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. 2021 May 4;16(5):e0249682. doi: 10.1371/journal.pone.0249682

Streptococcus suis serotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Chadaporn Chaiden 1, Janthima Jaresitthikunchai 2, Anusak Kerdsin 3, Nattakan Meekhanon 4, Sittiruk Roytrakul 2, Suphachai Nuanualsuwan 1,5,*
Editor: Joseph Banoub6
PMCID: PMC8096114  PMID: 33945547

Abstract

Streptococcus suis, particularly S. suis serotype 2 (SS2), is an important zoonotic pathogen causing meningitis in humans worldwide. Although the proper classification of the causative and pathogenic serotype is salutary for the clinical diagnosis, cross-reactions leading to the indistinguishability of serotypes by the current serotyping methods are significant limitations. In the present study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of extracted peptides was developed to improve the classification of serotype of S. suis. The peptide mass fingerprint (PMFs) database of S. suis was generated from the whole-cell peptides of 32 reference strains of S. suis isolates obtained from pigs. Thirty-two human S. suis isolates from clinical cases in Thailand were used to validate this alternative serotyping method in direct comparison to the multiplex (m)PCR approach. All reference strains, representing 32 serotypes of S. suis, exhibited their individual PMFs patterns, thus allowing differentiation from one another. Highly pathogenic SS2 and SS14 were clearly differentiated from the otherwise serologically closely related SS1/2 and SS1, respectively. The developed MALDI-TOF-MS serotyping method correctly classified the serotype in 68.8% (22/32) of the same serotype isolates generated from the PMFs database; while the validity for the clinical human isolates was 62.5% (20/32). The agreement between the MALDI-TOF-MS and mPCR serotyping was moderate with a Kappa score of 0.522, considering that mPCR could correctly serotype up to 75%. The present study demonstrated that PMFs from the developed MALDI-TOF-MS-based method could successfully discriminate the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1, respectively. Moreover, this serotyping method distinguished pathogenic SS6, and so is an alternative approach of choice to rapidly and reliably serotype clinically pathogenic S. suis isolates.

Introduction

Streptococcus (S.) suis is a zoonotic foodborne pathogen [1] that inhabits the nasal cavity and particularly the tonsils of pigs. People who work closely with live pigs can potentially become infected with this pathogen via open skin lesions leading to the clinical symptoms of septicemia, endocarditis, peritonitis, pneumonia, and meningitis [1, 2]. In the past, this bacterium has been classified by its capsular polysaccharide into up to 35 serotypes [1, 2], of which currently 29 serotypes are recognized [3, 4]. Of these, S. suis serotype (SS2) is the most significant serotype as it is predominantly associated with both diseased pigs and human clinical cases, and it has been reported as the causative agent in more than 70% of human clinical cases. Whereas SS14 and some other pathogenic serotypes are far less significant and account for only up to 3% of human clinical cases worldwide [5].

Although the prompt confirmation of the causative serotype as part of the clinical diagnosis is pivotal to the successful treatment rate of S. suis infection, more than 20% of human cases have gone undiagnosed due to inherent drawbacks in the current serotyping methods [5]. Initially, microbial identification of S.suis is presumed following biochemical tests. Subsequently, polymerase chain reaction (PCR) assay targeting the presence of the recombinant/repair protein gene recN [6] is then used to confirm S. suis. Serological methods have been considered as the standard procedures for serotyping S. suis. However, cross-reaction among pairs or groups of serotypes, such as SS1 and SS14; SS2 and SS1/2; SS2 and SS22; SS6 and SS16; and SS1, SS2, and SS1/2; can lead to the inconclusive or erroneous serotyping of S. suis. These indistinguishable serotypes are problematic since pathogenic serotypes in both pigs and humans, such as SS2, SS6, SS14, and SS16, are found among all the inconclusive pairs (or groups) of cross-reactions [5]. In addition, serotyping with all the typing antisera is laborious, time-consuming, and expensive, and preparing the antisera is difficult due to the high cost and labor associated with its production.

Even though multiplex (m)PCR has recently been developed and gained popularity as an alternative technique to serotype S. suis isolates, mPCR does not differentiate SS2 from SS1/2, or SS14 from SS1 due to the high capsular gene cluster similarity. So, additional or alternative methods are required to solve this issue. Lately, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been increasingly endorsed and adopted as an alternative approach to microbial identification [7], including S. suis identification [8, 9]. In this method, cellular proteins (and peptides) of S. suis are extracted and then used for species identification via peptide mass fingerprints (PMFs) formed using MALDI-TOF-MS.

Nevertheless, one previous study successfully examined few serotypes of SS using MALDI-TOF-MS still most of the serotypes were not classified especially undistinguishable serotypes by current serotyping methods [8], and so is tackled in this study. The peptide extraction method was modified from Bruker’s recommendation [10], and the extracted peptides of S. suis were then analyzed by MALDI-TOF-MS. The aim of this study was to demonstrate S. suis serotyping by means of the modified peptide extraction method coupled with MALDI-TOF-MS, and in particular to differentiate those inconclusive pathogenic and non-pathogenic serotypes.

