Zebrafish Infection Model Confirms Attenuation of Collection Strain and Points at Varying Virulence of the Clinical Isolates

Adult zebrafish were used as an infection model to determine the virulence of the individual strains. An intraperitoneal injection of 10⁸ cfu resulted in strain-dependent mortality rates (Figure 1), where UV74 was the most aggressive, with 84% mortality 48 h post-injection (p.i.), followed by strains M30 (55%), E77 (5%) and ATCC13637 (0%). Although the experimental period was during 7 days, in all cases mortality occurred during the first 48 h. Clinical symptoms of dead fish included cutaneous hemorrhage under the lower jaw, on the belly, and around the anus; all dead fish had a distended abdomen containing bloody or clear fluid and severe enteritis with intussusception in the lower intestine. All isolates obtained from post-mortem UV74 infected zebrafish were identified as S. maltophilia based on morphology, API (analytical profile index) and 16S rDNA sequence (data not shown). No zebrafish died when injected with 20 µl of sterile PBS (data not shown). Interestingly, injection of 10⁸ heat-inactivated UV74 bacteria did not produce mortality in zebrafish. This suggests that viable bacteria producing thermo-labile proteins are required for infection and that mortality is not caused by non-specific activation due to bacterial components such as lipopolysaccharides.

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Figure 1. Survival curves of adult zebrafish injected with S. maltophilia ATCC13637, E77, M30, UV74 (10⁸ cfu in 20 µl).

Control groups injected with heat-inactivated UV74 (10⁸ cfu in 20 µl) and sterile PBS (20 µl) presented no mortality (not shown). Zebrafish mortalities were recorded for 168 h post-infection (hpi) and survival curves analyzed using the Kaplan-Meier method (log-rank test: ***, p≤0.001; **, p≤0.01).

https://doi.org/10.1371/journal.pone.0067207.g001

Strain-genotyping Analysis Reveals a Distant Genetic Relationship between the Strains

The genetic relationship of the four S. maltophilia strains was assessed using multilocus sequence typing (MLST). The isolates were compared and organized based on similarities among seven housekeeping genes (atpD, gapA, guaA, mutM, nuoD, ppsA and recA). This analysis resulted in the three clinical strains being classified into new, different sequence types (M30 as ST-76, UV74 as ST-77 and E77 as ST-81), indicating they are not clonally related despite being isolated from a common hospital setting. Nevertheless, strains M30 and UV74 share the same allele for genes atpD and mutM. The collection strain ATCC13637 had been already characterized by MLST and assigned the ST-14 [28]. In addition, thirty-five concatenate sequences from different S. maltophilia strains, isolated from human clinical cases, were obtained from public databases and used to provide the context for the genotypic classification of the three clinical isolates and the collection strain. Strains were chosen such that they cover as far as possible the full genetic breadth of the species [28]. The phylogenetic analysis revealed distant genetic relationships between the three clinical strains and the reference that according to a previous classification based on AFLP fingerprinting [29] would belong to a different genomic group (Figure 2). Strains M30 and UV74 show the closest genetic relationship, clustering within genomic group C as previously described by Kaiser et al. [28]. The MLST results imply that the three pair wise (collection:clinical) proteomic analyses described below are non-redundant.

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Figure 2. Neighbor-joining radiation tree based on the concatenated sequences of the MLST loci, showing relationships among the three recent clinical isolates (solid circles), the ATCC13637 strain and 35 previously characterized S. maltophilia pathogenic strains [28].

Previously defined genomic groups [28], [29] are indicated (#1 to #7, C and D).

https://doi.org/10.1371/journal.pone.0067207.g002

The Most Virulent Strain, UV74, also shows the Most Resistant Phenotype

MIC determination included the following antibiotic families: tetracyclines, aminoglycosides, macrolides, sulfonamides, chloramphenicol and fluoroquinolones (Table 1). UV74 presented the most resistant phenotype to the antibiotics tested. The most distinguishing feature of this strain is its significantly higher MIC for ciprofloxacin (16 to 64-fold). In addition, the new clinical isolates were significantly more resistant to kanamycin than the ATCC13637 strain (8 to 32-fold).

