In vitro activities of antimicrobial agents, alone and in combinations, against Burkholderia cepacia isolated from blood

ELSEVIER

In Vitro Activities of Antimicrobial Agents, Alone and in Combinations, against Burkholderia cepacia Isolated from Blood Daniel C.-T. Lu, Shan-Chwen Chang, Yee-Chun Chen, Kwen-Tay Luh, and Wei-Chuan Hsieh

Burkholderia cepacia is a widespread, environmental gramnegative bacillus that is associated with nosocomial infections. This bacterium is considered to be an important pathogen in immunocompromised patients and is inherently resistant to multiple antimicrobial agents. To compare the activity of different antimicrobial agents and the potential of combinations against invasive strains of B. cepacia, we collected 36 isolates of B. cepaciafrom blood cultures and checked their susceptibilities to 13 antimicrobials by broth microdilution method. Most strains tested were susceptible to minocycline (94.4%), ceftazidime (86.1%)‘ ciprofloxacin (83.3%), and trimethoprimsulfamethoxazole (83.3%). All strains were resistant to amin-

oglycosides, and only some strains were susceptible to imipenem (16.7%), aztreonam (19.4%), moxalactam (25.0%), piperacillin (25.0%), and carbenicillin (47.2%). The effects of combinations of cefazidime with amikacin, ceftazidime with ciprofloxacin, and ciprofloxacin with amikacin were assayed by checkerboard titration method. Synergistic effect was found in 28 out of 36 tested strains (77.8%)‘ when ceftazidime was combined with amikacin, in 25 out of 36 strains (69.4%) when ceftazidime was combined with ciprofloxacin, and in only 8 out of 36 strains (22.2%) when ciprofloxacin was combined 0 1997 Elsevier Science Inc. with amikacin.

INTRODUCTION

Klinger 1986; Mortensen et al. 1995). The reservoir of this organism is considered to be the environment including soil, water, and plants (Ederer and Matsen 1972; Martone et al. 1981). Aqueous solutions have been found to be the source of infections with this bacterium (Ederer and Matsen 1972; Martone et al. 1987; Mortensen et al. 1995; Robin 1992). Over the past two decades, this species has been increasingly recognized as a nosocomial pathogen such as pneumonia, urinary tract infection, endocarditis, and bacteremia (Ederer and Matsen 1972; Pallent et al. 1983; Spencer 1995). Antimicrobial therapy for B. cepacia infections poses a significant challenge, because this pathogen is routinely resistant to many agents including aminoglycosides, first- and second-generation cephalosporins, and traditional antipseudomonal penicillin such as ticarcillin (Bhakta et al. 1992; Ferreira et al. 1985; Lewin et al. 1993; Rolston et al. 1987; Simpson

Burkholderia cepacia, previously named Pseudomonas cepacia, was renamed based on the 16s rRNA se-

quences and DNA-DNA homology (Yabuuchi et al. 1992). It is an aerobic, glucose-nonfermenting, gramnegative bacillus that proliferates under conditions of minimal nutrition and can survive even in the presence of certain disinfectants (Goldmann and From the Section of Infectious Diseases, Departments of Internal and Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan, Republic of China. Address reprinf requests to Dr. Shan-Chwen Chang, Department of Internal Medicine, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, Taiwan, Republic of China. Current address (DC-TL): Section of Infectious Diseases, Department of Internal Medicine, Lo-Tung I’oh-Ai Hospital, No. 83, Nan Chang Street, Lo-Tung, I-Lan, Taiwan, Republic of China. Received 21 August 1996; revised and accepted 4 April 1997.

DIAGN MICROBIOL INFECT DIS 1997;28:187-191 0 1997 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

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D.C.-T. Lu et al.

