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Mutation of a lipopolysaccharide synthesis gene results in increased biofilm ofStenotrophomonas maltophilia on plastic and glass surfaces

Mutation of a lipopolysaccharide synthesis gene results in increased biofilm ofStenotrophomonas... Annals of Microbiology, 58 (1) 35-40 (2008) Mutation of a lipopolysaccharide synthesis gene results in increased biofilm of Stenotrophomonas maltophilia on plastic and glass surfaces Joanna S. BROOKE*, Albert VO, Patrick WATTS, Nicholas A. DAVIS Department of Biological Sciences, DePaul University, 2325 N. Clifton, Chicago, IL 60614, USA Received 2 October 2007 / Accepted 28 December 2007 Abstract - Stenotrophomonas maltophilia is a biofilm-forming opportunistic pathogen that is emerging worldwide and demonstrates increasing rates of antibiotic resistance and significant case fatality/ratios in immunocompromised or debilitated patients. This study tested the hypothesis that mutation of a lipopolysaccharide synthesis gene results in increased biofilm of S. maltophilia. Transposon mutagenesis was used to disrupt genes of S. maltophilia clinical isolate X26332 and generated the JB12-23 mutant that produced increased biofilm in comparison to the isogenic parental isolate. Southern hybridisation revealed a single transposon insertion in the JB12-23 mutant. Rescue-cloning and DNA sequencing revealed that the transposon had inserted into the spgM gene of S. maltophil- ia mutant JB12-23. Biofilm assays revealed that the JB12-23 mutant produced more biofilm on polyvinyl chloride, polystyrene sur- faces, and on borosilicate glass in comparison to the parental isolate. Lipopolysaccharide SDS-PAGE showed that the JB12-23 mutant lacked high molecular weight LPS in comparison to the parental isolate. Key words: lipopolysaccharide, spgM, Stenotrophomonas, biofilm, transposon mutagenesis. INTRODUCTION al species. Stenotrophomonas maltophilia is often a co- pathogen and can be isolated with other pathogens, such Stenotrophomonas maltophilia is a worldwide emerging, as Pseudomonas aeruginosa in the cystic fibrosis lung envi- multi-antibiotic opportunistic pathogen associated with a ronment (Di Bonaventura et al., 2007). Given the possibil- significant case-fatality ratio in the immunocompromised ity of exchange of genetic information between bacteria patient population (Denton and Kerr, 1998). within biofilms, this emphasizes the need to investigate the Stenotrophomonas maltophilia has been recovered in hos- biofilm formed by S. maltophilia. This current study inves- pitals from respiratory devices, contaminated water-based tigated the effect of a transposon insertion in the gene, solutions and other materials (Denton and Kerr, 1998; spgM, on the biofilm of S. maltophilia. Senol, 2004). This pathogen is associated with a wide range of disorders including pneumonia, bacteraemia, cen- tral nervous system infections, ocular infections, endo- MATERIALS AND METHODS carditis, urinary tract infection, skin and soft tissue infec- tion, bone and joint infections, gastrointestinal infections, Maintenance of bacterial strains and plasmids. All and septic shock (Denton and Kerr, 1998). strains and plasmids used in this study are listed in Table 1. A significant characteristic of S. maltophilia is its ability Escherichia coli ER2420 (pACYC177) was maintained on to adhere to glass, Teflon, plastic surfaces and epithelial Luria-Bertani (LB) agar containing 50 µg/ml kanamycin and cells (Jucker et al., 1996; Vidipó et al., 2001; de Oliveira- 100 µg/ml ampicillin. Escherichia coli TransforMax EC100D Garcia et al., 2002, 2003). Stenotrophomonas maltophilia pir-116 (pJSB1) was maintained on LB agar containing 50 forms biofilms. Biofilms are microbial communities that are µg/ml kanamycin. Stenotrophomonas maltophilia isolate surrounded by a porous extracellular polysaccharide matrix X26332 was maintained on LB agar. The S. maltophilia material (Donlan, 2002; Stoodley et al., 2002). These JB12-23 mutant was maintained on LB agar containing 50 biofilms can serve as persistent sources of infection. Recent µg/ml kanamycin. All bacteria were grown at 37 ºC. studies report biofilms of S. maltophilia on stainless steel (Carpentier and Chassaing, 2004), glass and polystyrene Transposon mutagenesis. Transposon mutagenesis of surfaces (Di Bonaventura et al., 2004; Kavouras and Maki, the clinical parental isolate X26332 was performed using 2004; Huang et al., 2006). the EZ::TN<R6Kγori/KAN-2>Tnp Transposome mutagene- Frequently, biofilms are communities of mixed bacteri- sis kit (Epicenter Technologies, WI, USA). Transformants were selected on LB agar with 50 µg/ml kanamycin. The JB12-23 mutant was recovered after screening for biofilm * Corresponding author. Phone: (773) 325-1161; production as described below. Fax: (773) 325-7596; E-mail: jbrooke@depaul.edu 36 J.S. Brooke et al. TABLE 1 - Strains and plasmids used in this study Bacterial strains Description Reference or source Escherichia coli TransforMax EC100D pir-116 F mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80dlacZΔM15 Epicentre (Madison, WI) ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ rpsL nupG pir-116(DHFR). ER2420 F ara-14 leu fhuA2 Δ(gpt-proA)62 lacY1 glnV44 New England BioLabs (Beverly, MA) galK2 rpsL20 xyl-5 mtl-1 Δ(mcrC-mrr) Stenotrophomonas maltophilia X26332 Clinical isolate Children’s Memorial Hospital (Chicago, IL) JB12-23 Insertion derivative of X26332, Kan This study Plasmids r r pACYC177 Amp , Kan , 3.9 kb New England BioLabs (Beverly, MA) pJSB1 Kan This study r r Amp , ampicillin resistant; Kan , kanamycin resistant. DNA preparation and Southern hybridisation. obtained using custom-designed primers and pJSB1 DNA Chromosomal DNA from S. maltophilia X26332 and the as a template. JB12-23 mutant was isolated using a Wizard genomic DNA purification kit (Promega, WI, USA). Plasmid DNA was iso- Growth of cultures in shaken flasks and in stationary lated from E. coli ER2420 (pACYC177) and E. coli microtiter plates. The parental isolate X26332 was grown TransforMax EC100D pir-116 (pJSB1) using the QIAprep in LB broth and the JB12-23 mutant was grown in LB con- Spin Miniprep Kit (Qiagen, CA, USA). To detect the number taining 50 µg/ml kanamycin. Both cultures were grown at of EZ::TN<R6Kγ ori/KAN-2>Tnp transposon insertions pres- 37 ºC with agitation at 250 rpm in an orbital incubator ent in the chromosomal DNA of the mutant JB12-23, shaker (Amerex Instruments, CA, USA). Six 150 ml flask Southern hybridisations were performed (Sambrook et al., cultures each of X26332 and JB12-23 were used for the 1989). To detect the presence of the EZ::TN<R6Kγ ori/KAN- shaken flask growth