Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Identification of Stenotrophomonas maltophilia strains

isolated from environmental and clinical samples: a rapid
and efficient procedure
C. Pinot, A. Deredjian, S. Nazaret, E. Brothier, B. Cournoyer, C. Segonds and S. Favre-Bonte
Universite de Lyon, France Research Group on Bacterial Opportunistic Pathogens and Environment, UMR 5557 Ecologie Microbienne, CNRS,
VetAgro Sup and Universite Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France

Keywords
environment, identification,
Stenotrophomonas maltophilia.
Correspondence
Sabine Favre-Bonte, UMR CNRS 5557
Ecologie Microbienne, Universite Lyon 1, 43
bd du 11Novembre 1918, F-69622
Villeurbanne Cedex, France.
E-mail: sabine.favre-bonte@univ-lyon1.fr

2011 0174: received 28 January 2011,

revised 3 July 2011 and accepted 30 July
2011
doi:10.1111/j.1365-2672.2011.05120.x

Abstract
Aims: Aim of the study is to identify accurately Stenotrophomonas maltophilia
isolates recovered from environmental and clinical samples.
Methods and Results: Recovery of Sten. maltophilia-like isolates from soil samples using the vancomycin, imipenem, amphotericin B (VIA) selective agar
medium enabled distinction of various morphotype colonies. A set of soil and
clinical isolates was tested for species identification using different methods.
16S rDNA analyses showed the dark green with a blue halo morphotype to be
typical Sten. maltophilia strains. The API-20NE, Vitek-2 and Biolog phenotypic
analyses typically used for the identification of clinical isolates did not perform
well on these soil isolates. The species-specific PCR screening targeting
Sten. maltophilia 23S rDNA and the multiplex smeD ggpS PCR, differentiating
Sten. maltophilia from Stenotrophomonas rhizophila, were tested for improvement of these identification schemes. The latter multiplex PCR identified all
isolates tested in this study, whatever be their origin.
Conclusions: Isolation on VIA medium and confirmation of Sten. maltophilia
species membership by smeD PCR is proposed to identify environmental and
clinical isolates of Sten. maltophilia.
Significance and Impact of the Study: The proposed approach enables isolation
and identification of Sten. maltophilia from different environments in an easy
and rapid way. This approach will be useful to accurately manage studies on
the abundance and distribution of Sten. maltophilia in hospital and nonhospital
environments.

Introduction
The now known Stenotrophomonas maltophilia species was
originally classified as a member of the genus Pseudomonas
in 1961 (Hugh and Ryschenkow 1961) and then Xanthomonas in 1983, finally coming to rest in Stenotrophomonas
in 1993. The genus Stenotrophomonas belongs to the
c-proteobacteria and includes ten species: Sten. maltophilia
(Palleroni and Bradbury 1993), Stenotrophomonas nitritireducens (Finkmann et al. 2000), Stenotrophomonas acidaminiphila (Assih et al. 2002), Stenotrophomonas rhizophila
(Wolf et al. 2002), Stenotrophomonas koreensis (Yang et al.
2006), Stenotrophomonas humi and Stenotrophomonas

terrae (Heylen et al. 2007), Stenotrophomonas chelatiphaga

(Kaparullina et al. 2009), Stenotrophomonas ginsengisoli
(Kim et al. 2010) and Stenotrophomonas panacihumi
(Yi et al. 2010). These species occur ubiquitously in the
environment and have been isolated from a wide range of
sources including water, sediment, soil, rhizosphere and
plant tissues (Ryan et al. 2009). In contrast with the other
members of the genus, Sten. maltophilia can cause infections in cystic fibrosis (CF), immuno-compromised and
burned individuals.
Over the last two decades, the number of Sten. maltophilia isolates from hospitals has increased significantly. A
high mortality rate, up to 375%, was associated with

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C. Pinot et al.