et al. 1994). The resistance to ciprofloxacin, imipenem, and chloramphenicol is variable. But B. cepacia is usually susceptible to ceftazidime, piperacillin, and co-trimoxazole (Bhakta et al. 1992; Ferreira et al. 1985; Lewin et al. 1993; Rolston et al. 1987; Simpson et al. 1994; Spencer 1995). This resistance affects the selection of appropriate antibiotics for treating such patients. However, current available data about antimicrobial susceptibilities of B. cepacia mostly came from isolates from cystic fibrosis patients (Aronoff and Klinger 1984; Bhakta et al. 1992; Bosso et al. 1991; Kumar et al. 1989; Simpson et al. 1994) and/or from nonblood specimens such as sputum, tracheal aspiration, urine, pus, and nebulizer content (Bhakta et al. 1992; Bosso et al. 1991; Ferreira et al. 1985; Kumar et al. 1989; Simpson et al. 1994). B. cepaciu was found to account for 0.072% of the total blood culture and 0.90% of the total positive culture at the National Taiwan University Hospital (NTUH), both a primary- and tertiary-care medical center that had a 1,200-bed capacity before 1991 and had grown to 1,700 beds by 1994. To determine the antimicrobial susceptibility of B. cepuciu isolates that cause bacteremia and the combination effect of two agents against this organism, bacteria isolated from blood cultures of patients hospitalized at NTUH were collected to determine their in vitro susceptibility to various antimicrobial agents, alone and in combination with each other.

MATERIALS Bacterial

AND METHODS

Isolates

A total of 36 isolates from blood cultures of patients hospitalized at NTUH were used in this study. B. cepuciu was identified as a non-lactose-fermenting, oxidase variable, gram-negative bacillus that grew on MacConkey’s agar. Other confirmatory tests included oxidation of 1% glucose and 10% lactose, positive lysine decarboxylase activity, negative arginine decarboxylase activity, and absence of growth in 6.5% sodium chloride or at 42°C (Gilligan 1995). All isolates were stored in tryptic soy broth with 15% glycerol at -70°C. They were thawed, subcultured, and reconfirmed by using the Vitek automated system (Vitek-AMS 120; bioMerieux Vitek, Hazelwood, MD, USA) before susceptibility testing. No duplicate isolates from the same episode of a single patient was included in this study.

Antimicrobial

Agents

The 13 antimicrobial agents tested in this study were provided as standard powders for laboratory use from the manufacturers, including carbenicillin

(Pfizer, New York, NY, USA), piperacillin, minocycline (Cyanamide, New York, NY, USA), ceftazidime (Glaxo, Greenford, U.K.), aztreonam, amikacin, cefepime (Bristol-Myers Squibb, Syracuse, NY, USA), imipenem (Merck Sharp & Dohme, West Point, PA), gentamicin (Schering-Plough, Bloomfield, NJ), tobramycin (Eli Lilly, Indianapolis, IN), ciprofloxacin Wuppertal, moxalactam, Germany), (Bayer, trimethoprim-sulfamethoxazole (TMP-SMX, 1:19; Shionogi, Osaka Japan). All of them were prepared according to the manufacturers’ recommendations.

Antimicrobial

Susceptibility

Testing

Quantitative antibiotic susceptibility tests were performed by broth microdilution method according to guidelines by the National Committee for Clinical Laboratory Standards (NCCLS 1993). Reference strains of bacteria that served as controls in each susceptibility testing included Escherichiu coIi [American Type Culture Collection (ATCC) 259221 and Pseudomonas aeruginosu (ATCC 27853). Cationadjusted Mueller-Hinton broth supplemented with calcium and magnesium (final concentrations, 25 and 12 pg/mL, respectively) and incorporated with series twofold dilution of antibiotics (from 128 to 0.06 Fg/mL) were prepared in microtiter plates. The microtiter plates were stored at -70°C and used within 20 days. Microtiter wells were inoculated with an actively growing inoculum adjusted to suitable concentration, and the final concentration of bacteria was 5 x lo5 CFU/mL. The MIC was defined as the lowest concentration of drug that allowed no visible growth after 18-24 h of incubation at 35°C. Because there are no NCCLS-approved breakpoints specific for B. cepuciu currently, those breakpoints approved for P. aeruginosu were used in interpretation the susceptibility of B. cepuciu (NCCLS 1993).

Effect of Combining Agents

Two Antimicrobial

The checkerboard titration method, as previously described (Chang et al. 1995), was used to assess the effect of combining two antimicrobial agents. The antimicrobial combinations assayed included ceftazidime plus amikacin, ceftazidime plus ciprofloxatin, and ciprofloxacin plus amikacin. The fractional inhibitory concentration (FIC) was calculated for each combination that inhibited growth using the following formula: FIC = (MIC of drug A in combination/MIC of drug A alone) + (MIC of drug B in combination/MIC of drug B alone) (Eliopoulos and Moellering 1991). The minimum FIC of the calculated FICs was defined to be the FIC index. Synergism was defined as an FIC index of ~0.5. Antagonism was defined as an FIC index of 22.