assays. Each flask was inoculated with 2> transposon, a DNA probe was made to part of the overnight culture and adjusted to within the OD range of kanamycin resistance gene (kan ) between bases 2127 and 0.10-0.14. Flasks were incubated at 37 ºC with agitation at 2506 of the plasmid pACYC177 maintained in E. coli 250 rpm in a Labline incubator-shaker (Labline Instruments, ER2420. This region was amplified by using an A-Taq PCR IL, USA). Growth readings at OD were taken hourly up to kit (USB, OH, USA) in a GeneAmp PCR System 2400 (Perkin 11 h. Growth assays were performed in three independent Elmer, MA, USA) and labelled with digoxigenin-11-2’- experiments. deoxy-uridine-5’-triphosphate (DIG-11-dUTP) using the For the stationary microtiter plate growth assays, five DIG high prime DNA labelling and detection starter kit II cultures each of X26332 and JB12-23 were used (Head and (Roche, Germany). The forward primer sequence used was Yu, 2004). Overnight cultures were centrifuged at 4000 x g (5’-ATGGTCAGACTAAACTGGCTGACG-3’) and the reverse for 10 min. and pellets were resuspended to an OD of primer sequence was (5’-GTGACGACTGAATCCGGTGA- 0.25 in fresh LB or LB containing 50 µg/ml kanamycin. GAAT-3’). Southern blots were hybridised at 42 °C for 18- Wells of the microtiter plate containing 150 µl of culture 20 h and bands developed using the colorimetric detection medium were inoculated with 20 µl culture. Plates were system as recommended by the manufacturer. incubated stationary at 37 ºC. Growth readings at OD were taken at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 25, and Rescue cloning and sequencing of the transposon 48.5 h. Growth assays were performed in two independent insertion in the mutant JB12-23. The gene containing experiments. The generation times were determined for the transposon insertion was rescue-cloned from the chro- the batch culture and static cultures using the exponential mosomal DNA of mutant JB12-23 by self-ligation of EcoRI- growth phase from a graph of ln OD versus time (Ingraham digested chromosomal DNA and electroporation of E. coli et al., 1983). The specific growth rate was calculated as the TransforMax EC100D (pir ) electrocompetent cells accord- slope of the regression line (1/h). ing to manufacturer’s instructions (Epicenter). Plasmid pJSB1 contained the rescued mutated gene. Genomic DNA Biofilm assay on polyvinyl chloride, polystyrene, and surrounding the transposon insertion was sequenced using borosilicate glass surfaces. The biofilms of parental iso- an automated DNA sequencing apparatus (ABI prism 310 late X26332 and the JB12-23 mutant were assayed Genetic Analyzer, Perkin Elmer) with primers complemen- (Watnick and Kolter, 1999). Overnight cultures (16-18 h) tary to the transposon that were supplied in the mutagen- were centrifuged at 5000 x g and the cell pellets were esis kit. Extension of the nucleotide sequences was resuspended in new LB or LB containing 50 µg/ml Ann. Microbiol., 58 (1), 35-40 (2008) 37 kanamycin to the OD of 0.25. Twenty microlitres of each resuspended culture were used to inoculate each microtiter plate well that contained 150 µl LB medium (or 150 µl of LB medium containing 50 µg/ml kanamycin). Control wells contained 170 µl LB only (no cells). After stationary incu- bation at 37 ºC for 24 h, the medium was replaced with fresh medium, and then the plates were incubated station- ary for a further 24 h at 37 °C. Following incubation, cul- ture medium and nonadherent cells were removed, the wells were stained with 1% (wt/v) crystal violet, rinsed four times with phosphate buffered saline, and then the adher- ent cells were thoroughly resuspended in 80% (v/v) FIG. 1 - Southern blot of restriction enzyme-digested chromoso- mal DNA of Stenotrophomonas maltophilia X26332 and ethanol. OD readings were recorded of each well. mutant JB12-23. Lane 1: HindIII digested λDNA (frag- For the borosilicate glass tube biofilm assay, tubes were ment sizes in kb are indicated to the left of the blot), inoculated with the standardized cultures as described lane 2: uncut JB12-23, lane 3: EcoRI digested X26332, above for the microtiter plate assays. Tubes were incubat- lane 4: BamHI digested JB12-23, lane 5: EcoRV digest- ed stationary at 37 °C for 24 h. After incubation, the medi- ed JB12-23, lane 6: HindIII digested JB12-23, lane 7: um was replaced with fresh medium and the tubes were SalI digested JB12-23. incubated for a further 24.5 h at 37 °C. Biofilm production was detected using the crystal violet assay as described above for the microtiter plate assays. Each biofilm plate and tube assay was performed using five replicates. Two using primers to the EZ::TN<R6Kγ ori/KAN-2>Tnp transpo- independent biofilm assays were performed for each type son revealed that the JB12-23 mutant harboured the trans- of surface tested. poson at nucleotide 1476 within the spgM homologue. Recently it has been reported that the spgM homologue Lipopolysaccharide extraction and visualisation encodes the phosphoglucomutase enzyme that is used for using SDS-PAGE. Lipopolysaccharide was isolated from synthesis and assembly of LPS in S. maltophilia (McKay et overnight cultures of the parental isolate X26332 and the al., 2003). The transposon insertion is located at mutant JB12-23 (Marolda et al., 1990) and visualised using nucleotides 888/889 of the spgM gene downstream of both SDS-PAGE with silver staining (Fomsgaard et al., 1990). the active site (amino acid sequence TASHN residues 94- 98) at nucleotides 280-294, and the metal ion binding site Microbial adherence to hydrocarbon (MATH) assay. (amino acid sequence DGDFDR residues 236-241) at The parental isolate X26332 and the mutant JB12-23 were nucleotides 706-723, and is upstream of the sugar binding tested for their adherence to hydrocarbon (Déziel et al., site (amino acid sequence GEMS residues 318-321) at 2001). Overnight cultures (16-18 h) grown at 37 ºC with nucleotides 952-963. agitation in LB medium or LB medium with 50 µg/ml The gene, spgM, encodes a bifunctional enzyme with kanamycin were used for this assay. Each hydrophobicity phosphoglucomutase and phosphomannomutase that has assay was performed in replicates of six. been reported to be important for synthesis of LPS O-anti- gen in S. maltophilia (McKay et al., 2003). A recent study Statistical analyses of biofilm and MATH assays. To investigated the transposon mutagenesis of xanB, a gene compare the parental isolate X26332 with the mutant that lies downstream of spgM with both genes part of an JB12-23, the mean biofilm values and the MATH values for operon, and reported that the xanB mutant was defective these two were each analysed by unpaired t test using in