(This work was presented in part at the 18th Annual Meeting of the Surgical Infection Society, 30 April to 2 May 1998, abstr. P18, p. 93.

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2000, p. 1035 1040 Vol. 44, No. 4 0066-4804/00/$04.00 0 Copyright 2000, American Society for Microbiology. All Rights Reserved. In Vitro Activities of Nontraditional Antimicrobials against Multiresistant Acinetobacter baumannii Strains Isolated in an Intensive Care Unit Outbreak MARIA D. APPLEMAN, 1 * HOWARD BELZBERG, 2 DIANE M. CITRON, 1 PETER N. R. HESELTINE, 3 ALBERT E. YELLIN, 2 JAMES MURRAY, 2 AND THOMAS V. BERNE 2 Departments of Pathology, 1 Surgery, 2 and Medicine, 3 Los Angeles County-University of Southern California Medical Center, Los Angeles, California Received 11 January 1999/Returned for modification 29 April 1999/Accepted 13 December 1999 Fifteen multiresistant Acinetobacter baumannii isolates from patients in intensive care units and 14 nonoutbreak strains were tested to determine in vitro activities of nontraditional antimicrobials, including cefepime, meropenem, netilmicin, azithromycin, doxycycline, rifampin, sulbactam, and trovafloxacin. The latter five drugs were further tested against four of the strains for bactericidal or bacteriostatic activity by performing kill-curve studies at 0.5, 1, 2, and 4 times their MICs. In addition, novel combinations of drugs with sulbactam were examined for synergistic interactions by using a checkerboard configuration. MICs at which 90% of the isolates tested were inhibited for antimicrobials showing activity against the multiresistant A. baumannii strains were as follows (in parentheses): doxycycline (1 g/ml), azithromycin (4 g/ml), netilmicin (1 g/ml), rifampin (8 g/ml), polymyxin (0.8 U/ml), meropenem (4 g/ml), trovafloxacin (4 g/ml), and sulbactam (8 g/ml). In the kill-curve studies, azithromycin and rifampin were rapidly bactericidal while sulbactam was more slowly bactericidal. Trovafloxacin and doxycycline were bacteriostatic. None of the antimicrobials tested were bactericidal against all strains tested. The synergy studies demonstrated that the combinations of sulbactam with azithromycin, rifampin, doxycycline, or trovafloxacin were generally additive or indifferent. Acinetobacter baumannii is an aerobic, gram-negative, oxidase-negative, nonfermenting bacterium that has become an increasingly frequent cause of nosocomial infections, particularly in intensive care units (3, 4, 8, 11). It has a propensity to develop antibiotic resistance extremely rapidly (2). Successive surveys have shown increasing resistance in clinical isolates, and high proportions of strains have become resistant to older, commonly used antibiotics (6, 10, 15). Only newer antibiotics, such as broad-spectrum cephalosporins, imipenem, tobramycin, amikacin, and fluoroquinolones, remain useful. The recent development of more universally resistant strains of A. baumannii has made the search for effective therapies more important and urgent (9, 13, 14; M. Wolff, M. L. Joly-Gillou, R. Farionotti, and C. Carbon, Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. B-8, 1997). From 1996 through 1997, an outbreak of resistant A. baumannii infections involving 96 patients occurred in the intensive care units (ICUs) of Los Angeles County-University of Southern California (LAC-USC) Medical Center. The hospital clinical laboratory did routine susceptibility testing and reported that the isolates were resistant to imipenem, ceftazidime, cefotaxime, gentamicin, tobramycin, piperacillin-tazobactam, ticarcillin-clavulanate, ciprofloxacin, ofloxacin, and trimethoprim-sulfamethoxazole. The strains were moderately susceptible to ampicillin-sulbactam. Some strains were resistant to amikacin, and others were not. There was variability in susceptibility to amikacin among the strains, even among the same-patient strains, when testing was done by the clinical laboratory using an automated instrument. The amikacin susceptibility tests were repeated by using a reference method. * Corresponding author. Mailing address: Los Angeles County-University of Southern California Medical Center, 1200 N. State St., Room 2014, Los Angeles, CA 90033. Phone: (323) 226-7016. Fax: (323) 226-4075. E-mail: mapplema@hsc.usc.edu. Since the traditional antibiotics used against gram-negative aerobic bacteria were not effective