Veterinary Microbiology 176 (2015) 382–388
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Short communication
Species-level identification of staphylococci isolated from bovine mastitis in Brazil using partial 16S rRNA sequencing Carla C. Lange a,*, Maria A.V.P. Brito a, Daniele R.L. Reis a, Marco A. Machado a, Alessandro S. Guimara˜es a, Ana L.S. Azevedo a, E´rica B. Salles b, Mariana C.T. Alvim c, Fabiana S. Silva d, Igor R. Meurer c a
Embrapa Gado de Leite, Rua Eugeˆnio do Nascimento 610, 36038-330 Juiz de Fora, Minas Gerais, Brazil Rua Quintino Bocaiu´va 131/402, 31255-550 Belo Horizonte, Minas Gerais, Brazil Universidade Federal de Juiz de Fora, Rua Jose´ Lourenc¸o Kelmer, 36036-330 Juiz de Fora, Minas Gerais, Brazil d Centro de Ensino Superior de Juiz de Fora, Av. Luz Interior 345, 36030-776 Juiz de Fora, Minas Gerais, Brazil b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 December 2014 Received in revised form 29 January 2015 Accepted 31 January 2015
Staphylococci isolated from bovine milk and not classified as Staphylococcus aureus represent a heterogeneous group of microorganisms that are frequently associated with bovine mastitis. The identification of these microorganisms is important, although it is difficult and relatively costly. Genotypic methods add precision in the identification of Staphylococcus species. In the present study, partial 16S rRNA sequencing was used for the species identification of coagulase-positive and coagulase-negative staphylococci isolated from bovine mastitis. Two hundred and two (95%) of the 213 isolates were successfully identified at the species level. The assigning of an isolate to a particular species was based on 99% identity with 16S rRNA sequences deposited in GenBank. The identified isolates belonged to 13 different Staphylococcus species; Staphylococcus chromogenes, S. aureus and Staphylococcus epidermidis were the most frequently identified species. Eight isolates could not be assigned to a single species, as the obtained sequences showed 99% or 100% similarity to sequences from two or three different Staphylococcus species. The relatedness of these isolates with the other isolates and reference strains was visualized using a cladogram. In conclusion, 16S rRNA sequencing was an objective and accurate method for the proper identification of Staphylococcus species isolated from bovine mastitis. Additional target genes could be used in non-conclusive cases for the species-level identification of these microorganisms. ß 2015 Elsevier B.V. All rights reserved.
Keywords: S. aureus S. chromogenes S. agnetis S. devriesei Coagulase-negative staphylococci Tube coagulase test
1. Introduction Species of the genus Staphylococcus are among the agents most frequently isolated from cases of bovine mastitis worldwide. Staphylococcus aureus represents a
* Corresponding author. Tel.: +55 3233117452; fax: +55 3233117401. E-mail address: [email protected] (C.C. Lange). http://dx.doi.org/10.1016/j.vetmic.2015.01.024 0378-1135/ß 2015 Elsevier B.V. All rights reserved.
major agent of contagious bovine mastitis (Fox and Gay, 1993). On farms that have successfully controlled mastitis caused by S. aureus and Streptococcus agalactiae, opportunistic bacteria, such as coagulase-negative staphylococci (CoNS), are frequently associated with bovine mastitis (Ruegg, 2009). For practical purposes of mastitis control, the recommendation is to separate the genus Staphylococcus into S. aureus and the heterogeneous group CoNS. Currently, the Staphylococcus genus comprises 47 species, including 24 subspecies (LPSN, 2014).
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The identification of Staphylococcus species other than S. aureus is not routinely performed in veterinary diagnostic laboratories. However, the identification of these species might be important for epidemiological investigations, the assessment of the pathogenic significance of these organisms and the development of specific management practices to prevent mastitis (Sampimon et al., 2009). Although important, the identification of CoNS is difficult and relatively costly. Phenotypic and genotypic tests have been employed for the identification of Staphylococcus species isolated from bovine mastitis. The use of genotypic methods adds precision in the identification of these species and molecular tests, such as ribotyping and sequencing, have been used to identification of CoNS (Taponen et al., 2008; Capurro et al., 2009; Sampimon et al., 2009). The most common broad-range DNA target sequence to identify bacteria remains the first 500 bp of the 16S rRNA gene. The 16S rRNA gene serves as an excellent target for most staphylococci. For some coagulase-negative staphylococci, separation between species can be difficult because of lack of sufficient heterogeneity within the 16S rRNA gene (CLSI, 2008). In Brazil, studies have been conducted to identify CoNS isolated from bovine mastitis, and few of these studies (Santos et al., 2008; Lange et al., 2011) have used molecular techniques. Thus, the aim of the present study was to identify coagulase-positive and coagulase-negative Staphylococcus species isolated from bovine mastitis in Brazil using partial 16S rRNA gene sequencing. 2. Materials and methods 2.1. Bacterial isolates and control strains A total of 213 staphylococcal isolates, including 27 coagulase-positive and 186 coagulase-negative Staphylococcus spp., were included in this study. The isolates were randomly selected from a collection of bovine mastitis isolates obtained from the Dairy Cattle Research Center (Embrapa), Juiz de Fora, Brazil. These strains were isolated from Gir Holstein crossbred (ca. 80%) and purebred Holstein (approximately 20%) herds located in south and southeast Brazil, during an 11-year period (1996–2007). The isolates were identified through Gram staining and catalase, coagulase, and Voges–Proskauer (VP) tests, according to the National Mastitis Council (2004). Coagulase-positive and VP positive isolates were classified as S. aureus; coagulase-positive and VP negative isolates were classified as coagulase-positive staphylococci and coagulase-negative isolates were classified as CoNS. The samples were stored at 80 8C in skim milk (Difco, Sparks, MD, USA) containing 10% glycerol (Cromoline, Diadema, SP, Brazil). The following reference or type strains were included in the study: S. aureus ATCC 51651, Staphylococcus capitis ATCC 35661, Staphylococcus carnosus TM 300, Staphylococcus epidermidis ATCC 12228, Staphylococcus gallinarum ATCC 700401, Staphylococcus haemolyticus ATCC 29970T,