biosynthesis of LPS O-antigen (Huang et al., 2006). ProStat software (Version 4.0, Poly Software International, The JB12-23 mutant produced significantly more biofilm NY), with significance set at p-value ≤ 0.05. than the parental isolate X26332 on polyvinyl chloride microtiter plate wells [t(8) = 15.67; p < 0.001] (Fig. 2A), Congo red agar plate assay. The Congo red agar plate on polystyrene microtiter plate wells [t(8) = 4.51; p < assay was performed on the parental isolate X26332 and 0.05] (Fig. 2B), and on borosilicate glass tube surfaces the mutant JB12-23 (Chung et al., 2003). Overnight broth [t(8) = 3.70; p < 0.05] (Fig. 2C), as determined using cultures were streaked onto LB agar (or LB agar with 50 unpaired t tests. The biofilm of the JB12-23 mutant devel- µg/ml kanamycin) containing 0.05% (wt/v) Congo red dye. oped more quickly than the biofilm of the wild type culture. Plates were incubated at 37 °C for 48 h and colonies were A clearly visible crystal-violet stained biofilm could be then examined for absorption of Congo red dye. detected as early as 4 h (Fig. 3) in the JB12-23 mutant, in contrast to the wild type culture that forms a visible biofilm after 24-30 h. RESULTS AND DISCUSSION The doubling times of X26332 and the JB12-23 mutant were 59.7 min and 56.5 min respectively when cultures Southern hybridisation using a probe complementary to the were grown in the shaken flasks and were 96.8 min and EZ::TN<R6Kγori/KAN-2>Tnp transposon confirmed the 93.5 min respectively when cultures were grown in the sta- presence of a single insertion of this transposon within the tionary microtiter plates. These data suggest that transpo- chromosomal DNA of the JB12-23 mutant (Fig. 1). son insertion did not alter the doubling time of the mutant Hybridisation also confirmed that the chromosomal DNA of relative to the parental isolate. The observations eliminat- the X26332 parental isolate did not contain the ed the possibility that increased biofilm of the JB12-23 EZ::TN<R6Kγori/KAN-2>Tnp transposon. DNA sequencing 38 J.S. Brooke et al. The JB12-23 mutant expressed a lipopolysaccharide in which the high molecular weight LPS was absent in contrast to lipopolysaccharide expressed by isolate X26332 (Fig. 4). These results agree with those reported for the S. mal- tophilia spgM mutant K2049 that was defective in biosyn- thesis of LPS O-antigen (McKay et al., 2003). It is reason- able to suggest that the incomplete LPS produced on the cell surface of the mutant JB12-23 would unmask cell sur- face components that would otherwise be hidden by a com- plete LPS, thus enabling the cells to come into closer con- tact with the abiotic surface and increasing the ability of these cells to stick to the surface. This may be an explana- tion for the increased biofilm of the mutant JB12-23. FIG. 2 - Biofilm assays of Stenotrophomonas maltophilia X26332 and mutant JB12-23. Biofilm production of the cultures at 48 h of growth on (A) polyvinyl chloride microtiter well surfaces, (B) polystyrene microtiter well surfaces, and (C) on borosilicate glass tube surfaces. Mean biofilm values are shown. Vertical bars represent standard devi- ation around the mean. FIG. 4 - 10% SDS-PAGE silver stained gel of lipopolysaccharide extracted from Stenotrophomonas maltophilia. Lane 1: X26332, lane 2: JB12-23. McKay et al. (2003) proposed that the cell surface of the S. maltophilia spgM mutant would likely be more hydrophobic in comparison to that of the wild-type parental S. maltophilia isolate. This proposal would help explain the increased biofilm expression on the hydrophobic polyvinyl FIG. 3 - Biofilm formation of Stenotrophomonas maltophilia chloride wells by mutant JB12-23 in this current study. X26332 and mutant JB12-23 at 4 h of incubation on Such an increase in cell surface hydrophobicity in the polyvinyl chloride microtiter well surfaces. Each biofilm mutant JB12-23 should enable it to adhere more strongly assay was performed in triplicate. Mean biofilm values to the relatively hydrophobic polyvinyl chloride plastic sur- are shown. Vertical bars represent standard deviation face and thus form more biofilm in contrast to the biofilm around the mean. produced by the parental isolate X26332. However, this proposed increase in cell surface hydrophobicity does not explain the increased biofilm production by the mutant mutant was due to increased growth rate. Light microscopy JB12-23 on the relatively hydrophilic polystyrene and observations also showed no difference in cellular morphol- borosilicate glass surfaces (Figs. 2B and 2C). Thus, to ogy between the JB12-23 mutant and the parental isolate determine if cell surface hydrophobicity was a significant X26332, eliminating the possibility that gross morphologi- factor in cell adherence, the microbial adherence to hydro- cal changes produced by transposon insertion subsequent- carbon (MATH) assay was performed (Déziel et al., 2001). ly caused an increase in biofilm production by the mutant. Using unpaired t tests, no significant differences [t(8) = Disruption of the LPS in the JB12-23 mutant appeared 0.467; p > 0.05] in cell surface hydrophobicity between the to have enabled these cells to overcome the repulsive parental isolate and the mutant JB12-23 were observed forces at the abiotic surface and adhere to the surface more (Table 2). It is possible that the assay’s sensitivity could not rapidly than the cells of the parental isolate. These obser- distinguish subtle differences between the cell surface vations suggested that LPS length may help to regulate hydrophobicity of the parental isolate and the JB12-23 biofilm formation in S. maltophilia; this needs to be further mutant. The data suggested that the increased biofilm for- investigated with well defined LPS mutants of S. malto- mation by the JB12-23 mutant was not due to a significant philia. increase in cell surface hydrophobicity. Ann. Microbiol., 58 (1), 35-40 (2008) 39 TABLE 2 - Mean percentage (with standard deviation in parentheses) of cells adhered to hexadecane for Stenotrophomonas mal- tophilia X26332 and the JB12-23 mutant Volume of hexadecane 0 µl 100 µl 300 µl 500 µl 700 µl X26332 0 14.6 (9.4) 33.3 (1.7) 35.8 (10.4) 22.2 (9.3) JB12-23 0 10.1 (2.5) 33.0 (2.4) 23.6 (9.0) 19.0 (1.0) To observe any differences in exopolysaccharide pro- Acknowledgements duction by colonies of the parental isolate X26332 and the We thank B. Kabat (Children’s Memorial Hospital, Chicago, JB12-23 mutant, the Congo red agar plate assay was per- IL) for the gift of the S. maltophilia X26332 clinical isolate. formed (Chung et al., 2003). After 48 h at 37 °C, the iso- We thank M.E. Silliker of our Institution for assistance with late X26332 