Short Communication

Yonago Acta Medica 2017;60:67–70

Environmental Distribution and Drug Susceptibility of Achromobacter 		Xylosoxidans Isolated from Outdoor and Indoor Environments
Sachiko Nakamoto, Misaki Sakamoto, Kana Sugimura, Yuki Honmura, Yuki Yamamoto, Natsumi Goda, Hiroo
Tamaki and Naoto Burioka

Department of Pathobiological Science and Technology, School of Health Science, Tottori University Faculty of Medicine, Yonago 6838503, Japan

ABSTRACT
Achromobacter xylosoxidans is an environmental bacterium with multi-drug resistance. We isolated Achromobacter xylosoxidans and investigated its susceptibility
to 13 drugs. Seventy-eight water samples were collectedfrom rivers and ponds, and 11 samples were swabbed
from residential sinks and baths. Nine strains of Achromobacter xylosoxidans were isolated from the 89 samples. Five strains, including 2 that were sampled fromresidential homes, showed high resistance to multipleaminoglycosides. This indicated that Achromobacter xylosoxidans is widely distributed in various outdoor andindoor environments. Moreover, since these highly resistant bacteria were present in indoor environments, caution should be taken for elderly people living at home.
Furthermore, a careful assessment should be made fordiagnosing and treating compromised hosts.

chronic and refractory.2
We previously reported a rare case whereby astrain classified as multidrug-resistant Pseudomonas
aeruginosa (P. aeruginosa) was further identified as
drug-susceptible P. aeruginosa and multidrug-resistant
A. xylosoxidans.3 Based on this experience, we becameaware of the importance of surveying the environmental
distribution and drug-susceptibility of A. xylosoxidans
existing in nature. We devised an isolation procedure
suited for the properties of A. xylosoxidans, isolated itfrom various environmental samples, and investigatedthe environmental distribution and drug susceptibilityof the isolates. The results suggest that more attention tothis species is necessary.
MATERIALS AND METHODS
Culture media

Heart Infusion broth including Vancomycin and Aztreonam (HIVA) broth was prepared by adding 32 μg/mL ofvancomycin and aztreonam to heart infusion broth andwas used as an enrichment medium. Xylose-Macconkey
agar including Vancomycin and Aztreonam (X-MacVA)
agar plate was prepared by adding 1% xylose (X) and20 μg/mL of vancomycin and aztreonam to MacConkey
agar and was used as an isolation medium.2

Achromobacter xylosoxidans; environmental distribution; environmental resident bacterium;
multi-drug resistance; opportunistic infection
Key words

Achromobacter xylosoxidans (A. xylosoxidans) is a catalase-positive, oxidase-positive, and glucose-non-fermenting Gram-negative rod. Non-fermenting Gram-negativerods have similar properties and are difficult to differentiate.1 Furthermore, since A. xylosoxidans is an environmental pathogen, it is usually overlooked in routine
clinical examinations. A. xylosoxidans is an opportunistic bacterium with multi-drug resistance and infections
are known to cause clinical difficulties since it becomes

Method for isolation from the environment

Samples were collected from rivers, ponds, irrigation
ditches, and residential homes within an area of 20 kmof the Yumigahama Peninsula in the west area of Tottori
Prefecture, Japan. The samples were either collected involumes of 15 mL from outdoor water sources or obtained from drains of residential sinks and baths using
sterilized swabs, which was subsequently suspended
with 15 mL of distilled water. These samples were centrifuged at 4,000 × g for 15 minutes, and the sediment
was cultured in HIVA broth 72 h at 35 °C. Cultured
bacterial fluid was cultured on X-MacVA agar 72 h at
35 °C. The colonies that formed on the agar were typed,
and the number of colony types was regarded as thenumber of isolated colonies. A typical colony in eachcolony type was subjected to differentiation tests. Of
the bacteria determined to be Gram-negative rods, only

Corresponding author: Sachiko Nakamoto, PhD
naka827@med.tottori-u.ac.jp
Received 2016 December 2
Accepted 2017 January 18
Abbreviations: A. xylosoxidans, Achromobacter xylosoxidans;AG, aminoglycoside; AMK, amikacin; AZT, aztreonam; CFPM,
cefepime; CPFX, ciprofloxacin; GM, gentamicin; HIVA, Heart
Infusion broth including Vancomycin and Aztreonam; IPM, imipenem; KM, kanamycin; MIC, minimal inhibition concentration;
NEO, neomycin; NET, netilmicin; PIPC, piperacillin; P. aeruginosa, Pseudomonas aeruginosa; SM, streptomycin; SPCT, spectinomycin; TOB, tobramycin; X-MacVA, Xylose-Macconkey agar
including Vancomycin and Aztreonam