against the Acinetobacter isolates, we selected nontraditional antibiotics for potential use in eradicating these bacteria in cases where they were clinical pathogens. We determined bactericidal or bacteriostatic activities of the novel antibiotics by performing kill-curve studies on selected strains. In addition, we examined new combinations of drugs with a checkerboard configuration by looking for synergistic interactions. (This work was presented in part at the 18th Annual Meeting of the Surgical Infection Society, 30 April to 2 May 1998, abstr. P18, p. 93.) MATERIALS AND METHODS The A. baumannii strains were isolated from specimens obtained from patients in the ICUs at LAC-USC Medical Center. The specimens were processed by the clinical laboratory according to standard methods described in the Manual of Clinical Microbiology (7). Identification to genus and species levels and the original susceptibility tests were done with the Vitek automatic instrument (biomerieux Vitek, Inc., Hazelwood Mo.). The outbreak strains were sent to a reference laboratory for typing by pulse gel electrophoresis (PGE). The medical records of patients from whom one of these outbreak strains was isolated were reviewed. Fifteen of the A. baumannii isolates that were found to be multidrug resistant by the hospital clinical laboratory and 14 strains randomly selected were sent to the research laboratory and were tested for additional antibiotic susceptibilities by the agar dilution technique. Three of the outbreak strains plus one multiresistant, nonoutbreak strain were selected for kill-curve studies and synergy studies. Agar dilution test. Antibiotic laboratory standard powders of azithromycin, sulbactam, ampicillin, doxycycline, trovafloxacin (Pfizer, Inc., Groton, Conn.), cefepime, amikacin (Bristol-Myers Squibb, Princeton, N.J.), meropenem (Zeneca Pharmaceuticals, Wilmington, Del.), erythromycin (Eli Lilly, Indianapolis, Ind.), minocycline, tetracycline (Wyeth Laboratories, Philadelphia, Pa.), ciprofloxacin (Bayer Pharmaceuticals, West Haven, Conn.), netilmicin, gentamicin, isepamicin (Schering-Plough, Kenilworth, N.J.), rifampin (Novartis Pharmaceuticals, East Hanover, N.J.), and polymyxin B (Sigma Chemicals, St. Louis, Mo.) were obtained from their respective manufacturers and reconstituted according to their instructions. Stock solutions were stored at 70 C. On the day of the test, serial twofold dilutions were prepared and added to molten Mueller-Hinton agar 1035

1036 APPLEMAN ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Specimen sources of outbreak strains of A. baumannii Source of positive specimen a Total no. of patients (%) SICU b No. of patients in: Burn ICU Respiratory tract 69 (71) 54 15 Wound 36 (37) 15 21 Blood 27 (28) 11 16 Urine 9 (9) 6 3 Catheter tip 8 (8) 1 8 Total no. of patients 96 67 29 a Some patients had more than one source from which A. baumannii was isolated. b Patients in all ICUs except burn ICU. for preparation of plates. Ampicillin plus sulbactam were tested together in a 2:1 ratio and separately. The organisms were incubated overnight on blood agar at 35 C. Inocula were prepared by suspending cell paste in saline to equal the turbidity of a 0.5 McFarland standard. A 1:10 dilution was prepared prior to pipetting into a Steers replicating device. The organisms were applied to the plates at a final concentration of 1 10 4 to 5 10 4 CFU/spot. Plates without antibiotics were inoculated before and after each set of drug-containing plates as growth controls. After overnight incubation at 35 C, the MICs were interpreted. Fifteen outbreak strains and 14 randomly selected, nonoutbreak strains were tested by agar dilution. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were included as controls. The susceptibility results for most drugs were interpreted according to National Committee for Clinical Laboratory Standards standards (12). Results for polymyxin B were interpreted according to the third edition of the Manual of Clinical Microbiology (1). Trovafloxacin results were interpreted by guidelines provided by the manufacturer. Kill curves. Four strains were selected for further studies. Three were outbreak strains isolated over a period of 2 months, each from patients in different ICUs. Strain 13009 was isolated from a blood culture from a patient in the surgical ICU; 12770 was from a blood