Sten. maltophilia infection (Falagas et al. 2009). The

increasing occurrence of Sten. maltophilia infections is
partly because of the intrinsic resistance of Sten. maltophilia to many antibiotics, making selection of optimal
therapy difficult. Other factors could also have contributed at making Sten. maltophilia an emerging opportunistic
pathogens of increasing health concerns such as its ability
at secreting extracellular enzymes (Denton and Kerr
1998), to adhere and form biofilms (Di Bonaventura et al.
2007) and to stimulate factors involved in the inflammatory process (Waters et al. 2007).
The actual knowledge on the diversity and reorganization of the Stenotrophomonas genus requires a reevaluation of the Sten. maltophilia identification schemes.
Furthermore, a simple and reliable identification method
is needed to improve Sten. maltophilia diagnosis in a clinical context and also to investigate its prevalence outside
hospitals.
Isolation of Sten. maltophilia on agar plates was initially
performed on XMSM as described for isolation of
Xanthomonas maltophilia from bulk soil and plant
rhizosphere (Juhnke and des Jardin 1989). This culture
medium contains at least six antibacterial agents, two
antifungal agents, maltose and an indicator system (bromothymol blue) allowing the screening of orange colonies
with a yellow halo after 2 days of incubation. XMSM
preparation is complex, time-consuming, expensive and
was not found highly selective or specific enough for the
isolation of Sten. maltophilia recovered from the environment (Kerr et al. 1996). The vancomycin, imipenem,
amphotericin B (VIA) medium was more recently proposed for the isolation of Sten. maltophilia from clinical
and environmental samples (Kerr et al. 1996). VIA contains imipenem as a selective agent for Sten. maltophilia.
Furthermore, to inhibit the growth of Enterococcus faecium, another inherently imipenem resistant species, it
also includes vancomycin. Fungal growth is hampered by
the addition of amphotericin B. Mannitol, and bromothymol blue was added to VIA agar allowing distinction
between mannitol fermenting (i.e. vancomycin-resistant
Ent. faecium able to grow on VIA agar) and nonfermenting micro-organisms like Sten. maltophilia that do not
produce acid from mannitol and thus appears as green
colonies with a blue halo. VIA medium was demonstrated
to be less inhibitory to Sten. maltophilia and more
selective than XMSM in preventing the growth of other
bacteria like maltose fermenters. VIA medium has been
used to culture Sten. maltophilia from bottled water
(Wilkinson and Kerr 1998), salads (Qureshi et al. 2005),
as well as sputa (Denton et al. 2000), hospital (Denton
et al. 2003) and domestic environments (Denton et al.
1998). However, these studies did not report on
the specificity of the medium when used to select
1186

Sten. maltophilia for the screening of environmental samples. Taxonomic characterization of Sten. maltophilia isolates is mostly performed using a biochemical approach
(i.e.: Bollet et al. 1995; Denton and Kerr 1998; Qureshi
et al. 2005). Sometimes a molecular approach based on
the 23S rRNA gene amplification (Whitby et al. 2000)
completes the species identification scheme. This speciesspecific PCR (SS-PCR) approach was developed to overcome the problems associated with definitive identification. This PCR presented sensitivity and specificity of
100% for clinical strains or strains isolated from the environmental clinical area (Whitby et al. 2000; Giordano
et al. 2006; Foster et al. 2008a). However, there is a lack
of studies describing isolation and identification of
Sten. maltophilia isolated from environmental samples.
In the present paper, we reported on the use of the
VIA medium and 16S rDNA sequencing to select and
identify Sten. maltophilia-like isolates from soil samples.
We further applied various biochemical methods classically used in a clinical context and molecular-based
approaches reported from the literature to evaluate their
identification efficiencies. The accuracy of these methods
was also verified on a set of clinical isolates.
Materials and methods
Experimental sites
Soils were sampled from different locations in various areas
in France including agricultural sites in Burgundy and in
Ile de France, experimental waste water irrigation sites in
France and two sites in Tunisia; and agricultural sites
amended with organic matter in Burkina Faso. A total of
104 soil samples were collected from the upper layer (05,
010 or 020 cm), sieved (2 mm), stored at room temperature and analysed within 2 weeks. They were collected
during various campaigns between 2005 and 2008. Sites
and sampling strategy are briefly described in Table 1.
Isolation of Sten. maltophilia-like isolates from soil
samples
One hundred and four different soil samples were tested
for isolation of Sten. maltophilia-like isolates. Briefly, soil
samples (5 g) were blended with 50 ml of a saline solution (NaCl 08%) for 90 s in a Waring Blender (Eberbach
Corporation, Ann Arbor, MI, USA). Homogenized soil
suspensions were serially diluted in sterile saline solution
and dilutions were spread on VIA plates (Kerr et al.
1996) supplemented with cycloheximide (200 mg l)1) to
inhibit fungi development. Plates were incubated 2448 h
at 28C. Four morphotypes were obtained on VIA agar
plates including fermenting bacteria producing yellow or

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C. Pinot et al.