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Staphylococcus hyicusATCC 11249T, Staphylococcus intermedius ATCC 29663T, Staphylococcus lentus ATCC 700403, Staphylococcus lugdunensis ATCC 49576, Staphylococcus saprophyticus ATCC 15305T, Staphylococcus sciuri ATCC 29061, Staphylococcus simulans ATCC 27851, Staphylococcus warneri ATCC 49454 and Staphylococcus xylosus ATCC 29971T. These samples were stored in the same way as the clinical isolates. The isolates identified as S. aureus through 16S rRNA sequencing were further analyzed using the coagulase tube test with rabbit plasma from two different sources (Coagu-Plasma, Laborclin, Pinhais, PR, Brazil and Coagulase Test, Sigma-Aldrich, Buchs, Switzerland) and the latex agglutination test (PastorexTM Staph-Plus, BioRad Laboratories, Redmond, WA, USA), performed according to the manufacturer’s instructions. 2.2. Extraction of genomic DNA and PCR amplification Whole cell DNA was extracted from the isolates and the 15 reference/type strains. After overnight growth in Brain Hearth Infusion medium (BHI, Oxoid, Basingstoke, Hampshire, England), bacterial DNA was extracted from the pure cultures according to Rosec and Gigaud (2002). The extracted DNA was frozen at 20 8C until further use for PCR. The PCR reactions were performed on a GeneAmp1 PCR System 9700 thermocycler (Applied Biosystems, Foster City, CA). For 16S rRNA sequencing, forward (50 AGAGTTTGATCCTGGCTCAG-30 ) and reverse (50 -GTATTACCGCGGCTGCTG-30 ) primers were used to amplify a 536 bp product of the 16S rRNA gene. The PCR mixtures contained 45 mL PCR Master Mix [10 (100 mM Tris–HCl pH 8.8 and 500 mM KCl), 1.7 mM MgCl2, 0.2 mM dNTPs and 1.5 U of Taq DNA polymerase] (Ludwig Biotech, Alvorada, RS, Brazil), 2 mL of each primer (10 pmol/mL) and 1 mL of target DNA (100 ng/mL). The PCR cycling conditions included an initial denaturation step at 95 8C for 5 min, followed by 35 cycles at 95 8C for 30 s, 55 8C for 30 s, and 74 8C for 2 min, with a final extension step at 74 8C for 5 min. The PCR products were purified using the IllustraTM GFXTM PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK), according to the manufacturer’s instructions. The isolates identified as S. aureus through 16S rRNA sequencing were used in subsequent PCR analyses for the amplification of coa and femA genes. The primers COAG2 50 -CGAGACCAAGATTCAACAAG-30 and COAG3 50 -AAAGAAAACCACTCACAT-30 (Goh et al., 1992) were used for coa gene amplification and GFEMAR-1 50 -AAAAAAGCACATAACAAGCG-30 and GFEMAR-2 50 -GATAAAGAAGAAACCAGCAG-30 (Mehrotra et al., 2000) were used for femA gene amplification. The cycling conditions for coa PCR included an initial denaturation step at 94 8C for 2 min, followed by 30 cycles, at 94 8C for 30 s, 55 8C for 2 min, and 72 8C for 4 min, with a final extension step at 72 8C for 7 min. The conditions for femA PCR included a denaturation step at 94 8C for 4 min, followed by 5 cycles of 94 8C for 45 s, 56 8C for 45 s and 72 8C for 45 s, 20 cycles of 94 8C for 45 s, 52 8C for 45 s and 72 8C for 45 s, and a final extension step at 72 8C for 5 min.
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2.3. 16S rRNA sequencing A total of 200 ng of purified PCR DNA was used as a template in sequencing reactions performed using the DYEnamicTM ET Dye Terminator Cycle Sequencing Kit (GE Healthcare, Buckinghamshire, UK) and 0.5 mM of the same primer pairs used in the PCR amplification. The sequencing reactions were ethanol precipitated and loaded onto the MegaBACE 1000 DNA sequencer (GE Healthcare, Buckinghamshire, UK) at 9 kV for 180 min. Both forward and reverse sequencing reactions were performed with two sequencing reads per sample. The sequence pairs were edited and assembled using DNA Baser Sequence Assembler v3.5.4 (Heracle BioSoft SRL, www.DnaBaser.com), and the contigs were blasted against the Staphylococcus spp. nucleotide database of the National Center for Biotechnology Information (NCBI) available at http://www.ncbi.nlm. nih.gov/BLAST.
Table 1 Identification of the reference/type strains in the present study through partial 16S rRNA sequencing. Reference/type strain
16S rRNA sequencing result
% Sequence similaritya
S. aureus 51651 S. capitis 35661
S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S.
100 99
S. S. S. S. S. S. S. S. S. S. S. S. S.
carnosus TM 300 epidermidis 12228 gallinarum 700401 haemolyticus 29970T hyicus 11249T intermedius 29663T lentus 700403 lugdunensis 49576 saprophyticus 15305T sciuri 29061 simulans 27851 warneri 49454 xylosus 29971T
a
Similarity compared with sequences deposited in GenBank.
2.4. Cluster analysis A cladogram, based on partial 16S rRNA sequences, was generated using the MEGA 6.06 program (Tamura et al., 2013) based on the Kimura-2-parameter model. The sequences were aligned in ClustalW, and phylogenetic trees were constructed using the Maximum Likelihood method. The robustness of the tree was assessed with 1000 bootstrap replicates. 3. Results A total of 213 Staphylococcus isolates recovered from bovine mastitis and 15 Staphylococcus reference or type strains were subjected to partial 16S rRNA gene sequencing. All PCR products generated for sequence analysis showed the expected size, i.e., 536 bp. The 16S rRNA sequences were compared with sequences deposited in GenBank. Sequence similarity values greater than 99% (99%) were considered reliable, according to the CLSI (2008). In all but three cases, the sequence similarities with GenBank sequences ranged from 99% to 100%. The three isolates were identified as S. aureus, with 98% of sequence similarity, and were considered unidentified. Most of the identified reference strains were consistent with the ATCC nomenclature (Table 1). The exceptions included S. capitis ATCC 35661, named S. capitis or S. caprae or S. epidermidis, with 99% sequence similarity with strains deposited in GenBank; and S. warneri ATCC 49454 and S. xylosus ATCC 29971T, named S. warneri or Staphylococcus pasteuri and S. xylosus or S. saprophyticus, respectively. In both cases, the percentage of sequence similarity was 100%. Two hundred and thirteen Staphylococcus isolates were identified as Staphylococcus chromogenes (38.5%), S. aureus (19.2%), S. epidermidis (13.1%), S. haemolyticus (6.1%), S. sciuri (5.2%), S. hyicus (4.7%), S. simulans (3.7%), Staphylococcus agnetis (1.8%), Staphylococcus devriesei (0.5%), Staphylococcus auricularis (0.5%), S. intermedius (0.5%), Staphylococcus succinus (0.5%), S. pasteuri (0.5%) (Table 2). Three isolates (1.4%) were not identified and in eight cases, it was not possible to discriminate among
aureus capitis/S. caprae/ epidermidis carnosus epidermidis gallinarum haemolyticus hyicus intermedius lentus lugdunensis saprophyticus sciuri simulans pasteuri/S. warneri xylosus/S. saprophyticus
99 100 100 99 99 100 100 100 100 100 100 100 100
Table 2 Species-level identification of 213 Staphylococcus isolates from bovine mastitis through partial 16S rRNA sequencing. Species (% similaritya) S. chromogenes (99–100%) S. aureus (100%) S. epidermidis (99–100%) S. haemolyticus (99–100%) S. sciuri (100%) S. hyicus (99–100%) S. simulans (99–100%) S. agnetis (99–100%) S. devriesei (100%) S. auricularis (100%) S. intermedius (100%) S. succinus (100%) S. pasteuri (100%) S. hyicus/S. caprae/S. epidermidis (100%) S. xylosus/S. saprophyticus (100%) S. devriesei/S. haemolyticus (99%) S. warneri/S. pasteuri (100%) S. haemolyticus/S. warneri/S. auricularis (99%) Unidentified Total a
No. of isolates % 82 41 28 13 11 10 8 4 1 1 1 1 1 1 4 1 1 1 3 213
38.5% 19.2% 13.1% 6.1% 5.2% 4.7% 3.7% 1.8% 0.5% 0.5% 0.5% 0.5% 0.5% 0.5% 1.8% 0.5% 0.5% 0.5% 1.4% 100%
Similarity compared with sequences deposited in GenBank.