absorbed the dye and produced red colonies, DNA sequencing. We thank R. Tungekar and M. Zuniga for whereas the JB12-23 mutant did not absorb the dye and excellent technical assistance with the Congo red assay. produced cream-pink colonies (Fig. 5). After 72 h at 37 °C, This work was supported by an Academic Research both the parental isolate and mutant produced red Enhancement Award (AREA) grant 1R15AI062708-01A1 colonies. These observations suggested that there may be from the National Institutes of Health, and grants an increased expression of exopolysaccharide by the JB12- (University Research Council, College of Liberal Arts & 23 mutant. However, we have not eliminated the possibili- Sciences) from DePaul University. ty that the colony morphology differences may be due to additional cell surface structures in the parental isolate that REFERENCES are able to bind to Congo red dye. A mutant of Burkholderia cepacia strain C1394 showed a decreased absorption of Carpentier B., Chassaing D. (2004). Interactions in biofilms Congo red dye in comparison to the parental strain; the dif- between Listeria monocytogenes and resident microorgan- ference in absorption was reportedly due to the overex- isms from food industry premises. Int. J. Food Microbiol., 97: 111-122. pression of pili by the mutant (Chung et al., 2003). In our study, preliminary ultrastructural examinations of S. mal- Chung J.W., Altman E., Beveridge T.J., Speert D.P. (2003). Colonial morphology of Burkholderia cepacia complex tophilia X26332 using negative staining and transmission genomovar III: Implications in exopolysaccharide produc- electron microscopy have not revealed the presence of pili tion, pilus expression, and persistence in the mouse. Infect. in this isolate (data not shown). Further investigation is Immun., 71: 904-909. needed to see if overexpression of pili is present in the Denton M., Kerr K. (1998). Microbiological and clinical aspects of mutant JB12-23. infection associated with Stenotrophomonas maltophilia. In conclusion, transposon mutagenesis of the spgM Clin. Microbiol. Rev., 11: 57-80. gene resulted in significantly increased biofilm formation by de Oliveira-Garcia D., Dall’Agnol M., Rosales M., Azzuz A.C.G.S., S. maltophilia on polyvinyl, polystyrene, and borosilicate Martinez M.B., Girón J.A. (2002). Characterization of flagella produced by clinical strains of Stenotrophomonas maltophil- surfaces. Future research is required to determine if the ia. Emerg. Infect. Dis., 8: 918-923. lack of complete LPS in the transposon mutant exposed de Oliveira-Garcia D., Dall’Agnol M., Rosales M., Azzuz A.C.G.S., additional cell surface components that contributed to this Alcántara N., Martinez M.B., Girón J.A. (2003). Fimbriae and increased biofilm formation. adherence of Stenotrophomonas maltophilia to epithelial cells and to abiotic surfaces. Cell. Microbiol., 5: 625-636. Déziel E., Comeau Y., Villemur R. (2001). Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming, and twitching motilities. J. Bacteriol., 183: 1195-1204. A B Di Bonaventura G., Spedicato I., D’Antonio D., Robuffo I., Piccolomini R. (2004). Biofilm formation by Stenotrophomonas maltophilia: modulation by quinolones, trimethoprim-sulfamethoxazole, and ceftazidime. Antimicrob. Agents Chemother., 48: 151-160. Di Bonaventura G., Prosseda G., Del Chierico F., Cannavacciuolo S., Cipriani P., Petrucca A., Superti F., Ammendolia M.G., Concato C., Fiscarelli E., Casalino M., Piccolomini R., Nicoletti M., Colonna B. (2007). Molecular characterization of viru- lence determinants of Stenotrophomonas maltophilia strains isolated from patients affected by cystic fibrosis. Int. J. Immunopathol. Pharmacol., 20: 529-537. FIG. 5 - Growth of Stenotrophomonas maltophilia X26332 and Donlan R.M. (2002). Biofilms: Microbial life on surfaces. Emerg. mutant JB12-23 on 0.05% (wt/v) Congo red agar Infect. Dis., 8: 881-890. plates. At 48 h at 37 °C, (A) the X26332 colonies Fomsgaard A., Freudenberg M.A., Galanos C. (1990). absorbed the Congo red dye whereas (B) the JB12-23 Modification of the silver staining technique to detect mutant did not absorb the Congo red dye. This is par- lipopolysaccharide in polyacrylamide gels. J. Clin. Microbiol., ticularly evident for the isolated colonies. 28: 2627-2631. 40 J.S. Brooke et al. Head N.E., Yu H. (2004). Cross-sectional analysis of clinical and McKay G.A., Woods D.E., MacDonald K.L., Poole K. (2003). Role environmental isolates of Pseudomonas aeruginosa: biofilm of phosphoglucomutase of Stenotrophomonas maltophilia in formation, virulence, and genome diversity. Infect Immun., lipopolysaccharide biosynthesis, virulence, and antibiotic 72: 133-144. resistance. Infect. Immun., 71: 3068-3075. Huang T-P., Somers E.B., Lee Wong, A.C. (2006). Differential Sambrook J., Fritsch E.F., Maniatis T. (1989). Analysis and biofilm formation and motility associated with lipopolysac- cloning of eukaryotic genomic DNA. In: Ford N., Nolan C., charide/exopolysaccharide-coupled biosynthetic genes in Ferguson M., Eds., Molecular Cloning: A Laboratory Manual, nd Stenotrophomonas maltophilia. J. Bacteriol., 188: 3116- 2 edn., Cold Spring Harbor Laboratory Press, New York, pp. 3120. 9.31-9.59. Ingraham J.L., Maaloe O., Neidhardt F.C. (1983). Growth of cells Senol E. (2004). Stenotrophomonas maltophilia: the significance and cultures. In: Ingraham J.L., Ed., Growth of the Bacterial and role as a nosocomial pathogen. J. Hosp. Infect., 57: 1- Cell, Sinauer, Massachusetts, pp. 228-229. 7. Jucker B.A., Harms H., Zehnder A.J.B. (1996). Adhesion of the Stoodley P., Sauer K., Davies D.G., Costerton J.W. (2002). positively charged bacterium Stenotrophomonas Biofilms as complex differentiated communities. Annu. Rev. (Xanthomonas) maltophilia 70401 to glass and Teflon. J. Microbiol., 56: 187-209. Bacteriol., 178: 5472-5479. Vidipó L.A., Marques E.A., Puchelle E., Plotkowski M.C. (2001). Kavouras J.H., Maki J.S. (2004). Inhibition of the reattachment Stenotrophomonas maltophilia interaction with human of young adult zebra mussels by single-species biofilms and epithelial respiratory cells in vitro. Microbiol. Immunol., 45: associated exopolymers. J. Appl. Microbiol., 97: 1236-1246. 563-569. Marolda C.L., Welsh J., Dafoe L., Valvano M.A. (1990). Genetic Watnick P.I., Kolter R. (1999). Steps in the development of a analysis of the O7-polysaccharide biosynthesis region from Vibrio cholerae El Tor biofilm. Mol. Microbiol., 34: 586-595. the Escherichia coli O7:K1 strain VW187. J. Bacteriol., 172: 3590-3599. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Mutation of a lipopolysaccharide synthesis gene results in increased biofilm ofStenotrophomonas maltophilia on plastic and glass surfaces