Sten. maltophilia identification

Table 1 Site and soil description (total soil samples = 104)

Sites

Land
use

Clay Silt
(%) (%)

Sand pH
Number of
(%) water samples

France
Feucherolles
Pierrelaye
Balot
Venarey les laumes
Is sur Tille
Bourberain
Dompierre
en Morvan
Ruffey les Echirey
Marcilly Ogny
Commarin
Echevronne
Morey St Denis
St Aubin
Enesad

Wheat
Maize
Barley
Grassland
Rape
Bulk soil
Grassland

19
8
29
44
44
39
18

75
13
69
52
48
58
46

6
79
2
4
8
3
36

69
82
58
68
81
7
56

15
21
1
1
1
1
1

Rape
Triticale
Bulk soil
Cereal
Vineyard
Vineyard
Bulk soil

47
36
42
45
33
35
35

39
48
50
41
43
42
36

14
16
8
14
24
23
29

81
79
71
79
83
82
8

1
1
1
1
1
1
1

Burkina Faso
Tabtenga
Toudoubweogo
Zagtouli
Yagma NZ
Yagma Eapit

Bulk soil 11
Bulk soil
9
Sorghum nd
Sorghum
9
Sorghum 12

165
20
nd
30
18

693
71
nd
61
70

61
75
77
74
72

6
6
6
6
2

Tunisia
Souhil
Beni Khiar
Nabeul

Citrus
Orange
trees
Orange
trees

12
8

8
9

80
73

82
84

10
10

14

77

67

10

Each sample is a composite sampling made of 10 subsamples except

for Pierrelaye and the Tunisian sites. Samples from Pierrelaye were
obtained from a 3 km transect corresponding to a gradient of heavy
metal contamination. Samples from Souhil and Beni Khiar were
obtained from 10 sub-sites over a 2 km2 area. Other samples were
obtained at the field scale.

green colonies, and nonfermenting bacteria producing

either dark green colonies with a blue halo or blue colonies without a halo. Representatives of each morphotype
were conserved in 25% glycerol at )80C for further analyses Twenty-three isolates belonging to the four morphotypes were selected for this study (Table 2).
Bacterial strains
Reference strains of the genus Stenotrophomonas were
included in this study. The strains ATCC13637 and
DSM14405 were used as reference isolates of the
Sten. maltophilia and the Sten. rhizophila species, respectively. Stenotrophomonas terrae LMG23958 and Sten. humi
LMG23959
(Heylen
et al.
2007),
Sten. koreensis
LMG23369 (Yang et al. 2006), Sten. nitritireducens
LMG22074 (Finkmann et al. 2000) and Sten. acidamini-

phila AMX19 (Assih et al. 2002) were tested for their

ability at growing on VIA and for testing the specificity of
the PCR screenings.
A set of 28 clinical isolates from CF individuals and
infected patients hospitalized in intensive care, surgery or
ophthalmology units was investigated. These Sten. maltophilia-like isolates originated from CF sputum, tracheal
aspirate, broncho-alveolar washes, eye and wound infection. Clinical isolates were collected from 16 November
2006 to 4 January 2007 and from 19 March to 23 May
2008 in the university hospital of Toulouse, France. All
isolates were tested for oxidase and identified by use of
the Vitek2 GN cards (bioMerieux, Marcy lEtoile, France).
For CF sputa, a selective medium (Mueller-Hinton agar
containing 100 mg l)1 imipenem) was used for the recovery of Sten. maltophilia. Bacterial isolates were kept in
25% glycerol at )80C. To be studied, isolates were
seeded on TSA agar and incubated at 28 or 37C depending on their origin.
16S rDNA analysis
Bacterial DNA was extracted according to Pitcher et al.
(1989). The 16S rDNA was amplified using Taq polymerase (QBiogen, Illkirch, France), with 02 lM of 8F and
1492R primers in a 50 ll reaction (8F: AGAGTTTGATCMTGGCTCAG, 1492R: GGTTACCTTGTTACGACTT) (Weisburg et al. 1991). A negative control
(sterile water) and a positive control (genomic DNA of
Sten. maltophilia ATCC13637) were included in amplification runs. The optimal thermocycling conditions were as
follow: 95C hold for 15 min, 35 cycles of 94C for
1 min, 52C for 15 min and 70C for 15 min, followed
by 5 min at 70C. PCR products were visualized by electrophoresis using a 1% agarose gel and staining with ethidium bromide. The amplified DNA was purified using
the QIAquick PCR Purification kit (Qiagen, Courtaboeuf,
France) according to manufacturers instructions. The
concentration of the purified DNA products was estimated by comparison of the stained PCR products with
the Low DNA Mass Ladder (Invitrogen, Carlsbad, CA,
France), after electrophoresis.
Sequencing was performed on the 16S rDNA PCR
products using primers 16S-515F, 16S-906F and 16S-907R
primers (515F: GTGCCAGCMGCCGCGGTAA, 906F:
GAAACTTAAAKGAATTG and 907R: CCGTCAATTCCTTTRAGTTT) (Weisburg et al. 1991). Sequencing was performed by Beckman Coulter Genomics (Meylan, France).
The 16S rDNA sequences were compared with those
available in the NCBI GenBank Database using BlastN
at http://blast.ncbi.nlm.nih.gov/Blast.cgi. The 16S rDNA
sequence results were used as a reference for refining the
Sten. maltophilia identification scheme.