S. hyicus, S. caprae and S. epidermidis (1 isolate), S. xylosus and S. saprophyticus (4 isolates), S. devriesei and S. haemolyticus (1 isolate), S. warneri and S. pasteuri (1 isolate) and S. haemolyticus, S. warneri and S. auricularis (1 isolate). The partial 16S rRNA sequences from 20 isolates, representing the 13 species identified in the present study, were deposited in GenBank under accession numbers KP033211 to KPO33230. S. chromogenes was the coagulase negative species most frequently isolated in the present study, followed by S. epidermidis and S. haemolyticus. Although only 27 coagulase-positive isolates were included in the present study, 41 isolates were identified as S. aureus (coagulasepositive species), ten isolates were identified as S. hyicus (coagulase-variable species), four isolates were identified as
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S. agnetis (coagulase-variable species) and one isolate was identified as S. intermedius (coagulase-positive species). To investigate the unexpected large number of isolates identified as S. aureus, the 41 isolates identified as S. aureus were subjected to the coagulase tube test, using rabbit plasma from two different sources, Voges–Proskauer and latex agglutination tests, and the PCR amplification of femA and coa genes (Table 3). The 41 isolates identified as S. aureus through 16S rRNA sequencing showed 100% similarity with sequences deposited in GenBank. Although six isolates showed negative results in the tube coagulase test, both femA and coa genes were positively amplified through PCR. All 41 S. aureus isolates showed a positive result in the latex agglutination test. Three isolates that showed 98% of sequence similarity to S. aureus and were considered unidentified, showed negative results in coagulase and latex agglutination tests, and did not amplify the femA and coa genes (data not shown). To show the relatedness of the isolates, a cladogram, using the sequences obtained from the 213 clinical isolates and 15 reference/type strains, was constructed (Fig. 1). The analysis of the cladogram revealed that the isolates clustered into two major groups. Most isolates identified as S. aureus, S. epidermidis, S. haemolyticus and S. hyicus clustered around the reference or type strain of the species. All isolates identified as S. simulans and S. sciuri clustered together S. simulans and S. sciuri reference strains, respectively. Eight isolates identified as S. chromogenes clustered together separately from the other 74 isolates of this species. The eight isolates that could not definitively be identified through partial 16S rRNA sequencing and comparing with sequences deposited in GenBank were grouped closely with four ATCC reference/type strains in the cladogram. The isolate identified as S. hyicus/S. caprae/ S. epidermidis grouped closely with strain S. capitis ATCC 35661; similarly, the isolate identified as S. warneri/S. pasteuri grouped closely with reference strain S. warneri ATCC 49454. One isolate identified as S. haemolyticus/S. warneri/S. auricularis grouped closely with strain S. haemolyticus ATCC 29970T and the eleven isolates identified as S. haemolyticus. The isolate identified as S. devriesei/ S. haemolyticus also grouped with the strain S. haemolyticus ATCC 29970T. The four isolates identified as S. xylosus/S. saprophyticus grouped together the strain S. xylosus ATCC 29971T. 4. Discussion The accurate species identification of S. aureus and other staphylococcal species that cause bovine mastitis is
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important to determine the host-pathogen relationships involved in the disease caused by this group of microorganisms. Various DNA-based methods for the identification of Staphylococcus species have been developed, and the high sensitivity and specificity of these techniques provide an alternative method for the accurate identification and classification of Staphylococcus species. In the present study, we used partial 16S rRNA gene sequencing to identify coagulase-positive and coagulase-negative Staphylococcus spp. isolates of bovine mastitis at the species level. All type and reference strains were correctly identified. However, in three cases, S. capitis 35661, S. warneri 49454 and S. xylosus 29971T, the fragment sequenced showed 99% or 100% similarity with the sequences of other species. Ghebremedhin et al. (2008) reported that some Staphylococcus taxa have the same 16S rRNA gene sequences in variable regions, including S. capitis subsp. urealyticus and S. caprae. According to the CLSI (2008), 16S rRNA provides a poor separation between S. capitis, S. caprae and S. epidermidis, as S. warneri shares approximately 98.7% identity with S. pasteuri and S. saprophyticus and S. xylosus share approximately 100% identity. The differentiation among these species is possible using alternative DNA targets, such as dnaJ, sodA, tuf and rpoB genes, as distinct phylogenetic clusters were observed within these genes targets, resulting from heterogeneity within species (CLSI, 2008). Using the partial 16S rRNA gene sequencing, we obtained the definitive species-level identification of 202 (95%) of the 213 isolates examined. Thirteen different staphylococcal species were identified, including the two recently described species, S. devriesei (Supre´ et al., 2010) and S. agnetis (Taponen et al., 2012). S. chromogenes, S. epidermidis and S. haemolyticus were the most frequently observed coagulase-negative species. These results are consistent with those reported by Taponen et al. (2008), Capurro et al. (2009) and Sampimon et al. (2009), who identified CoNS from bovine mastitis using genotypic methods, and cited S. chromogenes, S. simulans, and S. epidermidis as the most common isolated species. In addition to three unidentified isolates, we were not able to definitively identify eight further isolates, as the obtained sequences showed 99% or 100% similarity to sequences from two or three different Staphylococcus species. These eight isolates grouped closely or together with reference/type strains in the cladogram, suggesting that these bacteria might belong to the species of the reference/type strains they grouped. Hellmark et al. (2009) also described inconclusive identification of the species
Table 3 Phenotypic and genotypic characteristics of the 41 isolates identified as S. aureus through partial sequencing of the 16S rRNA gene. No. of isolates 30 2 3 6 a b
Tubea coagulase 1
Tube coagulase 2
VPb
Latex agglutination
femA PCR
coa PCR
+ + – –
+ + + –
+ + +
+ + + +
+ + + +
+ + + +
Tube coagulase 1 and 2: tube coagulase tests performed using lyophilized rabbit plasma from two different sources. VP: Voges–Proskauer test.
% Similarity in sequencing 100% 100% 100% 100%
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Fig. 1. Cluster analysis of the partial 16S rRNA sequences from 213 Staphylococcus spp. isolated from bovine mastitis and 15 Staphylococcus reference/type strains. The bootstrap values of 1000 repeated trees are shown at major branches.
