SciELO - Scientific Electronic Library Online

vol.49 issue6Plant essential oils against bacteria isolated from fish: an in vitro screening and in vivo efficacy of Lippia origanoidesMultiparasitism in gills of Metynnis lippincottianus from the environmental protection area of Curiaú river, Eastern Amazon author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand




Related links


Ciência Rural

Print version ISSN 0103-8478On-line version ISSN 1678-4596

Cienc. Rural vol.49 no.6 Santa Maria  2019  Epub May 23, 2019 


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

Identificação acurada de cepas atípicas de Staphylococcus chromogenes que coagulam o plasma envolvidas com mastite bovina

Pedro Avellar-Costa1 

Danielle Cabral dos Santos1 

Carla Christine Lange2 

Marinella Silva Laport1 

Maria Aparecida Vasconcelos Paiva Brito2 

Ciro César Rossi1 

Marcia Giambiagi-deMarval1  *

1Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, RJ, Brasil.

2Embrapa Gado de Leite, Juiz de Fora, MG, Brasil..


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.

Key words: Staphylococcus chromogenes; mastitis; MALDI-TOF; tuf; groEL


Nós comparamos o potencial de técnicas rotineiras utilizadas para a identificação de espécies de Staphylococcus, com o objetivo de avaliar a acurácia delas na detecção de 43 isolados de Staphylococcus chromogenes envolvidos com mastite bovina que, apesar de ser uma espécie coagulase-negativa, são capazes de coagular o plasma. Essas cepas poderiam ser erroneamente suspeitas de serem S. aureus e levarem a um tratamento não adequado da doença. MALDI-TOF, PCR-RFLP do gene da chaperonina groEL e sequenciamento do gene do rRNA 16S e do gene do fator de elongação Tu, tuf, foram avaliados. Os resultados dos quatro métodos foram coincidentes para apenas metade das cepas, devido à baixa precisão da PCR-RFLP com groEL (51,2% de acurácia). Apesar de todos os resultados do sequenciamento serem idênticos, a alta precisão dos resultados do MALDI-TOF (97,7% de acurácia, com apenas uma cepa identificada incorretamente) encoraja o uso dessa técnica, pois, não requer preparação laboriosa de amostras, sendo rápida e simples de executar.

Palavras-chave: Staphylococcus chromogenes; mastite; MALDI-TOF; tuf; groEL.

Staphylococcus aureus, the major pathogen from its genus, causes a wide range of clinical infections in human beings and animals (TONG et al., 2015). Discrimination between S. aureus from most staphylococci is primarily made by evaluating its ability to produce the enzyme coagulase, which promotes clotting of plasma. This activity is the foundation of routine identification tests and helps in the differentiation between S. aureus and coagulase-negative staphylococci (CoNS), which were long considered as harmless environmental contaminants, but are emerging as important opportunist pathogens (BECKER et al., 2014). However, our group has recently described Staphylococcus chromogenes strains, isolated from dairy animals with mastitis, that are surprisingly 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-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).

Figure 1 Comparison of results obtained from four methods (MALDI-TOF, groEL-RFLP and sequencing of tuf and 16S rRNA genes) used for the identification of 43 coagulase-negative Staphylococcus strains showing atypical results in rabbit plasma coagulation assays. 

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, and BIOTECON Diagnostics (Germany,

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.


The authors are grateful for the financial support from Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, grants E-26/203.037/2016 and E-26/201.451/2014), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant 305940/2018-0), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Programa de Excelência Acadêmica-Finance Code 001 (CAPES ProEx, grant 23038.002486/2018-26).


ADEYEMI, A. I., et al. Bacterial bloodstream infections in HIV-infected adults attending a Lagos teaching hospital. J Health Popul Nutr, v.28, n.4, p.318-326, 2010. Available from: <Available from: >. Accessed: Feb. 27, 2019. [ Links ]

BACK-BRITO, G. N., et al. Staphylococcus spp., Enterobacteriaceae and Pseudomonadaceae oral isolates from Brazilian HIV-positive patients. Correlation with CD4 cell counts and viral load. Arch Oral Biol, v.56, n.10, p.1041-6, 2011. Available from: <Available from: >. Accessed: Feb. 27, 2019. doi: 10.1016/j.archoralbio.2011.02.016. [ Links ]