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Springer Journals
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Copyright © 2008 by University of Milan and Springer
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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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Abstract

Annals of Microbiology, 58 (1) 35-40 (2008) Mutation of a lipopolysaccharide synthesis gene results in increased biofilm of Stenotrophomonas maltophilia on plastic and glass surfaces Joanna S. BROOKE*, Albert VO, Patrick WATTS, Nicholas A. DAVIS Department of Biological Sciences, DePaul University, 2325 N. Clifton, Chicago, IL 60614, USA Received 2 October 2007 / Accepted 28 December 2007 Abstract - Stenotrophomonas maltophilia is a biofilm-forming opportunistic pathogen that is emerging worldwide and demonstrates increasing rates of antibiotic resistance and significant case fatality/ratios in immunocompromised or debilitated patients. This study tested the hypothesis that mutation of a lipopolysaccharide synthesis gene results in increased biofilm of S. maltophilia. Transposon mutagenesis was used to disrupt genes of S. maltophilia clinical isolate X26332 and generated the JB12-23 mutant that produced increased biofilm in comparison to the isogenic parental isolate. Southern hybridisation revealed a single transposon insertion in the JB12-23 mutant. Rescue-cloning and DNA sequencing revealed that the transposon had inserted into the spgM gene of S. maltophil- ia mutant JB12-23. Biofilm assays revealed that the JB12-23 mutant produced more biofilm on polyvinyl chloride, polystyrene sur- faces, and on borosilicate glass in comparison to the parental isolate. Lipopolysaccharide SDS-PAGE showed that the JB12-23 mutant lacked high molecular weight LPS in comparison to the parental isolate. Key words: lipopolysaccharide, spgM, Stenotrophomonas, biofilm, transposon mutagenesis. INTRODUCTION al species. Stenotrophomonas maltophilia is often a co- pathogen and can be isolated with other pathogens, such Stenotrophomonas maltophilia is a worldwide emerging, as Pseudomonas aeruginosa in the cystic fibrosis lung envi- multi-antibiotic opportunistic pathogen associated with a ronment (Di Bonaventura et al., 2007). Given the possibil- significant case-fatality ratio in the immunocompromised ity of exchange of genetic information between bacteria patient population (Denton and Kerr, 1998). within biofilms, this emphasizes the need to investigate the Stenotrophomonas maltophilia has been recovered in hos- biofilm formed by S. maltophilia. This current study inves- pitals from respiratory devices, contaminated water-based tigated the effect of a transposon insertion in the gene, solutions and other materials (Denton and Kerr, 1998; spgM, on the biofilm of S. maltophilia. Senol, 2004). This pathogen is associated with a wide range of disorders including pneumonia, bacteraemia, cen- tral nervous system infections, ocular infections, endo- MATERIALS AND METHODS carditis, urinary tract infection, skin and soft tissue infec- tion, bone and joint infections, gastrointestinal infections, Maintenance of bacterial strains and plasmids. All and septic shock (Denton and Kerr, 1998). strains and plasmids used in this study are listed in Table 1. A significant characteristic of S. maltophilia is its ability Escherichia coli ER2420 (pACYC177) was maintained on to adhere to glass, Teflon, plastic surfaces and epithelial Luria-Bertani (LB) agar containing 50 µg/ml kanamycin and cells (Jucker et al., 1996; Vidipó et al., 2001; de Oliveira- 100 µg/ml ampicillin. Escherichia coli TransforMax EC100D Garcia et al., 2002, 2003). Stenotrophomonas maltophilia pir-116 (pJSB1) was maintained on LB agar containing 50 forms biofilms. Biofilms are microbial communities that are µg/ml kanamycin. Stenotrophomonas maltophilia isolate surrounded by a porous extracellular polysaccharide matrix X26332 was maintained on LB agar. The S. maltophilia material (Donlan, 2002; Stoodley et al., 2002). These JB12-23 mutant was maintained on LB agar containing 50 biofilms can serve as persistent sources of infection. Recent µg/ml kanamycin. All bacteria were grown at 37 ºC. studies report biofilms of S. maltophilia on stainless steel (Carpentier and Chassaing, 2004), glass and polystyrene Transposon mutagenesis. Transposon mutagenesis of surfaces (Di Bonaventura et al., 2004; Kavouras and Maki, the clinical parental isolate X26332 was performed using 2004; Huang et al., 2006). the EZ::TN<R6Kγori/KAN-2>Tnp Transposome mutagene- Frequently, biofilms are communities of mixed bacteri- sis kit (Epicenter Technologies, WI, USA). Transformants were selected on LB agar with 50 µg/ml kanamycin. The JB12-23 mutant was recovered after screening for biofilm * Corresponding author. Phone: (773) 325-1161; production as described below. Fax: (773) 325-7596; E-mail: jbrooke@depaul.edu 36 J.S. Brooke et al. TABLE 1 - Strains and plasmids used in this study Bacterial strains Description Reference or source Escherichia coli TransforMax EC100D pir-116 F mcrA Δ(mrr-hsdRMS-mcrBC) ϕ80dlacZΔM15 Epicentre (Madison, WI) ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ rpsL nupG pir-116(DHFR). ER2420 F ara-14 leu fhuA2 Δ(gpt-proA)62 lacY1 glnV44 New England BioLabs (Beverly, MA) galK2 rpsL20 xyl-5 mtl-1 Δ(mcrC-mrr) Stenotrophomonas maltophilia X26332 Clinical isolate Children’s Memorial Hospital (Chicago, IL) JB12-23 Insertion derivative of X26332, Kan This study Plasmids r r pACYC177 Amp , Kan , 3.9 kb New England BioLabs (Beverly, MA) pJSB1 Kan This study r r Amp , ampicillin resistant; Kan , kanamycin resistant. DNA preparation and Southern hybridisation. obtained using custom-designed primers and pJSB1 DNA Chromosomal DNA from S. maltophilia X26332 and the as a template. JB12-23 mutant was isolated using a Wizard genomic DNA purification kit (Promega, WI, USA). Plasmid DNA was iso- Growth of cultures in shaken flasks and in stationary lated from E. coli ER2420 (pACYC177) and E. coli microtiter plates. The parental isolate X26332 was grown TransforMax EC100D pir-116 (pJSB1) using the QIAprep in LB broth and the JB12-23 mutant was grown in LB con- Spin Miniprep Kit (Qiagen, CA, USA). To detect the number taining 50 µg/ml kanamycin. Both cultures were grown at of EZ::TN<R6Kγ ori/KAN-2>Tnp transposon insertions pres- 37 ºC with agitation at 250 rpm in an orbital incubator ent in the chromosomal DNA of the mutant JB12-23, shaker (Amerex Instruments, CA, USA). Six 150 ml flask Southern hybridisations were performed (Sambrook et al., cultures each of X26332 and JB12-23 were used for the 1989). To detect the presence of the