Elsevier

Research in Veterinary Science

Volume 132, October 2020, Pages 426-438
Research in Veterinary Science

Discrimination of major and minor streptococci incriminated in bovine mastitis by MALDI-TOF MS fingerprinting and 16S rRNA gene sequencing

https://doi.org/10.1016/j.rvsc.2020.07.027Get rights and content

Highlights

  • Discrimination of BM-implicated streptococci was facilitated by both 16S rRNA GS and by MALDI-TOF MS fingerprinting

  • MALDI-TOF MS contributed to the discovery of protein biomarkers of streptococci, which is crucial for mastitis control.

  • MALDI-TOF MS also provided more information at the intra-species level for certain streptococci compared to 16S rRNA GS.

  • MALDI-TOF MS can be used efficiently and independently for the diagnosis of BM-implicated Streptococcus spp.

Abstract

The current work investigated the discriminatory potential of MALDI-TOF MS fingerprinting towards most-relevant major (Streptococcus agalactiae, S. dysgalactiae, S. uberis) and minor (S. canis, S. parauberis, S. salivarius, S. equinus and S. gallolyticus) streptococci involved in bovine mastitis (BM), in comparison to 16S rRNA gene sequencing (GS)-based identification. The MALDI-TOF MS-generated spectral fingerprints were recruited for eliciting a detailed proteomic map that demonstrated clear variability for inter- and intra-species-specific biomarkers. Besides, a phyloproteomic dendrogram was evolved and comparatively analyzed against the phylogenetic one obtained from 16S rRNA GS in order to assess the differentiation of streptococci of bovine origin based on variability of protein fingerprints versus the variation of 16S rRNA gene homology. Results showed that the discrimination of BM-implicated streptococci can be obtained by both approaches; however MALDI-TOF MS was superior, achieving more variability at both intra- and sub-species levels. MALDI-TOF MS spectral analytics revealed that Streptococcus spp. exhibited three genus-specific biomarkers (peaks with m/z values at 2112, 4452 and 5955) and all streptococci exhibited spectral variability at both species and subspecies levels. Remarkably, MALDI-TOF MS fingerprinting was found to be at least as robust as 16S rRNA GS-based identification, allowing much cheaper and faster analysis, and additionally exhibiting high reliability for characterization of BM-implicated streptococci, thus proving to be a powerful tool that can be used independently within dairy diagnostics.

Introduction

Mastitis is a serious, easily disseminated and highly multi-factorial complex issue that threatens dairy industry. It results in huge economic losses attributed to reduced milk yield and quality, diminished reproductive efficiency and culling of animals, coupled by increased use of drugs and veterinary services. This disease is mainly attributed to a wide spectrum of microbial species but, in particular, streptococci remain of crucial importance and the percentage of mastitic cases caused by them increased progressively worldwide.

The most common members of Streptococcus spp. that are implicated in mastitis cases include: (1) Streptococcus agalactiae, one of the major contagious mastitis pathogens (Raemy et al., 2013); (2) Streptococcus uberis, the most frequently-observed environmental Streptococcus that can be involved in clinical, subclinical, chronic unresponsive-to-treatment and toxic versions of mastitis (Raemy et al., 2013; Zadoks et al., 2003); and (3) Streptococcus dysgalactiae, which is strongly involved in the multi-etiological clinical entity referred to as “summer mastitis”, that affects dry cows and heifers during summer (Nam et al., 2009; Raemy et al., 2013; Schabauer et al., 2014). Unlike S. agalactiae, studies showed that S. uberis and S. dysgalactiae behave mainly as environmental pathogens and to lesser extent as contagious pathogens (Archer et al., 2017; Nam et al., 2009; Raemy et al., 2013; Riffon et al., 2001; Rossitto et al., 2002; Zadoks et al., 2003). Two subspecies of S. dysgalactiae were isolated from mastitic cases, mainly S. dysgalactiae subsp. dysgalactiae and the less frequent S. dysgalactiae subsp. equisimilis (Lee et al., 2004; Raemy et al., 2013; Rossitto et al., 2002).