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Table 2 Identification of study isolates according to the methods used

Isolates

16S-rRNA

API-20NE

Vitek-2

Biolog Gram-negative

Multiplex PCR

BPOE 5100
BPOE 5101
BPOE 5102
BPOE 5103
BPOE 5104
BPOE 5105
BPOE 5106
BPOE 5107
BPOE 5108
BPOE 5109
BPOE 5110
BPOE 5111
BPOE 5112
BPOE 5113
BPOE 5114
BPOE 5115
BPOE 5116
BPOE 5117
BPOE 5118
BPOE 5119
BPOE 5120
BPOE 5121
BPOE 5122
Clinical

Stenotrophomonas maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Stenotrophomonas rhizophila
Sten. rhizophila
Sten. rhizophila
Sten. rhizophila
Sten. rhizophila
Sten. rhizophila
Variovorax sp.
Dyella japonica
Sten. maltophilia

Burkholderia cepacia
B. cepacia
Pseudomonas luteola
Ps. luteola
Ps. luteola
B. cepacia
Ps. luteola
Ps. luteola
Ps. luteola
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Ps. luteola
Sten. maltophilia
Ps. luteola
Ps. luteola
Sten. maltophilia
Ps. luteola
Sten. maltophilia
Sten. maltophilia
Ps. luteola
Sten. maltophilia
Sten. maltophilia

Pseudomonas aeruginosa
Sten. maltophilia
Ps. fluorescens*
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Brevundimonas diminuta
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Ps. fluorescens
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
B. diminuta*
no id
Aeromonas salmonicida
Sten. maltophilia

Pseudomonas fluorescens
Sten. maltophilia
Pseudomonas syringae
Sten. maltophilia
Ps. fluorescens
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Sten. maltophilia
Flavobacterium tirrenicum
Pseudomonas synxantha
Sten. maltophilia
Ps. fluorescens
Sten. maltophilia
Sten. maltophilia
Pseudomonas
Sten. maltophilia
Sten. maltophilia
Ps. synxantha
Sten. maltophilia
Sten. maltophilia

smeD+
smeD+
smeD+
smeD+
smeD+
smeD+
smeD+
smeD+
smeD+
smeD+
smeD+
smeD+
smeD+
smeD+
smeD+
ggpS+
ggpS+
ggpS+
ggpS+
ggpS+
ggpS+
ggpS+
smeD+
smeD+

no id, no identification; B. cepacia: Burkholderia cepacia complex.

*Low discrimination.
Low identification.
One clinical isolate was misidentified by Biolog as it was identified as Xanthomonas campestris.

Biochemical characterization
API 20NE
The API test kit (bioMerieux) consists of enzymatic and
carbon compound assimilation test elements (20 tests).
For each isolate, inoculation of API 20NE strips was
performed as recommended by the manufacturer (bioMerieux). After incubation for 48 h at 28C, results were
read and analysed using the apilab plus software
package. Only confidence levels expressed as very good
identification or excellent identification were considered
in this study as described in Zbinden et al. (2007).
Vitek analysis
Identification was performed using ID-GNB Vitek2 cards
(bioMerieux). The Vitek-2 technology (bioMerieux) is
based on API technology but comprised 64 tests and is
automated. It leads to an identification result in 36 h for
Gram-negative (GN) bacteria. The Vitek cards were filled
according to manufacturers instructions for testing in the
Vitek2-compact automate (bioMerieux). Results were
described with a high confidence level (excellent identification or very good identification) except when
mentioned.
1188

