Comparação da identificação fenotípica e genotípica de espécies do gênero Staphylococcus isoladas de mastite bovina

pvb Pesquisa Veterinária Brasileira Pesq. Vet. Bras. 0100-736X 1678-5150 Colégio Brasileiro de Patologia Animal - CBPA. Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) RESUMO: Além de Staphylococcus aureus atualmente outros estafilococos coagulase positiva (SCP) e estafilococos coagulase-negativos (SCN), anteriormente considerados de menor relevância, são reconhecidos como importantes patógenos para humanos e animais. O envolvimento desses micro-organismos na etiologia da mastite bovina e a possibilidade da sua transmissão através do leite aos humanos justifica a utilização de métodos confiáveis para a identificação das espécies mais frequentes. O objetivo deste estudo foi comparar as técnicas fenotípicas com o método genotípico realizada por sequenciamento do gene rpoB na identificação de espécies do gênero Staphylococcus spp. isolados de mastite bovina. Um total de 300 estafilococos isolados de casos de mastite bovina em diferentes rebanhos leiteiros brasileiros foram estudados por técnicas fenotípicas e genotípicas, respectivamente: 150 linhagens de SCP e 150 linhagens de SCN. Foram identificados um total de 18 espécies de SCN e 4 espécies SCP. Entre os SCN as seguintes espécies identificadas: 48 (32%) Staphylococcus warneri, 22 (15%) S. epidermidis, 20 (13%) S. hyicus, 10 (7%) S. xylosus, 7 (5%) S. haemolyticus, 6 (4%) S. simulans, 6 (4%) S. schleiferi subsp schleiferi, 6 (4%) S. hominis, 5 (3%) S. pasteuri, 4 (2,7%) S. cohnii, 3 (2%) S. saprophyticus subsp. saprophyticus, 3 (2%) S. chromogenes, 3 (2%) S. sciuri, 2 (1%) S. saccharolyticus, 2 (1%) S. lugdunensi, 1 (0,7%) S. auricularis, 1 (70 %) S. saprophyticus subsp. bovis, 1 (0,7%) S. capitis. E entre as 150 SCP foram identificados, 105 (70%) S. aureus, 21 (14%), S. hyicus, 19 (13%) S. intermedius e 5 (3%) S. schleiferi subsp coagulans. Considerando-se os 150 SCN isolados, as identificações realizadas por testes fenotípicos e genotípicos apresentaram 96,7% de concordância, coeficiente de concordância kappa = 0,933, SE (erro padrão) de kappa = 0,021 (95% intervalo de confiança: 0,893-0,974), coeficiente de correlação de Pearson (r) = 0,9977, (intervalo de confiança de 95%: 0,9938 a 0,9992) e em relação a 150 SCP isolados foi observado uma concordância de 98,7%, kappa = 0,960, sE de kappa = 0,016, (95% de intervalo de confiança: 0,929 a 0,992) coeficiente de correlação de Pearson (r) = 0,9994 (95% intervalo de confiança: 0,9681-1,0000). A correlação entre os métodos de identificação pode ser considerada como excelente. Esses resultados demonstraram que de acordo com os recursos disponíveis no laboratório, poderia ser utilizada qualquer uma das metodologias. Introduction Knowledge of the phenotypic, genotypic characteristics of a microorganism is imperative for the detection of the pathogenicity and/or toxins that are detrimental to the health of plants, animals, humans and the environment. As well as, the accurate identification of the microorganism is a fundamental component for the establishment of safety measures (Hennekinne et al. 2010). Dairy cow mastitis is the most important disease in the dairy industry worldwide. This disorder causes relevant economical losses, due to reduced milk production and poor milk quality, increasing the cost of treatment, and the premature disposal of cows (McDougall et al. 2009). One of the main agents of bovine contagious mastitis is Staphylococcus aureus (Capurro et al. 2010). Nowadays, the intramammary infections caused by other species of the genus Staphylococcus become a serious problem for dairy herds (Ergün et al. 2009, Pyorala & Taponen 2009, Unal & Yildirim 2010, Kunz et al. 2011, Guimarães et al. 2013, Langoni et al. 2015). Furthermore the emergence of other species besides Staphylococcus aureus as human pathogens, as well as, reservoirs of antimicrobial resistance and other virulence factor, increases the necessity in the developing of reliable methods for the most frequent species identification, to establish the pathogen-host relationship, as well, development of epidemiological approaches (Silva