Although less common, other streptococcal species can also occasionally cause BM. Among them, Streptococcus canis, a common inhabitant bacterium usually found at skin, respiratory tract and genitals of canines and felines, can cause bovine subclinical mastitis (SCM) (Król et al., 2015; Tikofsky and Zadoks, 2005) for long duration and behave in a contagious manner (Król et al., 2015). Also, Streptococcus gallolyticus subsp. gallolyticus (formerly Streptococcus bovis biotype-1), which commonly inhabits the alimentary tract of various animals and has been isolated from blood and faeces of infected humans (Köhler, 2007), has also been reported as another minor bovine mastitic agent (Facklam, 2002). Further streptococci implicated to a lesser extent in sporadic BM cases are Streptococcus parauberis (Bentley et al., 1993; Facklam, 2002; Riffon et al., 2001), Streptococcus equinus (Lee et al., 2004; Watts, 1989) and Streptococcus salivarius (Nam et al., 2009).

A rapid and accurate typing method for mastitis-causing microbial strains is urgently required in order to detect epidemiological outbreaks of streptococcal infections and provide both appropriate and timely intervention in the control of the infection. Unfortunately, phenotypic and serological assays are labor-intensive and frequently provide unsatisfactory results for certain streptococci due to their phenotypic and biochemical similarities, even if complex analytical approaches are used (Facklam, 2002; Raemy et al., 2013). Thus, no single system based on phenotypic and serological assays suffices till now for the differentiation of this heterogeneous group. Consequently, molecular assays such as PCR targeted to different genes have provided more accurate identification schemes, overcoming identification problems attributed to phenotypic and biochemical similarities and allowing a more robust, highly reproducible and accurate discrimination within diagnostic sectors. However, PCR-based methods are in general not ideally suited for rapid diagnosis and are often time-consuming and require extensive costs (Glazunova et al., 2010; Poyart et al., 1998).

Among the conserved genes that encode rRNA, 16S “Svedberg sedimentation” rRNA genes are considered as a gold standard for microbial identification and taxonomic classification. PCR targeting either partial or whole 16S rRNA gene has been applied for the identification of several Streptococcus spp. of dairy concern (Alnakip et al., 2016; Hassan et al., 2001; Merl et al., 2003; Raemy et al., 2013; Wilson et al., 2019). Also, PCR targeted to genes other than 16S rRNA (i.e.: 23S rRNA, rpoB, gyrA, gyrB, etc.), has been proposed for certain streptococcal species (Chen et al., 2004; Glazunova et al., 2010; Kawata et al., 2004; Raemy et al., 2013), particularly for closely-related isolates, where 16S rRNA GS exhibited a limited discriminatory potential (Itoh et al., 2006; Kawamura et al., 1999).

More recently, as a promising alternative method, Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) fingerprinting, which is based on analysis of the protein composition of microbial cells, “particularly ribosomal proteins”, has been successfully applied for microbial identification and typing. The application of MALDI-TOF MS was initially proposed for the biomedicine sector, and lately has been applied to food (Böhme, et al., 2010; Fernández-No et al., 2012; Quintela-Baluja et al., 2014; Quintela-Baluja et al., 2013), and veterinary (Barreiro et al., 2010; Raemy et al., 2013; Santos et al., 2015; Schabauer et al., 2014) sectors, providing higher diagnostic potential, lesser cost, more rapidity and labor-saving as compared to other traditional phenotypic or PCR-based tools. Additionally, it provides more useful information for inter- and intra-specific analyses than genotypic-based methods (Barreiro et al., 2017; Braga et al., 2018; Moura et al., 2008).

