Prolonged exposure of Stenotrophomonas maltophilia biofilms to trace levels of clofibric acid alters antimicrobial tolerance and virulence.

The presence of pharmaceuticals in water sources, including in drinking water (DW), is increasingly being recognized as an emerging and global concern for the environment and public health. Based on the principles of the "One Health" initiative, the present work aims to understand the effects of clofibric acid (CA), a lipid regulator, on the behavior of a selected bacterium isolated from drinking water (DW). Biofilms of the opportunistic pathogen Stenotrophomonas maltophilia were exposed to CA for 12 weeks at 170 and 17000 ng/L. The effects of CA were evaluated on planktonic S. maltophilia susceptibility to chlorine and antibiotics (amoxicillin, ciprofloxacin, erythromycin, kanamycin, levofloxacin, oxacillin, spectinomycin, tetracycline and trimethoprim-sulfamethoxazole), biofilm formation, motility, siderophores production and on the adhesion and internalization of the human colon adenocarcinoma cell line (HT-29). It was found that CA did not affect planktonic S. maltophilia tolerance to chlorine exposure. Additionally, no effects were observed on biofilm formation, motility and siderophores production. However, biofilms formed after CA exposure were more tolerant to chlorine disinfection and lower CFU reductions were obtained. Of additional concern was the effect of CA exposure on S. maltophilia increased tolerance to erythromycin. CA exposure also slightly reduced S. maltophilia ability to invade HT-29 cells. In conclusion, this work reinforces the importance of studying the effects of non-antibiotic contaminants on the behavior of environmental microorganisms, particularly their role as drivers affecting resistance evolution and selection.

Membrane vesicle secretion and prophage induction in multidrug-resistant Stenotrophomonas maltophilia in response to ciprofloxacin stress.

Several bacterial species produce membrane vesicles (MVs) in response to antibiotic stress. However, the biogenesis and role of MVs in bacterial antibiotic resistance mechanisms have remained unclear. Here, we studied the effect of the fluoroquinolone ciprofloxacin on MV secretion by Stenotrophomonas maltophilia using a combination of electron microscopy and proteomic approaches. We found that in addition to the classical outer membrane vesicles (OMV), ciprofloxacin-stimulated cultures produced larger vesicles containing both outer and inner membranes termed outer-inner membrane vesicles (OIMV), and that such MVs are enriched with cytosolic proteins. Remarkably, OIMV were found to be decorated with filamentous structures identified as fimbriae. In addition, ciprofloxacin stress leads to the release of bacteriophages and phage tail-like particles. Prophage induction by ciprofloxacin has been linked to pathogenesis and horizontal gene transfer in several bacterial species. Together, our findings show that ciprofloxacin treatment of S. maltophilia leads to the secretion of a heterogeneous pool of MVs and the induction of prophages that are potentially involved in adverse side-effects during antibiotic treatment.

Genome Shuffling of Stenotrophomonas maltophilia OK-5 for Improving the Degradation of Explosive RDX (Hexahydro-1,3,5-trinitro-1,3,5-triazine).

A genome-shuffled Stenotrophomonas maltophilia strain showing the enhanced ability of RDX degradation was constructed, and its characteristics were compared with those of the wild-type one. The shuffled strain was able to completely degrade 25, 50, and 75 µM RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) within 10, 30, and 50 days, respectively. However, it took 30 and 70 days for the wild-type strain to degrade 25 and 50 µM RDX, respectively, and at day 70, the strain degraded only 67% of 75 µM RDX. The shuffled strain reached its maximum growth at 50-60 days and exhibited approximately 1.5-fold increased cell numbers. SEM revealed more severe damage on the surface of the wild-type cells compared to the genome-shuffled cells. The mRNA levels of dnaK and groEL encoding the heat shock proteins were increased by 2.5-fold and fourfold, and DnaK and GroEL proteins were more highly produced in the shuffled cells. In addition, the mRNA levels of pnrB encoding a TNT nitroreductase, and algA involved in exopolymer biosynthesis, were slightly higher in the shuffled strain, but not as high as those of dnaK and groEL. These results indicate that the genome shuffling rendered the shuffled cells more resistant to RDX stress. A proteomic comparison revealed changes in the production levels of certain proteins including nitrate and cell protection, particularly those involved in metabolism. These proteomic analyses provide clues for understanding the improved RDX degradation by the genome-shuffled S. maltophilia strain.