Materials and methods

Bacterial strains

In this study, 32 reference strains (SS1-14, SS16-20, SS22-32, SS34, and SS1/2) representing 32 different serotypes of S. suis (Table 1) along with local S. suis SS2 (n = 23) and SS14 (n = 9) isolates from humans were used, the strains were originally collected from hospital, distributed in 5 regions (north, northeast, central, east, and south) of Thailand. Sequencing of the 16S rRNA gene was used to confirm these 32 reference strains [11]. Bacteria were cultured on Columbia blood agar (Difco Laboratories, Detroit, Mich.) with 5% (v/v) sheep blood at 37°C in an anaerobic condition for 24 h. Reference strains of Staphylococcus aureus ATCC 25923 and E. coli DH5 alpha were included to study cross-reaction.

Table 1. Repeatability of MALDI-TOF-MS serotyping of the reference S. suis serotypes after anaerobic culture.

Serotype Strain Source MALDI-TOF-MS GenBank acc.no. of 16S rRNA gene
Serotype (best match) LSV
1 NCTC10237 Diseased pig 1 2.187 LR594043.1
2 NCTC10234 Diseased pig 2 2.453 LS483418.1
3 4961 Diseased pig 3 2.341 AF009478.1
4 6407 Diseased pig - - AF009479.1
5 11538 Diseased pig - - AF009480.1
6 2524 Diseased pig 6 2.207 AF009481.1
7 8074 Diseased pig 7 2.369 AF009482.1
8 14636 Diseased pig 8 2.302 AF009483.1
9 22083 Diseased pig 9 2.178 AF009484.1
10 4417 Diseased pig - - AF009485.1
11 12814 Diseased pig - - AF009486.1
12 8830 Diseased pig - - AF009487.1
13 10581 Diseased pig 13 2.354 AF009488.1
14 13730 Diseased pig 14 2.492 AF009489.1
16 2726 Diseased pig 16 2.504 AF009491.1
17 93A Healthy pig - - AF009492.1
18 NT77 Healthy pig 18 2.539 AF009493.1
19 42A Healthy pig 19 2.36 AF009494.1
20 86–5192 Diseased calf 20 2.566 AF009495.1
22 88–1861 Diseased pig - - AF009497.1
23 89–2479 Diseased pig - - AF009498.1
24 88-5299A Diseased pig 24 2.207 AF009499.1
25 89-3576-3 Diseased pig 25 2.419 AF009500.1
26 89-4109-1 Diseased pig - - AF009501.1
27 89–5259 Diseased pig 27 2.304 AF009502.1
28 89–590 Diseased pig - - AF009503.1
29 92–1191 Diseased pig 29 2.3 AF009504.1
30 92–1400 Diseased pig 30 2.256 AF009505.1
31 92–4172 Diseased calf 31 2.124 AF009506.1
32 EA1172.91 Diseased pig 32 2.336 AF009507.1
34 92–2742 Diseased pig 34 2.393 AF009509.1
1/2 2651 Diseased pig ½ 2.255 AF009476.1
Total 32 32 22 (68.8%) 2.34*
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*Average LSV of correct MALDI-TOF-MS classifications.

Peptide extraction

Pure colonies of S. suis for peptide extraction were prepared on Columbia blood agar as above. Then ethanol was added to 70% (v/v) to precipitate out the bacterial peptides from the whole bacterial cell. The bacterial suspension was centrifuged at 11,000 g for 5 min, and then the pellet was collected. Next, 5% (v/v) trifluoroacetic acid (TFA) in absolute acetonitrile (ACN) was added to the pellet and the suspension was dissolved by gentle vortex. The bacterial peptides dissolved in the supernatant were collected after clarification by centrifugation at 11,000 g for 5 min and then kept at -20°C prior to MALDI-TOF-MS analysis. Lowry assay was used to quantify the concentrations of extracted peptides [12].

Peptide analysis by MALDI-TOF-MS

The extracted peptides from the 32 reference strains of S. suis were mixed with the sinapinic matrix solution [sinapinic acid in 5% (v/v) TFA in absolute ACN] and then spotted onto the MALDI target plate and allowed to crystalize at room temperature before inserting the MALDI target plate into the MALDI-TOF-MS instrument. The mass spectrometry (MS) spectra were collected using an Ultraflex III TOF/TOF (Bruker Daltonik, GmbH) instrument in a linear positive mode with a mass range between 2–20 kDa. Five hundred shots were reiterated and accumulated with a 50 Hz laser per SS. Likewise, this MALDI-TOF-MS procedure was repeated for all 32 serotypes of S. suis. All MS spectra were analyzed for fingerprint spectra and subjected to PCA using the FlexAnalysis version 3.4 and ClinProTool version 3.0 software (Bruker Daltonik, GmbH). The ACTH fragment 18–39 (human), insulin oxidized B chain (bovine), insulin (bovine), cytochrome C (equine), and apomyoglobin (equine) were used as the external protein calibrations. Analysis of variance (ANOVA), Student’s t-test and statistics in the software, were used to assess the statistically significant differences in the PMFs across bacterial strains.

Generating of S. suis’s PMFs database

The S. suis PMFs database was generated by inserting 20 qualified MS spectra from each of the 32 individual reference serotypes into the MALDI Biotyper database system according to Bruker’s recommendation. The PMFs of 32 serotypes in the database will be used as the references PMFs for the serotype characterization and to evaluate the repeatability, reproducibility and validation.

Serotype analysis by mPCR

All reference strains of S. suis were serotyped by mPCR [13]. The results from the MALDI-TOF-MS analysis were compared to those from the mPCR of the same samples in terms of the repeatability validation and agreement of tests. Four sets of primers targeting the capsule (cps) loci across 28 serotypes of S. suis were used. The first primer sets targeted SS1/2, SS1-3, SS7, SS9, SS11, SS14, and SS16; the second primer set targeted SS4, SS5, SS8, SS12, SS18, SS19, SS24, and SS25; the third primer set targeted SS6, SS10, SS13, SS17, SS23, and SS31; and the fourth primer set targeted SS21 and SS27-30. The mPCR serotyping of S. suis were followed as previously described [13].

