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Journal of Clinical Microbiology, November 1999, p. 3594-3600, Vol. 37, No. 11
Department of Microbiology,
Received 17 March 1999/Returned for modification 4 May
1999/Accepted 31 July 1999
While the gram-negative bacterium Stenotrophomonas
maltophilia is used in biotechnology (e.g., for biological
control of plant pathogens and for bioremediation), the number of
S. maltophilia diseases in humans has dramatically
increased in recent years. A total of 40 S. maltophilia
isolates from clinical and environmental sources (plant associated and
water) was investigated to determine the intraspecies diversity of the
group and to determine whether or not the strains could be grouped
based on the source of isolation. The isolates were investigated by
phenotypic profiling (enzymatic and metabolic activity and antibiotic
resistance patterns) and by molecular methods such as
temperature-gradient gel electrophoresis of the 16S rRNA gene fragment,
PCR fingerprinting with BOX primers, and pulsed-field gel
electrophoresis (PFGE) after digestion with DraI. Results
of the various methods revealed high intraspecies diversity. PFGE was
the most discriminatory method for typing S. maltophilia
when compared to the other molecular methods. The environmental strains
of S. maltophilia were highly resistant to antibiotics, and
the resistance profile pattern of the strains was not dependent on
their source of isolation. Computer-assisted cluster analysis of the
phenotypic and genotypic features did not reveal any clustering
patterns for either clinical or environmental isolates.
Stenotrophomonas
maltophilia, previously called Pseudomonas maltophilia
and Xanthomonas maltophilia (33), is ubiquitous in the environment. It has been recovered from a number of water sources and from a wide range of nosocomial sources (8, 12, 37). S. maltophilia is often associated with plants
and has been isolated from the rhizosphere of wheat, oat, cucumber,
maize, oilseed rape, and potato (4, 10, 19, 25).
Investigations have indicated a potential role for this species in
biotechnology. It has been used as biological control agent of fungal
plant pathogens in agriculture (5, 13, 23, 26) and in
bioremediation (7, 30, 43). S. maltophilia has
also become important in the last decade as a nosocomial pathogen
associated with significant case/fatality ratios in certain patient
populations, particularly among individuals who are severely
debilitated or immunosuppressed (15, 29, 47). Long-term
hospitalization, fungal infections, antimicrobial pressure, and
catheterization are also contributory factors to the rise in the
S. maltophilia infection rate (11). The emergence
of new opportunistic pathogenic microorganisms is somehow linked to a
multiresistance phenotype that makes them refractory to the antibiotics
commonly used in clinical practice (11). The majority of
clinical strains of S. maltophilia are characterized by their multiresistance to common antibiotics (2, 41). With the exception of trimethoprim-sulfamethoxazole,
many post-therapy isolates of S. maltophilia
quickly become resistant to antimicrobial agents (1,
15). Molecular typing methods for this bacterial species, e.g.,
restriction fragment length polymorphism by pulsed-field gel
electrophoresis (PFGE) (6, 47, 48), random amplified
polymorphic DNA (RAPD) analysis by arbitrarily primed PCR
(48), enterobacterial repetitive intergenic consensus PCR (9, 12), arbitrarily primed PCR
(40), ribotyping (16), and repetitive extragenic
palindromic PCR (34), have been developed. All of these
fingerprinting methods have been used to detect relationships between
clinical strains in epidemiological studies. Despite the acknowledged
importance of S. maltophilia as a nosocomial pathogen,
little is known regarding its epidemiology. Presently, it is unclear
how S. maltophilia finds its way to clinical environments
(11, 16). Since certain strains of S. maltophilia may have considerable biotechnological potential, it would be desirable
to be able to distinguish those strains from ones obtained from
clinical sources. Differences in levels of antibiotic resistance and in
the ability to macerate onion tissue between clinical and environmental
isolates of Burkholderia cepacia, which is also used as
biocontrol or bioremediation agent but can be an opportunist pathogen,
were reported (44).
