10.1128/AAC.32.2.276.

1988, 32(2):276. DOI:Antimicrob. Agents Chemother. Y Glupczynski, W Hansen, J Freney and E Yourassowsky antimicrobial agents.denitrificans subsp. xylosoxidans to 24 In vitro susceptibility of Alcaligenes

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Vol. 32, No. 2ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1988, p. 276-2780066-4804/88/020276-03$02.00/0

In Vitro Susceptibility of Alcaligenes denitrificans subsp.xylosoxidans to 24 Antimicrobial Agents

Y. GLUPCZYNSKI,l* W. HANSEN,' J. FRENEY,2 AND E. YOURASSOWSKY'Department of Clinical Microbiology, Brugmann University Hospital, 1020 Brussels, Belgium,' and Laboratoire de

Bacteriologie, Faculte de Medecine A. Carrel, 69372 Lyon Cedex 8, France2

Received 20 August 1987/Accepted 2 November 1987

The in vitro susceptibilities of 37 clinical isolates of Alcaligenes denitrificans subsp. xylosoxidans to 24antimicrobial agents were determined. Imipenem was the only drug with consistent activity (MIC for 90% ofisolates, 2 ,ug/ml). Piperacillin, ticarcillin-clavulanic acid, ceftazidime, and co-trimoxazole were active againstmost strains. All the isolates were resistant to ampicillin, cefazolin, cefuroxime, cefamandole, cefotetan,ceftriaxone, cefotaxime, aztreonam, amdinocillin, and temocillin. Most isolates were resistant to the amino-glycosides tested, including amikacin. Lack of activity was also observed for all new 4-quinolone antimicrobialagents.

Alcaligenes denitrificans subsp. xylosoxidans (11), previ-ously designated Achromobacter xylosoxidans (24), is anaerobic, glucose-nonfermentative, gram-negative bacillus. Itis motile by means of peritrichous flagella and produces acidoxidatively from xylose. A. denitrificans subsp. xylosoxi-dans has been isolated from various environmental andhospital water sources (6, 9, 10) and also from clinicalspecimens, including ear discharge, blood, cerebrospinalfluid, wound, peritoneal fluid, urine, stool, throat, sputum,and tracheal aspirate specimens (21). In many instances ofisolation, this organism is considered as a colonizer and apotential pathogenic role is generally dismissed. However,well-documented infections have been reported on severaloccasions. These include neonatal meningitis (16), ventricu-litis after neurosurgery (22), fatal bacteremia (6, 9, 14, 20),pneumonia (2, 5, 20, 23), urinary tract infections (10),peritonitis (15, 20), aortic root graft infection (18), endocar-ditis (13), and otitis externa (19). Nosocomial outbreaksusually associated with an aqueous source (usually nonbac-teriostatic saline or hemodialysis fluid) have also been de-scribed (14, 20). Infected patients in these outbreaks fre-quently had compromised host defenses or undei'yingdiseases, such as malignancy or renal insufficiency.

Antimicrobial susceptibility data for A. denitrificanssubsp. xylosoxidans are scarce. The multiresistance patternof susceptibility of this organism to several antibiotics hasbeen emphasized in previous reports (2, 3, 5, 6, 10, 15, 20,22, 24). However, in these studies, either the number ofstrains tested was small or the antimicrobial agents selecteddid not include new agents. There are no data regarding thesusceptibility of A. denitrificans subsp. xylosoxidans to4-quinolone derivatives or to many of the modern beta-lactams.A total of 30 strains ofA. denitrificans subsp. xylosoxidans

isolated in Brugmann University Hospital since 1983 wereexamined. Six additional clinical isolates were received fromFrance and were the subject of a recent report (20). Refer-ence strain ATCC 27061 was also included in the study.Confirmation of the identification was performed by bio-chemical tests by the method of Gilardi (8) and by chromato-graphic determination of the fatty acid cell wall profile (4).

* Corresponding author.

The MICs of the antimicrobial agents were determined bya microdilution broth method (1), using commercial plasticmicrodilution trays (Biotest-Serum-Institut, Dreieich, Fed-eral Republic of Germany). Unsupplemented Mueller-Hinton broth was used in the microdilution trays. The trayswere sealed in plastic bags and stored frozen at -20°C for amaximum of 6 weeks. Amoxycillin-clavulanic acid concen-trations used were in a 2:1 proportion. Trays containingimipenem and ticarcillin-clavulanic acid were stored at-70°C. The inocula were prepared as follows. Four to fiveisolated colonies were picked from a 24-h culture on a bloodagar plate and inoculated into 0.5 ml of brain heart infusionbroth, which was then incubated for 4 h at 35°C. The activelygrowing broth culture was then diluted so as to match theturbidity of a McFarland 0.5 standard (ca. 108 CFU/ml).Further transfer of the bacterial suspension (200 ,ul) into ascrew-cap tube containing 20 ml of sterile distilled H20supplemented with 0.02% Tween 80 resulted in a 1/100dilution. Samples (5 ,ul) of inoculum were then depositedwith a multipoint plastic replicator into 0.1 ml of eachsolution in the tray wells. This last 1/20 dilution resulted in afinal cup concentration of approximately 105 CFU/ml. Theinoculated plates were incubated at 35°C for 18 h. The MICwas recorded as the concentration of antimicrobial agentthat totally inhibited growth.The susceptibilities of the A. denitrificans subsp. xylosox-

idans strains to the antimicrobial agents tested are shown inTable 1. Of 24 antibiotics tested, only 5 had some inhibitoryactivity: piperacillin, ticarcillin-clavulanic acid, ceftazidime,imipenem, and trimethoprim-sulfamethoxazole. Among thepenicillins, piperacillin and ticarcillin-clavulanic acid exhib-ited good activity against most of the isolates (91% of thestrains were inhibited by piperacillin at 64 ,ug/ml; 79% wereinhibited by 2 ,ug of ticarcillin-clavulanic acid per ml, and97% were inhibited by 32 ,ug/ml). All the strains weresusceptible to imipenem, whereas none was inhibited bytemocillin, amdinocillin, or aztreonam. Except ceftazidime(81% of isolates susceptible at 8 ,ug/ml), none of the expand-ed-spectrum cephalosporins tested was active against A.denitrificans subsp. xylosoxidans. Confirming the results ofprevious reports (2, 3, 5, 6, 10, 15, 20, 22, 24), all theaminoglycosides (including amikacin) were found to lackactivity against A. denitrificans subsp. xylosoxidans. Tri-