Repeatability

The same isolate of each serotype was repeatedly cultured and extracted in exactly the same manner. Eight spots from each serotype were applied to determine the precision of this developed serotyping method Bruker Biotyper. Log score values (LSVs) were used to evaluate the serotype classifications, where LSVs between 0–1.69 were merely reliable, between 1.70–1.99 were probable, and more than 2.00 were highly probable. Among 8 spots, the matched serotypes with highest LSVs will be considered as a correct classification.

Validation

To assess the validity of this serotyping method against known clinical isolates of S. suis, 32 isolates of S. suis from human cases in Thailand, comprised of SS2 (n = 23) and SS14 (n = 9), were cultured in the conventional anaerobic growth condition, extracted, and characterized by MALDI-TOF-MS as described earlier. Each individual isolate was spotted for eight entries. The serotype classification of the clinical isolates was evaluated by the same Bruker Biotyper LSVs and criteria.

Reproducibility or culture-condition repeatability

To evaluate the repeatability of this serotyping method with serotypes cultured under a different condition, then the 32 serotypes of S. suis were cultured as before except in an aerobic growth condition, and then peptide extracted and serotype classified by MALDI-TOF-MS using exactly the same MALDI-TOF-MS protocol as described earlier. The serotype classification of was evaluated by the same Bruker Biotyper LSVs and criteria.

Degree of test agreement

The 32 reference strains of S. suis were grown anaerobically or aerobically and subjected to both mPCR and developed MALDI-TOF-MS serotyping methods as described. The serotyping results are presented in a 2 × 2 contingency table of frequencies with the rows and columns representing the serotype results for both serotyping methods. The degree of agreement between these two methods was assessed using Cohen’s kappa statistic (κ) [14], as shown in Eq (1);

κ=PO−PE1−PE, (1)

where n is the total observed frequency, OD is the sum of observed frequencies along the diagonal (Table 2), ED is the sum of expected frequencies along the diagonal, PO is the OD/n, and PE is the ED/n.

Table 2. Contingency table of identification frequencies to evaluate S. suis serotypes using the MALDI-TOF-MS analysis of this study compared to mPCR test.

Observed Multiplex PCR
True serotype False serotype Sum
MALDI-TOF True serotype 59 3 62
False serotype 19 22 41
Sum 78 25 103
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Results

Peptide mass spectra of S. suis

After extraction, peptide masses between 2–20 kDa were collected and analyzed using the FlexAnalysis version 3.4 and ClinProTool version 3.0 software (Bruker Daltonik, GmbH). All of the extracted peptides delivered an adequate number and intensity (more than 104 a.u.) of peptide mass. Extracted peptides from each individual serotype generated particular PMFs that contained their individual unique mass(es) and so differentiated each serotype from one another (Fig 1A–1D). Overall, 4420, 5337, 5965, 6634, 6748, 6834, and 8260 Da peptide masses were commonly found in most of the SSs. Interestingly, SS1/2, SS11, and, SS13, had some other unique masses (2990 and 3005 Da) in common.

Fig 1.

Fig 1

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Representative MALDI-TOF peptide mass spectra of reference S. suis serotypes (A) SS1-7, (B) SS8-14 and SS16, (C) SS17-20 and SS22-25, and (D) SS26-32 and SS34. The X-axis represents the mass to charge ratio (m/z) and the Y-axis represents the intensity of the spectra.

The former SS32 and SS34 (S. orisratti) had a unique PMF pattern with the 4447, 6610, and 6775 Da peptide masses being common to these two serotypes and so Biotyper software classified SS32 and SS34 in another group, as shown in the dendrogram (Fig 2).

Fig 2. Dendrogram based on the PMFs of reference SS1/2, SS1-14, SS16-20, SS22-32, SS34, and SS1/2 grown anaerobically.

Fig 2

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SS32 and SS34 clustered in a group, while SS1/2, SS11, and SS13 clustered in another group and closer to the main SS PMFs. Staphylococcus aureus ATCC 25923 and E. coli DH5 alpha were included as the out group.

The ambiguous serotypes by mPCR, which were the highly pathogenic SS2 and SS1 that are serologically associated with SS1/2 and SS14, respectively, were clearly distinguished from one another (Fig 1A and 1B). The 6956 Da peptide mass discriminated between SS2 and SS1/2 (p < 0.01), while the 6919 Da peptide mass discriminated between SS1 and SS14 (p < 0.01), as shown in Table 3. Likewise, principal component analysis (PCA) revealed that the PMFs of SS2, SS1/2, SS1, and SS14 distinctly clustered around their individual serotypes (Fig 3). Therefore, the PCA results did support that the ambiguity of serotype pairs SS2 and SS1/2; and SS1 and SS14 could be clearly resolved by PMFs mapping using MALDI-TOF-MS. Additionally, cross-reacting serotypes based on serological methods were also classified, where the 6319, 6914, and 5056 Da peptides were index masses that differentiated between SS1 and SS2, between SS6 and SS16; and between SS2 and SS22, respectively, (Table 3).

Table 3. Differentiating between serologically ambiguous serotypes using the index mass obtained from MALDI-TOF-MS analysis of PMFs from anaerobically cultured S. suis serotypes.