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S. intermedius, S. delphini, S. pseudointermedius and S. schleiferi using 16S rRNA sequencing, probably because of the similarity among these species. All S. aureus isolates identified in the present study showed 100% similarity to sequences deposited in GenBank. Six S. aureus isolates did not clot rabbit plasma, but showed positive results in Voges–Proskauer and latex agglutination tests and amplified femA and coa genes. S. aureus strains isolated from bovine mastitis that failed to coagulate rabbit plasma have been previously described (Vieira-Da-Mota et al., 2001; Capurro et al., 2009; Akineden et al., 2011). Vieira-Da-Mota et al. (2001) reported S. aureus strains isolated from bovine mastitis that were negative in the tube coagulase test, but showed a positive result in the PCR amplification of the coa gene. Capurro et al. (2009) identified two coagulase-negative S. aureus strains isolated from bovine mastitis through tuf gene sequencing and PCR amplification of the nuc gene. Akineden et al. (2011) investigated two coagulasenegative variants of S. aureus isolated from the milk samples of a dairy cow diagnosed with subclinical mastitis using 16S rRNA sequencing and RT-PCR of coa, clfA and nuc genes. However, the reason to the lack of clotting ability in vitro from these strains remains unclear and need further investigation. It is likely that coagulase-negative S. aureus occur with low frequency in strains isolated from bovine mastitis and can be misidentified as CoNS in routine mastitis diagnoses. Therefore, mastitis laboratories and veterinarians should consider that coagulase-negative S. aureus can occur, and the use of additional molecular tests, that are independent of gene expression, has been recommended for the identification of S. aureus from bovine mastitis. The partial 16S rRNA sequencing differentiated 95% of the isolates tested for species identification in the present study. The identification rate of 95% obtained in this study was higher than those reported by Mellmann et al. (2006) and Shin et al. (2011). Using 16S rRNA sequencing to identify CoNS from human origin, these authors reported identification rates of 91% and 84%, respectively. Eleven isolates could not be definitively identified using partial 16S rRNA sequencing. According to the CLSI (2008), the 16S rRNA gene is an excellent target for most staphylococcal species, although discrimination among species can be difficult due to the lack of sufficient heterogeneity within the 16S rRNA gene. Thus, additional DNA targets have been recommended for the unquestionable resolution of staphylococcal species. According to Woo et al. (2009), the accuracy of identification of bacteria using 16S rRNA gene sequencing is further limited by the species included in the database, as bacterial species that are not included in the database would never be determined as the identity of an isolate. Heikens et al. (2005) proposed that a drawback of sequence-based genotypic methods might be the low quality of the sequences deposited in data banks. According to the same authors, genotyping identification based on 16S rRNA sequences has limited discriminating power for closely related Staphylococcus species, and these authors recommended the tuf gene sequencing for the identification of human CoNS. Similarly, Mellmann et al. (2006)
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compared partial 16S rRNA sequencing with partial rpoB gene sequencing to identify human staphylococcal strains and concluded that rpoB sequencing was superior to partial 16S rRNA identification in these strains. Shin et al. (2011) concluded that the tuf gene sequencing had an excellent identification capacity, but relatively few type strains sequences for tuf gene, such as for S. caprae and S. pasteuri, are available in public databases. We concluded that 16S rRNA sequencing was a powerful tool for the identification of Staphylococcus isolated from bovine mastitis, as this technique was successful in 95% of the cases tested. Nevertheless, inconclusive results might occur, requiring the sequencing of additional target genes to properly identify this group of microorganisms. Conflict of interest statement None. Acknowledgments This work was supported through funding from the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas Gerais (Fapemig APQ-00165-11) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). E.B. Salles received a scholarship from Microvet, Vic¸osa, Minas Gerais. References Akineden, O., Hassan, A.A., Schneider, E., Usleber, E., 2011. A coagulasenegative variant of Staphylococcus aureus from bovine mastitis milk. J. Dairy Res. 78, 38–42. Capurro, A., Artursson, K., Persson Waller, K., Bengtsson, B., EricssonUnnerstad, H., Aspa´n, A., 2009. Comparison of a commercialized phenotyping system, antimicrobial susceptibility testing, and a tuf gene sequence-based genotyping for species-level identification of coagulase-negative staphylococci isolated from cases of bovine mastitis. Vet. Microbiol. 134, 327–333. CLSI, 2008. Interpretative Criteria for Identification of Bacteria and Fungi by DNA Target Sequencing; Approved Guideline. CLSI document MM18-A Clinical and Laboratory Standards Institute, Wayne, PA. Fox, I.K., Gay, J.M., 1993. Contagious mastitis. Vet. Clin. North Am. Food Anim. Pract. 9, 475–487. Ghebremedhin, B., Layer, F., Ko¨nig, W., Ko¨nig, B., 2008. Genetic classification and distinguishing of Staphylococcus species based on different partial gap, 16S rRNA, hsp60, rpoB, sodA, and tuf gene sequences. J. Clin. Microbiol. 46 (3) 1019–1025. Goh, S.H., Byrne, S.Z., Zhang, J.L., Chow, A.W., 1992. Molecular typing of Staphylococcus aureus on the basis of coagulase gene polymorphisms. J. Clin. Microbiol. 30, 1642–1645. Heikens, E., Fleer, A., Paauw, A., Florijn, A., Fluit, A.C., 2005. Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci. J. Clin. Microbiol. 43 (5) 2286–2290. Hellmark, B., So¨derquist, B., Unemo, M., 2009. Simultaneous species identification and detection of rifampicin resistance in staphylococci by sequencing of the rpoB gene. Eur. J. Clin. Microbiol. Infect. Dis. 28, 183–190. Lange, C.C., Brito, M.A.V.P., Brito, J.R.F., Arcuri, E.F., Souza, G.N., Machado, M.A., Domingues, R., Salimena, A.P.S., 2011. Uso de PCR e sequenciamento do rDNA 16S para identificac¸a˜o de bacte´rias do geˆnero Staphylococcus isoladas de mastite bovina. Pesq. Vet. Bras. 31, 36–40. LPSN—List of prokaryotic names with standing in nomenclature, 2014. Available in: http://www.bacterio.net/-allnamessz.html. Accessed on: 20 January 2015. Mehrotra, M., Wang, G., Johnson, W.M., 2000. Multiplex PCR for the detection of genes for Staphylococcus aureus enterotoxins, exfoliative
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using 16S ribosomal RNA, tuf and sodA gene sequencing. Peritoneal Dialysis Int. 31, 340–346. Supre´, K., De Vliegher, S., Cleenwerck, I., Engelbeen, K., Van Trappen, S., Piepers, S., Sampimon, O.C., Zadoks, R.N., De Vos, P., Haesebrouck, F., 2010. Staphylococcus devriesei sp. nov., isolated from teat apices and milk of dairy cows. Int. J. Syst. Evol. Microbiol. 60, 2739–2744. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Taponen, S., Bjo¨rkroth, J., Pyo¨ra¨la¨, S., 2008. Coagulase-negative staphylococci isolated from bovine extramammary sites and intramammary infections in a single dairy herd. J. Dairy Res. 75, 422–429. Taponen, S., Supre´, K., Piessens, V., Van Coillie, E., De Vliegher, S., Koort, J.M.K., 2012. Staphylococcus agnetis sp. nov., a coagulase-variable species from bovine subclinical and mild clinical mastitis. Int. J. Syst. Evol. Microbiol. 62, 61–65. Vieira-Da-Mota, O., Folly, M.M., Sakyiama, C.C.H., 2001. Detection of different Staphylococcus aureus strains in bovine milk from subclinical mastitis using PCR and routine techniques. Braz. J. Microbiol. 32 (1) 27–31. Woo, P.C.Y., Teng, J.L.L., Wu, J.J.L., Leung, F.P.S., Tse, H., Fung, A.M.Y., Lau, S.K.P., Yuen, K., 2009. Guidelines for interpretation of 16S rRNA gene sequence-based results for identification of medically important aerobic Gram-positive bacteria. J. Med. Microbiol. 58, 1030–1036.