The impact of spgM, rpfF, rmlA gene distribution on biofilm formation in Stenotrophomonas maltophilia.

BACKGROUND: Stenotrophomonas maltophilia is emerging as one of the most frequently found bacteria in chronic pulmonary infection. Biofilm is increasingly recognized as a contributing factor to disease pathogenesis. In the present study, a total of 37 isolates of S. maltophilia obtained from chronic pulmonary infection patients were evaluated to the relationship between biofilm production and the relative genes expression. METHODS: The clonal relatedness of isolates was determined by pulse-field gel electrophoresis. Biofilm formation assays were performed by crystal violet assay, and confirmed by Electron microscopy analysis and CLSM analysis. PCR was employed to learn gene distribution and expression. RESULTS: Twenty-four pulsotypes were designated for 37 S. maltophilia isolates, and these 24 pulsotypes exhibited various levels of biofilm production, 8 strong biofilm-producing S. maltophilia strains with OD492 value above 0.6, 14 middle biofilm-producing strains with OD492 average value of 0.4 and 2 weak biofilm-producing strains with OD492 average value of 0.19. CLSM analysis showed that the isolates from the early stage of chronic infection enable to form more highly structured and multilayered biofim than those in the late stage. The prevalence of spgM, rmlA, and rpfF genes was 83.3%, 87.5%, and 50.0% in 24 S. maltophilia strains, respectively, and the presence of rmlA, spgM or rpfF had a close relationship with biofilm formation but did not significantly affect the mean amount of biofilm. Significant mutations of spgM and rmlA were found in both strong and weak biofilm-producing strains. CONCLUSION: Mutations in spgM and rmlA may be relevant to biofilm formation in the clinical isolates of S. maltophilia.

Molecular characterization and ultrastructure of a new amoeba endoparasite belonging to the Stenotrophomonas maltophilia complex.

Naegleria and Acanthamoeba spp. were recovered from biofilm of a flushing cistern in a lavatory and both were found to be infected by rod-shaped bacteria enclosed within a vacuole. These intracellular bacteria behave like parasites, causing lysis of host amoebae. The bacteria proved unculturable on bacteriological media, and but could be maintained as endocytobionts within Acanthamoeba on agar plates. A marked differential host preference was observed in co-culture assays with various strains of amoebae. Molecular phylogenetic analyses performed on almost complete 16S rDNA sequences showed that the bacteria emerged as an atypical rapidly-evolving strain within the Stenotrophomonas maltophilia complex (Gamma-Proteobacteria, Xanthomonadales).

Adhesion to and biofilm formation on IB3-1 bronchial cells by Stenotrophomonas maltophilia isolates from cystic fibrosis patients.