Serotyping method Ambiguous serotype Differentiating index mass (Da) by MALDI-TOF-MS
Multiplex PCR SS2 vs. SS1/2 6956
SS1 vs. SS14 6919
Serological SS1 vs. SS2 6319
SS2 vs. SS1/2 6956
SS6 vs. SS16 6914
SS2 vs. SS22 5056
SS1 vs. SS14 6919
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Fig 3. The PCA, based on the PMFs, of the ambiguous serotypes of S.suis SS2, SS1/2, SS1, and SS14.

Fig 3

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SS1, SS2, SS1/2, SS1, and SS14 represent the clusters of S. suis SS2, SS1/2, SS1, and SS14.

Repeatability

The same isolate of each serotype was repeatedly cultured, extracted, and classified in exactly the same manner to determine the precision of the developed serotyping method. Eight spots from each serotype were applied to classify comparing with the S. suis PMFs database generated as described earlier. The results showed that 22 out of 32 serotypes (68.8%; average LSV of 2.34) were correctly serotype classified. The ambiguous serotypes by mPCR (SS2 with SS1/2, and SS1 with SS14) were also correctly serotyped with LSVs of 2.453, 2.255, 2.187, and 2.492, respectively. The serological cross-reacting serotypes SS1 with SS2, and SS6 with SS16, were correctly classified with LSVs of 2.187, 2.453, 2.207, and 2.504, respectively (Table 1). However, SS22 (cross-reactive with SS2) was unable to be serotyped (distinguished) by this MALDI-TOF-MS approach method.

Validation

Thirty-two human isolates of S. suis from patients in Thailand, comprised of SS2 (n = 23) and SS14 (n = 9), were serotyped using the developed MALDI-TOF-MS method. The method correctly serotyped 20 out of 32 serotypes (62.5%) of human isolates, with an average LSV of 2.20 (Table 4). Serotype 2 was correctly classified in 13 out of 23 strains (56.5%, average LSV of 2.19) and SS14 was correctly classified in 7 out of 9 strains (77.8%, average LSV of 2.20).

Table 4. Validation of MALDI-TOF-MS for serotyping S. suis human strains isolated in Thailand compared to the mPCR approach.

Serotype Source MALDI-TOF-MS
Serotype (best match) LSV
2 Human 2 (13/23) 2.19
14 Human 14 (7/9) 2.20
Total 32 20 (62.5%) 2.20*
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*Average LSV of correct MALDI-TOF-MS classifications.

Reproducibility

Instead of using an anaerobic growth condition, 32 the serotypes of S. suis (SS1-14, SS16-20, SS22-32, SS34, and SS1/2) were cultured as before except under an aerobic growth condition. The strains were then peptide extracted and serotype classified by exactly the same MALDI-TOF-MS protocol. The MALDI-TOF-MS approach was found to correctly serotype 18 out of 32 strains of S. suis (56.3%) with average LSV of 2.34.

Degree of test agreement

Cohen’s unweighted Kappa statistic was used to elucidate the agreement between the mPCR and this developed MALDI-TOF-MS serotyping method, and gave a Kappa score of approximately 0.522. While the true serotype classifications of the MALDI-TOF-MS and mPCR were not statistically significant (p > 0.05) approximately 60% (95% CI: 51%-70%) and 76% (95% CI: 67%-84%), respectively.

Discussion

As a rapid high throughput technique, MALDI-TOF-MS has been increasingly used in microbiological studies. It provides improved accuracy and power of resolution to identify or even classify microbial isolates [7, 15]. In this study, we modified the peptide extraction method coupled with MALDI-TOF-MS to identify S. suis and further classify the SSs. Mass spectra yielded from this peptide extraction technique exhibited good qualities, a high intensity, and an adequate number of peptide masses that overall indicated that this method could be applicable for SS classification.

The PMFs presented 4420, 5337, 5965, 6634, 6748, 6834, and 8260 Da peptide masses that were commonly found across all the tested SSs. Two of these masses were close to the 4420 and 8266 Da peptide masses that have recently been reported in previous studies using whole cell extraction of S. suis, which mass 4133 and 8367 were reported as the indicative species-specific peaks [8, 9]. This congruent finding may reach a general assumption that the 4420 and 8260 Da peptide masses could act as species-specific markers for S. suis. As these two masses were absent in others closely related Streptococcus spp. e.g. S. plurextorum, S. porci and S.porcorum where their species-specific markers corresponded to 6164, 6133 and 4190/8381, respectively [9]. However, some other peptide masses were found to be different from those in previous studies, which can be explained by the different preparation protocols, such as the acid concentration, spotting method, and type of matrices [16–18]. In the present study, the principal difference in the preparation protocol was the modified peptides extraction method, that formic acid was not included, gentle dissolve pellet by vortex and vigorously mix peptides with sinapinic matrix, which likely accounted for some of the dissimilar PMFs. Moreover, dissimilar PMFs could be inherent in the intraspecies discrepancy as well [19].