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Short communication
Species-level identification of staphylococci isolated from bovine mastitis in Brazil using partial 16S rRNA sequencing Carla C. Lange a,*, Maria A.V.P. Brito a, Daniele R.L. Reis a, Marco A. Machado a, Alessandro S. Guimara˜es a, Ana L.S. Azevedo a, E´rica B. Salles b, Mariana C.T. Alvim c, Fabiana S. Silva d, Igor R. Meurer c a
Embrapa Gado de Leite, Rua Eugeˆnio do Nascimento 610, 36038-330 Juiz de Fora, Minas Gerais, Brazil Rua Quintino Bocaiu´va 131/402, 31255-550 Belo Horizonte, Minas Gerais, Brazil Universidade Federal de Juiz de Fora, Rua Jose´ Lourenc¸o Kelmer, 36036-330 Juiz de Fora, Minas Gerais, Brazil d Centro de Ensino Superior de Juiz de Fora, Av. Luz Interior 345, 36030-776 Juiz de Fora, Minas Gerais, Brazil b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 December 2014 Received in revised form 29 January 2015 Accepted 31 January 2015
Staphylococci isolated from bovine milk and not classified as Staphylococcus aureus represent a heterogeneous group of microorganisms that are frequently associated with bovine mastitis. The identification of these microorganisms is important, although it is difficult and relatively costly. Genotypic methods add precision in the identification of Staphylococcus species. In the present study, partial 16S rRNA sequencing was used for the species identification of coagulase-positive and coagulase-negative staphylococci isolated from bovine mastitis. Two hundred and two (95%) of the 213 isolates were successfully identified at the species level. The assigning of an isolate to a particular species was based on 99% identity with 16S rRNA sequences deposited in GenBank. The identified isolates belonged to 13 different Staphylococcus species; Staphylococcus chromogenes, S. aureus and Staphylococcus epidermidis were the most frequently identified species. Eight isolates could not be assigned to a single species, as the obtained sequences showed 99% or 100% similarity to sequences from two or three different Staphylococcus species. The relatedness of these isolates with the other isolates and reference strains was visualized using a cladogram. In conclusion, 16S rRNA sequencing was an objective and accurate method for the proper identification of Staphylococcus species isolated from bovine mastitis. Additional target genes could be used in non-conclusive cases for the species-level identification of these microorganisms. ß 2015 Elsevier B.V. All rights reserved.
Keywords: S. aureus S. chromogenes S. agnetis S. devriesei Coagulase-negative staphylococci Tube coagulase test
1. Introduction Species of the genus Staphylococcus are among the agents most frequently isolated from cases of bovine mastitis worldwide. Staphylococcus aureus represents a
* Corresponding author. Tel.: +55 3233117452; fax: +55 3233117401. E-mail address: [email protected] (C.C. Lange). http://dx.doi.org/10.1016/j.vetmic.2015.01.024 0378-1135/ß 2015 Elsevier B.V. All rights reserved.
major agent of contagious bovine mastitis (Fox and Gay, 1993). On farms that have successfully controlled mastitis caused by S. aureus and Streptococcus agalactiae, opportunistic bacteria, such as coagulase-negative staphylococci (CoNS), are frequently associated with bovine mastitis (Ruegg, 2009). For practical purposes of mastitis control, the recommendation is to separate the genus Staphylococcus into S. aureus and the heterogeneous group CoNS. Currently, the Staphylococcus genus comprises 47 species, including 24 subspecies (LPSN, 2014).
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The identification of Staphylococcus species other than S. aureus is not routinely performed in veterinary diagnostic laboratories. However, the identification of these species might be important for epidemiological investigations, the assessment of the pathogenic significance of these organisms and the development of specific management practices to prevent mastitis (Sampimon et al., 2009). Although important, the identification of CoNS is difficult and relatively costly. Phenotypic and genotypic tests have been employed for the identification of Staphylococcus species isolated from bovine mastitis. The use of genotypic methods adds precision in the identification of these species and molecular tests, such as ribotyping and sequencing, have been used to identification of CoNS (Taponen et al., 2008; Capurro et al., 2009; Sampimon et al., 2009). The most common broad-range DNA target sequence to identify bacteria remains the first 500 bp of the 16S rRNA gene. The 16S rRNA gene serves as an excellent target for most staphylococci. For some coagulase-negative staphylococci, separation between species can be difficult because of lack of sufficient heterogeneity within the 16S rRNA gene (CLSI, 2008). In Brazil, studies have been conducted to identify CoNS isolated from bovine mastitis, and few of these studies (Santos et al., 2008; Lange et al., 2011) have used molecular techniques. Thus, the aim of the present study was to identify coagulase-positive and coagulase-negative Staphylococcus species isolated from bovine mastitis in Brazil using partial 16S rRNA gene sequencing. 2. Materials and methods 2.1. Bacterial isolates and control strains A total of 213 staphylococcal isolates, including 27 coagulase-positive and 186 coagulase-negative Staphylococcus spp., were included in this study. The isolates were randomly selected from a collection of bovine mastitis isolates obtained from the Dairy Cattle Research Center (Embrapa), Juiz de Fora, Brazil. These strains were isolated from Gir Holstein crossbred (ca. 80%) and purebred Holstein (approximately 20%) herds located in south and southeast Brazil, during an 11-year period (1996–2007). The isolates were identified through Gram staining and catalase, coagulase, and Voges–Proskauer (VP) tests, according to the National Mastitis Council (2004). Coagulase-positive and VP positive isolates were classified as S. aureus; coagulase-positive and VP negative isolates were classified as coagulase-positive staphylococci and coagulase-negative isolates were classified as CoNS. The samples were stored at 80 8C in skim milk (Difco, Sparks, MD, USA) containing 10% glycerol (Cromoline, Diadema, SP, Brazil). The following reference or type strains were included in the study: S. aureus ATCC 51651, Staphylococcus capitis ATCC 35661, Staphylococcus carnosus TM 300, Staphylococcus epidermidis ATCC 12228, Staphylococcus gallinarum ATCC 700401, Staphylococcus haemolyticus ATCC 29970T,