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Table 1. MICs for several antibiotics against the S. maltophilia collection and clinical strains.

https://doi.org/10.1371/journal.pone.0067207.t001

The Collection Strain shows Reduced Biofilm-formation and Swimming Capacities

The strains presented similar growth curves except for E77, whose doubling time during the exponential phase was 1.3 h as opposed to 1 h for the other three strains (Figure S1). The capacity to form biofilms on polystyrene plates was tested (Table 2) and statistical analysis revealed that the collection strain had a reduced ability to form biofilms in comparison to the clinical strains. M30 presented the greatest biofilm-formation capacity.

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Table 2. Swimming, twitching and biofilm formation of the S. maltophilia collection and clinical strains.

https://doi.org/10.1371/journal.pone.0067207.t002

The analysis of different types of bacterial motility (swimming, twitching, swarming) showed that ATCC13637 displays significantly reduced swimming compared to the clinical strains (Table 2). Twitching migration capabilities however were similar among the four strains. Finally, all strains presented swarming motility consisting of branches or tentacles radiating from the inoculation point. Although E77 presented swarming under the conditions described in materials and methods, observation of this type of motility in the other strains required 12 days of incubation at 30°C in the medium described by Kholer et al. [30]. To our knowledge, this is the first time swarming has been described for S. maltophilia.

Serum Sensitivity Correlates with Virulence in Zebrafish

Resistance of the four strains to antibody/complement-mediated bactericidal action of serum was also tested (Figure 3). The ATCC13637 and E77 strains clearly showed a higher sensitivity to the bactericidal action of serum (0.0044% and 0.082% survival, respectively) compared to the M30 and UV74 (5% and 16% survival, respectively). As a control, when the incubation was performed with Hank’s balanced salt solution (HBSS) or inactivated serum no mortality was observed. Notably, these results correlate with zebrafish mortality (p = 0.059).

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Figure 3. S. maltophilia serum-sensitivity assay.

Percentage of surviving cells after 90 minutes of incubation in the presence of Hank’s balanced salt solution, human serum or inactivated serum. The values represent the mean of three replicas of the three independent experiments. **, p≤0.01; ***, p≤0.0001 significance of difference with ATCC13637 by unpaired t-test with Welch correction for unequal variances.

https://doi.org/10.1371/journal.pone.0067207.g003

Adhesion to HeLa Cells Correlates with Serum Sensitivity

To test the relative adhesion capacities of the S. maltophilia strains to eukaryotic cells, adhesion experiments with HeLa cells were performed (Figure 4). For ATCC13637, the number of adhered bacteria to HeLa cells after 2 h of incubation was 1.7% of the initial bacterial load. This was significantly lower than the percentages found for M30 (12%), E77 (5%) and UV74 (45%). This results correlate with serum sensitivity (p = 0.008) and also follow the strain-virulence order.

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Figure 4. S. maltophilia adhesion to HeLa cells.

Percentage of bacteria adhered to HeLa cells after 120 min of incubation in 24-well plates. The data correspond to the mean and standard deviation of three different assays carried out in triplicate. ***, p≤0.001 significance of difference with ATCC13637 by unpaired t-test with Welch correction for unequal variances.

https://doi.org/10.1371/journal.pone.0067207.g004

Differential Proteomics Points at Specific Processes and Highlights a Protein that Correlates with Virulence

To analyze potential differences in the protein profiles of the collection and clinical strains, independent samples were taken from each strain in the exponential-growth phase (OD = 1) and were Cy-dye labeled and pairwise separated by 2DE. An example of the resulting fluorescence images is shown in Figure S2, with the ATCC13637 sample in red and the clinical sample in green. A total of 1807, 1776 and 1677 protein spots were detected in the ATCC13637-M30, ATCC13637-E77 and ATCC13637-UV74 gels, respectively. Using a 1.5-fold threshold for clinical:collection abundance ratio, ca. 100 spots displayed significant differential content of protein from collection and clinical sources (p<0.05, ANOVA test). These spots were excised and in-gel digested with trypsin. Following MALDI-MS time-of-flight analysis, 38 proteins were identified for which the absolute value of the ratio was ≥1.5 for at least two clinical isolates. The low identification rate (38 out of 100 differential spots) stems partly from the fact that the identification is performed on the analytical gels, to avoid uncertainties with mismatching spots on the preparative silver-stained gels. The protein amount loaded on the analytical gels is low (25 µg per sample), making the identification of weak-intensity spots impossible. Some of the proteins were identified in several spots, indicating the presence of different post-translationally modified forms. A detailed list of the identified proteins and quantified abundance differences is given in Table S1, with the corresponding statistical analysis provided in Table S2. Example fluorescence images and expression profiles of some of the identified proteins are shown in Figures S2 and S3, respectively. In addition, a survey on orthologs of the 38 proteins that have been linked to pathogenesis, virulence or resistance has been performed and summarized in Table S3.

