NIH Public Access Author Manuscript Curr Protoc Microbiol. Author manuscript; available in PMC 2015 February 06.

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Published in final edited form as: Curr Protoc Microbiol. ; 32: Unit–6F.1.. doi:10.1002/9780471729259.mc06f01s32.

Stenotrophomonas maltophilia: Laboratory Culture and Maintenance Osama Mahdi1, Bridget Eklund1, and Nathan Fisher1 Osama Mahdi: [email protected]; Bridget Eklund: [email protected]; Nathan Fisher: [email protected] 1North

Dakota State University, Department of Veterinary and Microbiological Sciences, 1523 Cenntennial Blvd, Fargo, ND 58078, 734-646-0588

Abstract NIH-PA Author Manuscript

Stenotrophomonas maltophilia is a ubiquitous soil bacterium that is increasingly recognized as an emerging nosocomial pathogen. This unit includes protocols for the in vitro growth and maintenance of S. maltophilia.

Keywords Stenotrophomonas maltophilia; laboratory growth; nosocomial; hospital acquired infection

INTRODUCTION

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Stenotrophomonas maltophilia is a metabolically diverse species of gamma proteobacteria that inhabits a wide variety of environmental niches including moist soils and the plant rhizosphere. The species seems well suited to life in association with eukaryotic cells and a number of isolates have been characterized as endophytes of plants or endoparasites of amoeba. Recently, S. maltophilia, has been increasingly recognized as a significant opportunistic pathogen in a number of healthcare settings. Typically, nosocomial outbreaks are associated with contamination of a water source, from which S. maltophilia is spread to the patient population via routine healthcare procedures. S. maltophilia forms biofilms on a wide range of biotic and abiotic surfaces including indwelling medical devices. Initial device and patient colonization can progress to life-threatening infection, particularly in patients with significant comorbid conditions such as generalized immunodeficiency, neutropenia, chronic obstructive pulmonary disorder, cystic fibrosis, organ transplant or cancer. Intrinsic resistance to most classes of broad-spectrum antibiotics often complicates treatment of S. maltophilia infections. The reader is referred to Brooke, 2012, for an excellent review of S. maltophilia biology. This unit describes basic techniques to grow and maintain S. maltophilia in the laboratory. Fastidiousness appears to vary widely between isolates, but the majority of clinical and environmental isolates can be successfully cultivated on common laboratory media across a

Correspondence to: Nathan Fisher, [email protected]

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range of temperatures. These protocols are meant to enable the reader to grow and maintain S. maltophilia for in vitro study.

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CAUTION: Virulence mechanisms of S. maltophilia are not well understood and most isolates are considered Biosafety Level 1 (BSL-1) organisms. However, there are notable exceptions, including some commonly studied isolates, which are considered Biosafety Level 2 (BSL-2) pathogens. Therefore, it is recommended that readers follow all institutional protocols for the handling of BSL-2 organisms when handling S. maltophilia cultures. For general biosafety information, see UNIT 1A.1.

STRATEGIC PLANNING Strain Selection

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There are a number of S. maltophilia strains available from the American Type Culture Collection (www.atcc.org) and most large medical centers maintain clinical isolate collections. Many genetic studies are simplified by working with fully sequenced strains. The genome sequences from several strains are currently available (see Table 1). However, many phenotypes such as biofilm production or resistance to phagocytosis vary widely across isolates (Pompilio, et al, 2011; Rouf, et al, 2011; Fisher, unpublished data) and, depending on the goals of the study, investigators may need to consider working with isolates for which a sequenced genome is not yet available. Growth Conditions