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Staphylococcus hyicusATCC 11249T, Staphylococcus intermedius ATCC 29663T, Staphylococcus lentus ATCC 700403, Staphylococcus lugdunensis ATCC 49576, Staphylococcus saprophyticus ATCC 15305T, Staphylococcus sciuri ATCC 29061, Staphylococcus simulans ATCC 27851, Staphylococcus warneri ATCC 49454 and Staphylococcus xylosus ATCC 29971T. These samples were stored in the same way as the clinical isolates. The isolates identified as S. aureus through 16S rRNA sequencing were further analyzed using the coagulase tube test with rabbit plasma from two different sources (Coagu-Plasma, Laborclin, Pinhais, PR, Brazil and Coagulase Test, Sigma-Aldrich, Buchs, Switzerland) and the latex agglutination test (PastorexTM Staph-Plus, BioRad Laboratories, Redmond, WA, USA), performed according to the manufacturer’s instructions. 2.2. Extraction of genomic DNA and PCR amplification Whole cell DNA was extracted from the isolates and the 15 reference/type strains. After overnight growth in Brain Hearth Infusion medium (BHI, Oxoid, Basingstoke, Hampshire, England), bacterial DNA was extracted from the pure cultures according to Rosec and Gigaud (2002). The extracted DNA was frozen at 20 8C until further use for PCR. The PCR reactions were performed on a GeneAmp1 PCR System 9700 thermocycler (Applied Biosystems, Foster City, CA). For 16S rRNA sequencing, forward (50 AGAGTTTGATCCTGGCTCAG-30 ) and reverse (50 -GTATTACCGCGGCTGCTG-30 ) primers were used to amplify a 536 bp product of the 16S rRNA gene. The PCR mixtures contained 45 mL PCR Master Mix [10 (100 mM Tris–HCl pH 8.8 and 500 mM KCl), 1.7 mM MgCl2, 0.2 mM dNTPs and 1.5 U of Taq DNA polymerase] (Ludwig Biotech, Alvorada, RS, Brazil), 2 mL of each primer (10 pmol/mL) and 1 mL of target DNA (100 ng/mL). The PCR cycling conditions included an initial denaturation step at 95 8C for 5 min, followed by 35 cycles at 95 8C for 30 s, 55 8C for 30 s, and 74 8C for 2 min, with a final extension step at 74 8C for 5 min. The PCR products were purified using the IllustraTM GFXTM PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK), according to the manufacturer’s instructions. The isolates identified as S. aureus through 16S rRNA sequencing were used in subsequent PCR analyses for the amplification of coa and femA genes. The primers COAG2 50 -CGAGACCAAGATTCAACAAG-30 and COAG3 50 -AAAGAAAACCACTCACAT-30 (Goh et al., 1992) were used for coa gene amplification and GFEMAR-1 50 -AAAAAAGCACATAACAAGCG-30 and GFEMAR-2 50 -GATAAAGAAGAAACCAGCAG-30 (Mehrotra et al., 2000) were used for femA gene amplification. The cycling conditions for coa PCR included an initial denaturation step at 94 8C for 2 min, followed by 30 cycles, at 94 8C for 30 s, 55 8C for 2 min, and 72 8C for 4 min, with a final extension step at 72 8C for 7 min. The conditions for femA PCR included a denaturation step at 94 8C for 4 min, followed by 5 cycles of 94 8C for 45 s, 56 8C for 45 s and 72 8C for 45 s, 20 cycles of 94 8C for 45 s, 52 8C for 45 s and 72 8C for 45 s, and a final extension step at 72 8C for 5 min.
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2.3. 16S rRNA sequencing A total of 200 ng of purified PCR DNA was used as a template in sequencing reactions performed using the DYEnamicTM ET Dye Terminator Cycle Sequencing Kit (GE Healthcare, Buckinghamshire, UK) and 0.5 mM of the same primer pairs used in the PCR amplification. The sequencing reactions were ethanol precipitated and loaded onto the MegaBACE 1000 DNA sequencer (GE Healthcare, Buckinghamshire, UK) at 9 kV for 180 min. Both forward and reverse sequencing reactions were performed with two sequencing reads per sample. The sequence pairs were edited and assembled using DNA Baser Sequence Assembler v3.5.4 (Heracle BioSoft SRL, www.DnaBaser.com), and the contigs were blasted against the Staphylococcus spp. nucleotide database of the National Center for Biotechnology Information (NCBI) available at http://www.ncbi.nlm. nih.gov/BLAST.
Table 1 Identification of the reference/type strains in the present study through partial 16S rRNA sequencing. Reference/type strain
16S rRNA sequencing result
% Sequence similaritya
S. aureus 51651 S. capitis 35661
S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S.
100 99
S. S. S. S. S. S. S. S. S. S. S. S. S.
carnosus TM 300 epidermidis 12228 gallinarum 700401 haemolyticus 29970T hyicus 11249T intermedius 29663T lentus 700403 lugdunensis 49576 saprophyticus 15305T sciuri 29061 simulans 27851 warneri 49454 xylosus 29971T
a
Similarity compared with sequences deposited in GenBank.
2.4. Cluster analysis A cladogram, based on partial 16S rRNA sequences, was generated using the MEGA 6.06 program (Tamura et al., 2013) based on the Kimura-2-parameter model. The sequences were aligned in ClustalW, and phylogenetic trees were constructed using the Maximum Likelihood method. The robustness of the tree was assessed with 1000 bootstrap replicates. 3. Results A total of 213 Staphylococcus isolates recovered from bovine mastitis and 15 Staphylococcus reference or type strains were subjected to partial 16S rRNA gene sequencing. All PCR products generated for sequence analysis showed the expected size, i.e., 536 bp. The 16S rRNA sequences were compared with sequences deposited in GenBank. Sequence similarity values greater than 99% (99%) were considered reliable, according to the CLSI (2008). In all but three cases, the sequence similarities with GenBank sequences ranged from 99% to 100%. The three isolates were identified as S. aureus, with 98% of sequence similarity, and were considered unidentified. Most of the identified reference strains were consistent with the ATCC nomenclature (Table 1). The exceptions included S. capitis ATCC 35661, named S. capitis or S. caprae or S. epidermidis, with 99% sequence similarity with strains deposited in GenBank; and S. warneri ATCC 49454 and S. xylosus ATCC 29971T, named S. warneri or Staphylococcus pasteuri and S. xylosus or S. saprophyticus, respectively. In both cases, the percentage of sequence similarity was 100%. Two hundred and thirteen Staphylococcus isolates were identified as Staphylococcus chromogenes (38.5%), S. aureus (19.2%), S. epidermidis (13.1%), S. haemolyticus (6.1%), S. sciuri (5.2%), S. hyicus (4.7%), S. simulans (3.7%), Staphylococcus agnetis (1.8%), Staphylococcus devriesei (0.5%), Staphylococcus auricularis (0.5%), S. intermedius (0.5%), Staphylococcus succinus (0.5%), S. pasteuri (0.5%) (Table 2). Three isolates (1.4%) were not identified and in eight cases, it was not possible to discriminate among
aureus capitis/S. caprae/ epidermidis carnosus epidermidis gallinarum haemolyticus hyicus intermedius lentus lugdunensis saprophyticus sciuri simulans pasteuri/S. warneri xylosus/S. saprophyticus
99 100 100 99 99 100 100 100 100 100 100 100 100
Table 2 Species-level identification of 213 Staphylococcus isolates from bovine mastitis through partial 16S rRNA sequencing. Species (% similaritya) S. chromogenes (99–100%) S. aureus (100%) S. epidermidis (99–100%) S. haemolyticus (99–100%) S. sciuri (100%) S. hyicus (99–100%) S. simulans (99–100%) S. agnetis (99–100%) S. devriesei (100%) S. auricularis (100%) S. intermedius (100%) S. succinus (100%) S. pasteuri (100%) S. hyicus/S. caprae/S. epidermidis (100%) S. xylosus/S. saprophyticus (100%) S. devriesei/S. haemolyticus (99%) S. warneri/S. pasteuri (100%) S. haemolyticus/S. warneri/S. auricularis (99%) Unidentified Total a
No. of isolates % 82 41 28 13 11 10 8 4 1 1 1 1 1 1 4 1 1 1 3 213
38.5% 19.2% 13.1% 6.1% 5.2% 4.7% 3.7% 1.8% 0.5% 0.5% 0.5% 0.5% 0.5% 0.5% 1.8% 0.5% 0.5% 0.5% 1.4% 100%
Similarity compared with sequences deposited in GenBank.