Among the proteins with altered abundance in clinical strains there are enzymes involved in the biosynthesis of fatty acids and other cell-wall components, enzymes involved in energy-related and other metabolic pathways, proteases, an RNA polymerase subunit, outer membrane proteins with receptor and transport activities and two completely uncharacterized proteins. The first 12 proteins (enzymes) in Table S1 are assigned to a common network by predicted pairwise interactions (see Table S3). Likewise, proteins 13 to 17 in Table S1 appear also cross connected. Indeed, some of them are known to share a role in biofilm formation and quorum sensing. Note that the ortholog information provided in Table S3 is not necessarily exportable to S. maltophilia. The molecular mechanisms underlying pathogenesis, virulence and resistance may vary not only between species but also between strains within a species. Furthermore, ortholog proteins involved in parallel mechanisms in two closely related species might still do so in different ways. For example, various proteins in Table S1, with known or suggested role in pathogenesis in other species appear significantly upregulated in one clinical isolate and downregulated in another, the most striking case being the LptD transporter. This protein is essential for the translocation of Lipid A across the outer membrane and, by extension, of LPS presence in the surface, and is strongly upregulated in E77 and downregulated in UV74 (remarkably the most virulent strain). While bearing this limitation in mind, comparison of Tables S1 and S3 highlights the following proteins as potentially relevant to pathogenesis, virulence or resistance: FabD, AccC, AcsA, PdhB, AcnB, enoyl-CoA hydratase, MurA, RpoA, LptD, putative TonB-dependent receptors Smlt3444 and Smlt4151, putative FadL, putative OmpA Smlt0955, PepO and putative Ax21. The latter protein, quorum-sensing factor Ax21, shows a strong correlation with the results of virulence in zebrafish challenges (p = 0.022) as well as with the strain order for adhesion to HeLa cells and serum sensitivity. In addition to these candidates, on the sole basis of the proteomics data (Table S1) the following proteins also deserve special attention: FadI, SucB, Bcd, OprP, putative exported peptidase S9, NuoG, UPF0234 family protein and hypothetical protein Smlt3796. Note that FadI and SucB, which are predicted to interact (Table S3), have a similar expression behaviour (Table S1), strongly downregulated in UV74 and weakly upregulated in M30 and E77.

Bacterial Strains, Media, and Growth Conditions

The three clinical S. maltophilia strains characterized in this work were isolated from different patients at the Hospital Municipal de Badalona (Barcelona, Spain) during the year 2009. The clinical origin of M30, E77 and UV74 strains were decubitus ulcer, sputum and vascular ulcer, respectively. Species identification was confirmed biochemically using the API NE system (bioMérieux). The collection strain ATCC13637, isolated in 1960 from the oropharyngeal region of a patient with mouth cancer [32], was also included in the experiments. S. maltophilia strains were routinely cultured o/n in Luria-Bertani (LB) media at 37°C and 250 rpm unless otherwise stated, and growth curves were monitored following the optical density at 550 nm (Figure S1).

Zebrafish Infection Assay

Adult (9–12 months) wild-type zebrafish (D. rerio) were kept in a 12 h light:12 h dark cycle at 28°C and fed twice daily with dry feed. All fish used in infection experiments were transferred to an isolated system and acclimated for three days before infection. Adult zebrafish (n = 12/each condition) were infected by intraperitoneal injection (i. p.) [41] with 20 µl of a 5×10⁹ cfu/ml suspension of S. maltophilia strain ATCC13637 and the clinical isolates M30, E77 and UV74. These strains were previously grown at 28°C in blood agar plates (BioMérieux) for 20 h and collected directly from the plates with phosphate buffered saline (PBS). Two control groups were injected with PBS and with a heat-inactivated UV74 strain (incubation at 100°C for 30 min, at which time no viable bacteria were detectable), respectively. Fish were observed daily for signs of disease and mortality. All living injected fish were sacrificed after 7 days by MS-222 overdose. All experiments were repeated independently twice.