BARKEMA, H. W., et al. Invited Review: The role of cow, pathogen, and treatment regimen in the therapeutic success of bovine Staphylococcus aureus mastitis. J Dairy Sci, v.89, n.6, p.1877-95, 2006. Available from: <Available from: >. Accessed: Feb. 27, 2019. doi: 10.3168/jds.S0022-0302(06)72256-1. [ Links ]

BECKER, K., et al. Coagulase-negative staphylococci. Clin Microbiol Rev, v.27, n.4, p.870-926, 2014. Available from: <Available from: >. Accessed: Feb. 27, 2019. doi: 10.1128/CMR.00109-13. [ Links ]

CATANO, J. C., et al. Bacterial contamination of clothes and environmental items in a third-level hospital in Colombia. Interdiscip Perspect Infect Dis, v.2012, p.507640, 2012. Available from: <Available from: >. Accessed: Feb. 27, 2019. doi: 10.1155/2012/507640. [ Links ]

DUBOI, D., et al. Identification of a variety of Staphylococcus species by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol, v.48, n.3, p.941-945, 2010. Available from: <Available from: > Accessed: Apr. 1, 2019. doi: 10.1128/JCM.00413-09. [ Links ]

ROSSI, C.C., et al. The oral microbiota of domestic cats harbors a wide variety of Staphylococcus species with zoonotic potential. Vet Microbiol, v.201, p.136-140, 2017. Available from: <Available from: > Accessed: Apr. 1, 2019. doi: 10.1016/j.vetmic.2017.01.029. [ Links ]

SANTOS, D. C., et al. Staphylococcus chromogenes, a coagulase-negative Staphylococcus species that can clot plasma. J Clin Microbiol, v.54, n.5, p.1372-1375, 2016. Available from: <Available from: > Accessed: Feb. 27, 2019. doi: 10.1128/jcm.03139-15. [ Links ]

SANTOS, O. C., et al. Identification of coagulase-negative staphylococci from bovine mastitis using RFLP-PCR of the groEL gene. Vet Microbiol, v.130, n.1-2, p.134-40. 2008. Available from: <Available from: >. Accessed: Feb. 27, 2019. doi: 10.1016/j.vetmic.2007.12.009. [ Links ]

SILVA, M.B., et al. An evaluation of matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the identification of Staphylococcus pseudintermedius isolates from canine infections. J Vet Diagn Invest, v.27, n.2, p. 231-255, 2015. Available from: <Available from: >. Accessed: Apr. 1, 2019. doi: 10.1177/1040638715573297. [ Links ]

TOMAZI, T., et al. Identification of coagulase-negative staphylococci from bovine intramammary infection by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol, v.52, n.5, p.1658-63. 2014. Available from: <Available from: >. Accessed: Feb. 27, 2019. doi: 10.1128/JCM.03032-13. [ Links ]

TONG, S. Y., et al. Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev, v.28, n.3, p.603-61. 2015. Available from: <Available from: >. Accessed: Feb. 27, 2019: 10.1128/CMR.00134-14. [ Links ]

VANDERHAEGHEN, W., et al. Invited review: effect, persistence, and virulence of coagulase-negative Staphylococcus species associated with ruminant udder health. J Dairy Sci, v.97, n.9, p.5275-93. 2014. Available from: <Available from: >. Accessed: Feb. 27, 2019: 10.3168/jds.2013-7775. [ Links ]

ZEMOURI, C., et al. A scoping review on bio-aerosols in healthcare and the dental environment. PLoS One, v.12, n.5, p.e0178007. 2017. Available from: <Available from: >. Accessed: Feb. 27, 2019. doi: 10.1371/journal.pone.0178007. [ Links ]

ZHENZHEN, D., et al. An alarming presence of methicillin-resistant Staphylococcus aureus in a major hospital in the Henan province, China: A five-year observation. Asian Pac J Health Sci, v.5, n.1, p.111-113. 2018. Accessed: Feb. 27, 2019. doi: 10.21276/apjhs.2018.5.1.27. [ Links ]

ZHU, W., et al. Evaluation of the Biotyper MALDI-TOF MS system for identification of Staphylococcus species. J Microbiol Methods, v.117, p.14-7. 2015. Available from: <Available from: >. Accessed: Feb. 27, 2019. doi: 10.1016/j.mimet.2015.07.014. [ Links ]


Received: February 27, 2019; Accepted: April 10, 2019; Revised: May 06, 2019

E-mail: *Corresponding author


The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.


The authors contributed equally to the manuscript.

Creative Commons License This is an open-access article distributed under the terms of the Creative Commons Attribution License