The PMFs of S. suis-like serotypes (SS32 and SS34) had a unique pattern with 4447, 6610, and 6775 Da peptide masses commonly found in these two serotypes. The 6775 Da peptide mass was somewhat close to the 6772 Da peptide mass that has previously been reported as a representative mass of S. porcorum PMFs [9]. Previous studies have suggested that SS32 and SS34 are likely to be Streptococcus orisratti [3]. In accord, the obtained PMFs dendrogram in this study (Fig 2) clustered both SS32 and SS34 together but separate from the other S. suis isolates, indicating that these two serotypes possibly possessed a high genetic dissimilarity from the other examined SSs. The phylogenetic tree based on 16s rRNA gene (Fig 4) also supported this finding as SS32 and SS34 were also clustered separately from the main group of S. suis. Furthermore, SS1/2, SS11, and SS13 revealed some specific peptide masses that exclusively clustered together in the dendrogram (Fig 2). Thus, we presumed that these three reference serotype strains were divergent from the other tested reference serotype strains, in accord with a previous study that reported that SS13 was also divergent from other S. suis strains [20]. However, these divergent characters were not displayed in phylogenetic tree (Fig 4). In terms of the relatedness of PMFs, the cluster of SS1/2, SS11, and SS13 was closer to the main cluster than the cluster of SS32 and SS34 (Fig 2).

Fig 4. Phylogenetic relationships of reference SS1/2, SS1-14, SS16-20, SS22-32, SS34, and SS1/2.

Fig 4

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Maximum Likelihood method was used based on 16s rRNA gene.

For SS classification, the PCA results demonstrated that the use of extracted peptides subject to MALDI-TOF-MS analysis was capable of discriminating between the high similarity serotypes pairs of (i) SS2 and SS1/2 and (ii) SS1 and SS14. Notably that, these two pairs of serotypes closely clustered together in phylogenetic tree (Fig 4) but separately in peptide dendrogram (Fig 2). This finding could be used to explained why our modified method could differentiate these two pairs of high similarity serotypes. Importantly, SS2 and SS14 are the two serotypes that accounted for most major S. suis infections [5, 21], where the misidentification of the causative pathogen could delay the appropriate treatment [22]. Moreover, the currently available classification methods are sometimes inconclusive. Hence, this modified peptide extraction method from Bruker’s recommendation coupled with MALDI-TOF-MS analysis could be an alternative approach to discriminate between serologically cross-reactive SSs and so is a potentially significant advantage for S. suis infection diagnosis.

According to the repeatability test, the extracted PMFs coupled with our S. suis database was capable of discriminating 22 out of 32 (68.8%) reference SSs. Given that some serotypes of S. suis share the same structural components [23], then the structural peptides acquired by the present study could be similar and so have led to the misclassification of the other 10 serotypes (31.3%). Moreover, only 8 spots were applied for repeatability test, while 20 qualified spots were used to generate the S. suis PMFs database. Similarly, only 4 spots are sufficient to identify bacteria by means of MALDI-TOF-MS [15]. However, serotype classification is more subtle than bacterial identification thus requiring more replication of MS spectra to differentiate the more closely related serotype within the same species. In order to improve the correct serotype classification rate, we suggested that the repeatability test of the serotype classification, including unknown serotyping, should require at least 8 MS spectra (spots). Likewise, LSVs criteria of correct serotype classification are probable between 1.70–1.99. More PMFs included in the database from each individual serotype would warrant a higher degree of correct classification.

Clinical human isolates of S.suis were included in this study to validate this method. Just over half (56.5%) and three-quarters (77.8%) of human SS2 and SS14 strains, respectively, were correctly classified. This moderate level of validity of the serotyping method implied that the S. suis reference strains and field strains posed a somewhat dissimilar peptide background. Of relevance then is it has previously been reported suggested that different backgrounds of the same serotype of S. suis could result in different phenotype expressions [24]. For further study, more PMFs from field strains of S. suis should be included in the database to improve the reliability of the serotype classification.

The reproducibility or culture-condition repeatability of the serotype classification was merely acceptable (56.3%) when S. suis was cultured in an aerobic condition. The different background derived from different growth conditions of the same isolates could yield the dissimilar peptides, such as in this case, where the anaerobic vs. aerobic growth conditions of S. suis resulted in a mismatched classification. Therefore, the reproducibility of this serotyping method was growth-condition specific, as previously reported for Burkholderia pseudomallei, where altering the incubation condition lowered the identification scores of Burkholderia pseudomallei [18]. Therefore, various growth conditions, including the anaerobic growth of S. suis should be recommended and emphasized when using this MALDI-TOF-MS classification coupled with corresponding PMFs database.

That only a moderate agreement between the MALDI-TOF-MS and mPCR analyses (unweighted Kappa score of 0.522) pointed out that the results among these methods were controversial. The mPCR is not without limitations, such as ambiguous serotype classifications corresponding to the moderate degree (76%) of the true serotype classification [13], while However, these divergent characters were not displayed in phylogenetic tree SS2 and SS14 from SS1/2 and SS1, respectively. The degree of agreement measured how well the two tests agreed with each other, but not on the reliability to correctly serotype S. suis. Nevertheless, 95% CI of both methods indicated that the true serotype classification rate of both methods was not statistically different. Theoretically, in order to improve the kappa score, more PMFs of S. suis should be included, since the accuracy of the classification depends on the number of reference spectra present in the database [25].

Conclusions

In present study, we successfully differentiated the ambiguous serotypes of S. suis. Thus, providing an alternative method for S. suis classification that could be useful for S. suis infection diagnosis and epidemiological study of this zoonotic pathogen.

Supporting information

S1 Table. The complete list of m/z of S. suis serotyping by MALDI-TOF-MS identified and used in this study.

(XLSX)

Click here for additional data file. (18.3KB, xlsx)

Acknowledgments

We thank the staffs Center of Excellence for Emerging and Re-emerging Infectious Diseases in Animals, Chulalongkorn University, Bangkok, Thailand for supporting the PCR thermocycler and techniques.