Antibiotics

against Burkholderia

189

cepacia

RESULTS Antimicrobial Agent

Susceptibility

to a Single

The comparative activities in terms of MI&, MI&,, range of MIC, and susceptible percentage for the 13 antimicrobial agents tested are shown in Table 1. Piperacillin and carbenicillin, two antipseudomonal penicillins, had activity against only some strains of the isolates (36.1% and 47.2%, respectively), and the MIC,, values were 128 pg/mL. Of three cephalosporins tested, ceftazidime had the best activity with an MIC,, of 4 pg/mL and an MIC,, of 16 pg/mL. The monobactam (aztreonam) and the carbapenem (imipenem) both had poor activity. They were active against only 19.4% and 16.6% of the isolates, respectively, and both of them had an MIC,, of 128 pg/mL. All three aminoglycosides that were tested had poor activity. Gentamicin was active against 2.8%; tobramycin, 0%; and amikacin, 13.9% of the isolates, respectively, and all had a high MIC,, of 2128 pg/mL. The 4-quinolone derivative, ciprofloxacin, was active against 83.3% of the isolates. It had a low MIC,, of 1 pg/mL and an MIC,, of 4 pg/mL. Minocycline had a very good activity against the isolates. More than 94.4% of the isolates were susceptible to minocycline. The MIC,, was 1 pg/mL and the MIC,, was 4 pg/ mL. TMP-SMX also had a good activity with an MIC,, of 0.5 Fg/mL, MIC,, of 4 pg/mL, and a susceptible percentage of 83.3%.

Effect of Combining Agents

Two Antimicrobial

The FIC indexes of 50% and 90% (FIG,,, FIG,,) and percentage of synergism for the combinations of

TABLE

1 In Vitro Activities

of 13 Antimicrobial Isolates from Bacteremia Patients

ceftazidime, ciprofloxacin, and amikacin with each other are shown in Table 2. When ceftazidime was combined with amikacin, synergism was found in 28 out of 36 tested strains (77.8%) with an FIG,, of 0.38 and an FIG,, of 0.63. Ceftazidime plus ciprofloxacin showed synergism in 25 out of 36 strains (69.4%) with an FIG,, of 0.5 and an FIG,, of 0.75. When ciprofloxacin was combined with amikacin, only eight strains (22.2%) showed synergism, the FIG,, was 0.75, and the FIG,, was 1.02.

DISCUSSION B. cepacia was first described as a cause of soft rot in onions by Burkholder (1950). Human infections with B. cepacia tend to occur in immunocompromised patients and in those who have acquired the organism from contaminated equipment in hospitals (Goldmann and Klinger 1986; Pallent et al. 1983; Spencer 1995). Because infections caused by this bacterium have increased recently, choosing suitable agents to treat patients with B. cepacia infection is a great concern. From this study, the activity of 13 antimicrobial agents against 36 isolates of B. cepacia has led us to note important differences in the spectrum of susceptibility of this species in comparison with the usual susceptibility of Pseudomonas spp. (Aronoff 1984; Bosso 1991; Prince 1986; Rolston 1987). The isolates in this study showed different genomic patterns by polymerase chain reaction typing, except four isolates clustered in a burn unit demonstrated the same genomic type. These isolates from blood culture showed resistance to various antimicrobial agents, especially p-lactam antibiotics. Among P-lactam antibiotics studied, ceftazidime had the best activity against B. cepacia (86.1% susceptibil-

Agents against 36 B. cepacia

MIC (I*g/mL) Drug

Range

Carbenicillin Piperacillin Moxalactam Ceftazidime Cefepime Aztreonam Imipenem Gentamicin Tobramycin Amikacin Ciprofloxacin Minocycline TMP-SMX”

32->128 2-> 128 4->128 l-128 l->128 4-128 l-128 4->128 8-128 S->128 0.25-64 50.06-S 50.06-S

a Trimethoprim-sulfamethoxazole

50%

(1:19).