Biolog
The Biolog Omnilog Identification System (AES Chemunex,
Bruz, France) utilizes automated biochemical tests. It is a
walk-away instrument that tests the ability of bacteria to
use a panel of 95 carbon sources. Growth is detected by
using a tetrazolium violet salt which is incorporated into
each well of a 96-wells Omnilog microtiter plate. Reduction in tetrazolium violet gives a purple colour indicating
the presence of growing bacteria. Prior inoculation of the
Omnilog plates, each isolate was cultured at 28 or 37C
on a Biolog Universal Growth agar plate with 5% sheep
blood. After an incubation period of 1824 h, a bacterial
colony was suspended at the specified density using Omnilog inoculating fluid as described by the manufacturer.
The suspension was subsequently used to inoculate wells
of GN2 microplates (AES Chemunex). According to their
origin, the microplates were incubated at 28 or 37C.
Each metabolic profile was compared with the appropriate GN Omnilog Biolog database which contains
biochemical fingerprints of 526 GN species. Biolog identifications were considered if the similarity index of the
genus or species was 0750 or greater after an incubation
of the plates for 4 h. When a lower similarity value was

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C. Pinot et al.

obtained, incubation was performed for 24 h. If bacterial

identification could not be achieved after 24 h, a no ID
result was given.
Genotypic characterization based on PCR analyses
SS-PCR directed to the 23S rDNA of Sten. maltophilia
was applied to DNA extracted from VIA isolates collected
from environmental and clinical samples using SM1 (5CAGCCTGCGAAAAGTA-3) and SM4 (5-TTAAGCTTGCCACGAACAG-3) primers (Whitby et al. 2000). The
presence of a 531-bp product on 10% agarose gel was
considered as a Sten. maltophilia strain according to the
authors.
Multiplex PCR to amplify partial gene sequences of
ggpS (885 bp) and smeD (150 bp) was used to distinguish
Sten. rhizophila strains from Sten. maltophilia strains
(Ribbeck-Busch et al. 2005). PCRs were performed using
gene-specific primers (ggpSG3i: 5-CAACGGCGTGATCCACAT-3; ggpSG5i: 5-GATGCTGCATGT GTTCTG-3,
smeD3: 5-CCAAGAGCCTTTCCGTCAT-3; smeD5: 5TCTCGG ACTTCAGCGTGAC-3) (Alonso and Martinez
2001). Sten. maltophilia ATCC13637 and Sten. rhizophila
DSM14405 were used as PCR-positive control strains for
smeD and ggpS genes amplification, respectively. The PCR
products were visualized as described above.
Results
Morphotypes and 16S rDNA sequencing
The use of VIA medium enabled isolation of colonies
from soil samples of 91 sites of 104 sites (i.e. 875%).
Thirteen sites (125% of tested soils) did not lead to VIA
bacterial colony growth. This screening revealed that
when VIA allowed the growth of bacterial colonies, one
to four morphotypes were obtained after plating soil suspensions. The dominant morphotype was dark green with
a blue halo as it constituted about 80100% of the colonies. The yellow morphotype represented from 10 to 20%
of the VIA colonies depending on the soil sample. The
green and blue morphotypes were rarely observed. For
each soil sample, 10 colonies (when it was possible) were
conserved and screened for further analysis. Sequencing
of 16S rDNA was performed on representative isolates of
the VIA morphotypes. The 16S rDNA sequencing allowed
allocation of each isolate to species belonging or not to
the Stenotrophomonas genus. Based on the sequencing
results, we selected 51 isolates to perform comparative
analyses of biochemical and molecular identification
schemes. As expected, isolates showing a dark green and
blue halo morphotype belonged to the Sten. maltophilia
species (16S rDNA sequences within 99% identity). The

Sten. maltophilia identification

yellow, green and blue colonies were identified as

Sten. rhizophila, Variovorax sp and Dyella japonica, respectively. Twenty-three environmental isolates, of which 15
were belonging to Sten. maltophilia, six to Sten. rhizophila,
one to Variovorax sp and one to D. japonica, were
selected for further comparisons (Table 2). Morphotypic
analysis of reference strains of species belonging to the
Stenotrophomonas genus growing on VIA was perfomed.
Stenotrophomonas koreensis did not grow on VIA medium. Stenotrophomonas terrae, Sten. humi and Sten. acidaminiphila grew as white colonies with a blue halo and
Sten. nitritireducens as light green colonies with a blue
halo. These last morphotypes were not recovered on VIA
medium from our soil samples.
Biochemical analyses
Three biochemical identification systems classically used
in clinical context were then applied to the 23 selected
isolates (Table 2). In most cases, the API system, the
Vitek-2 and the Biolog were able to attribute a species
name to each isolate with a high level of confidence (i.e.
very good to excellent according to each database).
Among the 15 Sten. maltophilia isolates validated by 16S
rDNA sequencing, 11 were properly identified by the
Vitek-2 and 10 by the Biolog system, but only five were
identified as Sten. maltophilia by API 20NE (Table 3).
Among the isolates misidentified by Vitek-2 and Biolog
systems, three were in common and identified as
belonging to the Pseudomonas genus. Stenotrophomonas
maltophilia isolates misidentified by API 20NE were identified as Burkholderia cepacia or Pseudomonas luteola. The
Sten. rhizophila isolates were not correctly identified by
these systems and were considered as being Sten. maltophilia (5 6) or Brevundimonas diminuta but with low
confidence for the isolate BPOE 5120, by the Vitek-2 system, as Sten. maltophilia (4 6) and Pseudomonas species
Table 3 Number (percentage) of Stenotrophomonas maltophilia (Sm)
isolates correctly identified by API-20NE, Vitek-2, Biolog and smeD
PCR methods (16S-rRNA gene sequencing results were used as reference species affiliation after comparison to GenBank database)
Confirmation of
Sten. maltophilia species
No (%) isolates
Environmental
isolates

