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113（ 33 ）〈Brief Report〉

Properties of Achromobacter xylosoxidans highly
resistant to aminoglycoside antibiotics
SACHIKO NAKAMOTO1, NATSUMI GODA1, TATSUYA HAYABUCHI2,HIROO TAMAKI1, AYAMI ISHIDA1, AYAKA SUZUKI1, KAORI NAKANO1,SHOKO YUI1, YUTO KATSUMATA1, YUKI YAMAGAMI1,NAOTO BURIOKA1, HIROKI CHIKUMI3 and EIJI SHIMIZU4
1

Department of Pathobiological Science and Technology,
School of Health Science, Tottori University Faculty of Medicine
2
Matsue City Hospital3
Department of Infection Control and Prevention, Tottori University Hospital
4
Division of Medical Oncology and Molecular Respirology,
Department of Multidisciplinary Internal Medicine, School of Medicine,
Tottori University Faculty of Medicine(Received for publication September 10, 2015)

We herein discovered a highly resistant clinical isolate of Pseudomonas aeruginosawith MICs to amikacin, gentamicin, and arbekacin of 128 μg/mL or higher in a drug
sensitivity survey of 92 strains isolated from the specimens of Yoka hospital patients
between January 2009 and October 2010, and Achromobacter xylosoxidans wasseparated from this P. aeruginosa isolate. The sensitivity of this bacterium to 29
antibiotics was investigated. The MICs of this A. xylosoxidans strain to 9
aminoglycoside antibiotics were: amikacin, gentamicin, arbekacin, streptomycin,
kanamycin, neomycin, and spectinomycin, 1,024 μg/mL or ≧1,024 μg/mL;
netilmicin, 512 μg/mL; and tobramycin, 256 μg/mL. This strain was also resistant todibekacin. This aminoglycoside antibiotic resistant phenotype is very rare, and weare the ﬁrst report the emergence of A. xylosoxidans with this characteristic.

In the present study, we investigated the antibiotic sensitivities of 92 clinical isolates of
Pseudomonas aeruginosa collected from Yoka hospital between January 2009 and October 2010.
The agar plate dilution and disc diffusion methods speciﬁed by the Clinical and Laboratory Standards Institute were employed for the antibiotic sensitivity test. Ninety-two strains were tested,
and the agents used were 16 antibiotics to treat P. aeruginosa infections: piperacillin, piperacillin-114（ 34 ）THE JAPANESE JOURNAL OF ANTIBIOTICS

