Usefulness of MALDI-TOF MS as a Diagnostic Tool for the Identification of <i>Streptococcus</i> Species Recovered from Clinical Specimens of Pigs

<div><p>The application of MALDI-TOF MS for identifying streptococcal isolates recovered from clinical specimens of diseased pigs was evaluated. For this proposal, the MALDI BDAL Database (Bruker Daltoniks, Germany) was supplemented with the main spectrum profiles (MSP) of the reference strains of <i>S</i>. <i>porci</i>, <i>S</i>. <i>porcorum</i> and <i>S</i>. <i>plurextorum</i> associated with pneumonia and septicemia. Although these three species showed similar MALDI profiles, several peaks were recognized that can be useful for their differentiation: <i>S</i>. <i>porci</i> (4113, 6133, 7975 and 8228 m/z Da), <i>S</i>. <i>plurextorum</i> (3979, 4078, 4665, 6164, 6491, 6812, 7959 and 9330 m/z Da) and <i>S</i>. <i>porcorum</i> (3385, 3954, 4190, 6772, 7908, and 8381 m/z Da). After adding these MSPs, an evaluation was conducted to determine the accuracy of MALDI-TOF MS for the identification of streptococci from diseased pigs using 74 field isolates. Isolates were identified as <i>S</i>. <i>suis</i>, <i>S</i>. <i>porcinus</i>, <i>S</i>. <i>dysgalactiae</i>, <i>S</i>. <i>hyovaginalis</i>, <i>S</i>. <i>porcorum</i>, <i>S</i>. <i>alactolyticus</i>, <i>S</i>. <i>hyointestinalis</i> and <i>S</i>. <i>orisratti</i>. This is the first time that the latter three species have been reported from clinical specimens of pigs. Overall, there was good concordance (95.9%) between the results obtained from MALDI-TOF MS identification (best hint) and those from genotyping. Our results demonstrate the good performance of MALDI-TOF MS (100% sensitivity and specificity) for identifying most of the species of streptococci that can frequently be isolated from diseased pigs. However, conflicting results were observed in the correct identification of some isolates of <i>S</i>. <i>dysgalactiae</i> and <i>S</i>. <i>alactolyticus</i>.</p></div

Results of MALDI-TOF MS and genotype identification of the 74 streptococcal clinical isolates from diseased pigs examined in this study.

<p>Results of MALDI-TOF MS and genotype identification of the 74 streptococcal clinical isolates from diseased pigs examined in this study.</p

Box-and-whisker plots of the distribution of mean score values of <i>Streptococcus</i> species potentially isolated from clinical specimens of swine.

<p>Two cut-off values were established for excellent (≥2.300) and good (2.000–2.299) identification at species level.</p

MALDI-TOF MS spectra of whole-cell extracts of <i>S</i>. <i>porcorum</i> (A), <i>S</i>. <i>plurextorum</i> (B) and <i>S</i>. <i>porci</i> (C) reference strains.

<p>The relative intensities of the ions are shown on the <i>y</i> axis, and the masses (in Da) of the ions are shown on the <i>x</i> axis.</p

MALDI-TOF MS indicative peak masses of the different streptococcal species isolated from clinical specimens in pigs.

<p>MALDI-TOF MS indicative peak masses of the different streptococcal species isolated from clinical specimens in pigs.</p

Cross-antagonism between D₄ and α_1B or β₁ receptors in transfected cells and in pineal gland.

<p>In (A to D) CHO cells were transiently co-transfected with 2 µg of plasmid coding for D₄ receptors and with 3 µg of plasmid coding for α_1B receptors (A and B) or β₁ receptors (C and D). In (E and F) rat pineal glands were extracted at 9:00 h and processed as indicated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>. Cells were treated for 7 min and pineal glands were treated for 10 min with 500 nM of RO 10-5824 (RO), phenylephrine (Phenyl), or isoproterenol (Iso) or with 1 µM of L-745,870 (L-745), REC 15/2615 (REC), or CGP 20712 (CGP), alone or in combination. The immunoreactive bands, corresponding to ERK 1/2 (Thr¹⁸³-Tyr¹⁸⁵) phosphorylation (A, C, and E) and Akt (Ser⁴⁷³) phosphorylation (B, D, and F) of four experiments were quantified and values represent the mean ± S.E.M. of the fold increase with respect to basal levels found in untreated cells. Significant differences were calculated by a one-way ANOVA followed by post hoc Bonferroni's tests (***<i>p</i><0.001, as compared to the basal level; ^#<i>p</i><0.001, as compared to the sample treated with RO 10-5824; ^$<i>p</i><0.001, as compared to the sample treated with phenylephrine; ^&<i>p</i><0.001, as compared to the sample treated with isoproterenol). A representative Western blot is shown at the top of each panel.</p