In this study, 40 isolates of S. maltophilia (including some
strains of biotechnological interest) obtained from different origins
(clinical and environmental sources) were investigated by various
phenotypic (enzymatic and metabolic activity and antibiotic resistance
pattern) and genotypic (temperature gradient gel electrophoresis [TGGE], BOX-PCR, and PFGE) fingerprinting methods in order to find a
system that characterizes the variability among this species and that
can distinguish between clinical and environmental isolates.
Bacterial strains.
A total of 40 isolates were investigated
in this study. The isolates c1 through c20 were obtained from Britta
Bruun and P. Gerner-Smid (Statens Seruminstitut, Copenhagen, Denmark).
The strains were isolated in the Rigshospitalet Copenhagen from various sites (tracheal aspirates, sputa, blood, throat, wounds, skin, ulcers,
drainage fluids and aspirates, catheters, urine, etc. [16]). Marine isolates e1 and e2 were obtained from
Arite Minkwitz (University of Rostock, Rostock, Germany). Strain e3 was
used as biocontrol agent against phytopathogenic fungi (4),
and strain e8 showed antifungal properties (5). S. maltophilia DSM 50170 (ATCC 13637, type strain t20, isolated from
a patient with oral carcinoma [20]) was used as a
reference strain for comparison. Environmental strains were isolated on
X. maltophilia selective medium (22). The medium
contained the following: maltose (10 g liter Identification and metabolic fingerprinting.
All isolates
were identified by API (BioMérieux, Marcyl' Etoile, France) and
BIOLOG (Biolog Inc., Hayward, Calif.). In addition, some strains were
identified by fatty acid analysis and 16S rRNA gene fragment sequencing
(data not shown). Bacterial cells cultivated at 30°C on nutrient agar
(Sifin, Berlin, Germany) for 24 h were transferred into the API 20 NE gallery (system for nonenteric rods) and incubated for 24 h at
30°C. Results were read visually and compared to the statistical
databank (BioMérieux). To obtain the metabolic fingerprints by
BIOLOG, strains were cultivated on tryptic soy agar (Gibco, Eggenstein,
Germany) for 24 h at 30°C. Bacterial cells were harvested and
suspended in a 0.85% NaCl solution. A 125-µl volume of the
suspension with an optical density of 0.2 was transferred with a
multichannel pipette into BIOLOG GN microplates (system for
gram-negative bacteria). The results were read visually after 24 h
of incubation at 30°C and compared to the statistical databank
(MicroLog system). All strains were tested in duplicate.
Antibiotic resistance pattern.
The assay (breakpoint
determination) to determine the susceptibility of bacteria to relevant
antibiotics with a semisolid medium, which was similar to the reference
method (agar dilution method), was carried out with ATB Antibiogram PSE
1 (BioMérieux) according to the manufacturer's recommendations.
Two concentrations of each antibiotic were used, as follows (in mg
liter Molecular typing by TGGE.
Total DNA was extracted from the
bacterial pellet as previously described (46). PCR
amplification of the bacterial 16S rRNA fragment was done with primers
spanning V6 to V8 (F968 and R1401 [Escherichia coli
numbering system]). Separation of the PCR products was done by TGGE
analysis as previously described (18). Approximately equal
amounts of PCR products (1 to 2 µl), as determined from an ethidium
bromide-stained agarose gel, were applied to TGGE gels. A temperature
gradient from 38 to 52°C was used for TGGE (Qiagen, Hilden, Germany)
which was performed in TAE buffer (40 mM Tris-acetate, 1 mM EDTA [pH
8.0]) at a constant voltage of 180 V for 4 h. DNA was visualized
in TGGE gels by acid silver staining (36).
Molecular typing by BOX-PCR.
Genomic DNA from each strain
was extracted by the method of Wilson (46). BOX element
oligonucleotide primers with the sequence of
5'-CTACGGCAAGGCGACGCTGACG-3' were synthesized by MWG Biotech (Ebersberg, Germany). The PCR were performed as previously described by
Rademaker and De Bruijn in duplicate for each isolate (35).
Molecular typing by PFGE.
