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

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

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

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

Microorganisms isolated from lesions.

<p>^<b>a</b><i>Rhodococcus boritolerans</i>, <i>Dietzia timorensis</i>, <i>Pseudoclavibacter</i> spp, <i>Brevibacterium</i> spp and <i>Actinomyces masicol</i> (1 isolate/each)</p><p>^b<i>Streptococcus</i> spp. (3 isolates), <i>S</i>. <i>mitis</i>, <i>S</i>. <i>rattus</i> and <i>S</i>. <i>bovis</i> (1 isolate/each)</p><p>^c<i>E</i>. <i>faecium</i> (8 isolates) <i>E</i>. <i>durans</i> (3 isolates), <i>E</i>. <i>faecalis</i> (6 isolates), <i>E</i>. <i>gallinarum</i> and <i>E</i>. <i>avium</i> (1 isolate/each)</p><p>^d<i>C</i>. <i>maltaromaticum</i> (16 isolates) and <i>C</i>. <i>divergens</i> (1 isolate)</p><p>^e<i>A</i>. <i>urinae</i> (7 isolates), <i>A</i>. <i>viridans</i> (4 isolates) and <i>A nurinaequi</i> (2 isolates)</p><p>^f<i>S</i>. <i>sciuri</i>, <i>S</i>. <i>xylosus</i> (2 isolates/each), <i>Staphylococcus</i> spp., <i>S</i>. <i>aureus</i> and <i>S</i>. <i>haemolyticus</i> (1 isolate/each)</p><p>^<b>g</b><i>Leuconostoc</i> spp. (4 isolates), <i>Escherichia coli</i> (2 isolates) <i>Mezorhizobium</i> spp., <i>Halospirulina</i> spp., <i>Glanulicatella</i> spp. and <i>Lactococcus lactis</i> (1 isolate/each)</p><p>Microorganisms isolated from lesions.</p

PFGE patterns generated after Bsp120I macrorestriction of <i>Flavobacterium plurextorum</i> sp. nov.

<p>Lane 1, DNA molecular size marker; Lanes 2 to 6, strains 1126-1H-08^T, 51B-09, 986-08, 1084B-08 and 424-08, respectively.</p

Distribution of granulomas per examined organ and stage of development.

<p>^aSLN: submandibular lymph nodes.</p><p>Distribution of granulomas per examined organ and stage of development.</p

Phylogenetic tree based on 16S rRNA gene sequence comparisons, obtained with the neighbour-joining algorithm, showing the relationships of <i>Flavobacterium plurextorum</i> sp. nov. with related species.

<p><i>Flexibacter flexilis</i> ATCC 23079^T was used as an outgroup. Bootstrap values (expressed as a percentage of 1,000 replications) greater than 70% are given at the nodes. Solid circles indicate that the corresponding nodes (groupings) are also obtained on the maximum-likelihood tree. Open circles indicate that the corresponding nodes (groupings) are also obtained on the maximum-likelihood and parsimony trees. Sequence accession numbers are indicated in brackets. Bar, 1% sequence divergence.</p

A-G.

<p><b>A)</b> TBL in the submandibular lymph node of an affected pig. Bar, 1cm. B) TBL in the spleen of an affected pig. Bar, 1cm. C) Microscopic image of a TBL lesions in the lymph node of an affected animal showing a profuse infiltrate of degenerated neutrophils. HE. Bar, 200μm. D) Clustered epithelioid macrophages surrounded by lymphocytes and erythrocytes in a stage I granuloma in the liver. HE. Bar, 50μm. E) Coalescent stage II granulomas in the lymph node of a pig showing epithelioid macrophages completely enclosed by a thin capsule, with peripheral infiltration of scattered lymphocytes. HE. Bar, 100μm. F) Stage III granuloma with a central necrotic core, partially mineralized, surrounded by a dense connective tissue capsule infiltrated by lymphocytes and scattered neutrophils. HE. Bar, 100μm. G) Thickly encapsulated, large, irregular, multicentric granulomas with prominent caseous necrosis and multifocal islands of mineralization (stage IV granulomas). HE. Bar, 500μm.</p