EZ::TN<R6Kγ ori/KAN- shaken flask growth assays. Each flask was inoculated with 2> transposon, a DNA probe was made to part of the overnight culture and adjusted to within the OD range of kanamycin resistance gene (kan ) between bases 2127 and 0.10-0.14. Flasks were incubated at 37 ºC with agitation at 2506 of the plasmid pACYC177 maintained in E. coli 250 rpm in a Labline incubator-shaker (Labline Instruments, ER2420. This region was amplified by using an A-Taq PCR IL, USA). Growth readings at OD were taken hourly up to kit (USB, OH, USA) in a GeneAmp PCR System 2400 (Perkin 11 h. Growth assays were performed in three independent Elmer, MA, USA) and labelled with digoxigenin-11-2’- experiments. deoxy-uridine-5’-triphosphate (DIG-11-dUTP) using the For the stationary microtiter plate growth assays, five DIG high prime DNA labelling and detection starter kit II cultures each of X26332 and JB12-23 were used (Head and (Roche, Germany). The forward primer sequence used was Yu, 2004). Overnight cultures were centrifuged at 4000 x g (5’-ATGGTCAGACTAAACTGGCTGACG-3’) and the reverse for 10 min. and pellets were resuspended to an OD of primer sequence was (5’-GTGACGACTGAATCCGGTGA- 0.25 in fresh LB or LB containing 50 µg/ml kanamycin. GAAT-3’). Southern blots were hybridised at 42 °C for 18- Wells of the microtiter plate containing 150 µl of culture 20 h and bands developed using the colorimetric detection medium were inoculated with 20 µl culture. Plates were system as recommended by the manufacturer. incubated stationary at 37 ºC. Growth readings at OD were taken at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 25, and Rescue cloning and sequencing of the transposon 48.5 h. Growth assays were performed in two independent insertion in the mutant JB12-23. The gene containing experiments. The generation times were determined for the transposon insertion was rescue-cloned from the chro- the batch culture and static cultures using the exponential mosomal DNA of mutant JB12-23 by self-ligation of EcoRI- growth phase from a graph of ln OD versus time (Ingraham digested chromosomal DNA and electroporation of E. coli et al., 1983). The specific growth rate was calculated as the TransforMax EC100D (pir ) electrocompetent cells accord- slope of the regression line (1/h). ing to manufacturer’s instructions (Epicenter). Plasmid pJSB1 contained the rescued mutated gene. Genomic DNA Biofilm assay on polyvinyl chloride, polystyrene, and surrounding the transposon insertion was sequenced using borosilicate glass surfaces. The biofilms of parental iso- an automated DNA sequencing apparatus (ABI prism 310 late X26332 and the JB12-23 mutant were assayed Genetic Analyzer, Perkin Elmer) with primers complemen- (Watnick and Kolter, 1999). Overnight cultures (16-18 h) tary to the transposon that were supplied in the mutagen- were centrifuged at 5000 x g and the cell pellets were esis kit. Extension of the nucleotide sequences was resuspended in new LB or LB containing 50 µg/ml Ann. Microbiol., 58 (1), 35-40 (2008) 37 kanamycin to the OD of 0.25. Twenty microlitres of each resuspended culture were used to inoculate each microtiter plate well that contained 150 µl LB medium (or 150 µl of LB medium containing 50 µg/ml kanamycin). Control wells contained 170 µl LB only (no cells). After stationary incu- bation at 37 ºC for 24 h, the medium was replaced with fresh medium, and then the plates were incubated station- ary for a further 24 h at 37 °C. Following incubation, cul- ture medium and nonadherent cells were removed, the wells were stained with 1% (wt/v) crystal violet, rinsed four times with phosphate buffered saline, and then the adher- ent cells were thoroughly resuspended in 80% (v/v) FIG. 1 - Southern blot of restriction enzyme-digested chromoso- mal DNA of Stenotrophomonas maltophilia X26332 and ethanol. OD readings were recorded of each well. mutant JB12-23. Lane 1: HindIII digested λDNA (frag- For the borosilicate glass tube biofilm assay, tubes were ment sizes in kb are indicated to the left of the blot), inoculated with the standardized cultures as described lane 2: uncut JB12-23, lane 3: EcoRI digested X26332, above for the microtiter plate assays. Tubes were incubat- lane 4: BamHI digested JB12-23, lane 5: EcoRV digest- ed stationary at 37 °C for 24 h. After incubation, the medi- ed JB12-23, lane 6: HindIII digested JB12-23, lane 7: um was replaced with fresh medium and the tubes were SalI digested JB12-23. incubated for a further 24.5 h at 37 °C. Biofilm production was detected using the crystal violet assay as described above for the microtiter plate assays. Each biofilm plate and tube assay was performed using five replicates. Two using primers to the EZ::TN<R6Kγ ori/KAN-2>Tnp transpo- independent biofilm assays were performed for each type son revealed that the JB12-23 mutant harboured the trans- of surface tested. poson at nucleotide 1476 within the spgM homologue. Recently it has been reported that the spgM homologue Lipopolysaccharide extraction and visualisation encodes the phosphoglucomutase enzyme that is used for using SDS-PAGE. Lipopolysaccharide was isolated from synthesis and assembly of LPS in S. maltophilia (McKay et overnight cultures of the parental isolate X26332 and the al., 2003). The transposon insertion is located at mutant JB12-23 (Marolda et al., 1990) and visualised using nucleotides 888/889 of the spgM gene downstream of both SDS-PAGE with silver staining (Fomsgaard et al., 1990). the active site (amino acid sequence TASHN residues 94- 98) at nucleotides 280-294, and the metal ion binding site Microbial adherence to hydrocarbon (MATH) assay. (amino acid sequence DGDFDR residues 236-241) at The parental isolate X26332 and the mutant JB12-23 were nucleotides 706-723, and is upstream of the sugar binding tested for their adherence to hydrocarbon (Déziel et al., site (amino acid sequence GEMS residues 318-321) at 2001). Overnight cultures (16-18 h) grown at 37 ºC with nucleotides 952-963. agitation in LB medium or LB medium with 50 µg/ml The gene, spgM, encodes a bifunctional enzyme with kanamycin were used for this assay. Each hydrophobicity phosphoglucomutase and phosphomannomutase that has assay was performed in replicates of six. been reported to be important for synthesis of LPS O-anti- gen in S. maltophilia (McKay et al., 2003). A recent study Statistical analyses of biofilm and MATH assays. To investigated the transposon mutagenesis of xanB, a gene compare the parental isolate X26332 with the mutant that lies downstream of spgM with both genes part of an JB12-23, the mean biofilm values and the MATH values for operon, and reported that the xanB mutant was defective these two were