et al. 2013, Silva et al. 2014, Rall et al. 2014, Robles et al. 2014). Coagulase-negative Staphylococcus (CoNS) have become increasingly important in human and veterinary medicine, therefore it is mandatory accurately identify the isolates to species level in order to define the clinical significance of these bacteria, to carry out a proper epidemiological surveillance, and to manage patients infected with CoNS in case of relapse (Poyart et al. 2001). The genus Staphylococcus currently includes about 50 species e subspecies (Hennekinne et al. 2010). The identification of staphylococci species other than S. aureus is not regularly made in clinical laboratories of microbiology, because it is expensive, even with automation, and is time-consuming when carried out manually (Edwards et al. 2001). Therefore, the purpose of this study was to compare phenotypic techniques with genotypic method in the identification of several different species of genus Staphylococcus isolated from bovine mastitis cases. Materials and Methods A total of 300 staphylococci isolates of bovine mastitis cases collected from several Brazilian dairy herds were studied, respectively: 150 CoPS and 150 CoNS strains. The methodology and interpretation criteria used for diagnosis of clinical and subclinical mastitis were based on examination of animals before each milking by the strip cup and the California Mastitis Test - CMT (Schalm & Noorlander 1957). Milk samples were collected in sterile tubes aseptically, and they were transported to the laboratory under refrigeration (4-8°C) in cool boxes with ice packs. Bacterial isolates. The samples were plated on blood agar (5%) (Oxoid) and incubated under aerobic conditions at 37°C, and readings were performed after 24, 48 and 72 hours of incubation. Identification of staphylococci was based on colony morphology, Gram staining, catalase, coagulase and DNase activities (Koneman et al. 2008). Staphylococcus spp. were differentiated from Micrococcus spp. based on oxidation and fermentation of glucose, resistance to bacitracin (0.04 U), and susceptibility to furazolidone (100μg) (Baker 1984). Identification of Staphylococcus spp. was carried out according to the criteria suggested by Kloos & Schleifer (1975) modified by Cunha et al. (2004). Series of biochemical tests were performed such as sugar fermentation (xylose, arabinose, sucrose, trehalose, maltose, mannitol, lactose, xylitol, ribose, fructose, and mannose), production of hemolysin, nitrate reduction, presence of urease and ornithine decarboxylase, and resistance to novobiocin. To confirm the identification of individual species, the following international reference CoNS strains were used as controls: Staphylococcus auricularis (ATCC 33753), S. capitis subsp. capitis (ATCC 27843), S. capitis subsp. urealyticus (ATCC 49325) S. caprae (ATCC 35538), S. cohnii (ATCC49330), S. cohnii subsp. cohnii (ATCC 29974), S. epidermidis (ATCC12228 e 35983), S. haemolyticus (ATCC 29970), S. hominis (ATCC 27844), S. hominis subsp. novobiosepticus (ATCC 700237), S. lentus (ATCC 700403), S. lugdunensis (ATCC 700328), S. saprophyticus (ATCC 15305), S. schleiferi subsp. schleiferi (ATCC 43808), S. sciuri subsp. sciuri (ATCC 29062) S. simulans (ATCC 27851), S. warneri (ATCC 10209), S. xylosus (ATCC 29979) and S. aureus (ATCC 33591 and ATCC 25923). Gene rpoB detection. In all the isolates CoNS and CoPS were performed gene rpoB detection, the gene encoding the beta subunit of RNA polymerase, by sequencing amplified region, using the primers (Mellmann et al. 2006) the following parameters was used: 899 pb, rpob1418for 5’-CAATTCATGGACCAAGC-3’ and rpob3554rev 5’-CCGTCCAAGTCATGAAAC-3’, in 25 μL there was 10 pmol of each primer, 2,5 U de Taq DNA polimerase, 200 μM de desoxirribonucleotides thrifosfate, 20 mM de Tris-HCL (pH 8,4), 0,75mM de MgCl2 e 3 μL de DNA. The amplification was performed by Mastercycler gradient® (Eppendorf) termal cycler during 5 minutes at 94 ºC, followed by 35-seconds cycles of denaturation at 94 ºC during 45 minutes, and the primers annealing one minute at 52oC and, one minute and thirty seconds at 72 °C. The program was completed with an additional extension of 