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NOTES 277

TABLE 1. Antimicrobial susceptibilities of 33 clinical isolatesof A. denitrificans subsp. xylosoxidans

Antimicrobial MIC (1Lg/ml)aagent Range 50o 90%

Ampicillin 16-> 128 32 > 128Amoxycillin- 4-> 128 16 64

clavulanic acidPiperacillin 0.5->128 0.5 64Ticarcillin- 0.25-64 1 32

clavulanic acidTemocillin 64->256 >256 >256Amdinocillin 32->256 256 >256Imipenem 0.25-4 0.5 2Cefazolin 16-128 128 128Ceforanide 4-256 256 256Cefamandole 4-128 16 32Cefotetan 16-256 128 256Cefuroxime 32-128 64 64Cefotaxime 8-64 32 64Ceftriaxone 2-128 32 64Ceftazidime 1-64 4 16Trimethoprim- 0.12/2.4 64/1,216 0.12/2.4 16/304

sulfamethoxazoleGentamicin 4-64 64 64Tobramycin 16-64 32 64Netilmicin 8-64 64 64Amikacin 8-128 128 128Norfloxacin 8-64 32 64Pefloxacin 1-64 4 32Ofloxacin 1-64 4 32Ciprofloxacin 1-64 4 16

a 50% and 90%, MIC for 50 and 90% of isolates tested, respectively.

methoprim-sulfamethoxazole, generally considered one ofthe most reliable antimicrobial agents against this species (2,3, 5), was not uniformly active, one-third of our isolatesbeing resistant to this drug. The new 4-quinolone derivativesdisplayed virtually no activity against A. denitrificans subsp.xylosoxidans. This last observation is worth mentioningbecause there is a growing interest in using these compoundsfor the treatment of different types of infections, includingseptic episodes of unknown origin. Of all the agents tested,imipenem alone showed consistent activity (70% of strainsinhibited at 0.5 ,ug/ml; 91% at 2 ,ug/ml).Mechanisms of resistance to beta-lactam antibiotics in

Achromobacter species were studied previously. Levesqueet al. (12) described three different types of constitutive13-lactamases (cephalosporinases) mediated either by non-conjugative plasmids or by genes of chromosomal origin.Fujii et al. (7) characterized a different ,-lactamase (of thepenicillinase type) in 6 of 11 clinical isolates of A. denitrifi-cans subsp. xylosoxidans. Interestingly, Fujii et al. foundthat sulbactam, clavulanic acid, and imipenem had lowaffinities for the ,B-lactamase and that the enzymatic activitywas inhibited by clavulanic acid and sulbactam (7). More-over, a diminished beta-lactam-antibiotic affinity for penicil-lin-binding protein 3 has been suggested and could alsoaccount for the lack of activity of most beta-lactams againstthis species. The uniform pattern of resistance to all newfluoroquinolone compounds and to the aminoglycosides sug-gests that a plasmid-mediated form of resistance is not theonly mechanism involved but rather that the selection ofbacterial mutants could also lead to resistance because ofeither an alteration of the DNA gyrase or defective transportof the agents through the cell envelope.

Overall, the antimicrobial susceptibility profile of A. deni-trificans subsp. xylosoxidans demonstrated that this organ-

ism is resistant to many antibiotics, including the new4-quinolone derivatives, the aminoglycosides, and most ex-panded-spectrum beta-lactams, except imipenem. Therapyagainst this uncommon opportunistic organism should beguided by an awareness of its unique antimicrobial suscep-tibility profile.

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11. Kersters, K., and J. De Ley. 1984. Genus Alcaligenes Castellaniand Chalmers 1919, 936AL, p. 361-373. In N. R. Krieg and J. G.Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1.The Williams & Wilkins Co., Baltimore.

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15. Morrison, A. J., Jr., and K. Boyce IV. 1986. Peritonitis causedby Alcaligenes denitrificans subsp. xylosoxydans: case reportand review of the literature. J. Clin. Microbiol. 24:879-881.

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18. Olson, D. A., and P. D. Hoeprich. 1982. Postoperative infectionof an aortic prosthesis with Achromobacter xylosoxidans. West.J. Med. 136:153-157.

19. Pien, F. D., and H. Y. Higa. 1978. Achromobacter xylosoxidansisolates in Hawaii. J. Clin. Microbiol. 7:239-241.

20. Reverdy, M. E., J. Freney, J. Fleurette, M. Coulet, M. Surgot,

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21. Rubin, S. J., P. A. Granato, and B. L. Wasilauskas. 1985.Glucose-nonfermenting gram-negative bacteria, p. 330-349. InE. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J.Shadomy (ed.), Manual of clinical microbiology, 4th ed. Amer-ican Society for Microbiology, Washington, D.C.

22. Shigeta, S., Y. Yasunaga, K. Honzumi, H. Okamura, R. Ku-mata, and S. Endo. 1978. Cerebral ventriculitis associated withAchromobacter xylosoxidans. J. Clin. Pathol. 31:156-161.

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