S. hyicus, S. caprae and S. epidermidis (1 isolate), S. xylosus and S. saprophyticus (4 isolates), S. devriesei and S. haemolyticus (1 isolate), S. warneri and S. pasteuri (1 isolate) and S. haemolyticus, S. warneri and S. auricularis (1 isolate). The partial 16S rRNA sequences from 20 isolates, representing the 13 species identified in the present study, were deposited in GenBank under accession numbers KP033211 to KPO33230. S. chromogenes was the coagulase negative species most frequently isolated in the present study, followed by S. epidermidis and S. haemolyticus. Although only 27 coagulase-positive isolates were included in the present study, 41 isolates were identified as S. aureus (coagulasepositive species), ten isolates were identified as S. hyicus (coagulase-variable species), four isolates were identified as
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S. agnetis (coagulase-variable species) and one isolate was identified as S. intermedius (coagulase-positive species). To investigate the unexpected large number of isolates identified as S. aureus, the 41 isolates identified as S. aureus were subjected to the coagulase tube test, using rabbit plasma from two different sources, Voges–Proskauer and latex agglutination tests, and the PCR amplification of femA and coa genes (Table 3). The 41 isolates identified as S. aureus through 16S rRNA sequencing showed 100% similarity with sequences deposited in GenBank. Although six isolates showed negative results in the tube coagulase test, both femA and coa genes were positively amplified through PCR. All 41 S. aureus isolates showed a positive result in the latex agglutination test. Three isolates that showed 98% of sequence similarity to S. aureus and were considered unidentified, showed negative results in coagulase and latex agglutination tests, and did not amplify the femA and coa genes (data not shown). To show the relatedness of the isolates, a cladogram, using the sequences obtained from the 213 clinical isolates and 15 reference/type strains, was constructed (Fig. 1). The analysis of the cladogram revealed that the isolates clustered into two major groups. Most isolates identified as S. aureus, S. epidermidis, S. haemolyticus and S. hyicus clustered around the reference or type strain of the species. All isolates identified as S. simulans and S. sciuri clustered together S. simulans and S. sciuri reference strains, respectively. Eight isolates identified as S. chromogenes clustered together separately from the other 74 isolates of this species. The eight isolates that could not definitively be identified through partial 16S rRNA sequencing and comparing with sequences deposited in GenBank were grouped closely with four ATCC reference/type strains in the cladogram. The isolate identified as S. hyicus/S. caprae/ S. epidermidis grouped closely with strain S. capitis ATCC 35661; similarly, the isolate identified as S. warneri/S. pasteuri grouped closely with reference strain S. warneri ATCC 49454. One isolate identified as S. haemolyticus/S. warneri/S. auricularis grouped closely with strain S. haemolyticus ATCC 29970T and the eleven isolates identified as S. haemolyticus. The isolate identified as S. devriesei/ S. haemolyticus also grouped with the strain S. haemolyticus ATCC 29970T. The four isolates identified as S. xylosus/S. saprophyticus grouped together the strain S. xylosus ATCC 29971T. 4. Discussion The accurate species identification of S. aureus and other staphylococcal species that cause bovine mastitis is
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important to determine the host-pathogen relationships involved in the disease caused by this group of microorganisms. Various DNA-based methods for the identification of Staphylococcus species have been developed, and the high sensitivity and specificity of these techniques provide an alternative method for the accurate identification and classification of Staphylococcus species. In the present study, we used partial 16S rRNA gene sequencing to identify coagulase-positive and coagulase-negative Staphylococcus spp. isolates of bovine mastitis at the species level. All type and reference strains were correctly identified. However, in three cases, S. capitis 35661, S. warneri 49454 and S. xylosus 29971T, the fragment sequenced showed 99% or 100% similarity with the sequences of other species. Ghebremedhin et al. (2008) reported that some Staphylococcus taxa have the same 16S rRNA gene sequences in variable regions, including S. capitis subsp. urealyticus and S. caprae. According to the CLSI (2008), 16S rRNA provides a poor separation between S. capitis, S. caprae and S. epidermidis, as S. warneri shares approximately 98.7% identity with S. pasteuri and S. saprophyticus and S. xylosus share approximately 100% identity. The differentiation among these species is possible using alternative DNA targets, such as dnaJ, sodA, tuf and rpoB genes, as distinct phylogenetic clusters were observed within these genes targets, resulting from heterogeneity within species (CLSI, 2008). Using the partial 16S rRNA gene sequencing, we obtained the definitive species-level identification of 202 (95%) of the 213 isolates examined. Thirteen different staphylococcal species were identified, including the two recently described species, S. devriesei (Supre´ et al., 2010) and S. agnetis (Taponen et al., 2012). S. chromogenes, S. epidermidis and S. haemolyticus were the most frequently observed coagulase-negative species. These results are consistent with those reported by Taponen et al. (2008), Capurro et al. (2009) and Sampimon et al. (2009), who identified CoNS from bovine mastitis using genotypic methods, and cited S. chromogenes, S. simulans, and S. epidermidis as the most common isolated species. In addition to three unidentified isolates, we were not able to definitively identify eight further isolates, as the obtained sequences showed 99% or 100% similarity to sequences from two or three different Staphylococcus species. These eight isolates grouped closely or together with reference/type strains in the cladogram, suggesting that these bacteria might belong to the species of the reference/type strains they grouped. Hellmark et al. (2009) also described inconclusive identification of the species
Table 3 Phenotypic and genotypic characteristics of the 41 isolates identified as S. aureus through partial sequencing of the 16S rRNA gene. No. of isolates 30 2 3 6 a b
Tubea coagulase 1
Tube coagulase 2
VPb
Latex agglutination
femA PCR
coa PCR
+ + – –
+ + + –
+ + +
+ + + +
+ + + +
+ + + +
Tube coagulase 1 and 2: tube coagulase tests performed using lyophilized rabbit plasma from two different sources. VP: Voges–Proskauer test.
