents against all test strains Organism (no. of strains) Drug MIC (Lg/ml) Range 50WI 90% Salmonella spp. (50) <0.5-> <0.5-> -> 6 co.5- c0.5 c0.5 c0.5 3- <0.5- so.5- c0.5 50.5 <0.5-> c0.5 co.5 a <0.5-> Shigella spp. (0) <0.5-3 :0.5 <0.5-> > 4-3 6 3 <0.5-3 4 6 c0.5 O0.5 co.5 6-> 50.5-4 <0.5-0.5 o0.5 c0.5- so.5 c0.5 /SMX <O.5-4 ET E. coli (50) so.5-a4 0.5 -> > 6-3 3 Doxycycine c0.5-3 3 c0.5 o0.5 6-3 so.5-4 <0.5- o0.5 co.5 50.5-> c0.5 <0.5-> Downloaded from http://aac.asm.org/ on September 9, 0 by guest

VOL. 4, 93 C. jejuni (30) A. hydrophila (6) P. shigelloides (4) V. parahaemolyticus (9) ANTIBIOTIC ACTIVITY AGAINST ENTEROPATHOGENS 5 TABLE -Continued MIC (tlgim) Organism (no. of strains) Drug Range 50% 90 Range 50tlo 90% Y. enterocolitica (4) <0.5-> > 6-3 3- <0.5- c0.5 3-6- 3 --O.5 '<0.5 -> - 3 I Doxycyctine -6 _0.5-4-6-3 - O0.5- <:0.5-3-> 6-> so.5-4 -> > -3 - <0.5- <0.5-3 -3 6 _:0.5 6-3 - -4 > - - was tested at a ratio of :9; MICs of sulfamethoxazole are shown. 50.5. :so0.5 > > so.5 s-0.5 6 _0.5 6 3 O.5 > so.5 6 _:0.5 > > > 5:0.5 s:0.5 6 s:0.5 6 <0.5 3 4 > <:0.5 Downloaded from http://aac.asm.org/ on September 9, 0 by guest test drugs, in addition to amdinocillin, had MIC9os for the Shigella and ET E. coli isolates of c p.g/ml. The drugs showing the highest degree of activity against C. jejuni strains were enoxacin, erythromycin, furazolidone, amdinocilin, and norfloxacin (MIC90, c,ug/ml)., enoxacin, norfloxacin,, and - SMX were highly active (MIC90, c,ug/ml)

5 CARLSON ET AL. against the remaining four genera of bacteria consisting of Yersinia, Aeromonas, Plesiomonas, and Vibrio spp. showed low activity against the test strains of Salmonella and Shigella spp., ET E. coli strains, and Aeromonas and Yersinia spp. (MIC90,.,ug/ml). The Shigella spp. that were resistant to ampicillin included 0 Shigella sonnei, Shigella dysenteriae, and one Shigella flexneri. For Plesiomonas, Vibrio, and Campylobacter spp., however, the MlCgo of ampicillin was s ptg/ml. The drugs with the highest activities against ampicillin-resistant strains of Salmonella and Shigella spp. and ET E. coli were enoxacin (MIC90, c0.5,ug/ml), furazolidone (MIC90, c,ug/ml), and norfloxacin (MIC90, s,ug/ml). inhibited all the test strains at an intermediate to low level, with MIC90 ranges of 6 to >,ug/ml. DISCUSSION Antimicrobial therapy has been shown to be useful in the treatment of diarrhea caused by Shigella spp. (4-6) and V. cholerae strains (4). It has also been demonstrated to be effective in both the treatment and prophylaxsis of traveler's diarrhea (6,, 6). The value of antimicrobial therapy has not been established for diarrhea caused by Salmonella spp. or C. jejuni. Antimicrobial treatment of diarrhea caused by Salmonella spp. has been shown to prolong the carrier state (, 5). has been recommended for the treatment of campylobacteriosis. However, the only controlled study of this drug failed to show any effect on the course of this disease (), although C. jejuni organisms were eradicated from 00%o of the treated patients.,, and doxycycline are the three most common antimicrobial agents now used to treat or prevent diarrhea of unknown etiology. This study has shown that resistance to ampicillin is prevalent in bacterial enteropathogens, and ampicillin probably should no longer be used in the empirical treatment of acute diarrhea. and remain the drugs of choice for treatment of shigellosis, and they have also been shown to be effective in treating traveler's diarrhea (3,, ). The efficacy of doxycycline has been established only in the treatment of cholera (4), but in view of the intermediate activity of this antimicrobial agent against the majority of enteropathogens tested in this study and the high intraluminal concentration of the drug when taken orally, it may continue to be useful in the prophylaxsis of traveler's diarrhea. There is little doubt that antimicrobial agents will continue to be employed in the treatment and prevention of bacterial diarrhea. This use of currently effective agents may also contribute to ANTIMICROB. AGENTS CHEMOTHER. the development of resistance to these agents by bacterial enteropathogens in the future, thus necessitating a continual search for new, effective drugs for use in the treatment and prevention of diarrhea. Our results suggest that four antimicrobial agents that were tested are highly active against the enteropathogen strains in this study and should be examined more closely as potentially useful therapeutic drugs. These agents include a preparation that has been available for some time, furazolidone, and three unlicensed preparations, enoxacin, norfloxacin, and amdinocillin. These antibiotics may be administered as oral medications and should achieve high intraluminal concentrations. In addition, all of these antimicrobial agents have demonstrated excellent broad-spectrum activities against the enteropathogens that cause the treatable forms of diarrhea, as well as the Campylobacter isolates. Therefore, clinical trials with these agents should be initiated to evaluate their efficacy in the treatment and prevention of diarrheal diseases. ACKNOWLEDGMENTS This work was supported by Public Health Service contract NO-AI 066 from the National Institutes of Health and grants from Merck Sharp & Dohme Research Laboratories, Norwich-Eaton Pharmaceuticals, and Hoffmann-La Roche Inc Ẇe gratefully acknowledge the following colleagues who supplied bacterial isolates: Rubin Wende, Houston City Health Department, Houston, Texas; Emma Galindo, Hospital Infantil de Mexico and Hospital Central Militar, Mexico City, Mexico; and Safwat Mohieldin, Central Health Laboratory, Cairo, Egypt. LITERATURE CITED. Anders, B. J., B. A. Lav9r, J. W. Paely, and L. B. Reller. 9. Double-blind placebo controlled trial of erythromycin for treatment of campylobacter enteritis. Lancet :3-34.. Aserkoff, B., and J. V. Bennett. 969. 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