culture in a patient in the burn ICU; and 13801 was from a sputum specimen from a medical ICU patient. The three strains were from patients in different environments who had been subjected to different antibiotic therapies and selective pressures. The fourth strain (12676) was a randomly selected blood culture isolate that exhibited a different susceptibility pattern from the pattern of the outbreak strains (susceptible to imipenem and resistant to azithromycin). Tests were performed in flasks containing 100 ml of Mueller-Hinton broth and Antimicrobial agent(s) either azithromycin, rifampin, sulbactam, trovafloxacin, or doxycycline at concentrations of 0.5, 1, 2, and 4 times the MIC for each organism. Starting inocula were 3 10 5 to 6 10 5 CFU/ml. The prewarmed broths were placed on an orbital shaker at 100 rpm and incubated at 35 C. Aliquots were removed after 2, 4, 8, 24, and 48 h and 7 days. The aliquots were diluted and plated quantitatively onto Mueller-Hinton agar. After overnight incubation, colonies were counted. Organisms that persisted after 48 h and 7 days were subcultured, and MICs were determined by using the agar dilution method, as described above. Synergy studies. The checkerboard method (5) was used to assess the activity of the antimicrobial combinations. Test tubes containing sulbactam plus azithromycin, doxycycline, trovafloxacin, or rifampin in 1 ml of Mueller-Hinton broth were prepared in a checkerboard configuration. The test strains were added at a final concentration of approximately 10 5 CFU/ml. After overnight incubations, the MICs were interpreted. Tubes showing no growth were subcultured onto Mueller-Hinton agar to determine the minimal bactericidal concentration. The fractional inhibitory concentration index (FIC index) for each drug was derived by dividing the concentration of that drug necessary to inhibit growth in a given row by the MIC of the drug alone for the test organism (5). The FIC index was then calculated by summing the separate FICs for each of the drugs present in that tube. Synergism was defined as an FIC index of 0.5; additivity was an FIC index of 1.0; indifference was an FIC index of 1.0 and 4; and antagonism was an FIC index of 4.0. RESULTS There are 14 medical and 40 surgical ICU beds for adult patients at the LAC-USC Medical Center. The surgical beds include those in the general surgical, neurosurgical, and burn services. Ninety-six patients who had been in one of these ICUs had at least one specimen from which multiresistant A. baumannii was isolated. In Table 1, the specimen sources of the isolates are listed. The most frequent source of the isolate was the respiratory tract, with 41% of the patients having this as their only source of positive specimen. Wound and blood specimens accounted for 37 and 28% of specimen sources, respectively. PGE patterns of the outbreak strains showed the presence of one clone with eight subtypes based on a difference of one to three minor bands. In the burn surgical ICU, there were 29 patients with A. baumannii infections. However, in this service, only one patient had the respiratory tract as the sole source of the organism. The majority, 96%, had more than one specimen site TABLE 2. In vitro activities of 18 antimicrobial agents against outbreak and randomly selected strains of A. baumannii MICs of antimicrobial agents for: Outbreak strains (n 15) a Random strains (n 14) Range 50% 90% Range 50% 90% Ampicillin-sulbactam b 8 32 16 16 1 8 2 4 Sulbactam 4 16 8 8 0.5 8 2 4 Ampicillin 128 128 128 2 128 32 64 Cefepime 32 128 64 128 0.5 4 2 2 Meropenem 4 4 4 1 2 1 1 Azithromycin 2 4 4 4 2 32 2 32 Erythromycin 16 32 32 32 8 64 16 64 Doxycycline 1 1 1 0.06 0.25 0.125 0.25 Minocycline 1 2 1 2 0.25 5 0.25 0.5 Tetracycline 16 32 16 32 2 16 4 16 Trovafloxacin 4 8 4 4 0.25 4 0.25 4 Ciprofloxacin 32 64 64 64 0.25 64 0.25 64 Netilmicin 0.5 1 1 1 0.5 64 0.25 32 Gentamicin 16 64 16 32 0.5 128 0.5 128 Amikacin 8 32 16 16 1 64 1 32 Isepamicin 16 64 32 32 1 128 1 128 Polymyxin B c 0.4 0.8 0.8 0.8 0.4 0.8 0.8 0.8 Rifampin 4 128 8 8 4 8 8 8 a These strains were resistant to imipenem, ceftazidime, cefotaxime, gentamicin, tobramycin, piperacillin-tazobactam, ticarcillin-clavulanate, ciprofloxacin, ofloxacin, and trimethoprim-sulfamethoxazole. b Ampicillin-sulbactam were tested in a ratio of 2:1. c Data are expressed in units per milliliter.