Multilocus Sequence Typing and Phylogenetic Analysis

PCR amplification and sequencing of the seven housekeeping genes included in the MLST scheme was performed as previously described [28], [42]. The detailed MLST procedure and set of primers used were obtained from the S. maltophilia MLST database (http://pubmlst.org/smaltophilia/). Briefly, genomic DNA was extracted with GenElute Bacterial Genomics DNA Kit (Sigma-Aldrich). Fragments of the seven genes atpD, gapA, guaA, mutM, nuoD, ppsA, and recA were amplified using FastStart Taq DNA Polymerase (Roche, Diagnostics) as follows: initial denaturation at 95°C for 9 min followed by 30 cycles of denaturation at 94°C for 20 s; annealing at the appropriate temperature for 1 min; extension at 72°C for 50 s; final elongation at 72°C for 5 min. PCR products were treated with ExoSAP-IT (USB Products) and sequenced at Macrogen Inc. (Seoul, Korea) in both directions using standard conditions. The allele numbers for each locus and sequence type (ST) were determined by comparison with the available sequences at the S. maltophilia MLST database, where the data has been deposited. Phylogenetic relationships were established on the basis of the concatenated seven gene sequences without further corrections. Cluster analysis was performed with the Neighbour-Joining method, using uncorrected p-distance. The phylogenetic tree of S. maltophilia strains was constructed by use of MEGA software version 4 [43], together with 35 already analyzed pathogenic S. maltophilia strains [28] with sequence information available at the S. maltophilia MLST database.

Determination of MICs

The susceptibility of S. maltophilia to the following antimicrobial agents was tested: tetracycline, minocycline, gentamicin, kanamycin, trimethoprim (from Apollo Scientific Ltd), sulfamethoxazole, norfloxacin, ciprofloxacin, erythromycin, levofloxacin (from Sigma-Aldrich), and chloramphenicol (from Roche Diagnostics). The MICs for these antibiotics were determined by microdilution test using 96-well plates by serial two-fold dilutions of each drug in 100 µl of LB. 100 µl of bacterial suspension (final OD_550nm = 0,005) were added and the antibiotic dilutions and the organism suspension were mixed and incubated at 37°C for 16 h before developing with resazurin (30 µl 0.01%) [44]. The MIC was defined as the lowest drug concentration that prevented bacterial growth. The microdilution assay followed the Clinical and Laboratory Standards Institute (CLSI, www.clsi.org) guidelines for antimicrobial susceptibility testing.

HeLa Cell Adherence Assay

HeLa cells were grown for 24 h on 24-well tissue culture plates (TPP Techno Plastic Products AG) containing 2 ml of Minimum Essential Medium α (Invitrogen) with 10% (v/v) inactivated fetal bovine serum (Invitrogen) and Glutamax™ (Invitrogen) to 90–95% of confluence. Bacterial cultures of ATCC13637, M30, E77 and UV74 strains were grown o/n in LB medium at 37°C without agitation and resuspended in HeLa medium. Bacteria were added at a multiplicity of infection (MOI) of 50∶1 in triplicate to the confluent 24-well plates and were incubated at 37°C in a humidified atmosphere of 5% carbon dioxide for 120 minutes. Wells were gently washed five times with 2 ml of Dulbecco's Phosphate-Buffered Saline (Invitrogen) to remove non-adherent bacteria. The number of cell-attached bacteria was quantified by lysis with 0.1% Triton X-100 and serial dilution were plated onto LB plates. Adhesion was measured as a percentage between adhered and the initial cells.

Serum Sensitivity Assay

Human serum sensitivity assay was performed following the protocol described by Waters et al. [17]. Briefly, bacteria were grown on LB medium to an OD_550nm of 0.5, washed in Hanks' balanced salt solution (HBSS) (Invitrogen), and incubated in HBSS, 60% serum or 60% heat-inactivated serum with agitation at 37°C with agitation for 90 min. The serum inactivation was performed at 56°C during 30 min. After incubation, bacteria were plated on LB plates after serial dilution. The survival was measured as a percentage between the surviving and initial cells.