% Similarity in sequencing 100% 100% 100% 100%
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Fig. 1. Cluster analysis of the partial 16S rRNA sequences from 213 Staphylococcus spp. isolated from bovine mastitis and 15 Staphylococcus reference/type strains. The bootstrap values of 1000 repeated trees are shown at major branches.
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S. intermedius, S. delphini, S. pseudointermedius and S. schleiferi using 16S rRNA sequencing, probably because of the similarity among these species. All S. aureus isolates identified in the present study showed 100% similarity to sequences deposited in GenBank. Six S. aureus isolates did not clot rabbit plasma, but showed positive results in Voges–Proskauer and latex agglutination tests and amplified femA and coa genes. S. aureus strains isolated from bovine mastitis that failed to coagulate rabbit plasma have been previously described (Vieira-Da-Mota et al., 2001; Capurro et al., 2009; Akineden et al., 2011). Vieira-Da-Mota et al. (2001) reported S. aureus strains isolated from bovine mastitis that were negative in the tube coagulase test, but showed a positive result in the PCR amplification of the coa gene. Capurro et al. (2009) identified two coagulase-negative S. aureus strains isolated from bovine mastitis through tuf gene sequencing and PCR amplification of the nuc gene. Akineden et al. (2011) investigated two coagulasenegative variants of S. aureus isolated from the milk samples of a dairy cow diagnosed with subclinical mastitis using 16S rRNA sequencing and RT-PCR of coa, clfA and nuc genes. However, the reason to the lack of clotting ability in vitro from these strains remains unclear and need further investigation. It is likely that coagulase-negative S. aureus occur with low frequency in strains isolated from bovine mastitis and can be misidentified as CoNS in routine mastitis diagnoses. Therefore, mastitis laboratories and veterinarians should consider that coagulase-negative S. aureus can occur, and the use of additional molecular tests, that are independent of gene expression, has been recommended for the identification of S. aureus from bovine mastitis. The partial 16S rRNA sequencing differentiated 95% of the isolates tested for species identification in the present study. The identification rate of 95% obtained in this study was higher than those reported by Mellmann et al. (2006) and Shin et al. (2011). Using 16S rRNA sequencing to identify CoNS from human origin, these authors reported identification rates of 91% and 84%, respectively. Eleven isolates could not be definitively identified using partial 16S rRNA sequencing. According to the CLSI (2008), the 16S rRNA gene is an excellent target for most staphylococcal species, although discrimination among species can be difficult due to the lack of sufficient heterogeneity within the 16S rRNA gene. Thus, additional DNA targets have been recommended for the unquestionable resolution of staphylococcal species. According to Woo et al. (2009), the accuracy of identification of bacteria using 16S rRNA gene sequencing is further limited by the species included in the database, as bacterial species that are not included in the database would never be determined as the identity of an isolate. Heikens et al. (2005) proposed that a drawback of sequence-based genotypic methods might be the low quality of the sequences deposited in data banks. According to the same authors, genotyping identification based on 16S rRNA sequences has limited discriminating power for closely related Staphylococcus species, and these authors recommended the tuf gene sequencing for the identification of human CoNS. Similarly, Mellmann et al. (2006)
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compared partial 16S rRNA sequencing with partial rpoB gene sequencing to identify human staphylococcal strains and concluded that rpoB sequencing was superior to partial 16S rRNA identification in these strains. Shin et al. (2011) concluded that the tuf gene sequencing had an excellent identification capacity, but relatively few type strains sequences for tuf gene, such as for S. caprae and S. pasteuri, are available in public databases. We concluded that 16S rRNA sequencing was a powerful tool for the identification of Staphylococcus isolated from bovine mastitis, as this technique was successful in 95% of the cases tested. Nevertheless, inconclusive results might occur, requiring the sequencing of additional target genes to properly identify this group of microorganisms. Conflict of interest statement None. Acknowledgments This work was supported through funding from the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas Gerais (Fapemig APQ-00165-11) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq). E.B. Salles received a scholarship from Microvet, Vic¸osa, Minas Gerais. References Akineden, O., Hassan, A.A., Schneider, E., Usleber, E., 2011. A coagulasenegative variant of Staphylococcus aureus from bovine mastitis milk. J. Dairy Res. 78, 38–42. Capurro, A., Artursson, K., Persson Waller, K., Bengtsson, B., EricssonUnnerstad, H., Aspa´n, A., 2009. Comparison of a commercialized phenotyping system, antimicrobial susceptibility testing, and a tuf gene sequence-based genotyping for species-level identification of coagulase-negative staphylococci isolated from cases of bovine mastitis. Vet. Microbiol. 134, 327–333. CLSI, 2008. Interpretative Criteria for Identification of Bacteria and Fungi by DNA Target Sequencing; Approved Guideline. CLSI document MM18-A Clinical and Laboratory Standards Institute, Wayne, PA. Fox, I.K., Gay, J.M., 1993. Contagious mastitis. Vet. Clin. North Am. Food Anim. Pract. 9, 475–487. Ghebremedhin, B., Layer, F., Ko¨nig, W., Ko¨nig, B., 2008. Genetic classification and distinguishing of Staphylococcus species based on different partial gap, 16S rRNA, hsp60, rpoB, sodA, and tuf gene sequences. J. Clin. Microbiol. 46 (3) 1019–1025. Goh, S.H., Byrne, S.Z., Zhang, J.L., Chow, A.W., 1992. Molecular typing of Staphylococcus aureus on the basis of coagulase gene polymorphisms. J. Clin. Microbiol. 30, 1642–1645. Heikens, E., Fleer, A., Paauw, A., Florijn, A., Fluit, A.C., 2005. Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci. J. Clin. Microbiol. 43 (5) 2286–2290. Hellmark, B., So¨derquist, B., Unemo, M., 2009. Simultaneous species identification and detection of rifampicin resistance in staphylococci by sequencing of the rpoB gene. Eur. J. Clin. Microbiol. Infect. Dis. 28, 183–190. Lange, C.C., Brito, M.A.V.P., Brito, J.R.F., Arcuri, E.F., Souza, G.N., Machado, M.A., Domingues, R., Salimena, A.P.S., 2011. Uso de PCR e sequenciamento do rDNA 16S para identificac¸a˜o de bacte´rias do geˆnero Staphylococcus isoladas de mastite bovina. Pesq. Vet. Bras. 31, 36–40. LPSN—List of prokaryotic names with standing in nomenclature, 2014. Available in: http://www.bacterio.net/-allnamessz.html. Accessed on: 20 January 2015. Mehrotra, M., Wang, G., Johnson, W.M., 2000. Multiplex PCR for the detection of genes for Staphylococcus aureus enterotoxins, exfoliative
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