10 minutes at 72 °C. The efficiency of the amplifications was monitored by electrophoresis of the reaction in 2% UltrapureTM agarose gel prepared in o.5 Tris-Borate-EDTA(TBE) buffer. A marker with molecular weight of 100bp was used as a standard. DNA was stained by Sybr Safe® and later photographed under ultraviolet illumination. Sequencing. The rpoB regions amplicons of CoNS and CoPS were first submitted to purification by GFX PCR kit and Gel Band Purification (GE Healthcare®), subsequently the sequencing reactions were carried out in DNA ABI (Applied Biosystems) Prism model 377. The rpoB sequences were aligned by using the multisequence alignment Mega 5.2 program analyzed in GenBank using Blat tool in order of compare the sequences. Statistical analysis. Statistical analyzes were performed with GRAPHPAD INSTAT software (Statistical Analysis Systems for personal computers, 1990-1993), using the test pearson’s correlation coefficient (r) and it were analyzed the level of agreement, obtaining the kappa value (<0 = no agreement; 0-0.2 = slight agreement; 0.21-0.4 = fair agreement; 0.41-0.6 = moderate agreement; 0.61-0.8 = substantial agreement; 0.8-1 = almost perfect agreement). The K linear weighting test was calculated directly in the following site http://www.graphpad.com/quickcalcs/kappa. Results Among the 150 CoNS isolates from bovine mastitis cases eighteen different species were identified respectively: Staphylococcus warneri, S. epidermidis, S. hyicus, S. xylosus, S. haemolyticus, S. simulans, S. schleiferi subsp schleiferi, S. hominis, S. pasteuri, S. cohnii, S. saprophyticus subsp. saprophyticus, S. chromogenes, S. sciuri, S. saccharolyticus, S. lugdunensi, S. auricularis, S. saprophyticus subsp. bovis, S. capitis. The results of phenotypic and genotypic identifications were presented on Table 1, the methods showed a 96.7% of concordance, and it was verified Pearson’s correlation coefficient (r) = 0.9977 (95% confidence interval: 0.9938 to 0.9992) and, Kappa = 0.933, SE of kappa = 0.021 (95% confidence interval: 0.893 to 0.974). Table 1: Phenotypic and genotypic identification of the 150 CoNS of bovine mastitis cases isolates from Brazillian dairy herds. 2016 Species Phenotypic Genotypic Nº % Nº % Staphylococcus auricularis 1 0.7 1 0.7 S. capitis 1 0.7 1 0.7 S. chromogenes 1 0.7 3 2.0 S. cohnii subsp cohnii 4 2.7 4 2.7 S. epidermidis 22 14.7 22 14.7 S. haemolyticus 7 4.70 7 4.7 S. hominis 6 4.0 6 4.0 S. hyicus 22 14.7 20 13.3 S. lugdunensis 2 1.3 2 1.3 S. pasteuri 5 3.3 5 3.3 S. saccharolyticus 2 1.3 2 1.3 S. saprophyticus subsp bovis 1 0.7 1 0.7 S. saprophyticus subsp. saprophyticus 3 2.0 3 2.0 S. schleiferi subsp schleiferi 5 3.3 6 4.0 S. sciuri subsp sciuri 3 2.0 3 2.0 S. simulans 4 3.0 6 4.0 S. warneri 50 33.0 48 32.0 S. xylosus 11 7.3 10 6.7 Total 150 150 Concordance = 96.7%; Kappa = 0.933; SE of kappa = 0.021(95%; Confidence interval = 0.893 to 0.974) And four different species were identified among the 150 CoPS isolates: S. aureus, S. hyicus, S. intermedius and S. schleiferi subsp coagulans. The results of phenotypic and genotypic identifications were presented on Table 2, the methods showed 98.7% of concordance, Pearson’s correlation coefficient (r) = 0.9994 (95% confidence interval: 0.9681 to 1.0000) and, Kappa = 0.960, SE of Kappa = 0.016 (95% confidence interval: 0.929 to 0.992). Table 2: Phenotypic and genotypic identification of the 150 CoPS isolates of bovine mastitis cases isolates from Brazilian dairy herds. 2016 Species Phenotypic Genotypic Nº % Nº % Staphylococcus hyicus 21 14.0 19 12.7 S. intermedius 19 12.7 21 14.0 S. schleiferi subsp coagulans 5 3.3 5 3.3 S. aureus 105 70.0 105 70.0 Total 150 150 Concordance = 98.01%; Kappa = 0.960; SE of kappa = 0.016 (95%; Confidence interval = 0.929 to 0.992) Discussion In the last years, CoNS has attracted larger attention due to her pathogenicity and involvement in human and animal diseases. The importance of identifying the species of CoNS in the clinical laboratories, for the determination of their physiopathological and epidemiological characteristics has been increasing. However, it is not an easy task, since phenotypic tests can present similar