67

S. Nakamoto et al.

Drug susceptibility tests

those that were positive for catalase and oxidase tests
were further tested for their non-fermentability, growthability in stab culture, citrate utilization ability, andnitrate-reducing ability. These differential test-positivebacteria were identified using ID test NF-18 (Nissui
Pharmaceutical, Tokyo, Japan), a simple identification
kit for glucose-non-fermenting Gram-negative rods. A
final identification was made according to a 16S rDNA
base sequence.4

The minimal inhibition concentration (MIC) was determined by the agar plate dilution method using theClinical and Laboratory Standard Institute procedure.
The following drugs were tested: piperacillin (PIPC),
cefepime (CFPM), aztreonam (AZT), imipenem (IPM),
ciprofloxacin (CPFX), amikacin (AMK), gentamicin(GM), streptomycin (SM), kanamycin (KM), netilmicin(NET), spectinomycin (SPCT), neomycin (NEO) andtobramycin (TOB).

Bacterial strains

Nine environmentally isolated A. xylosoxidans and A.
xylosoxidans 600S strains3 were used.

RESULTS
Table 1 shows the isolation state of A. xylosoxidans fromvarious samples and the MICs of 13 antibacterial drugs

Table 1. The state of isolation of Achromobacter xylosoxidans from environmental materials and the MICs of
13 antibacterial drugs against environmentally isolated Achromobacter xylosoxidans
Number of colonies
Antibiotics MIC (µg/mL)
AGs
Samples
Isolated Differen- Identified Identifiednnnname PIPC CFPM AZT IMP CPFX AMK GM SPCT SM KM TOB NET NEO
tiated n
W1
0.5
8 128
1
1
8
4 128 ≥ 128
8
4
4
16
W2
0.25
8 128
1
2 128
32 12816 128
8
4 128
W3
0.5
4 128
1
1
4
2 ≥ 128
64
8
2
2
8
Water
57
45
6
78
W4
1
32
64
4
2 ≥ 128 ≥ 128 ≥ 128 ≥ 128 128
64 128 ≥ 128
W5
0.25
8 128
1
2
4
2 128
32
8
2
2
8
W6
16
32 1281
46464 ≥ 128 ≥ 128 128
16
32 128S1
0.5
32 1282
8 ≥ 128 128 ≥ 128 ≥ 128 ≥ 128
64 128 ≥ 128
Swabs
8
8
3
S2
0.5
32
64
2
2 ≥ 128 ≥ 128 ≥ 128 128
64
64 128 ≥ 128
11
S3
0.25
8 128
1
1
4
2 128
64
8
2
4
8
Achromobacter xylosoxidans 600S
0.5
128 1281
16 ≥ 128 ≥ 128 ≥ 128 ≥ 128 ≥ 128 ≥ 128 ≥ 128 ≥ 128
Samples: water; rivers, ponds, irrigation ditches, swabs; the drains of residential sinks and baths.
Drugs:AG; aminoglycoside; AMK, amikacin; AZT, aztreonam; CFPM, cefepime; CPFX, ciprofloxacin; GM, gentamicin; IMP, imipenem; KM, kanamycin; NEO, neomycin; NET, netilmicin; PIPC, piperacillin; SPCT, spectinomycin; SM, streptomycin; TOB, tobramycin.
MIC, minimal inhibition concentration.

Sakaiminato city	
42	
42
41	

30	
32	
32
31	
31
33	
28	
29	
25	
25
64	
64
27	
27
23	
23
63	
63
22	
22
24	
24
86	
84	
84
66	
66
83	
83
71	
72	
58	
58
34	
34
26	
26
85	
85
68	
68
67	
 651
67
70	
69	
69
36	
34	
35	
37	
62	
62
74	
39	
39
38	
38
3
81	
73	
40	
40
61	
76	
75	
59	
88	
88
87	
89	
18	
10	
11	
19	
49	
20	
19
20
9	
 12	
12	
15	
16	
49	
49
50	
50
44	
51	
 52	
44
43
43	
8	
14	
51	
 14
Hiezu son	
46	
46
45	
 55	
45
17	
2	
53	
7	
82	
77	
60	
78	
54	
21	
 3	
79	
81	
80	
56	
6	
57	
13	
5	
 4	
Yonago city	

5 km	

68

Fig. 1. Map of the sampling
points and A. xylosoxidans
isolation. Map of the Yumigahama Peninsula extending
from Yonago City to Sakaiminato City, Tottori Prefecture,
Japan. The sampling points ofA. xylosoxidans-positive and-negative materials are indicatand, respectively.
ed as
Sample numbers are shown inthe symbols. A. xylosoxidans,Achromobacter xylosoxidans.