Clinical
isolates

Identification system

Sm (15)

Sm (28)

API-20NE
Vitek-2 compact
Biolog Gram-negative plates
smeD PCR

5
11
10
15

28
28
27
28

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Journal of Applied Microbiology 111, 11851193 2011 The Society for Applied Microbiology

(33)
(73)
(67)
(100)

(100)
(100)
(96)
(100)

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C. Pinot et al.

(BPOE 5115 and BPOE 5118) by the Biolog system and

finally as Sten. maltophilia (3 6) and Ps. luteola (BPOE
5115, BPOE 5116 and BPOE 5118) by API 20NE. The
Variovorax isolate was unidentified by Vitek-2 and identified as a Pseudomonas by API 20NE and Biolog systems.
The D. japonica isolate was identified as Sten. maltophilia
by Biolog and API 20NE systems but was identified as
Aeromonas salmonicida by the Vitek-2 system.
Further testing the growth of the 28 clinical isolates on
the VIA medium evidenced that they were able to grow
on this medium and showed the dark green with a blue
halo morphotype. The 16S rDNA sequencing confirmed
their classification in the Sten. maltophilia species. Identification based on API 20NE system and Vitek-2 agreed
with the 16S rDNA data set (Table 2). One clinical isolate
was misidentified by the Biolog system and was allocated
to Xanthomonas campestris (Table 3). This isolate originated from the catheter of a non-CF patient.
Identification based on PCR tests
As PCR screenings were previously reported for allocation
of bacterial strains to Sten. maltophilia species, we used
these approaches on the environmental and clinical isolates of our study. The 51 isolates were allocated to
Sten. maltophilia by the SS-PCR targeting the 23S rDNA
whether they were identified by 16S rDNA sequencing as
Sten. maltophilia or not. The 43 isolates (15 environmental
isolates + 28 clinical isolates) identified as Sten. maltophilia based on 16S rDNA sequencing were correctly identified by the multiplex smeD ggpS PCR (i.e. positive PCR
amplification of smeD gene and negative PCR amplification of ggpS gene). The six Sten. rhizophila isolates were
confirmed to belong to this species leading to a positive
amplification of the ggpS fragment and no PCR product
for smeD. Unexpectedly, PCR analysis of Variovorax sp
and D. japonica isolates allowed amplification of the
ggpS gene and smeD gene, respectively. Reference strains
of other Stenotrophomonas species were screened for
the multiplex smeD ggpS PCR. No amplification was
obtained using both primer sets. The SS-PCR and smeD
PCR were applied on a panel of 250 soil isolates belonging to the dark green with a blue halo VIA morphotype
and demonstrated that both PCRs were highly sensitive
by giving positive PCR products for all of these isolates
(data not shown).
Identifications of isolates growing on VIA medium and
allocated to Sten. maltophilia using either the biochemical
tests or the PCR screenings were always reliable for the
clinical isolates. Nevertheless, one exception was recorded
using the Biolog system (Table 3). Misidentifications were
more frequently recorded on environmental isolates when
using biochemical tests. The least efficient system was the
1190