Strains were grown overnight in
10-ml volumes of Luria broth (Difco). After centrifugation at 13,600 × g for 1 min, each cell pellet was washed (twice) and
resuspended in 1 ml of SE buffer (25 mM EDTA [pH 7.4], 75 mM NaCl).
Agarose plugs were made from a 1:1 mixture of 1.6% low-melting-point
agarose (Biometra, Göttingen, Germany) and the cell suspension.
Each plug was placed in 5 ml of lysis buffer (10 mM Tris-HCl [pH
7.6], 100 mM EDTA [pH 8.0], 50 mM NaCl, 0.2% deoxycholic acid, 1%
N-lauroyl sarcosine, 2 mg of lysozyme) for 3 h at
35°C. Samples were then treated for 16 h at 42°C with the same
volume of proteinase K solution containing 50 µg of proteinase K per
ml, 100 mM EDTA (pH 8.0), 0.2% deoxycholic acid, and 1%
N-lauroyl sarcosine. After three 1-h washes with TE buffer
(10 mM Tris-HCl, 0.1 mM EDTA [pH 8.0]), the agarose plugs were stored
in TE buffer at 4°C for subsequent PFGE. Agarose plugs were digested
with restriction enzyme DraI (New England Biolabs,
Schalbach, Germany) for 20 h at 35°C according to the manufacturer's recommendations. DNA fragments were separated on a 1%
PFGE agarose (peqLab, Erlangen, Germany) gel in a contour-clamped homogeneous electrical field by using the Rotaphor V system of Biometra
with 0.5× TBE buffer (45 mM Tris-borate, 1 mM EDTA [pH 8.0]) at
12°C. The voltage was set at 200 V/cm, and pulse times ranged from 5 to 45 s over 20 h, with linear ramping. The procedure was
repeated at least twice for each isolate to determine the reproducibility of the results. The gel was stained with ethidium bromide.
Statistical analysis.
Differences between the antibiotic
resistance patterns of clinical and environmental isolates were
determined by a two-sided test of binomial proportion
(P < 0.05). Data were converted to a binary code, and
interisolate relationships were measured by the Euclidian metric
unweighted pair-group average method by using the STATISTICA program
(StatSoft, Hamburg, Germany). Molecular fingerprint patterns generated
for each strain were compared and grouped by using the Gelcompare
program (Kortrijk, Belgium). Data describing susceptibility against
antibiotics were analyzed by BioMath GmbH company (Rostock, Germany).
Phenotypic characterization and identification.
A total of 40 S. maltophilia isolates from clinical and environmental
sources were compared by their enzymatic and metabolic activity by
using the API and the BIOLOG systems. Both methods identified all 40 strains correctly to the species level. With the API system, the
positive identification rate ranged from 97.7 to 99.9% (Table
1). Seven different API profiles were
detected (Table 1), and the isolates were grouped according to these
profiles. The largest profile groups (I, II, and IV) comprised 34 isolates and contained isolates from clinical and environmental
sources. The oxidative utilization of 95 different carbon sources was
tested for each isolate with BIOLOG GN plates. The isolates exhibited heterogeneity in their carbon utilization profiles. Most isolates varied from the typical Stenotrophomonas pattern in the
utilization of 1 to 10 carbon sources, as determined by their
individual carbon utilization profiles compared to the average pattern
of the investigated isolates (data not shown). Of the carbon sources
tested, 34 were not utilized by any of the S. maltophilia
strains analyzed. All the other carbon sources (61) were
utilized by the majority (>80%) of isolates. However, the BIOLOG
system identified all strains as S. maltophilia (Table 1).
Relationships between isolates were analyzed statistically by cluster
analysis. On the basis of similarity it was possible to arrange all the
isolates into six groups (Table 1). Three of these groups were
homogenous, and each contained only a single isolate of clinical
origin; the other three were heterogenous groups with isolates of both
origins. Altogether, the grouping of isolates was independent of
origin.
Antibiotic resistance pattern.
All 40 of the selected strains
were resistant to several antibiotics (Fig.