Distribution and drug susceptibility of A. xylosoxidans

for the isolated A. xylosoxidans. Figure 1 shows the mapof the sampling points and A. xylosoxidans isolation.

menting Gram-negative Pseudomonas and A. xylosoxidans. Pin-point colonies, in particular, were considered
to be an important criterion for detecting A. xylosoxidans.2 Although the differential tests are effective for
confirming major properties of bacteria, they are not able
to precisely determine the species. Therefore, a definitive
diagnosis based on the 16S rDNA base sequence is important. Since Pseudomonas and A. xylosoxidans have
similar properties, they are often misidentified or overlooked due to differences in their growth ability.3 Proper
knowledge regarding the properties of A. xylosoxidans
is necessary to avoid misdiagnoses in routine examinations.
Amoureux et al.2 isolated A. xylosoxidans in a wide
area and reported identification rates of 28% and 12%
from samples collected from the outdoor and indoor environments, respectively. In our study, the identification
rate for samples collected from indoor residences washigh at approximately 30% for bath drains. In contrast,
the identification rate was relatively low for outdoor water sources, which may have been due to the small sample volume of 15 mL. In addition, A. xylosoxidans has abiofilm-forming ability.5 Biofilm can form in the drainsof sinks and baths, which may have led to the high bacterial concentrations in the swab samples. Considering
these points, we deem our bacterial isolation procedure
appropriate. Furthermore, the wide distribution of thisspecies in indoor environments was demonstrated.
The 9 strains of A. xylosoxidans isolated from thevarious environments resembled the common multidrug resistance pattern,6 and some drugs showed forhigh MICs, AGs in particular. The MICs of the 8 AGs
for the A. xylosoxidans 600S strain 3 were ≥ 128 μg/
mL, and 5 (W2, W4, W6, S1, S2) strains were isolated
from both the outdoor and indoor samples. Since A.
xylosoxidans can be mistaken for P. aeruginosa, care isnecessary when selecting drugs for Gram-negative rod
infections. In addition, this species is drug-resistant andis distributed in wet areas. The latter feature may cause
problems since the bacteria may accumulate in the sinksof residential kitchens and baths. Currently, residencesof elderly people in need of medical and nursing careare increasingly transitioning from medical facilities toresidential homes, especially care homes for the elderly.
Sufficient attention is necessary for vulnerable elderly
people and compromised hosts who live at home.

State of isolation

Of the 89 samples, 9 strains of A. xylosoxidans wereidentified. The identification rate was 10.1% (6 strains(7.7%) in outdoor water samples and 3 strains (27.3%)
in swab samples from residential homes) (Table 1). The
species was identified in a wide sampling area (Fig. 1).
Evaluation of the isolation and identification methods

The number of isolated colonies that formed on X-MacVA agar was low, at around 70% per sample. The differentiation tests showed that 81.5% of the isolated colonies
were positive for oxidase and catalase. Based on thebacterial identification tests, 10.1% of the colonies wereidentified as A. xylosoxidans. Furthermore, according to
the isolation and identification protocol, of the red colonies that formed on X-MacVA agar, the large colonies
were identified as Pseudomonas fluorescens and the pinpoint colonies as A. xylosoxidans. Finally, A. xylosoxidans was confirmed using the 16 rDNA base sequence.
Drug susceptibility tests

The strains were susceptible to β-lactams (PIPC, IMP,
CFPM) except for monobactam (AZT), and new quinolone (CPFX). Of the 8 aminoglycosides (AGs; AMK,
GM, SPCT, SM, KM, TOB, NET, NEO) that were
tested, the MIC of SPCT was 128~ ≥ 128 μg/mL for
all strains. However, drugs other than SPCT and SM
had low MICs for 4 strains (W2, W3, W5, S3) and highMICs for 5 strains (W1, W4, W6, S1, S2), which closely
resembled the resistance pattern of the A. xylosoxidans
600S strain. Of the 5 multidrug-resistant strains (W2,
W4, W6, S1, S2) in all, 2 were isolated from residential
homes (S1, S2) (Table 1).
DISCUSSION
Since A. xylosoxidans is an environmental bacterium, it
is rarely detected in routine clinical examinations, and,
if it is detected, its differentiation and identification isdifficult as it is often mistaken for P. aeruginosa. A. xylosoxidans has multidrug resistance and erroneous identification of the bacteria will lead to inappropriate selection of drugs. Therefore, information concerning the
distribution of this species and the drug susceptibility ofbacteria distributed in the environment is important.
The enrichment and isolation media used in thisstudy, which included AZT resistance and xylose degradability, showed high selectivity. Red colonies thatformed on X-MacVA agar were likely glucose-non-fer-

The authors declare no conflict of interest.

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S. Nakamoto et al.
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