API 20NE as only 33% of the isolates were successfully

identified as Sten. maltophilia. Vitek-2 and Biolog systems
were fairly reliable, giving a 73 and 67% rate of proper
identification, respectively. The most reliable identification approach was found to be the multiplex PCR screening which enabled 100% of the tested isolates to be
correctly identified.
Discussion
Stenotrophomonas maltophilia is an emergent bacterial
opportunistic pathogen. Despite its presence in a wide
range of environmental habitats, few data are available on
its prevalence and its potential dissemination to the hospital. In the present work, we chose the VIA medium
supplemented with cycloheximide to investigate the presence of Sten. maltophilia in soil samples and further evaluated the efficiency of various biochemical and molecular
methods to rapidly and efficiently allocate isolates to this
species. Fungi being abundant in soil, cycloheximide was
added to prevent their growth. Fungal growth on VIA
would make bacterial colonies difficult to observe. Dark
green VIA colonies with a blue halo were expected to be
related to Sten. maltophilia. To validate this trend, 16S
rDNA sequencings and analyses were performed. 16S
rDNA is the marker of reference in bacterial classification
and was used to classify nonfermenting GN bacteria
(Morgan et al. 2009). 16S rDNA analyses showed dark
green VIA colonies with a blue halo to belong to the
Sten. maltophilia species.
Colonies obtained on VIA medium after spreading of
104 bacterial soil dilutions were dominated by dark green
Sten. maltophilia colonies with a blue halo. This morphotype represented between 80 and 100% of the VIA
isolates. It is noteworthy that sometimes no VIA colony
was recovered from the soil dilutions demonstrating the
high selectivity of the VIA medium. Selective media are
classically used for the isolation of opportunistic bacterial
species such as the cetrimide-nalidixic acid agar for
Pseudomonas aeruginosa, the TB-T media for B. cepacia
complex (Hagedorn et al. 1987) or the LAM media for
Acinetobacter spp. (Jawad et al. 1994). Whereas they have
been found appropriate in a clinical context, they often
showed limitations for their application to environmental
samples. As observed for the VIA medium, the TB-T one
exhibited a high selectivity rate of 7075% for B. cepacia
complex isolates. On the opposite, the cetrimide-nalidixic
acid agar used to select Ps. aeruginosa from water samples
(open ocean, bay, etc) yielded only 62 colonies of this
species over 1670 colonies obtained from 17 sampling
sites according to Khan et al. (2007). The percentage of
Ps. aeruginosa-like strains recovered from that medium
did not exceed 10%, whatever be the sample. Over these

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C. Pinot et al.

selective media, VIA has the advantage of allowing a differentiation of Sten. maltophilia isolates from nonSten. maltophilia isolates which is often not the case for
selective media. On cetrimide-nalidixic acid agar,
Ps. aeruginosa can exhibit different morphotypes (Lavenir
et al. 2008) but on the LAM medium, the different
Acinetobacter species and B. cepacia complex isolates can
show similar ones (Jawad et al. 1994). It is noteworthy
that a high selectivity of a culture medium can sometimes
be a disadvantage as it could lead to an under-estimation
of bacterial diversity as evidenced in the study of Tarnawski et al. (2003) who compared the diversity of fluorescent Pseudomonas populations recovered from two
different selective media.
We were interested in identifying species exhibiting
other morphotypes on VIA than the Sten. maltophilia
dark green colonies with a blue halo to better define the
extent of the diversity of the species able to grow on that
medium. Three of these morphotypes were found to
belong to D. japonica, Sten. rhizophila and Variovorax sp.
by 16S rDNA sequence analysis. However, Sten. terrae,
Sten. humi, Sten. acidaminiphila or Sten. nitritireducens,
which were previously isolated from soils e. g. Heylen
et al. (2007), were not recovered from our soils. This was
not surprising because neither white bacterial colonies
with a blue halo nor light green colonies with a blue halo,
typical of these species growing on VIA, were detected
during our platings.
Screenings on VIA medium and analyses of 16S rDNA
sequences appeared reliable approaches for identifying
Sten. maltophilia. However, such a strategy is not usually
applied for routine identification especially in hospital
laboratories which prefer high throughput, less timeconsuming and inexpensive methods. Biochemical
approaches fit these requirements and can be easily
automated. Sensitivity analyses of these approaches on a
selection of 23 environmental isolates obtained from soil
samples and representative of each morphotype were performed. API20NE was the least efficient method, with only
33% of Sten. maltophilia isolates being properly identified.
API-20NE sometimes allocated the isolates to B. cepacia
and Ps. luteola. Not least, some Sten. maltophilia morphotypes closely related to Sten. rhizophila or D. japonica,
were allocated to the Xanthomonadaceae family. Previous
studies reported Sten. maltophilia isolation on VIA
medium followed by the identification with API-20NE
from environmental samples including faucets, domestic
sink drains or salads (Denton et al. 1998; Qureshi et al.
2005) but no description of the morphotypes was available
to relate these data with our observations. The accuracy of
the automated Vitek-2 system was also checked because it
involves a higher number of biochemical tests than
API20NE. This system was found more efficient than