List of abbreviations

MALDI-TOF-MS

matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

PCA

principal component analysis

mPCR

multiplex polymerase chain reaction

PMFs

peptide mass fingerprints

SS

Streptococcus suis serotype

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This study was supported by The 100th Anniversary Chulalongkorn University Fund for Doctoral Scholarship; The 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund); The Scholarship from the Graduate School, Chulalongkorn University to commemorate the 72nd anniversary of his Majesty King Bhumibol Adulyadej; The Agricultural Research Development Agency (Public Organization).

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Decision Letter 0

Joseph Banoub

25 Feb 2021

PONE-D-21-02524

Streptococcus suis serotyp ing by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

PLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: In the manuscript titled “Streptococcus suis serotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry”, Chaiden et al. used MALDI-TOF mass spectrometry to serotype Streptococcus suis from reference samples and compare against the use of multiplex-PCR, then validate the mass spectrometry method using clinical isolates. Specific comments are as follows:

1. On line 83, it is a bit misleading to state that “the classification of SSs has never been reported”. In reading through reference #8, it seems the authors of that paper have been able to examine serotypes 2, 7, and 9, as well as what they referred to as “less prevalent” serotypes. Though not a complete listing of serotypes (as it seems the authors assessed at least eight serotypes), the authors of the current manuscript cannot state that any classification/serotype attempts have not been reported previously. This needs to be corrected through the manuscript, where appropriate.

2. Is multiplex-PCR really considered a “gold standard”, when it has a reasonably poor rate of identifying serotypes? Sequencing, and likely of just the 16S RNA, would really provide near 100% serotype identity. (After all, the authors in the current manuscript indicate that the 32 reference strains were determined by sequencing of the 16S rRNA). It is strongly recommended to remove reference to “gold standard” throughout the manuscript regarding multiplex-PCR.

3. Under “Validation”, provide the source/location of the clinical isolates used (i.e., which hospital or clinic, and what parts of the country the subjects came from).

4. Figure 2 is not clear at all at the resolution provided.

5. In the Discussion, the authors state that “other peptide masses were found to be different from those in previous studies, which can be explained by the different preparation protocols…”. This is of concern if the purpose of the study is to develop a new, reproducible method for serotype detection. Some different preparation protocols should be considered and examined with the current study, to attempt to provide common spectra that could be used by other groups and their own protocols.

6. The authors state in the Discussion that “the obtained PMFs dendrogram in this study (Fig 2) clustered both SS32 and SS34 together but separate from the other S. suis isolates, indicating that these two serotypes possibly possessed a high genetic dissimilarity from the other examined SSs”. Provide a dendrogram based on 16S RNA to show this, and any dissimilarity of other serotypes. Make a comparison between this and the dendrogram for the mass spectrometry data.

7. Within the Discussion, the authors state that “In order to improve the correct serotype classification rate, we suggested that the repeatability test of the serotype classification, including unknown serotyping, should require at least two times more than 8 MS spectra (spots)”. This reviewer recommends that the authors carry this out.

8. The complete list of m/z identified and used should be provided as a data supplement.

Reviewer #2: This manuscript describes a novel matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of the extracted peptides obtained from Streptococcus suis, and in particularly S. suis serotype (SS2). In this method, cellular proteins (and peptides) of S. suis are extracted and then used for species identification via peptide mass fingerprints (PMFs) formed using MALDI-TOF-MS.

The authors showed that this MALDI-TOF-MS analysis permitted to improve the classification of 32 serotypes of S. suis. Indeed, each individual MALDI-TOF-MS exhibited an individual PMFs patterns, which allowed to differentiate each serotype. This was followed by developing an exclusive peptide mass fingerprint (PMFs) database of S. suis, which was generated from the whole-cell peptides of 32 reference strains of S. suis. This PMFs database was generated by inserting 20 qualified MS spectra from each of the 32 individual reference serotypes into the MALDI Biotyper database system according to Bruker’s recommendation.

It should be noted that although, it was suggested that multiplex (m)PCR analysis could be used to serotype S. suis isolates. However, it was shown that it did not allow the differentiation of SS2 from SS1/2, or SS14 from SS1 due to the high capsular gene cluster similarity. The authors showed that this discrepancy could be resolved by using the PMFs blueprints to discriminate the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1. Indeed, serotyping using MALDI-TOF-MS correctly classified SS2 from SS1/2, or SS14 from SS1serotypes in 68.8% (22/32); while the validity for the clinical human isolates was 62.5% (20/32). The agreement between the MALDI-TOF MS and mPCR serotyping was moderate with a Kappa score of 0.522, considering that mPCR could correctly serotype up to 75%.

In conclusion, the authors have demonstrated that PMFs from the developed MALDI-TOF-MS based method can successfully discriminate the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1, respectively. Moreover, this serotyping method distinguished pathogenic SS6, and so is an alternative approach of choice to rapidly and reliably serotype clinically pathogenic S. suis isolates.

This manuscript is generally well written and the authors have shown that by using MALDI-TOF-MS serotyping using PMFs it was possible to classify the correct serotypes of S. suis isolates.

Nevertheless, few changes are requested to improve the scientific flow of this manuscript.