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Most S. maltophilia isolates grow well under standard laboratory conditions (nutrient-rich media, 37°C). However, some environmental isolates exhibit considerably enhanced growth at lower temperatures between 20°C and 30°C. Most isolates are able to survive at 4°C for an extended period of time, but the reader should verify survival for their strain of interest prior to relying upon refrigeration for long-term storage. S. maltophilia tolerates a wide pH range but is more sensitive to salt concentrations than most common laboratory bacteria and this should be taken into consideration when planning growth conditions or buffers used for in vitro assays. In rich medium, the doubling time for most S. maltophilia isolates is between 20 and 40 minutes during log phase. Overnight cultures reach densities of >109 cfu/ml. However, a significant death phase is observed in some isolates between 20–24 hours postinoculation and the reader should take care to avoid extending incubation times longer than is necessary. Most S. maltophilia isolates also form significant biofilms on a wide range of abiotic materials including glass and most plastics used in the construction of common culture vessels. Media Most S. maltophilia isolates can be cultivated on a wide range of media and we suggest LB (Luria or Lennox) or nutrient agar in the following protocols. However, a few isolates have proven resistant to axenic culture and must be maintained in association with host cells (amoeba; Corsaro, Muller and Michel, 2013). Growth in defined minimal media varies widely across isolates and the reader should empirically test any particular media of interest with the isolates or strains under analysis. In all cases, it is important to be cognizant of the

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salt sensitivity of S. maltophilia and adjust recipes accordingly. For that reason, we suggest the Luria (0.5g/L NaCl) or Lennox (5g/L NaCl) formulations of LB and not the Miller recipe (10g/L NaCl). On agar plates, S. maltophilia will appear as small, circular, raised colonies with a yellow tint that varies in strength (from nearly white or grey to a deep yellow) depending on the particular isolate. S. maltophilia colonies are non-hemolytic on blood agar. They do not ferment lactose, and appear colorless on MacConkey agar. Many S. maltophilia isolates exhibit natural resistance to multiple classes of antibiotics, leaving few choices for in vitro use. Table 1 details common antibiotic supplementation, but readers should verify sensitivities before beginning experiments. Note that many antibiotics are used at concentrations above those typical for Escherichia coli and other common laboratory species.

BASIC PROTOCOL 1 GROWTH OF S. MALTOPHILIA FROM A FROZEN STOCK

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Long-term storage of S. maltophilia should be accomplished by means of frozen (-80°C or below) stocks in rich media with 30–40% glycerol. Prolonged or indefinite laboratory passage should be avoided. Thus, experiments should generally begin by reviving cells from a frozen stock. We recommend reviving cells by streaking onto a fresh agar plate in order to directly assess the purity of the culture at this critical stage. Materials S. maltophilia frozen stock (see Basic Protocol 3) LB (Luria) agar plates (APPENDIX 4A) Note: do not use high NaCl (>5 g/l) recipes of LB (see above) Inoculating loop or needle, sterile 37°C incubator

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1.

Without allowing the frozen stock to thaw, use the inoculating loop or needle to remove a small clump of bacteria.

2.

Streak the clump of bacteria onto an agar plate to isolate colonies (see APPENDIX 4A).

3.

Incubate up to 24 hours at 37°C.

BASIC PROTOCOL 2 GROWTH OF S. MALTOPHILIA IN LIQUID MEDIUM S. maltophilia grows well in liquid culture. In order to prevent a significant portion of cells from adhering to the walls of the culture vessel, cultures should be agitated vigorously. Growth in liquid culture is used in a wide variety of experiments including growth curves and isolation of nucleic acids. For general purposes, LB (Lennox or Luria), nutrient, or trypticase soy broth is suggested.

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Materials

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S. maltophilia, grown on LB (Lennox or Luria) or nutrient agar, containing antibiotics if appropriate (see Basic Protocol 1) LB (Lennox or Luria), nutrient, or trypticase soy broth, sterile (see APPENDIX 4A) Note: do not use high NaCl (>5 g/l) recipes of LB (see above) Antibiotics, if required (Table 2) Inoculating loop or needle, sterile Capped test tubes or flask, sterile Vortex 37°C shaking incubator 1.

Aseptically, add sterile broth media to a sterile test tube or flask (e.g., 3 ml to a 15 ml screw-cap conical tube or one tenths volume to a flask)

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Baffled flasks are recommended for large volume cultures in order to achieve maximum agitation and aeration. 2.