each analysed by unpaired t test using in biosynthesis of LPS O-antigen (Huang et al., 2006). ProStat software (Version 4.0, Poly Software International, The JB12-23 mutant produced significantly more biofilm NY), with significance set at p-value ≤ 0.05. than the parental isolate X26332 on polyvinyl chloride microtiter plate wells [t(8) = 15.67; p < 0.001] (Fig. 2A), Congo red agar plate assay. The Congo red agar plate on polystyrene microtiter plate wells [t(8) = 4.51; p < assay was performed on the parental isolate X26332 and 0.05] (Fig. 2B), and on borosilicate glass tube surfaces the mutant JB12-23 (Chung et al., 2003). Overnight broth [t(8) = 3.70; p < 0.05] (Fig. 2C), as determined using cultures were streaked onto LB agar (or LB agar with 50 unpaired t tests. The biofilm of the JB12-23 mutant devel- µg/ml kanamycin) containing 0.05% (wt/v) Congo red dye. oped more quickly than the biofilm of the wild type culture. Plates were incubated at 37 °C for 48 h and colonies were A clearly visible crystal-violet stained biofilm could be then examined for absorption of Congo red dye. detected as early as 4 h (Fig. 3) in the JB12-23 mutant, in contrast to the wild type culture that forms a visible biofilm after 24-30 h. RESULTS AND DISCUSSION The doubling times of X26332 and the JB12-23 mutant were 59.7 min and 56.5 min respectively when cultures Southern hybridisation using a probe complementary to the were grown in the shaken flasks and were 96.8 min and EZ::TN<R6Kγori/KAN-2>Tnp transposon confirmed the 93.5 min respectively when cultures were grown in the sta- presence of a single insertion of this transposon within the tionary microtiter plates. These data suggest that transpo- chromosomal DNA of the JB12-23 mutant (Fig. 1). son insertion did not alter the doubling time of the mutant Hybridisation also confirmed that the chromosomal DNA of relative to the parental isolate. The observations eliminat- the X26332 parental isolate did not contain the ed the possibility that increased biofilm of the JB12-23 EZ::TN<R6Kγori/KAN-2>Tnp transposon. DNA sequencing 38 J.S. Brooke et al. The JB12-23 mutant expressed a lipopolysaccharide in which the high molecular weight LPS was absent in contrast to lipopolysaccharide expressed by isolate X26332 (Fig. 4). These results agree with those reported for the S. mal- tophilia spgM mutant K2049 that was defective in biosyn- thesis of LPS O-antigen (McKay et al., 2003). It is reason- able to suggest that the incomplete LPS produced on the cell surface of the mutant JB12-23 would unmask cell sur- face components that would otherwise be hidden by a com- plete LPS, thus enabling the cells to come into closer con- tact with the abiotic surface and increasing the ability of these cells to stick to the surface. This may be an explana- tion for the increased biofilm of the mutant JB12-23. FIG. 2 - Biofilm assays of Stenotrophomonas maltophilia X26332 and mutant JB12-23. Biofilm production of the cultures at 48 h of growth on (A) polyvinyl chloride microtiter well surfaces, (B) polystyrene microtiter well surfaces, and (C) on borosilicate glass tube surfaces. Mean biofilm values are shown. Vertical bars represent standard devi- ation around the mean. FIG. 4 - 10% SDS-PAGE silver stained gel of lipopolysaccharide extracted from Stenotrophomonas maltophilia. Lane 1: X26332, lane 2: JB12-23. McKay et al. (2003) proposed that the cell surface of the S. maltophilia spgM mutant would likely be more hydrophobic in comparison to that of the wild-type parental S. maltophilia isolate. This proposal would help explain the increased biofilm expression on the hydrophobic polyvinyl FIG. 3 - Biofilm formation of Stenotrophomonas maltophilia chloride wells by mutant JB12-23 in this current study. X26332 and mutant JB12-23 at 4 h of incubation on Such an increase in cell surface hydrophobicity in the polyvinyl chloride microtiter well surfaces. Each biofilm mutant JB12-23 should enable it to adhere more strongly assay was performed in triplicate. Mean biofilm values to the relatively hydrophobic polyvinyl chloride plastic sur- are shown. Vertical bars represent standard deviation face and thus form more biofilm in contrast to the biofilm around the mean. produced by the parental isolate X26332. However, this proposed increase in cell surface hydrophobicity does not explain the increased biofilm production by the mutant mutant was due to increased growth rate. Light microscopy JB12-23 on the relatively hydrophilic polystyrene and observations also showed no difference in cellular morphol- borosilicate glass surfaces (Figs. 2B and 2C). Thus, to ogy between the JB12-23 mutant and the parental isolate determine if cell surface hydrophobicity was a significant X26332, eliminating the possibility that gross morphologi- factor in cell adherence, the microbial adherence to hydro- cal changes produced by transposon insertion subsequent- carbon (MATH) assay was performed (Déziel et al., 2001). ly caused an increase in biofilm production by the mutant. Using unpaired t tests, no significant differences [t(8) = Disruption of the LPS in the JB12-23 mutant appeared 0.467; p > 0.05] in cell surface hydrophobicity between the to have enabled these cells to overcome the repulsive parental isolate and the mutant JB12-23 were observed forces at the abiotic surface and adhere to the surface more (Table 2). It is possible that the assay’s sensitivity could not rapidly than the cells of the parental isolate. These obser- distinguish subtle differences between the cell surface vations suggested that LPS length may help to regulate hydrophobicity of the parental isolate and the JB12-23 biofilm formation in S. maltophilia; this needs to be further mutant. The data suggested that the increased biofilm for- investigated with well defined LPS mutants of S. malto- mation by the JB12-23 mutant was not due to a significant philia. increase in cell surface hydrophobicity. Ann. Microbiol., 58 (1), 35-40 (2008) 39 TABLE 2 - Mean percentage (with standard deviation in parentheses) of cells adhered to hexadecane for Stenotrophomonas mal- tophilia X26332 and the JB12-23 mutant Volume of hexadecane 0 µl 100 µl 300 µl 500 µl 700 µl X26332 0 14.6 (9.4) 33.3 (1.7) 35.8 (10.4) 22.2 (9.3) JB12-23 0 10.1 (2.5) 33.0 (2.4) 23.6 (9.0) 19.0 (1.0) To observe any differences in exopolysaccharide pro- Acknowledgements duction by colonies of the parental isolate X26332 and the We thank B. Kabat (Children’s Memorial Hospital, Chicago, JB12-23 mutant, the Congo red agar plate assay was per- IL) for the gift of the S. maltophilia X26332 clinical isolate. formed (Chung et al., 2003). After 48 h at 37 °C, the iso- We thank M.E. Silliker of our