1). The e13 strain isolated from the
rhizosphere of oilseed rape was most susceptible to antibiotics. Two
clinical isolates (c2 and c4) and two environmental isolates (e5 and
e19) were found to have resistance to 16 antibiotics. Three of the multiresistant isolates (c4, e5, and e19) had identical antibiotic resistance profiles. On average, strains were susceptible to 11 of the
antibiotics tested. No significant differences (P = 0.624071) in the number of resistances between the two groups of
isolates, clinical isolates (mean, 11.45; standard deviation, ± 2.85)
and environmental isolates (mean, 11.30; standard deviation, ± 4.0), were found. The higher standard deviation obtained for the
environmental isolates indicated the heterogeneity of resistance
profiles in this group. Altogether, 34 different antibiotic resistance
patterns were observed. Thirty profiles were unique, but four equal
profiles were determined for two or more strains. Identical resistance patterns were observed for the clinical strain c3 and the environmental strains e1 and e2 with resistance against 14 antibiotics. None of the
strains had resistance to both trimethoprim and sulfamethoxazole (co-trimoxazole). The percentages of resistant strains varied among the
antibiotics (Fig. 1).
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Genotypic and Phenotypic Relationships between
Clinical and Environmental Isolates of Stenotrophomonas
maltophilia
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1; Sigma,
Deisenhofen, Federal Republic of Germany), tryptone (Gibco, Paisley,
Scotland), bromthymol blue (4 ml of 2% aqueous solution liter1; Sigma), and Bacto-Agar (15 g
liter1; Difco, Detroit, Mich.). The medium was adjusted
to a pH of 7.1 with 1 N NaOH, and the following antibiotics (all from
Sigma) were added: cycloheximide (100 µg liter1),
nystatin (50 µg liter1), cephalexin (50 µg
liter1), bacitracin (25 µg liter1),
penicillin G (25 µg liter1), novobiocin (10 µg
liter1), neomycin sulfate (30 µg liter1),
and tobramycin (1 µg liter1). The environmental strains
are available from the culture collection of the Department of
Microbiology at the University of Rostock. The control strain used was
Pseudomonas aeruginosa ATCC 15441.
1): azlocillin, 16 and 64; piperacillin, 4 and 32;
piperacillin-tazobactam, 4 and 32; ticarcillin-clavulanic acid, 8 and
32; cefsulodin, 2 and 16; ceftazidime, 4 and 16; gentamicin, 1 and 4;
tobramycin, 1 and 4; amikacin, 4 and 16; doxycycline, 1 and 4;
ofloxacin, 1 and 2; ciprofloxacin, 1 and 2; imipenem, 1 and 4;
aztreonam, 2 and 16; and cotrimoxazol, 16 and 128. Strains were
classified as either susceptible (no growth or resultant turbidity at
either concentration), intermediate (growth and turbidity only at the lower concentration), or resistant (growth or turbidity at both concentrations). The disk diffusion test was performed with antibiotic sensitivity disks (BioMérieux) on Mueller-Hinton media (Difco). The final antibiotic concentrations were as follows (in micrograms disk1): chloramphenicol, 30; kanamycin, 30; tetracycline,
30; and erythromycin, 15 (all from Sigma).
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Taxonomic, phenotypic, and genotypic classification of
S. maltophilia isolates
View larger version (76K):
[in a new window]
FIG. 1.
Percentages of clinical and environmental isolates
resistant to antibiotics. AZL, azlocillin; PIP, piperacillin; TZP,
piperacillin-tazobactam; TIM, ticarcillin-clavulanic acid; CFS,
cefsoludin; CAZ, ceftazidime; GEN, gentamicin; TOB, tobramycin; AMK,
amikacin; DOX, doxycycline; OFX, ofloxacin; CIP, ciprofloxacin; IPM,
imipenem; ATM, aztreonam; SXT, trimethoprim-sulfamethoxazole
(co-trimoxazole); CHL, chloramphenicol; KAN, kanamycin; ERY,
erythromycin; TET, tetracycline.