>128 128 >128 4 128 64 8 32 64 64 1 1 0.5

90%

% Susceptible

>128 >128 >128 16 >128 128 128 >128 128 >128 4 4 4

47.2 36.1 25.0 86.1 19.4 19.4 16.7 2.8 0.0 13.9 83.2 94.4 83.3

D.C.-T. Lu et al.

190

TABLE

2

of FIC Index and Percentage of Synergy for Antimicrobial Agent Combinations against 36 B. cepacia Isolates from Blood Cultures

Comparison

FIC index

Antimicrobial agent Ceftazidime plus amikacin Ceftazidime plus ciprofloxacin Civrofloxacin vlus amikacin

Range

50%

90%

% Synergy

0.25-l 0.19-l 0.38-1.25

0.38 0.50 0.75

0.63 0.75 1.02

77.8 69.4 22.2

ity). The antipseudomonal penicillin, carbenicillin, and piperacillin and other cephalosporins studied had activity against only some isolates. The resistant rates were higher than those found in other studies (Aronoff and Klinger 1984; Bhakta et al. 1992; Bosso et al. 1991; Ferreira et al. 1985). In addition, the monobactam, aztreonam, and the carbapenem, imipenam, had poor activity. The prevalence of resistance to these two antibiotics also was higher in our isolates than in those found in previous studies from the United States and Europe (Aronoff and Klinger et al. 1984; Bosso et al. 1991; Prince 1986). Because /3-lactam antibiotics are easily available over the counter and have been widely used in Taiwan, it is not surprising that resistance to /3-lactam antibiotics is very common not only in B. cepacia but also in various other bacteria isolated from patients in Taiwan, including E. coli, Enterobncter spp., Haemophilus infuenzae, P. aeruginosa, and Acinetobacter baumannii (Chang et al. 1994, 1995). The high resistance rate to the three aminoglycosides confirmed previous reports (Aronoff and Klinger 1984; Bhakta et al. 1992; Ferreira et al. 1985; Kumar et al. 1989; Prince 1986; Simpson et al. 1994). In this study, ciprofloxacin showed activity against most of our isolates (83.3%). This result differed in other reports on cystic fibrosis patients (Boss0 et al. 1991; Ferreira et al. 1985; Lewin et al. 1993). Ciprofloxacin was widely used in cystic fibrosis patients for repeated P. aerqinosa infection. This fact may explain why strains from cystic fibrosis patients had higher resistant rates. In Taiwan, cystic fibrosis patients are very rare, and this probably is the reason why the susceptibilities to ciprofloxacin are better than those in other studies. Minocycline and TMPSMX also had good activity against our isolates (94.4% and 83.3%, respectively). This is the same as

the results in a previous study on cystic fibrosis patients (Ferreira et al. 1985). Combinations utilizing an antipseudomonal cephalosporin or a new quinolone with an aminoglycoside are frequently used for the treatment of lifethreatening, multiple-drug-resistant, gram-negative bacilli infections, such as P. neruginosa and B. cepacia (Aronoff and Klinger 1984; Bosso et al. 1991; Hallander et al. 1982; Kumar et al. 1989). It has been reported that combining a p-lactam with a quinolone may have a synergistic effect against B. cepacia isolates (Boss0 et al. 1991; Kumar et al. 1989). In the present study, it was demonstrated that the combination of ceftazidime with amikacin or ciprofloxacin could be synergistic against most isolates from blood (77.8% and 69.4%, respectively), but ciprofloxacin combined with amikacin had only additive effects against most isolates, and only a few strains showed synergistic effects. There was no correlation between susceptibility to individual antibiotics of tested strains and results of the combination effect. Combinations could be synergistic for both resistant and susceptible strains. In summary, isolates of B. cepacia from blood of patients with bacteremia in Taiwan were generally resistant to aminoglycosides. Many strains were also resistant to /3-lactam antibiotics, including piperacillin, cefepime, moxalactam, and imipenem. A small percentage was resistant to ceftazidime and ciprofloxacin. However, despite such resistance, combination therapy using amikacin or ciprofloxacin with ceftazidime may still be synergistic against most strains, and ceftazidime in combination with amikatin may be the best choice of combination treatment for B. cepacia infections among the three combinations tested.

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