Biofilm Formation on Polystyrene Plates

Quantification of S. maltophilia biofilm formation was assessed by crystal violet (CV) staining in 96-well polystyrene plates. Bacterial cultures of the four different S. maltophilia strains were grown o/n at 37°C. Absorbance at 550 nm was adjusted to 0.1 and 200 µl were grown in a 96-well plate during 24 h at 30°C. Cells were washed three times with water, fixed at 60°C for 1 h and stained during 15 min with 200 µl of 0.1% CV. The dye was discarded, and the plate was rinsed in standing water and allowed to dry for 30 min at 37°C. Stained biofilms were exposed to 250 µl of 95% ethanol for 15 min, and the OD of the extracted dye was measured at 620 nm.

Motility Assays

Overnight cultures of the different S. maltophilia strains were grown (plate or liquid medium) in LB under standard conditions. The swimming motility was determined in TrA plates (1% tryptone, 0.5% NaCl, 0.25% agar) [45]. Thus, 5 µl of adjusted o/n cultures of the different S. maltophilia strains were spotted on TrA plates. The twitching and swarming motility plates were LB plates at 1% agar and BM2 at 0.5% agar [46], respectively. Twitching was assessed via subagar stab inoculations (stab assay) from o/n fresh plates as previously described [47]. The twitching zones were then visualized by staining with 1% (wt/vol) crystal violet and their diameters measured. Noble agar (Difco™) was used in the preparation of the three motility assay plates. The growth halos were measured in cm after 24 h of incubation at 30°C for the swimming and twitching motilities and after 7 days at 30°C for swarming.

Statistics

Statistical analyses were performed using the GraphPad Prism program version 5.00. Comparison of strain phenotypic data was performed by one-way analysis of variance (ANOVA) with a Bonferroni’s multiple comparison post-test or unpaired t-test with Welch correction for unequal variances, as indicated in figure and table captions. Survival curves of zebrafish infection experiments were analyzed using the Kaplan-Meier method. Differences were evaluated using the log-rank test. The relationship between relative protein abundance in the different strains and pathogenicity variables, such as zebrafish mortality rates and adhesion to human cells, was evaluated with Pearson’s chi-squared test. p≤0.05 was considered significant.

Sample Preparation for Two-dimensional Gel Electrophoresis

20 ml of exponential culture (OD = 1) were washed with PBS 1X three times and resuspended in lysis solution (8 M urea, 2 M thiourea, 2.5% 3-[(3-cholamidopropyl) dimethylammonio]-1 propanesulfonate (CHAPS), 2% ASB-14, 40 mM Tris-HCl, pH 8.8). ASB-14 was used to increase the presence of membrane proteins in the 2DE [48]. Then, samples were disrupted by sonication and centrifuged in order to discard any insoluble cellular debris. In order to remove salts and other contaminants, samples were cleaned with 2D Clean-Up Kit (GE Healthcare). Resulting pellets were resuspended in the above-mentioned lysis solution. Protein concentration was determined with 2D-Quant Kit (GE Healthcare) and adjusted to 2 mg/ml by the addition of a DIGE labeling buffer (7 M urea, 2 M thiourea, 2.5% w/v CHAPS, 40 mM Tris, pH 8.8). A pool consisting of equal amounts of each of the two samples analyzed in the experiment was prepared as an internal standard for quantitative comparisons [49]. The clinical isolates were labeled with Cy3 and the collection strain ATCC13637 was labeled with Cy5. A third fluorescent dye, Cy2, was used to label the internal standard sample. Labeling was carried out by the addition of 400 pmol of the required Cydye in 1 µl of anhydrous N,N-dimethylformamide per 50 µg of protein. After 30 min of incubation on ice in the dark, the reaction was quenched with 10 mM lysine and the samples incubated for a further 10 min. Samples were combined according to the experimental design, using 50 µg of protein per Cy dye per gel, and diluted two-fold with isoelectric focusing (IEF) sample buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, 2% dithiothreitol [DTT], 2% pharmalytes, pH 3–10). One clinical strain and the ATCC13637 strain sample, together with an aliquot of the internal standard pool, were then separated by two-dimensional electrophoresis (2-DE) in each of the gels. This experimental design allows the accurate quantification and statistical assessment of the differences in protein abundances observed between the two sample groups.