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tazobactam, ceftazidime, cefepime, cefozopran, aztreonam, imipenem, meropenem, biapenem,doripenem, ciproﬂoxacin, levoﬂoxacin, pazuﬂoxacin, amikacin, gentamicin, and arbekacin. The
MIC distribution of these 16 antibiotics is shown in Table 1. Their antimicrobial activities wereretained at relatively high levels. Of the aminoglycoside antibiotics tested, there were only 4
strains for which the MIC values of gentamicin and amikacin were 128 μg/mL or higher, and only
one strain for which the MIC values of all 3 antibiotics including arbekacin were ＞128 μg/mL.
We focused on the P. aeruginosa strain that showed high-level resistance to the 3 antibiotics withthe high MIC value of arbekacin not indicated for treatment against P. aeruginosa. When thisstrain was subjected to disc diffusion tests with aminoglycoside antibiotics, bacteria grew in thegrowth inhibition ring. At that time, we detected contaminating P. aeruginosa. Hence, we employed the disk diffusion method to check for other P. aeruginosa (bacterial) strains. However, noother strains were identiﬁed. Thus, the P. aeruginosa strain was subjected to re-isolation on Drigalski medium, which resulted in the formation of large and pin-point colonies. Based on biochemical tests, the ID test, NF-18 simple identiﬁcation kit ʻNissuiʼ, and the 16S rRNA basesequence1), the large colony was identiﬁed as P. aeruginosa, and the pin-point colony as
Achromobacter xylosoxidans. The clinical isolate of P. aeruginosa and separated strains were
designated as the P. aeruginosa 60 and 600 L strains and A. xylosoxidans 600S strain, respectively.
The sensitivities of these 3 strains to streptomycin, kanamycin, netilmicin, spectinomycin,neomycin, tobramycin, dibekacin, chloramphenicol, tetracycline, erythromycin, vancomycin,oxacillin, and sulfamethoxazole-trimethoprim were investigated, in addition to the 16 antibiotics
described above. The MIC values for and antibiotic sensitivity phenotypes of the 3 strains on thedisc diffusion tests are shown in Table 2. The high MIC values of cefepime, cefozopran, aztreonam, and the 3 aminoglycoside antibiotics for the P. aeruginosa 60 strain were derived from theA. xylosoxidans 600S strain. This multidrug resistance pattern was consistent with the previously
reported pattern for A. xylosoxidans2,3). The MIC values of the aminoglycoside antibiotics were:
tobramycin, 256 μg/mL; netilmicin, 512 μg/mL; and the 7 other antibiotics, 1,024 μg/mL or
higher. The aminoglycoside resistance of A. xylosoxidans is well-known, whereas the MIC value
varies and was previously reported to be approximately 128 μg/mL4). Non-fermentative Gramnegative rods have RND-type efﬂux pumps5,6,7). In 2013, BADOR et al. reported that the drug
efﬂux pump, AxyXY-OprZ, is involved in the aminoglycoside resistance of A. xylosoxidans, forwhich the MIC values of amikacin and gentamicin are greater than 256 μg/mL8). The MIC valuesof streptomycin and spectinomycin were greater (1,024 μg/mL or higher) for the related bacterium, Burkholderia pseudomallei 9). The MIC value (1,024 μg/mL) for the A. xylosoxidans 600S
strain was high and equivalent to those for related bacteria. Based on the phylogenetic treeof bacteria with drug efﬂux pumps10) and previous ﬁndings by BADOR et al.8), A. xylosoxidans,Pseudomonas, and Burkholderia were found to be very closely related. Therefore, the high MIC
value for the A. xylosoxidans 600S strain suggests the involvement of drug efﬂux pumps.

Table 1. MIC distribution of 16 antibiotics for 92 clinical isolates of Pseudomonas aeruginosa

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Table 2. MICs and sensitivity phenotypes of 29 antibiotics for Pseudomonas aeruginosa 60strain, Pseudomonas aeruginosa 600 L strain and Achromobacter xylosoxidans 600S strain

However, no strain with high MIC values for many types of aminoglycoside antibiotics has previously been reported. This resistance pattern of the A. xylosoxidans 600S stain may be rare, andseveral resistance mechanisms, such as acquired 16S rRNA methylase genes11) and aacA4 gene
cassettes12), including drug efﬂux pumps may be involved.
A. xylosoxidans is an aerobic, Gram-negative, non-fermentative rod that is indigenous13,14). It
is rarely detected in routine tests, and issues associated with misidentiﬁcation as P. aeruginosa on

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69―2117（ 37 ）microbial identiﬁcation and drug resistance information are not widely shared. The characteristics
of aminoglycoside antibiotics and the disc diffusion method allowed us to identify mixed bacteria. The prevention of misjudgments in routine tests is important, for which it may be necessary
to investigate simple methods in order to isolate/differentiate mixed bacteria from related bacteria. Moreover, it is unclear whether the A. xylosoxidans 600S strain is involved in the route of
mixing and infection. However, it is important to note that the A. xylosoxidans 600S strain wasmixed with a clinical isolate of P. aeruginosa. The bacterium A. xylosoxidans has been isolated
from dialysates, incubators, and antiseptic solutions in hospitals at familiar sites15,16,17). It is a newaerobic, Gram-negative rod that requires attention, particularly in infection-sensitive individuals.
Conﬂicts of interest
None to declare.

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