Using a sterile inoculating loop or needle, pick a single colony from the agar plate.

3.

Suspend the colony in the broth. If inoculating a large culture volume, it may be preferable to suspend the colony in 1 ml of sterile broth media prior to transfer of the entire suspension to the culture flask.

4.

Tightly cap the test tube and vortex to evenly distribute the bacterial cells throughout the broth.

5.

Incubate the test tube in an orbital shaking incubator (225–300 rpm) for 12 to 16 hours at 37°C.

BASIC PROTOCOL 3 PREPARATION OF S. MALTOPHILIA FROZEN STOCKS NIH-PA Author Manuscript

S. maltophilia should not be stored for long periods of time at room temperature or 4°C. Nor should cultures be continuously passaged on plates or in broth media. Instead, experiments should begin by streaking for isolation from a frozen stock (see protocol 1). We recommend preparing an original seed stock immediately upon receipt of a new isolate and generating, from that seed stock, a number of working stocks to be accessed as needed for experiments. The original seed stock is then only accessed to replenish working stocks when needed. Materials S. maltophilia, grown on LB (Lennox or Luria) or nutrient agar, containing antibiotics if appropriate (see Basic Protocol 1) LB (Lennox or Luria) broth (see APPENDIX 4A)

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Note: do not use high NaCl (>5 g/l) recipes of LB (see above) 50% (v/v) glycerol, sterile

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1.5-ml freezer vials, sterile Vortex 37°C shaking incubator −80°C freezer Inoculating loop or needle, sterile

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1.

Using aseptic technique, transfer a single colony of S. maltophilia into 3 ml liquid LB (see Basic Protocol 2) in a 15 ml screw-cap conical tube.

2.

Incubate 12 hours at 37°C, 225 rpm.

3.

Add 0.5 ml of the culture to a 1.5 ml freezer vial containing 0.75 ml 50% glycerol solution.

4.

Vortex for 20 seconds to ensure cells are well distributed throughout the suspension.

5.

Immediately transfer the vial to storage at −80°C.

REAGENTS AND SOLUTIONS Deionized, distilled water should be used in all recipes and protocols. APPENDIX 2A provides recipes for common stock solutions.

COMMENTARY Background Information

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S. maltophilia is an emerging, multi-drug resistant, healthcare-associated pathogen of significant concern for patients with comorbidities that compromise the normal function of the immune system. In its environmental niche, S. maltophilia colonizes aquatic and soil microcosms where it forms extensive biofilms (Brooke, 2012). This propensity toward biofilm colonization of aquatic niches allows S. maltophilia to contaminate and persist in a wide variety of healthcare settings including municipal water supplies, taps, and drains; water purification systems; air conditioning and other cooling systems; and an extensive list of both indwelling and external medical devices. Nosocomial spread from these sites of contamination can result in point-source outbreaks among susceptible patient populations. Short of the requirement for some immune compromised status of the patient, very little is known regarding the specific virulence mechanisms utilized by S. maltophilia during colonization and infection of patients and that is a current area of active investigation. S. maltophilia is also recognized as an endosymbiont or endophyte of certain plants where it is thought to promote growth and contribute to disease protection (Taghavi, et al, 2009; Zhu, et al, 2012). Finally, certain S. maltophilia isolates are used in biotechnological applications where their unique metabolic activities can be harnessed for biosynthesis of organic compounds or bioremediation of contaminated soil or water (Allen, et al, 2012).

