Accurate identification of atypical Staphylococcus chromogenes plasma-clotting strains causing bovine mastitis. Accurate identification of atypical Staphylococcus chromogenes plasma-clotting strains causing bovine mastitis

: We compared the potential of routine techniques used for the identification of Staphylococcus species, aiming to evaluate their accuracy in the detection of 43 Staphylococcus chromogenes strains isolated from bovine mastitis that, despite being a coagulase-negative species, are able to clot plasma. These strains could be mistakenly suspected to be S. aureus and lead to an unappropriated treatment of the disease. MALDI-TOF, PCR-RFLP of the chaperonine gene groEL, and sequencing of the 16S rRNA and elongation factor Tu gene tuf were employed. Results from the four methods were coincident for only half of the strains because of the low accuracy of the groEL PCR-RFLP (51.2% accuracy). Even though all the sequencing results were identical, the high accuracy of the MALDI-TOF results (97.7% accuracy, with only one strain misidentified) encourage the use of this technique, since it does not require laborious sample preparation, being fast and simple to perform.

capable of clotting plasma, besides being a coagulase-negative species (SANTOS et al., 2016). Among CoNS, Staphylococcus chromogenes is one of the major pathogens involved with mastitis in dairy animals (VANDERHAEGHEN et al., 2014), while S. aureus is the major coagulase-positive pathogen causing the same disease (BARKEMA et al., 2006). For a long time overlooked, S. chromogenes has also been isolated from hospitals and other healthcare facilities (CATANO et al., 2012;ZEMOURI et al., 2017;ZHENZHEN et al., 2018), in addition to being shown colonizing HIV-positive patients (BACK-BRITO et al., 2011), and causing bloodstream infections in patients with AIDS (ADEYEMI et al., 2010). Then, the coagulase- Avellar-Costa et al. positive phenotype of S. chromogenes shown by our group can easily lead to misidentification and inappropriate treatment of the disease caused by them.
Aiming to search for accurate methods to identify strains showing atypical results in the coagulation assays, we estimated the identification potential of four different techniques: Matrix-Assisted Laser Desorption Ionization-Times of Flight Mass Spectometry (MALDI-TOF), PCR-restriction fragment length polymorphism (PCR-RFLP) of the chaperonine gene groEL, and DNA sequencing of the 16S rRNA and elongation factor Tu gene tuf.
The S. chromogenes strains (n=43) studied in our previous research (SANTOS et al., 2016), were first analyzed in triplicate by MALDI-TOF as described before (TOMAZI et al., 2014). Mass spectral data were collected within the m/z range of 2,000 to 20,000, and the data were acquired and analyzed using the FlexControl software 3.3 (Bruker Daltonics, MA, USA). A PCR-RFLP of the groEL gene strategy, standardized by our group to differentiate several relevant Staphylococcus species (SANTOS et al., 2008), was then performed. The identification data obtained were compared to those from the previous DNA sequencing of the 16S rRNA and tuf genes (SANTOS et al., 2016).
Only 23 (51.2%) strains displayed coincident results for all the four methods (Figure 1), indicating strain variation, as previously observed (SANTOS et al., 2016). However, because all results from DNA sequencing were identical, all strains were confirmed as being S. chromogenes. All strains had MALDI-TOF scores≥2.3, indicating reliable identification of the species. The groEL PCR-RFLP was the less accurate (51.2% accuracy) method, as 21 (48.8%) strains were either identified as Staphylococcus caprae (n=20) or undetermined. The MALDI-TOF had 97.7% accuracy, with only one strain misidentified. This result is consistent with the high accuracy of the technique reported for several Staphylococcus species obtained from different origins, such as clinical, food samples and plants (DUBOI et al., 2010), domestic dogs (SILVA et al., 2015) and domestic cats (ROSSI et al., 2017).
For being faster, cost-effective and easier to perform, allowing diagnosis using intact cells or cell extracts, this technique is an emergent identification method (ZHU et al., 2015). The equipment is available in most major university centers and many large hospitals in Brazil and the cost per analysis of each sample is less than fifty cents of dollar. In addition, the MALDI-TOF analysis service is offered by international companies, such as MIDI Labs (USA, http://midilabs.com) and BIOTECON Diagnostics (Germany, http://www.bc-diagnostics.com).
Our results, together with the aforementioned advantages, encourage the use of MALDI-TOF as the identification method for Staphylococcus species isolated from cows with mastitis even for atypical S. chromogenes strains that are able to clot plasma and are circulating in Brazilian farms.