To the best of our knowledge, little information (Barreiro et al., 2010; Barreiro et al., 2018; Barreiro et al., 2017; Braga et al., 2018; Raemy et al., 2013; Schabauer et al., 2014; Wilson et al., 2019) is available as regards the applicability of MALDI-TOF MS for identification purposes of mastitis-implicated streptococci. Unfortunately, these studies only included the major streptococcal species (S. agalactiae, S. dysgalactiae, S. uberis), providing different protocols for cells lysis and/or preparation of protein extracts for MALDI-TOF MS analysis. Additionally, MALDI-TOF MS instrumental and software settings differed between these studies. On one hand, all these reasons resulted in discrepancies in the results provided by different studies, which generated doubts about the identification potential of this technique for streptococci. On the other hand, none of these studies revealed detailed spectral analyses or provided significant differences for data assigned by such spectra to different streptococci, which should be more useful for inter- and intra-species classification. Additionally, none of these studies provided the peaks corresponding to characteristic m/z values for cellular proteins “known as protein fingerprints”, that would allow a differential phyloproteomic analysis that could be compared with the phylogenetic dendrogram obtained by GS of a targeted gene and corresponding to the same strain.

Accordingly, this study aims to investigate the potential of both 16S rRNA GS and MALDI-TOF MS fingerprinting for the identification of BM-implicated streptococci, since the application of the latter approach in routine analysis in the dairy sector is still rare. To the best of our knowledge, the present study represents the first application of MALDI-TOF MS to the identification of nearly all major and minor streptococci, not only for discriminatory purposes, but also for providing the analysis of the spectral fingerprints. Because the rapid diagnosis of streptococci is crucial for mastitis control, especially in case of outbreaks, this study could be of special interest for the dairy industry, particularly for the dairy diagnostic sector. Additionally, it could be of interest for further proteomic and taxonomical investigations in the Streptococcus genus. Furthermore, this study could be valuable for other diagnostic sectors, where minor streptococci can be implicated in several diseases in other animals and/or humans.

Section snippets

Sample collection

Composite milk samples were collected from cows on a commercial dairy farm located in Lugo Province, Galicia region, Spain. Briefly, milk samples were aseptically collected from all quarters of previously culture-positive cows in sterile containers and transported under refrigeration (4 °C) to the laboratory to be immediately submitted to microbial analysis.

Bacterial strains used in this study

Thirty eight streptococcal strains were considered in this study. Twenty one strains were isolated from milk of mastitic cows (Table 1),

Phylogenetic analysis based on 16S rRNA GS

All bacterial strains were subjected to genetic analysis based on an approximately 800-bp fragment of the 16S rRNA gene using a universal 16S rRNA primer pair. The obtained sequences were analyzed and compared against strains from GenBank using the BLAST tool. All strains of Streptococcus spp. and the out-group were successfully identified to the species level, and in the case of twelve strains (CECT 758, USC 13, USC 17, CECT 926, DSM 23147, DSM 6176, ATCC 9542, USC 83, USC 84, DSM 16831, LHICA

Discussion

A major challenge in dairy diagnostic sector, particularly in the case of outbreaks, is to rapidly and precisely identify the mastitis-causative pathogens, preferably in a fast, easy and inexpensive way. This aspect is crucial and a prerequisite for infection control, and for initiating antibiotic treatment and efficient prophylaxes strategies.

Unfortunately, the routine phenotypic, serological and automated biochemical tools do not allow to achieve the unequivocal identification of certain

Conclusion

The discrimination of the most relevant streptococci involved in BM was accomplished by both 16S rRNA GS and by MALDI-TOF MS fingerprinting. Remarkably, MALDI-TOF MS proved to be a valuable and more potent tool for diagnostic purposes targeting in this bacterial group, also being less expensive and time-consuming than 16S rRNA GS. Moreover, MALDI-TOF MS also provided more discriminatory power at the intra-specific level for certain streptococci as compared to 16S rRNA GS. Finally, it can be

Funding

This work was funded by (1) Project No. 25457 funded by Science and Technology Development Fund (STDF), Egypt (2) projects no. 10PXIB261045PR from Xunta de Galicia and AGL2010-19646 and Subprogram INNPACTO 2011/PN037 IPT-2011-1290-010000 from the Spanish Ministry of Science and Innovation.

Declaration of Competing Interest

The authors declare that they have no conflict of interests. None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper.

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