D₄ receptors form heteromers with α_1B and β₁ receptors in the pineal gland.

<p>In (A to C), pinealocytes were isolated from pineal glands extracted at 9:00 h (top) or at 20:00 h (bottom) and stained using anti-S-arrestin antibody (green) and anti-D₄ (A), anti-α_1B (B), or anti-β₁ (C) antibodies (red) as indicated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>. Scale bar, 5 µm. In (D to F), pinealocytes were isolated from pineal glands extracted at 9:00 h (top) or at 20:00 h (bottom) and the expression of α_1B-D₄ (D) and β₁-D₄ (E) receptor heteromers was visualized as punctate red fluorescent spots detected by confocal microscopy using the proximity ligation assay (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>). Any expression of α_1B-β₁ receptor heteromers was seen (F). Scale bar, 20 µm. In (G and H), co-immunoprecipitation of D₄ and α_1B or D₄ and β₁ receptors from pineal gland extracted at 9:00 h (sunrise) or at 20:00 h (sunset) was performed. Glands were solubilized and processed for immunoprecipitation as described under <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a> using goat anti-D₄, rabbit anti-α₁, or goat anti-β₁ receptor antibodies or goat anti-adenosine A_2B receptor antibody as a negative control (N.C.). Solubilized gland membranes (G) and immunoprecipitates (H) were analyzed by SDS-PAGE and immunoblotted using rabbit anti-α₁, rabbit anti-β₁ receptor antibodies, or goat anti-β₁ receptor antibody. Immunoprecipitation experiments with anti-α₁ or anti-β₁ receptor antibodies (right image in H) were performed with pineal glands extracted at 9:00 h. IP, immunoprecipitation; WB, western blotting; MW, molecular mass.</p

Functional characteristics of α_1B-D₄ and β₁-D₄ receptor heteromers in pineal gland.

<p>Pineal glands extracted at 9:00 h (A and B) or at 20:00 h (C and D) were treated for 10 min with RO 10-5824 (RO), phenylephrine (Phenyl), or isoproterenol (Iso) at 1 µM concentration alone or in combination. The immunoreactive bands, corresponding to ERK 1/2 (Thr¹⁸³-Tyr¹⁸⁵) (A and C) or Akt (Ser⁴⁷³) (B and D) phosphorylation, of three experiments performed in duplicates were quantified, and values represent the mean ± S.E.M. of the fold increase with respect to basal levels found in untreated pineal glands. Significant differences were calculated by a one-way ANOVA followed by post hoc Bonferroni's tests (**<i>p</i><0.01 and ***<i>p</i><0.001, as compared to the basal level. ^#<i>p</i><0.05 and ^##<i>p</i><0.01, as compared to the sample treated with phenylephrine; ^$<i>p</i><0.05 and ^$$$<i>p</i><0.001, as compared to the sample treated with isoproterenol). A representative Western blot is shown at the bottom of each panel.</p

D₄ receptors form heteromers with α_1B and β₁ receptors in transfected cells.