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Critical Parameters

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Salt concentration is critical to S. maltophilia growth, with some isolates exhibiting sensitivity as low as ~1% (w/v) NaCl. If growth is slow or is not observed, the reader should verify that the concentration of salt in the media is below the threshold for the strain of interest. Troubleshooting Frozen stock suspensions of S. maltophilia typically maintain viability for extended periods of time. If no growth is observed upon revival on agar plates, it is possible that an insufficient number of cells were obtained or that the agar plates contain an antibiotic to which the isolate is sensitive. Start another culture using a larger ice chip and verify antibiotics, if any, added to the agar. Do not keep glycerol stocks at −20°C because cells can settle to the bottom due to incomplete freezing. Recovery and growth should generally be conducted at the optimum temperature for the S. maltophilia isolate(s) under analysis, which may be less than 37°C in some cases. Anticipated Results

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Basic Protocol 1 describes how to grow S. maltophilia on agar plates, starting from a frozen stock. After 16 hours of incubation, colonies will appear large, opaque, convex, smooth, round and white to yellow in color. Basic Protocol 2 describes how to grow S. maltophilia in liquid media starting from isolated colonies on an agar plate. Following 12 or more hours of growth, cultures should appear turbid. Basic Protocol 3 describes preparation of frozen stock suspensions of S. maltophilia cells. Revival of cells onto fresh agar plates should result in a large number of colonies. Time Considerations The protocols described in this unit require only a minimal amount of time—1 to 2 minutes each—excluding the time required to prepare reagents and incubation time. However, planning should be adjusted when handling large numbers of isolates or samples.

Acknowledgments NIH-PA Author Manuscript

OM, BE and NAF are supported by NIH Grant Number 5P30 GM103332 from the National Institute of General Medicine (NIGMS) and the NDSU Agricultural Research Station. The contents of this unit are solely the responsibility of the authors and do not necessarily represent the official views of the NIH or the NDSU ARS.

LITERATURE CITED Allen MJ, Tait K, Muhling M, Weynberg K, Bradley C, Trivedi U, Gharbi K, Nissimov J, Mavromatis K, Jensen CN, Grogan G, Ali ST. Genome sequence of Stenotrophomonas maltophiliaPML168, which displays Baeyer-Villiger monooxygenase activity. J Bacteriol. 2012; 194(17):4753–4. [PubMed: 22887661] Brooke JS. Stenotrophomonas maltophilia: an emerging global opportunistic pathogen. Clin Micro Rev. 2012; 25(1):e1–e41.10.1128/CMR.00019-11 Chauhan A, Green S, Pathak A, Thomas J, Venkatramanan R. Whole-genome sequence of five oysterassociated bacteria show potential for crude oil hydrocarbon degradation. Genome Announc. 2013; 1(5):e00802–13. [PubMed: 24092793]

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Corsar D, Muller KD, Michel R. Molecular characterization and ultrastructure of a new amoeba endoparasite belonging to the Stenotrophomonas maltophilia complex. Exp Parasitol. 2013; 133(4): 389–90. Crossman LC, Gould VC, Dow JM, Vernikos GS, Okazaki A, Sebaihia M, Saunders D, Arrowsmith C, Carver T, Peters N, Adlem E, Kerhornou A, Lord A, Murphy L, Seeger K, Squares R, Rutter S, Quail MA, Rajandream M, Harris D, Churcher C, Bentley SD, Parkhill J, Thomson NR, Avison MB. The complete genome, comparative and functional analysis of Stenotrophomonas maltophilia reveals an organism heavily shielded by drug resistance determinants. Genome Biol. 2008; 9:R74.10.1186/gb-2008-9-4-r74. [PubMed: 18419807] Lira F, Hernandez A, Belda E, Sanches MB, Moya A, Silva FJ, Martinez JL. Whole genome sequence of Stenotrophomonas maltophilia D457, a clinical isolate and a model strain. J Bacteriol. 2012; 194(13):3563–4. [PubMed: 22689246] Pompilio A, Pomponio S, Crocetta V, Gherardi G, Verginelli F, Fiscarelli E, Dicuonzo G, Savini V, D’Antonio D, Di Bonaventura G. Phenotypic and genotypic characterization of Stenotrophomonas maltophilia isolates from patients with cystic fibrosis: genome diversity, biofilm formation, and virulence. BMC Microbiol. 2011; 11:159. [PubMed: 21729271] Rouf R, Karaba SM, Dao J, Cianciotto NP. Stenotrophomonas maltophilia strains replicate and persist in the murine lung, but to significantly different degrees. Microbiology. 2011; 157(7):2133–42. [PubMed: 21546584] Sasserra D, Leardini I, Villa L, Comandatore F, Carta C, Almeida A, do Ceu Sousa M, Gaiarsa S, Marone P, Pozio E, Caccio SM. Draft genome sequence of Stenotrophomonas maltophilia strain EPM1, found in association with a culture of the human parasite Giardia duodenalis. Genome Announc. 2013; 1(2):e00182–13. Song S, Yuan X, Liu S, Zhang N, Wang Y, Ke Y, Xu J, Huang L, Chen Z, Li Y. Genome sequence of Stenotrophomonas maltophilia S028, an isolate harboring the AmpR-L2 resistance module. J Bacteriol. 2012; 194(23):6696. [PubMed: 23144428] Taghavi S, Garafola C, Monchy S, Newman L, Hoffman A, Weyens N, Barac T, Vangrosveld J, van der Lelie D. Genome survey and characterization of endophytic bacteria exhibiting a beneficial effect on growth and development of poplar trees. Appl Environ Microbiol. 2009; 75(3):748–57. [PubMed: 19060168] Zhang L, Morrison M, O Cuiv P, Evans P, Rickard CM. Genome sequence of Stenotrophomonas maltophilia strain AU12-09, isolated from an intravascular catheter. Genome Announc. 2013; 1(3):e00195–13. [PubMed: 23640378] Zhu B, Liu H, Tian WX, Fan XY, Li B, Zhou XP, Jin GL, Xie GL. Genome sequence of Stenotrophomonas RR-10, isolated as an endophyte from rice root. J Bacteriol. 2012; 194(5): 1280–1. [PubMed: 22328769]