Sten. maltophilia identification

API-20NE; 73% of the Sten. maltophilia environmental

isolates were correctly identified. The Biolog system initially designed for environmental isolates was also found
as accurate as the Vitek-2 technology with 67% of proper
identification. Isolates misidentified by both systems were
often the same ones and allocated isolates to the Pseudomonas genus or B. diminuta or Flavobacterium tirrenicum
species. As previously reported for the API20NE system,
some VIA isolates with a non-Sten. maltophilia morphotype were sometimes allocated to Sten. maltophilia by the
Vitek-2 and Biolog systems whereas analyses of 16S rDNA
sequences indicated that they were closely related to
Sten. rhizophila, D. japonica or Variovorax sp. Although
numerous commercial identification systems have been
developed during the last decade, the poor inclusion of
environmental strains in the validation processes introduced biases in the database and led to some misidentifications. To improve the accuracy of these tests in
identifying Sten. maltophilia, these biochemical systems
need also to include data from Sten. maltophilia closest
relatives such as Sten. rhizophila. This latter species can be
abundant in nonhospital environments such as soil and
the rhizosphere (Berg et al. 2005). These conclusions are
in agreement with our data on clinical isolates. All clinical
isolates but one was properly identified with these methods. The 16S rDNA sequencing confirmed that they were
correctly affiliated. Testing the growth on VIA medium
evidenced the typical Sten. maltophilia morphotype, i.e.,
dark green with a blue halo.
The biochemical methods being insufficiently reliable
for environmental studies, PCR screenings were tested for
improvement of the identification schemes. The SS-PCR
based on 23S rDNA identified environmental isolates as
Sten. maltophilia but sometimes gave false-positive results
allocating Sten. rhizophila, Variovorax sp and D. japonica
strains (according to 16S rDNA analyses) to this species.
This PCR screening was previously validated on clinical
strains isolated from CF sputum (Whitby et al. 2000) and
its use should thus be limited to this context where less
overall bacterial diversity has been observed than the one
found among a soil sample. Indeed Sten. maltophilia clinical isolates from our collection were correctly identified by
SS-PCR. This result is in agreement with those obtained
by Giordano et al. (2006) which confirmed the sensitivity
of this PCR screening on strains isolated from CF and
non-CF patients. On the contrary and in agreement with
our results, cross-reactions with strains belonging to the
Stenotrophomonas species other than Sten. maltophilia and
their closely related genera as Xanthomonas were previously reported by Foster et al. (2008b). The smeD and
ggpS multiplex PCR based on differential amplification of
smeD and ggpS genes in Sten. maltophilia and Sten. rhizophila, respectively (Ribbeck-Busch et al. 2005) confirmed

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Sten. maltophilia identification

C. Pinot et al.

with our environmental isolate collection that we were

always able to distinguish both species when applied on
the dark green with a blue halo morphotype colony. A single false-positive smeD PCR result was obtained but this
isolate was found to belong to a rare Sten. maltophilia
morphotype recovered on VIA and allocated to D. japonica. The absence of amplification of the smeD gene
on Sten. terrae, Sten. nitritireducens, Sten. humi and
Sten. acidaminiphila confirmed the relevance of this
screening. Moreover, the screening of around 250 isolates
belonging to the dark green with blue halo morphotype
revealed amplification of the smeD fragment in all cases.
The multiplex PCR properly identified all Sten. maltophilia
clinical isolates of this study. Our data evidenced that this
molecular method is simple and efficient to identify
Sten. maltophilia-like isolates.
Several soil samples were tested in this study but other
environmental samples as water samples should be
screened to evaluate the efficiency of the procedure. Such
a tool would be useful to investigate the occurrence of
Sten. maltophilia vs Sten. rhizophila for example in aquatic
habitats such as a marine environment as started by other
authors (Romanenko et al. 2008).
In this study, the VIA medium allowed to select a
dominant morphotype easily confirmed as Sten. maltophilia using the smeD PCR screening, whatever be the
isolate origin. This cheap, rapid and easy to use procedure could be considered a method of choice to
conduct high throughput detection of Sten. maltophilia
for ecological studies.
Acknowledgements
The authors thank Gerard Chabanon for providing
clinical strains, Francoise Maurin and Laurence Loiseau
(PARMIC and CRB EML) for technical help. This work
was supported by the BQR (Bonus Qualite Recherche)
program from the University Lyon 1 and the Agence
Nationale de la Recherche (ANR) 05-SEST project
009-01. Amelie Deredjian was supported by a BDI PhD
fellowship from the CNRS.
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