<p>(A) BRET saturation curves were performed in HEK-293T cells co-expressing a constant amount of D₄-RLuc construct (2 µg of plasmid transfected) and increasing amounts of β₁-YFP construct (0.4–5 µg plasmid transfected, red), α_1B-YFP construct (0.4–5 µg of plasmid transfected, blue), or D₁-YFP construct (1–4 µg of plasmid transfected, gray) or with cells co-expressing a constant amount of α_1B-RLuc construct (3 µg of plasmid transfected) and increasing amounts of β₁-YFP construct (0.4–5 µg of plasmid transfected, green). Both fluorescence and luminescence of each sample were measured prior to every experiment to confirm equal expression of Rluc construct (∼100,000 luminescence units) while monitoring the increase of YFP construct expression (2,000 to 40,000 fluorescence units). Milli BRET Units (mBU) are BRET ratio (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a>)×1,000 and are expressed as means ± S.D. of five different experiments grouped as a function of the amount of BRET acceptor normalized with respect to the BRET donor (YFP/RLuc). (B and C) BRET was determined in HEK-293T cells expressing a constant amount of D₄-RLuc construct (2 µg of plasmid transfected) and (B) α_1B-YFP construct (4 µg of plasmid transfected) or (C) β₁-YFP construct (4 µg of plasmid transfected) and increasing amounts (2–12 µg of plasmid transfected) of (B) α_1B receptor (red) or β₁ receptor (blue) or (C) β₁ receptor (red) or α_1B receptor (blue). Both fluorescence and luminescence of each sample were measured prior to every experiment to confirm that there were no changes in the expression of D₄-RLuc, α_1B-YFP, or β₁-YFP constructs. BRET data (see above) are expressed as means ± S.D. of three different experiments. Significant differences with respect to cells not expressing α_1B or β₁ receptors were calculated by one-way ANOVA followed by a Dunnett's multiple comparison post hoc test (*<i>p</i><0.05 and **<i>p</i><0.01). (D) Confocal microscopy images of HEK-293T cells transfected with 1 µg of plasmid coding for D₄-RLuc and 0.5 µg of plasmid coding for α_1B-YFP or β₁-YFP. Proteins were identified by fluorescence or by immunocytochemistry. D₄-RLuc receptor is shown in red, α_1B-YFP and β₁-YFP receptors are shown in green, and co-localization is shown in yellow. Scale bar, 5 µm. (E and F) Co-immunoprecipitation of D₄ and α_1B or D₄ and β₁ receptors expressed in HEK-293T cells. Membranes from cells transfected with the indicated receptors were solubilized and processed for immunoprecipitation as described under <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001347#s4" target="_blank">Materials and Methods</a> using goat anti-D₄R, rabbit anti-α₁ or goat anti-β₁ receptor antibodies, or as negative controls (NC), goat anti-adenosine A_2B receptor antibody (top in F) or rabbit anti-adenosine A₁ receptor antibody (bottom in F). Solubilized membranes (E) and immunoprecipitates (F) were analyzed by SDS-PAGE and immunoblotted using rabbit anti-YFP, rabbit anti-α₁, or goat anti-β₁ antibody. IP, immunoprecipitation; WB, Western blotting (numbers are included to delineate the different lanes on the SDS-PAGE); MW, molecular mass.</p

Functional characteristics of α_1B-D₄ and β₁-D₄ receptor heteromers in transfected cells.

<p>CHO cells were transfected with 2 µg of plasmid coding for D₄ receptors or with 3 µg of plasmid coding for α_1B receptors or β₁ receptors alone (A) or in combination (B to G). In (A), the selectivity of ligands was tested by measuring ERK 1/2 (Thr¹⁸³-Tyr¹⁸⁵) and Akt (Ser⁴⁷³) phosphorylation in cells expressing D₄, α_1B, or β₁ receptors, treated for 7 min with 1 µM RO 10-5824, phenylephrine, or isoproterenol. In (B to E), cells expressing D₄ and α_1B receptors (B and C) or D₄ and β₁ receptors (D and E) were treated for 7 min with increasing concentrations of phenylephrine (B and C) or isoproterenol (D and E) in the presence (○) or in the absence (•) of 500 nM RO 10-5824. The immunoreactive bands, corresponding to ERK 1/2 (B and D) and Akt (C and E) phosphorylation of four experiments, were quantified and expressed as mean ± S.E.M. of arbitrary units. In (F and G) membranes of cells expressing D₄ and α_1B receptors (F) or D₄ and β₁ receptors (G) were used to perform competition binding experiments of α₁ receptor antagonist [³H]prazosin (1 nM) versus increasing concentrations of phenylephrine (1 nM to 1 mM) (F) or β₁ receptor antagonist [³H]CGP-12177 (1 nM) versus increasing concentrations of isoproterenol (1 nM to 1 mM) (G) in the presence (○) or in the absence (•) of 500 nM RO 10-5824.</p