Institution for assistance with late X26332 absorbed the dye and produced red colonies, DNA sequencing. We thank R. Tungekar and M. Zuniga for whereas the JB12-23 mutant did not absorb the dye and excellent technical assistance with the Congo red assay. produced cream-pink colonies (Fig. 5). After 72 h at 37 °C, This work was supported by an Academic Research both the parental isolate and mutant produced red Enhancement Award (AREA) grant 1R15AI062708-01A1 colonies. These observations suggested that there may be from the National Institutes of Health, and grants an increased expression of exopolysaccharide by the JB12- (University Research Council, College of Liberal Arts & 23 mutant. However, we have not eliminated the possibili- Sciences) from DePaul University. ty that the colony morphology differences may be due to additional cell surface structures in the parental isolate that REFERENCES are able to bind to Congo red dye. A mutant of Burkholderia cepacia strain C1394 showed a decreased absorption of Carpentier B., Chassaing D. (2004). Interactions in biofilms Congo red dye in comparison to the parental strain; the dif- between Listeria monocytogenes and resident microorgan- ference in absorption was reportedly due to the overex- isms from food industry premises. Int. J. Food Microbiol., 97: 111-122. pression of pili by the mutant (Chung et al., 2003). In our study, preliminary ultrastructural examinations of S. mal- Chung J.W., Altman E., Beveridge T.J., Speert D.P. (2003). Colonial morphology of Burkholderia cepacia complex tophilia X26332 using negative staining and transmission genomovar III: Implications in exopolysaccharide produc- electron microscopy have not revealed the presence of pili tion, pilus expression, and persistence in the mouse. Infect. in this isolate (data not shown). Further investigation is Immun., 71: 904-909. needed to see if overexpression of pili is present in the Denton M., Kerr K. (1998). Microbiological and clinical aspects of mutant JB12-23. infection associated with Stenotrophomonas maltophilia. In conclusion, transposon mutagenesis of the spgM Clin. Microbiol. Rev., 11: 57-80. gene resulted in significantly increased biofilm formation by de Oliveira-Garcia D., Dall’Agnol M., Rosales M., Azzuz A.C.G.S., S. maltophilia on polyvinyl, polystyrene, and borosilicate Martinez M.B., Girón J.A. (2002). Characterization of flagella produced by clinical strains of Stenotrophomonas maltophil- surfaces. Future research is required to determine if the ia. Emerg. Infect. Dis., 8: 918-923. lack of complete LPS in the transposon mutant exposed de Oliveira-Garcia D., Dall’Agnol M., Rosales M., Azzuz A.C.G.S., additional cell surface components that contributed to this Alcántara N., Martinez M.B., Girón J.A. (2003). Fimbriae and increased biofilm formation. adherence of Stenotrophomonas maltophilia to epithelial cells and to abiotic surfaces. Cell. Microbiol., 5: 625-636. Déziel E., Comeau Y., Villemur R. (2001). Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming, and twitching motilities. J. Bacteriol., 183: 1195-1204. A B Di Bonaventura G., Spedicato I., D’Antonio D., Robuffo I., Piccolomini R. (2004). Biofilm formation by Stenotrophomonas maltophilia: modulation by quinolones, trimethoprim-sulfamethoxazole, and ceftazidime. Antimicrob. Agents Chemother., 48: 151-160. Di Bonaventura G., Prosseda G., Del Chierico F., Cannavacciuolo S., Cipriani P., Petrucca A., Superti F., Ammendolia M.G., Concato C., Fiscarelli E., Casalino M., Piccolomini R., Nicoletti M., Colonna B. (2007). Molecular characterization of viru- lence determinants of Stenotrophomonas maltophilia strains isolated from patients affected by cystic fibrosis. Int. J. Immunopathol. Pharmacol., 20: 529-537. FIG. 5 - Growth of Stenotrophomonas maltophilia X26332 and Donlan R.M. (2002). Biofilms: Microbial life on surfaces. Emerg. mutant JB12-23 on 0.05% (wt/v) Congo red agar Infect. Dis., 8: 881-890. plates. At 48 h at 37 °C, (A) the X26332 colonies Fomsgaard A., Freudenberg M.A., Galanos C. (1990). absorbed the Congo red dye whereas (B) the JB12-23 Modification of the silver staining technique to detect mutant did not absorb the Congo red dye. This is par- lipopolysaccharide in polyacrylamide gels. J. Clin. Microbiol., ticularly evident for the isolated colonies. 28: 2627-2631. 40 J.S. Brooke et al. Head N.E., Yu H. (2004). Cross-sectional analysis of clinical and McKay G.A., Woods D.E., MacDonald K.L., Poole K. (2003). Role environmental isolates of Pseudomonas aeruginosa: biofilm of phosphoglucomutase of Stenotrophomonas maltophilia in formation, virulence, and genome diversity. Infect Immun., lipopolysaccharide biosynthesis, virulence, and antibiotic 72: 133-144. resistance. Infect. Immun., 71: 3068-3075. Huang T-P., Somers E.B., Lee Wong, A.C. (2006). Differential Sambrook J., Fritsch E.F., Maniatis T. (1989). Analysis and biofilm formation and motility associated with lipopolysac- cloning of eukaryotic genomic DNA. In: Ford N., Nolan C., charide/exopolysaccharide-coupled biosynthetic genes in Ferguson M., Eds., Molecular Cloning: A Laboratory Manual, nd Stenotrophomonas maltophilia. J. Bacteriol., 188: 3116- 2 edn., Cold Spring Harbor Laboratory Press, New York, pp. 3120. 9.31-9.59. Ingraham J.L., Maaloe O., Neidhardt F.C. (1983). Growth of cells Senol E. (2004). Stenotrophomonas maltophilia: the significance and cultures. In: Ingraham J.L., Ed., Growth of the Bacterial and role as a nosocomial pathogen. J. Hosp. Infect., 57: 1- Cell, Sinauer, Massachusetts, pp. 228-229. 7. Jucker B.A., Harms H., Zehnder A.J.B. (1996). Adhesion of the Stoodley P., Sauer K., Davies D.G., Costerton J.W. (2002). positively charged bacterium Stenotrophomonas Biofilms as complex differentiated communities. Annu. Rev. (Xanthomonas) maltophilia 70401 to glass and Teflon. J. Microbiol., 56: 187-209. Bacteriol., 178: 5472-5479. Vidipó L.A., Marques E.A., Puchelle E., Plotkowski M.C. (2001). Kavouras J.H., Maki J.S. (2004). Inhibition of the reattachment Stenotrophomonas maltophilia interaction with human of young adult zebra mussels by single-species biofilms and epithelial respiratory cells in vitro. Microbiol. Immunol., 45: associated exopolymers. J. Appl. Microbiol., 97: 1236-1246. 563-569. Marolda C.L., Welsh J., Dafoe L., Valvano M.A. (1990). Genetic Watnick P.I., Kolter R. (1999). Steps in the development of a analysis of the O7-polysaccharide biosynthesis region from Vibrio cholerae El Tor biofilm. Mol. Microbiol., 34: 586-595. the Escherichia coli O7:K1 strain VW187. J. Bacteriol., 172: 3590-3599.

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Published: Jan 13, 2010

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