BACKGROUND: Stenotrophomonas maltophilia has recently gained considerable attention as an important emerging pathogen in cystic fibrosis (CF) patients. However, the role of this microorganism in the pathophysiology of CF lung disease remains largely unexplored. In the present study for the first time we assessed the ability of S. maltophilia CF isolates to adhere to and form biofilm in experimental infection experiments using the CF-derived bronchial epithelial IB3-1cell line. The role of flagella on the adhesiveness of S. maltophilia to IB3-1 cell monolayers was also assessed by using fliI mutant derivative strains. RESULTS: All S. maltophilia CF isolates tested in the present study were able, although at different levels, to adhere to and form biofilm on IB3-1 cell monolayers. Scanning electron and confocal microscopy revealed S. maltophilia structures typical of biofilm formation on bronchial IB3-1 cells. The loss of flagella significantly (P < 0.001) decreased bacterial adhesiveness, if compared to that of their parental flagellated strains. S. maltophilia CF isolates were also able to invade IB3-1 cells, albeit at a very low level (internalization rate ranged from 0.01 to 4.94%). Pre-exposure of IB3-1 cells to P. aeruginosa PAO1 significantly increased S. maltophilia adhesiveness. Further, the presence of S. maltophilia negatively influenced P. aeruginosa PAO1 adhesiveness. CONCLUSIONS: The main contribution of the present study is the finding that S. maltophilia is able to form biofilm on and invade CF-derived IB3-1 bronchial epithelial cells, thus posing a rationale for the persistence and the systemic spread of this opportunistic pathogen in CF patients. Experiments using in vivo models which more closely mimic CF pulmonary tissues will certainly be needed to validate the relevance of our results.

Factors associated with adherence to and biofilm formation on polystyrene by Stenotrophomonas maltophilia: the role of cell surface hydrophobicity and motility.

We tested 40 clinical Stenotrophomonas maltophilia strains to investigate the possible correlation between adherence to and formation of biofilm on polystyrene, and cell surface properties such as hydrophobicity and motility. Most of the strains were able to adhere and form biofilm, although striking differences were observed. Eleven (27.5%) of the strains were hydrophobic, with hydrophobicity greatly increasing as S. maltophilia attached to the substratum. A positive correlation was observed between hydrophobicity and levels of both adhesion and biofilm formation. Most of the isolates showed swimming and twitching motility. A highly significant negative correlation was observed between swimming motility and level of hydrophobicity. Hydrophobicity is thus a significant determinant of adhesion and biofilm formation on polystyrene surfaces in S. maltophilia.

Biofilm formation by Stenotrophomonas maltophilia: modulation by quinolones, trimethoprim-sulfamethoxazole, and ceftazidime.

We investigated the in vitro effects of seven fluoroquinolones (ciprofloxacin, grepafloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin, and rufloxacin), compared to those of trimethoprim-sulfamethoxazole (SXT) and ceftazidime on total biomass and cell viability of Stenotrophomonas maltophilia biofilm. S. maltophilia attached rapidly to polystyrene, within 2 h of incubation, and then biofilm formation increased over time, reaching maximum growth at 24 h. In the presence of fluoroquinolones at one-half and one-fourth the MIC, biofilm biomass was significantly (P < 0.01) reduced to 55 to 70% and 66 to 76% of original mass, respectively. Ceftazidime and SXT did not exert any activity. Biofilm bacterial viability was significantly reduced by all antibiotics tested at one-half the MIC. At one-fourth the MIC all antibiotics, except levofloxacin, significantly reduced viability. Treatment of preformed biofilms with bactericidal concentrations (500, 100, and 50 micro g/ml) of all fluoroquinolones caused, except for norfloxacin, significant reduction of biofilm biomass to 29.5 to 78.8, 64.1 to 83.6, and 70.5 to 82.8% of original mass, respectively. SXT exerted significant activity at 500 micro g/ml only. Ceftazidime was completely inactive. Rufloxacin exhibited the highest activity on preformed biofilm viability, significantly decreasing viable counts by 0.6, 5.4, and 17.1% at 500, 100, and 50 micro g/ml, respectively. Our results show that (i) subinhibitory (one-half and one-fourth the MIC) concentrations of fluoroquinolones inhibit adherence of S. maltophilia to polystyrene and (ii) clinically achievable concentrations (50 and 100 micro g/ml) of rufloxacin are able to eradicate preformed S. maltophilia biofilm.

Fimbriae and adherence of Stenotrophomonas maltophilia to epithelial cells and to abiotic surfaces.