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Table 1

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Selected S. maltophilia isolates for which a genome sequence is available. Strain

Source

Accession Number

Reference

S028

Human

ALYK02000000

Song, et al, 2012

EPM1

Giardia culture

AMXM01000000

Sassera, et al, 2013

AU12-09

Huma—IV catheter

APIT00000000

Zhang, et al, 2013

RR-10

Rice Root

AGRB0000000

Zhu, et al, 2012

D457

Human

HE798556

Lira, et al, 2012

K279a

Human—cystic fibrosis

NC_010943

Crossman, et al, 2008

R551-3

Poplar endophyte

NC_011071

Taghavi, et al, 2009

MF89

Oyster microflora

ATAP00000000

Chauhan, et al, 2013

PML168

Environmental

CAJH01000097

Allen, et al, 2012

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Table 2

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Antibiotic Stock Solutions Used with S. maltophilia Antibiotic

Solventa

Stock Conc.

Working Conc. b

Storage

Chloramphenicol

Ethanol

40 mg/ml

40 μg/ml

−20°C

Kanamycinc

Water

200 mg/ml

200 μg/ml

4°C

Polymyxin B

Water

50,000 U/ml

50 U/ml

4°C

Norfloxacinc

DMSO

10 mg/ml

10 μg/ml

4°C

Tetracycline

Ethanol

40 mg/ml

40 μg/ml

−20°C

Trimethoprimc

DMSO

200 mg/ml

200 μg/ml

4°C

a

With the exception of norfloxacin and trimethoprim, all antibiotic solutions should be sterilized by filtration with a 0.22 μm filter. It is not necessary to filter sterilize antibiotics dissolved in DMSO. b

Dilute stock solution 1:1000 into media to produce media with the desired final concentration. Allow broth or agar to cool to 55°C or cooler before adding antibiotics. Protect tetracycline from light at all times. c

Most clinical isolates are resistant to kanamycin, norfloxacin, and trimethoprim. However, they can be useful for counter-selection of donor strains during mating experiments or as selection markers in sensitive environmental isolates.

NIH-PA Author Manuscript NIH-PA Author Manuscript Curr Protoc Microbiol. Author manuscript; available in PMC 2015 February 06.

Laboratory culture and maintenance of Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is a ubiquitous soil bacterium that is increasingly recognized as an emerging nosocomial pathogen. This unit includes pro...
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