2D Difference Gel Electrophoresis

The 2-DE was performed using GE Healthcare reagents and equipment. First-dimension isoelectric focusing was performed on immobilized pH gradient strips (24 cm, pH 3–10) using an Ettan IPGphor System (GE Healthcare). Samples were applied near the basic end of the strips by cup-loading, after being incubated o/n in 450 µl of rehydration buffer (7 M urea, 2 M thiourea, 2.5% w/v CHAPS, 2% ASB-14 w/v, 0.5% pharmalytes, pH 3–10, 100 mM DeStreak reagent). After focusing at 70 kVh, strips were equilibrated, first for 15 min in 10 ml of reducing solution (6 M urea, 100 mM Tris-HCl, pH 8, 30% v/v glycerol, 2% w/v SDS, 5 mg/ml dithiothreitol [DTT]) and then in 10 ml of alkylating solution (6 M urea, 100 mM Tris-HCl, pH 8, 30% v/v glycerol, 2% w/v SDS, 22.5 mg/ml iodoacetamide) for 15 min on a rocking platform. Second dimension SDS-PAGE was performed by laying the strips on 12.5% isocratic Laemmli gels (24×20 cm), cast in low fluorescence glass plates, on an Ettan DALT Six system. Gels were run at 20°C at a constant power of 2.5 W per gel for 60 min followed by 17 W per gel until the bromophenol blue tracking front had run off the end of the gel. Triplicate gels were run for each sample using independent biological replicates. Fluorescence images of the gels were obtained on a Typhoon 9400 scanner (GE Healthcare). Cy2, Cy3 and Cy5 images were scanned at excitation/emission wavelengths of 488/520 nm, 532/580 nm and 633/670 nm, respectively, at a resolution of 100 µm. Both image analysis and statistical quantification of relative protein levels were performed using Progenesis SameSpots V.4 (Nonlinear Dynamics) (See Table S1 for detailed statistics of each spot). The data were analyzed as pairwise comparisons.

Protein Identification by Mass Spectrometry

In order to excise the spots of interest, gels were silver stained as described elsewhere [50]. Protein spots of interest were excised from the gel using a cut tip. The selected spots are those that are differentially expressed in at least two clinical isolates. In-gel trypsin digestion was performed as described previously [51]. MALDI-MS analysis of tryptic peptides was performed on an Ultraflex time-of-flight instrument (Bruker Daltonics). Samples were prepared using α-cyano-4-hydroxy-cinnamic acid. Calibration was performed in the external mode using a peptide calibration standard kit (Bruker Daltonics). The spectra were processed using Flex Analysis 2.2 software (Bruker Daltonics). Peak lists were generated using the signals in the 800–4000 mass:charge ratio (m/z) range, with a signal:noise threshold >3. The SNAP algorithm included in the software was used to select the monoisotopic peaks from the isotopic distributions observed. After removing m/z values corresponding to commonly observed matrix cluster ions, an internal statistical calibration was applied. Peaks corresponding to frequently seen keratin and trypsin autolysis peptides were then removed. The resulting final peak list was used for the identification of the proteins by peptide mass fingerprint. The Mascot 2.0 program (Matrix Science) was used to search the NCBI non-redundant database (http://ncbi.nlm.nih.gov, March 2010), with no limitation on taxonomy. Search parameters were as follows: trypsin cleavages excluding N-terminal to P, one missed cleavage permission, carbamidomethylation and methionine oxidation as variable modification, mass tolerance <50 ppm, monoisotopic mass values. Criteria for positive identification were a significant Mascot probability score (p<0.05), and at least five matching peptide masses. A minimum score of 83, and a >50-point difference between this score and the score of the second ranked non-homologous match was obtained for all the identified differentially expressed proteins.

Ethics Statement

Stenotrophomonas maltophilia strains were obtained from the internal collection of Hospital Municipal de Badalona (Barcelona, Spain) and have no link with patient data.

Zebrafish were handled in compliance with Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes and with Decree 214/1997 of the Government of Catalonia, which regulates the use of animals for experimental and other scientific purposes. Experimental protocols have been reviewed and approved by the Animal and Human Experimentation Ethics Committee (CEEAH) of the Universitat Autònoma de Barcelona (UAB), Spain (ref # CEEAH-1968).