Genotypic characterization. Three different DNA-based fingerprinting methods were used to compare the isolates at the molecular level. With TGGE it was possible to separate 16S rRNA gene fragments of the same length but of different sequences according to their melting properties. A linearly increasing temperature gradient run in the presence of a constantly high concentration of urea and formamide was used for separation of PCR products in TGGE. Each isolate investigated had one band (Fig. 2). The isolates were arranged into five groups according to the position of the band on the gel (Table 1). Group I, the largest group, comprised 85% of the isolates. In most cases groups I, II, III, and V contained isolates from clinical as well as environmental sources. The BOX-PCR method was also used to differentiate the isolates. The PCR products obtained with BOX primers yielded DNA profiles with sufficient numbers of DNA bands to differentiate the 40 isolates (Fig. 3). The method was more discriminating than TGGE of 16S rRNA gene fragments, and most of the isolates showed unique PCR fingerprints. Very similar BOX-PCR banding patterns (relative position and intensity of bands) were observed for the environmental strain e9 and the clinical strain c12. The different BOX-PCR profiles were compared by numerical methods and the resultant dendrogram (Fig. 3), based on percent similarity between the isolates, showed a high degree of genetic diversity. At a 90% similarity level five major groups were defined. All groups were heterogenous and contained environmental and clinical isolates.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Each of the DNA-based fingerprinting methods was suitable for distinguishing and grouping the isolates, although the sensitivity of the methods varied. Of the two physiological methods tested, BIOLOG was more discriminatory than the API system. However, the systems did prove useful for the accurate identification of S. maltophilia strains. This is in accordance with findings of previous studies (28, 31).
Molecular fingerprinting methods yielded rapid, reproducible, and discriminatory fingerprints for clinical and environmental isolates of S. maltophilia. However, the methods displayed different discriminatory effects. Macrorestriction analysis of digested DNA by PFGE and BOX-PCR typing was found to be more discriminatory than TGGE of the 16S rRNA gene fragments. The majority of strains were shown to possess unique genotypes by PFGE and BOX-PCR (38). Different discriminatory effects of molecular typing methods have also been described by others (35, 48). TGGE analysis revealed some sequence variation in the 16S rRNA gene fragment containing the variable V6 to V8 region. Not surprisingly, TGGE analysis of 16S rRNA was less discriminatory than the PFGE and BOX-PCR methods. However, TGGE has great potential for differentiation of species within natural microbial communities with PCR-amplified 16S rRNA gene fragments obtained from total bacterial community DNA (17). A strategy for linking 16S rRNA from bacterial community fingerprints to pure culture isolates from the same habitat has been recently developed (17).
In recent epidemiological studies of S. maltophilia, PFGE yielded reproducible and easily identifiable patterns (24, 48). Thus, PFGE may be the more superior method for epidemiological typing of S. maltophilia. Although Yao et al. (48) have compared S. maltophilia DNA by PFGE and RAPD analysis methods, this study has demonstrated, for the first time, the intraspecies diversity of S. maltophilia by PCR-dependent fingerprinting with BOX primers and TGGE analysis. In conclusion, all three molecular methods proved useful for typing S. maltophilia. However, PCR analysis with BOX primers was the fastest method used.
The great diversity of S. maltophilia isolates observed in this study is consistent with findings of other previous typings. Palleroni and Bradbury (33) mentioned the diversity of the species in the type description of S. maltophilia. A wide range of heterogeneity in physiological parameters was also shown by Swings et al. (37). This heterogeneity was confirmed by genotypic studies. In further epidemiological studies, the majority of patients had unique types, and only occasionally have small clusters of indistinguishable strains been identified (11, 24, 40). In a recent study, ribotyping was used to characterize the 20 clinical strains used in this study which were isolated from a Danish hospital environment. Considerable diversity among the ribotypes of hospital strains was found, and no single-strain outbreak was detected (16). Potential reservoirs for these strains were not determined. Results by Chatelut et al. (9) obtained with ERIC-PCR and RAPD-PCR showed 29 different profiles among 38 isolates. A consistent observation of all the genotypic studies has been that a wide diversity of strains has been isolated from patients (11). Presently, little is known regarding the source of harmful S. maltophilia strains occurring in hospital environments (12). Investigations by other authors have reported no evidence of patient-to-patient transmission, and they suggest that multiple independent acquisitions from environmental sources could be an important mode of transmission of S. maltophilia (12). The most common sites of contamination were blood sampling tubes, dialysis machines, ice-making machines, nebulizers, shower heads, sink traps, water faucets, and other items frequently in contact with water (12).