Stenotrophomonas maltophilia is an emerging nosocomial bacterial pathogen associated with several infectious diseases and opportunistic infections, especially in immunocompromised patients. These bacteria adhere avidly to medical implants and catheters forming a biofilm that confers natural protection against host immune defences and different antimicrobial agents. The nature of the bacterial surface factors involved in biofilm formation on inert surfaces and in adherence of S. maltophilia to epithelial cells is largely unknown. In this study, we identified and characterized fimbrial structures produced by S. maltophilia grown at 37 degrees C. The S. maltophilia fimbriae 1 (SMF-1) are composed of a 17 kDa fimbrin subunit which shares significant similarities with the N-terminal amino acid sequences of several fimbrial adhesins (G, F17, K99 and 20K) found in Escherichia coli pathogenic strains and the CupA fimbriae of Pseudomonas aeruginosa. All of the clinical S. maltophilia isolates tested produced the 17 kDa fimbrin. Antibodies raised against SMF-1 fimbriae inhibited the agglutination of animal erythrocytes, adherence to HEp-2 cells and biofilm formation by S. maltophilia. High resolution electron microscopy provided evidence of the presence of fimbriae acting as bridges between bacteria adhering to inert surfaces or to cultured epithelial cells. This is the first characterization of fimbriae in this genus. We provide compelling data suggesting that the SMF-1 fimbriae are involved in haemagglutination, biofilm formation and adherence to cultured mammalian cells.

Transformations of selenate and selenite by Stenotrophomonas maltophilia isolated from a seleniferous agricultural drainage pond sediment.

A Gram-negative bacterium, identified as Stenotrophomonas maltophilia by fatty acid analysis and 16S rRNA sequencing, was isolated from a seleniferous agricultural evaporation pond sediment collected in the Tulare Lake Drainage District, California. In cultures exposed to the atmosphere, the organism reduces selenate (SeO4(2-)) and selenite (SeO3(2-)) to red amorphous elemental selenium (Se degrees ) only upon reaching stationary phase, when O2 levels are less than 0.1 mg l(-1). In 48 h, S. maltophilia removed 81.2% and 99.8% of added SeO4(2-) and SeO3(2-) (initial concentration of 0.5 mM), respectively, from solution. Anaerobic growth experiments revealed that the organism was incapable of using SeO4(2-), SeO3(2-), SO4(2-) or NO3- as a terminal electron acceptor. Transmission electron microscopy of cultures spiked with either Se oxyanion were found to contain spherical extracellular deposits. Analysis of the deposits by energy-dispersive X-ray spectroscopy revealed that they consist of Se. Furthermore, S. maltophilia was active in producing volatile alkylselenides when in the presence of SeO4(2-) and SeO3(2-). The volatile products were positively identified as dimethyl selenide (DMSe), dimethyl selenenyl sulphide (DMSeS) and dimethyl diselenide (DMDSe) by gas chromatography-mass spectrometry. Our findings suggest that this bacterium may contribute to the biogeochemical cycling of Se in seleniferous evaporation pond sediments and waters. This organism may also be potentially useful in a bioremediation scheme designed to treat seleniferous agricultural wastewater.

Characterization of flagella produced by clinical strains of Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is an emerging nosocomial pathogen associated with opportunistic infections in patients with cystic fibrosis, cancer, and HIV. Adherence of this organism to abiotic surfaces such as medical implants and catheters represents a major risk for hospitalized patients. The adhesive surface factors involved in adherence of these bacteria are largely unknown, and their flagella have not yet been characterized biochemically and antigenically. We purified and characterized the flagella produced by S. maltophilia clinical strains. The flagella filaments are composed of a 38-kDa subunit, SM(FliC), and analysis of its N-terminal amino acid sequence showed considerable sequence identity to the flagellins of Serratia marcescens (78.6%), Escherichia coli, Proteus mirabilis, Shigella sonnei (71.4%), and Pseudomonas aeruginosa (57.2%). Ultrastructural analysis by scanning electron microscopy of bacteria adhering to plastic showed flagellalike structures within the bacterial clusters, suggesting that flagella are produced as the bacteria spread on the abiotic surface.