1. To be compliant with the IUPAC MS nomenclature, please change all your MALDI-TOF MS into MALDI-TOF-MS.

2. Your Figure 2 is illegible, change for a better and crisper one.

3. I am confused and cannot understand the following argument of page 14:

Cohen’s unweighted Kappa statistic was used to elucidate the agreement between the mPCR, as the gold standard method, and this developed MALDI-TOF MS serotyping method, and gave a Kappa score of approximately 0.522. While the true serotype classifications of the mPCR and MALDI-TOF MS, based on the results from Table 4, were approximately 76% (78/103) and 60% (62/103), respectively

Query: Is the serotyping using mPCR (76 %) better than the MALDI-TOF-MS (60%)? Then I suggest that for the validation of your manuscript, you make it clearer. You must stress the benefit of your PMFs method allows better discrimination between the serotypes especially for the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1, respectively

**********

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Reviewer #2: No

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PLoS One. 2021 May 4;16(5):e0249682. doi: 10.1371/journal.pone.0249682.r002

Author response to Decision Letter 0

7 Mar 2021

Response to reviewers of PLOS ONE

We would like to thank the editor and the reviewers for their thoughtful comments. We have carefully addressed all the comments. The corresponding changes and refinements made in the revised paper are summarized in our response below.

Reviewer#1: In the manuscript titled “Streptococcus suis serotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry”, Chaiden et al. used MALDI-TOF mass spectrometry to serotype Streptococcus suis from reference samples and compare against the use of multiplex-PCR, then validate the mass spectrometry method using clinical isolates. Specific comments are as follows:

1. On line 83, it is a bit misleading to state that “the classification of SSs has never been reported”. In reading through reference #8, it seems the authors of that paper have been able to examine serotypes 2, 7, and 9, as well as what they referred to as “less prevalent” serotypes. Though not a complete listing of serotypes (as it seems the authors assessed at least eight serotypes), the authors of the current manuscript cannot state that any classification/serotype attempts have not been reported previously. This needs to be corrected through the manuscript, where appropriate.

RESPONSE : We thank the reviewer for pointing this out. After considering, we decided to remove the phrase “never been reported” to “Nevertheless, one previous study successfully examined few serotypes of SS using MALDI-TOF-MS still most of the serotypes were not classified especially undistinguishable serotypes by current serotyping methods [8].”

2. Is multiplex-PCR really considered a “gold standard”, when it has a reasonably poor rate of identifying serotypes? Sequencing, and likely of just the 16S RNA, would really provide near 100% serotype identity. (After all, the authors in the current manuscript indicate that the 32 reference strains were determined by sequencing of the 16S rRNA). It is strongly recommended to remove reference to “gold standard” throughout the manuscript regarding multiplex-PCR.

RESPONSE : The reviewer has made some excellent points and we sincerely appreciated these well-thought comments. However, sequencing of the 16S rRNA gene was performed only to confirm the reference strains used in this study. As our objective was to determine the ability of serotyping methods commonly used by the laboratories, thus mPCR detecting cps gene was chosen for agreement testing between two serotyping methods. Nevertheless, we recognized this insightful suggestion from reviewer, so reference to “gold standard” throughout the manuscript regarding mPCR was removed. Moreover, to make it clearer, the mPCR results in Table 2-3 were also removed.

3. Under “Validation”, provide the source/location of the clinical isolates used (i.e., which hospital or clinic, and what parts of the country the subjects came from).

RESPONSE : We agreed and have updated the source/location of the clinical isolates used in materials and methods section line 101, “local S. suis SS2 (n = 23) and SS14 (n = 9) isolates from humans were used, the strains were originally collected from hospital, distributed in 5 regions (north, northeast, central, east, and south) of Thailand.”

4. Figure 2 is not clear at all at the resolution provided.

RESPONSE : We apologized for our original Figure 2. We have modified the figure and hope that it is now clear.

5. In the Discussion, the authors state that “other peptide masses were found to be different from those in previous studies, which can be explained by the different preparation protocols…”. This is of concern if the purpose of the study is to develop a new, reproducible method for serotype detection. Some different preparation protocols should be considered and examined with the current study, to attempt to provide common spectra that could be used by other groups and their own protocols.

RESPONSE : We agreed with the reviewer that further elaborating on this point would be helpful. However, the peptide extraction method was only modified from Bruker’s recommendation, since it could not provide satisfying signals for serotyping. We also pointed out some different preparation protocols to Paragraph 2 in Discussion “In the present study, the principal difference in the preparation protocol was the modified peptides extraction method, that formic acid was not included, gentle dissolve pellet by vortex and vigorously mix peptides with sinapinic matrix, which likely accounted for some of the dissimilar PMFs. Moreover, dissimilar PMFs could be inherent in the intraspecies discrepancy as well.

6. The authors state in the Discussion that “the obtained PMFs dendrogram in this study (Fig 2) clustered both SS32 and SS34 together but separate from the other S. suis isolates, indicating that these two serotypes possibly possessed a high genetic dissimilarity from the other examined SSs”. Provide a dendrogram based on 16S RNA to show this, and any dissimilarity of other serotypes. Make a comparison between this and the dendrogram for the mass spectrometry data.

RESPONSE : We agreed and have updated a phylogenetic tree base on 16s rRNA gene as Figure 4. We also made a comparison to Paragraph 3 in Discussion “The phylogenetic tree based on 16s rRNA gene (Fig 4) also supported this finding as SS32 and SS34 were also clustered separately from the main group of S. suis”, to Paragraph 3 in Discussion “However, these divergent characters were not displayed in phylogenetic tree (Fig 4)” and to Paragraph 4 in Discussion “Notably that, these two pairs of serotypes closely clustered together in phylogenetic tree (Fig 4) but separately in peptide dendrogram (Fig 2). This finding could be used to explain why our modified method could differentiate these two pairs of high similarity serotypes”

7. Within the Discussion, the authors state that “In order to improve the correct serotype classification rate, we suggested that the repeatability test of the serotype classification, including unknown serotyping, should require at least two times more than 8 MS spectra (spots)”. This reviewer recommends that the authors carry this out.