Our results indicate that the antibiotic resistance profile of S. maltophilia isolates was not associated with their origin (e.g., clinical and environmental). Such findings suggest that strains of S. maltophilia did not acquire their antibiotic resistance during antibiotic therapy in the clinical or hospital environment. Wüst et al. (47) demonstrated that a single strain of S. maltophilia (typed by PFGE and ribotyping) became increasingly resistant to antimicrobial agents during 15 months after rigorous antimicrobial combination therapy. Their results suggested that isolates became resistant through antibiotic therapy with imipenem. Interestingly, our environmental isolates exhibited a high level of resistance to the antibiotic imipenem.
S. maltophilia is often a dominant member of the rhizosphere
microbial community of plants (22) and is known to be a
plant root-associated bacterium. S. maltophilia can also
produce high amounts of the plant growth hormone indole-3-acetic acid
(3, 5). Many of the strains investigated in this study were
isolated from the rhizosphere. This particular microenvironment is rich in nutrients due to the exudation of organic compounds from plants (27). Thus, the competition between microorganisms for these ecological sites is very high. Many other rhizobacteria, like the
fluorescent pseudomonads and Streptomyces species, produce an extended list of antibiotics (32). Antibiotics produced
by rhizobacteria include pyrrolnitrin, pyoluteorin, and herbicolin A,
which have also been detected in the rhizosphere (39). When antibiotics have been detected in nature, it has been in material obtained from these microhabitats, which are localized areas of intense
microbial interaction (39). Furthermore, S. maltophilia produces two macrocyclic lactam antibiotics, alteramid
A and maltophilin (21). Rhizobacteria including S. maltophilia are protected from their own antibiotics and those
produced by other rhizobacteria by resistance mechanisms most likely to
enhance competition in such natural microenvironments. Perhaps not
surprisingly, strains of S. maltophilia often exhibit
multiple antibiotic resistances (41). As has been previously
reported, we have found that the combination of trimethoprim and
sulfamethoxazole has an exceptionally high activity against S. maltophilia (2, 11, 14). Thus, it is the drug(s) of
choice for the treatment of severe S. maltophilia infections. Presently, the mechanisms of drug resistance in S. maltophilia are not well characterized. 
-Lactam drugs,
including imipenem, are not effective against this bacterium because
S. maltophilia produces several -lactamases
(metallo--lactamase and L2-cephalosporinase) (42).
Tetracycline resistance in S. maltophilia is the consequence
of an active efflux of the antibiotics, and it is associated with
resistance to quinolone and chloramphenicol but not to aminoglycosides
or -lactam antibiotics (1). Temperature-regulated gentamicin resistance has been correlated with the expression of outer
membrane proteins (45).
In conclusion, isolates from diseased humans (c1 to c20 [16]), biological control agents (e3 and e4), marine strains (e1 and e2), the type strain (t20), and other plant-associated bacteria (e4 to e19) have exhibited a high intraspecific diversity and did not cluster by origin, as determined by the different DNA-based fingerprinting methods used.
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ACKNOWLEDGMENTS |
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We thank Hella Goschke for valuable technical assistance; Britta Bruun (Copenhagen), Arite Minkwitz, Petra Marten, and Jana Lottmann (Rostock) for providing Stenotrophomonas strains; and J. Hacker (Würzburg) for helpful discussion. We thank J. Jungkurth (Braunschweig) for reading the manuscript.
This study was partially supported by the Fonds der Chemischen Industrie.
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FOOTNOTES |
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* Corresponding author. Mailing address: University of Rostock, Department of Biology, Microbiology, Gertrudenstrasse 11a, D-18051 ROSTOCK, Germany. Phone: 49 381 494 2049. Fax: 49 381 494 2244. E-mail: gabriele.berg{at}biologie.uni-rostock.de.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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