VOL. 44, 2000 NONTRADITIONAL AGENTS AGAINST RESISTANT ACINETOBACTER 1037 FIG. 1. The activity of trovafloxacin against strain 12676 typifies all four strains. While a slight decrease in CFU per milliliter was noted after 8 h, the drug was essentially bacteriostatic after 24 to 48 h. positive for the resistant bacteria. Twenty-one (72%) and 16 (55%) had positive wound and blood specimens, respectively. In Table 2, the determinations of the MICs at which 50 and 90% of the isolates tested were inhibited (MIC 50 s and MIC 90 s) for 18 antimicrobials are listed for the outbreak strains and for the randomly selected strains. The outbreak strains of A. baumannii were resistant to ampicillin, cefepime, erythromycin, tetracycline, ciprofloxacin, and gentamicin. However, not all the strains were resistant to amikacin, with a MIC range between 8 and 32 g/ml. The randomly selected nonoutbreak strains of Acinetobacter were resistant to ampicillin, erythromycin, tetracycline, ciprofloxacin, and gentamicin, just as the outbreak strains were. However, there were differences. Some of the randomly selected strains were resistant to azithromycin, netilmicin, and amikacin while the outbreak strains were not. Both groups of strains were susceptible to ampicillin-sulbactam, sulbactam alone at 16 g/ml, meropenem, doxycycline, minocycline, and polymyxin B and were moderately susceptible to trovafloxacin. Ampicillin alone was not active. The nonoutbreak stains were also susceptible to cefepime in this study, and many of them were susceptible to other cephalosporins and imipenem, as determined in the hospital laboratory. In the kill-curve studies, trovafloxacin showed some decrease in the number of organisms initially during the first 8 h (Fig. 1). However, after 24 h, the drug was essentially bacteriostatic. The same effect was seen with doxycycline, with an initial drop in numbers of organisms in the first 8 h of incubation followed by a subsequent static effect for the outbreak strains while having a bactericidal effect after 24 h on the nonoutbreak strain (Fig. 2). There were 28 kill-curve broths from which persistent colonies were grown when subcultured after 48 h and 7 days of exposure to the antibiotics. Two of the strains that survived after 1 week of exposure to trovafloxacin at one-half the MIC showed a twofold dilution increase in trovafloxacin MICs. The MICs of all antibiotics for the remaining 26 cultures were within 1 dilution of the MICs of the unexposed strains. MICs FIG. 2. The activity of doxycycline against strain 13801 typifies three of the strains. A slight decrease in CFU/milliliter was noted after 4 to 8 h, but regrowth started after 24 h, and the drug was essentially bacteriostatic. Strain 12770 was different in that doxycycline exerted a bactericidal effect after 24 h.

1038 APPLEMAN ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 3. Rifampin showed rapid bactericidal activity against two of the outbreak isolates: strains 13081 and 12770. for none of the strains exposed to doxycycline were more than 1 dilution different from those for the preexposed strains. Rifampin did not have consistent effects on all four strains tested. It showed rapid bactericidal activity against two of the outbreak strains (Fig. 3). Bacteriostatic effects were detected on the nonoutbreak strain and one outbreak strain (Fig. 4). Sulbactam had bactericidal effects only initially against all strains tested. After 48 h, there was regrowth (Fig. 5). MICs of sulbactam for 25 of the 28 regrowth strains were within 1 dilution of those for the preexposed strains. The MIC for one strain was 4 dilutions higher, one was 2 dilutions higher, and one was 2 dilutions lower. Azithromycin tested as bactericidal on the outbreak strains that were susceptible to the antibiotic (Fig. 6). On the nonoutbreak strain, which was resistant to azithromycin (MIC, 32 g/ml) the effect was bacteriostatic. MICs for 27 of the 28 persistent isolates were within 1 dilution of those for the preexposure strains. The MIC for one strain was 2 dilutions higher after 1 week of incubation at two times the MIC. The results of the checkerboard synergy testing are in Table 3. Using the checkerboard combinations of azithromycin, rifampin, trovafloxacin, or doxycycline with sulbactam, only the combinations of azithromycin with sulbactam and rifampin with sulbactam showed a synergistic effect with the nonoutbreak strain. The other combinations resulted in additive or indifferent effects. DISCUSSION Of all the species in the genus, A. baumannii is the main species associated with outbreaks of nosocomial infection. In recent years, the species has emerged as particularly important in nosocomial infections in ICUs, probably related to the increasingly invasive diagnostic procedures used and the increasingly greater quantity of broad-spectrum antimicrobials used. In our institution, 40 of the 96 patients from whom a resistant Acinetobacter organism was isolated had the organism in a single site. It can be argued that these patients may have been colonized as opposed to infected. The other 56 patients had more than a respiratory source positive for the organism. The FIG. 4. Rifampin was bacteriostatic against the nonoutbreak strain 12676 and the outbreak strain 13009. The third outbreak strain was resistant to rifampin (MIC 128 g/ml).