RESPONSE : We have fixed the error by changing the word “more than” to “at least” to Paragraph 5 in Discussion.

8. The complete list of m/z identified and used should be provided as a data supplement.

RESPONSE : We agreed and have submitted the complete list of m/z identified as a supplement data.

Reviewer#2: This manuscript describes a novel matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of the extracted peptides obtained from Streptococcus suis, and in particularly S. suis serotype (SS2). In this method, cellular proteins (and peptides) of S. suis are extracted and then used for species identification via peptide mass fingerprints (PMFs) formed using MALDI-TOF-MS.

The authors showed that this MALDI-TOF-MS analysis permitted to improve the classification of 32 serotypes of S. suis. Indeed, each individual MALDI-TOF-MS exhibited an individual PMFs patterns, which allowed to differentiate each serotype. This was followed by developing an exclusive peptide mass fingerprint (PMFs) database of S. suis, which was generated from the whole-cell peptides of 32 reference strains of S. suis. This PMFs database was generated by inserting 20 qualified MS spectra from each of the 32 individual reference serotypes into the MALDI Biotyper database system according to Bruker’s recommendation. It should be noted that although, it was suggested that multiplex (m)PCR analysis could be used to serotype S. suis isolates. However, it was shown that it did not allow the differentiation of SS2 from SS1/2, or SS14 from SS1 due to the high capsular gene cluster similarity. The authors showed that this discrepancy could be resolved by using the PMFs blueprints to discriminate the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1. Indeed, serotyping using MALDI-TOF-MS correctly classified SS2 from SS1/2, or SS14 from SS1serotypes in 68.8% (22/32); while the validity for the clinical human isolates was 62.5% (20/32). The agreement between the MALDI-TOF MS and mPCR serotyping was moderate with a Kappa score of 0.522, considering that mPCR could correctly serotype up to 75%. In conclusion, the authors have demonstrated that PMFs from the developed MALDI-TOF-MS based method can successfully discriminate the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1, respectively. Moreover, this serotyping method distinguished pathogenic SS6, and so is an alternative approach of choice to rapidly and reliably serotype clinically pathogenic S. suis isolates. This manuscript is generally well written and the authors have shown that by using MALDI-TOF-MS serotyping using PMFs it was possible to classify the correct serotypes of S. suis isolates. Nevertheless, few changes are requested to improve the scientific flow of this manuscript.

1. To be compliant with the IUPAC MS nomenclature, please change all your MALDI-TOF MS into MALDI-TOF-MS.

RESPONSE : We have fixed the error by changing the words from MALDI-TOF MS into MALDI-TOF-MS

2. Your Figure 2 is illegible, change for a better and crisper one.

RESPONSE : We apologize for our original Fig 2. We have modified the figure and hope that it is now clear.

3. I am confused and cannot understand the following argument of page 14:

Cohen’s unweighted Kappa statistic was used to elucidate the agreement between the mPCR, as the gold standard method, and this developed MALDI-TOF MS serotyping method, and gave a Kappa score of approximately 0.522. While the true serotype classifications of the mPCR and MALDI-TOF MS, based on the results from Table 4, were approximately 76% (78/103) and 60% (62/103), respectively

Query: Is the serotyping using mPCR (76 %) better than the MALDI-TOF-MS (60%)? Then I suggest that for the validation of your manuscript, you make it clearer. You must stress the benefit of your PMFs method allows better discrimination between the serotypes especially for the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1, respectively

RESPONSE : We recognized this insightful suggestion from reviewer, thus to Paragraph 8 in Discussion these following sentences were added “this MALDI-TOF-MS serotyping method allows better discrimination between the serotypes especially for the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1, respectively.” To make it clear, we also decided to remove the word “gold standard” and mPCR results in Table 2. Moreover, we also add 95% CI value of both methods in line 258 “While the true serotype classifications of the MALDI-TOF-MS and mPCR were not statistically significant (p > 0.05) approximately 60% (95% CI: 51%-70%) and 76% (95% CI: 67%-84%), respectively.” We also discussed more in line 364 “Nevertheless, 95% CI of both methods indicated that the true serotype classification rate of both methods was not statistically different”.

Attachment

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Decision Letter 1

Joseph Banoub

23 Mar 2021

Streptococcus suis serotyp ing by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

PONE-D-21-02524R1

Dear Dr. Nuanualsuwan,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Joseph Banoub, Ph,D., D. Sc.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The authors have answered all the demanded queries of both referees.

This manuscript is now acceptable.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #2: This is my second review . I noticed that answers from authors to my previous comments bring the required elemnts. I have not more comments.

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Reviewer #2: No

Acceptance letter

Joseph Banoub

23 Apr 2021

PONE-D-21-02524R1

Streptococcus suis serotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

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on behalf of

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. The complete list of m/z of S. suis serotyping by MALDI-TOF-MS identified and used in this study.

    (XLSX)

    Click here for additional data file. (18.3KB, xlsx)
    Attachment

    Submitted filename: Response to reviewers.docx

    Click here for additional data file. (20.5KB, docx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

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