Stenotrophomonas maltophilia: Laboratory Culture and Maintenance

Abstract

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.

INTRODUCTION

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

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

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.

Table 1.

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

Growth Conditions

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 post-inoculation 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 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

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

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.

Materials

• 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)</disp-quote>

• Antibiotics, if required (Table 2)

• Inoculating loop or needle, sterile

• Capped test tubes or flask, sterile

• Vortex

• 37°C shaking incubator

Table 2.

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

^aWith 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.

^bDilute 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.

^cMost 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.

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)

   Baffled flasks are recommended for large volume cultures in order to achieve maximum agitation and aeration.</disp-quote>

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.</disp-quote>

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

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)

  Note: do not use high NaCl (>5 g/l) recipes of LB (see above)</disp-quote>

• 50% (v/v) glycerol, sterile

• 1.5-ml freezer vials, sterile

• Vortex

• 37°C shaking incubator

• −80°C freezer

• Inoculating loop or needle, sterile

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

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).

Critical Parameters

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

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.