VOL. 44, 2000 NONTRADITIONAL AGENTS AGAINST RESISTANT ACINETOBACTER 1039 FIG. 5. The activity of sulbactam against strain 12270 typifies its activity against all the strains. There was initial bactericidal activity after 4 to 24 h, with regrowth occurring at 48 h. patients with A. baumannii isolated from a wound, blood, or urine source can be described as infected with that pathogen. The 15 strains included in this study were isolated during the first 3 months of the outbreak that occurred throughout all the ICUs. Although their antibiograms showed some variation among them, the PGE demonstrated them to belong to a single clone with eight subgroups. The control strains were of different PGE types and had different antibiograms. The striking characteristic of the outbreak strains was their resistance to imipenem as opposed to the imipenem-susceptible control strains. In the early 1970s, nosocomial Acinetobacter infections were treated successfully with gentamicin, ampicillin, naladixic acid, or carbenicillin either as single agents or in combinations (2). Successive surveys have demonstrated increasing resistance. The survey presented in this study reports an Acinetobacter organism resistant to most antibiotics, with few therapeutic choices remaining. The outbreak strains tested in this survey were resistant to erythromycin and sensitive to azithromycin. Nonoutbreak strains showed resistance to erythromycin and variable susceptibility to azithromycin. In the kill-curve studies, azithromycin, with a MIC of 4 g/ml, demonstrated rapid bactericidal activity for most strains and, with a MIC of 32 g/ml, demonstrated a bacteriostatic effect on one strain. However, the potential activity of azithromycin may not be predicted by comparing MICs to levels in serum since the drug is concentrated within the intracellular and interstitial compartments of tissues (13, 14). The extravascular MICs may be a better measure of the potential use of this drug since levels in tissue can be much higher than levels in serum according to pharmacokinetics studies. Neu has suggested that achievable tissue levels be used as breakpoints for susceptibility. Ultimately, the susceptibility breakpoints will be established by clinical studies. In the synergy results presented in this study, synergy was seen with azithromycin plus sulbactam, with a MIC of 32 g/ml for one strain. Trovafloxacin was moderately active against multiresistant A. baumannii. It had much better activity against both the outbreak and nonoutbreak strains than ciprofloxacin tested by FIG. 6. Azithromycin was bactericidal against strain 13009 and the other outbreak strains after 8 to 24 h. Azithromycin was not as active against the nonoutbreak strain (MIC 32 g/ml) and was very slowly bactericidal after 48 h and 7 days.

1040 APPLEMAN ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 3. Antimicrobial combination studies on multiresistant A. baumannii Strain Antibiotic combination FIC index Interpretation 12676 Rifampin sulbactam 0.31 Synergy Azithromycin sulbactam 0.38 Synergy Trovafloxacin sulbactam 0.75 Additive Doxycycline sulbactam 1.5 Indifferent 12770 Rifampin sulbactam 1.5 Indifferent Azithromycin sulbactam 2 Indifferent Trovafloxacin sulbactam 1.5 Indifferent Doxycycline sulbactam 2 Indifferent 13009 Rifampin sulbactam ND a Azithromycin sulbactam 1 Additive Trovafloxacin sulbactam 0.75 Additive Doxycycline sulbactam 2 Indifferent 13081 Rifampin sulbactam 0.75 Additive Azithromycin sulbactam 1 Additive Trovafloxacin sulbactam 1 Additive Doxycycline sulbactam 2 Indifferent a ND, not determined (because the rifampin MIC was 128 g/ml). agar dilution and ofloxacin tested with the Vitek instrument. The drug was bacteriostatic. Synergy studies with trovafloxacin and sulbactam demonstrated additive effects. Rifampin has been reported to have a strong in vitro bactericidal effect and a synergistic effect with beta-lactamase inhibitors on multiresistant A. baumannii (Wolff et al., 37th ICAAC). In our study, rifampin did not have a bactericidal effect on all strains tested and had a synergistic effect only on the nonoutbreak strain. Doxycycline, minocycline, and tetracycline were tested. Tetracycline was found to be ineffective while the other two were active. Further studies were done with doxycycline because it would be a nontoxic, inexpensive agent to use. Doxycycline had a bacteriostatic effect against three of the four outbreak strains tested. No effect was seen by combining doxycycline with sulbactam. Of the aminoglycosides tested, gentamicin, tobramycin, amikacin, and netilmicin, only netilmicin was effective against all the outbreak strains. There were several outbreak and nonoutbreak strains that demonstrated resistance to amikacin. 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