results, hindering the obtaining of a result, as well as a great expense of time in the identification of the species (Bannerman 2003, De Paulis et al. 2003). On the other hand, PCR sequencing of the rpoB gene effectively identify staphylococcal species. In contrast to the probe hybridization technique and the RFLP approach, sequencing enables any isolate to be characterized, including new species by their phylogenetic relationships (Drancourt & Raoult 2002). And these authors concluded that the partial sequencing of the rpoB gene as a suitable new tool for the accurate identification of Staphylococcus isolates. Concordance analysis is needed to establish the validity of a new diagnostic measuring or rating technique or to demonstrate the near-equivalence of multiple measuring or rating techniques. In the present study the comparison of the results obtained using the biochemical phenotypic method and sequencing of the rpoB gene for isolates identification showed a high concordance, 96.7% for the 150 CNS isolates and the kappa equal 0.933. As well as, in respect to the 150 CPS isolates it was observed a concordance of 98.1% and kappa equal 0.960. According to Landis & Koch (1977) these obtained values of kappa were indicative of high agreement between the methods. Landis & Koch (1977) have characterized different ranges of values for kappa with respect to the degree of agreement, they suggest for most purposes, values greater than 0.75 or so may be taken to represent excellent agreement beyond chance, values below 0.40 or so may be taken to represent poor agreement beyond chance, and values between 0.40 and 0.75 may be taken to represent fair to good agreement beyond chance. When a new assay or instrument was developed it is of interest to evaluate whether the new assay can reproduce the results based on a traditional the gold-standard assay (Bauer & Kennedy 1981), such validation processes are often evaluated using the Pearson correlation coefficient. The obtained identification results showed a Pearson’s correlation coefficient (r) = 0.9977, (confidence interval 95%: 0.9938 to 0.9992) to CoNS and, Pearson correlation coefficient (r) = 0.9994, (95% confidence interval: 0.9681 to 1.0000) to CoPS. Dancey & Reidy (2006) referred that is considered a strong correlation when the value of Pearson correlation coefficient was = 0.70 to 1, consequently the obtained results indicated a high correlation between the two methods. From the 300 staphylococci isolates of bovine mastitis cases twenty-two different species were identified through the phenotypic technique proposed by Kloos & Schleifer (1975), modified by Cunha et al. (2004) and genotypic technique carried out by sequencing of the rpoB gene. It is important to point out the great diversity of CoNS species in the etiology of bovine mastitis and that some of them were also relevant in human nosocomial cases, for example the most frequent species identified in the present study S. warneri, S. epidermidis and S. hyicus, among the CoNS (Table 1), S. aureus and S. intermedius among the CoPS (Table 2). In the current study the highest frequencies observed were S. aureus 70% among the 150 CoPS, S. warneri 32% and S. epidermidis 15% among the 150 CoNS isolates of bovine mastitis cases. The significance of S. aureus was well recognized not only to to animal health as well to human health. It is also imperative to point out the relevance of those other species to public health in view of the fact that S. epidermidis frequency among human isolates ranged from 43% to 92%, depending on the geographical region where the study was conducted (Ieven et al. 1995, Couto et al. 2001, Vuong & Otto 2002, De Paulis et al. 2003, Spanu et al. 2003, Cunha et al. 2004, Caierão et al. 2006). Among hospitalized Brazilian patients Pereira (2014) studying 300 CoNS isolated by blood cultures from verified that S. epidermidis was the most frequent identified specie (74.3%) and followed by S. haemolyticus 27 (9.0%), S. hominis 22 (7.3%), S. warneri 14 (4.7%), S. lugdunensis 9 (3.0%) e S. capitis 5 (1.7%). Kamath et al. (1992) reported twenty-seven episodes of bacteremia nosocomially acquired caused by S. warneri. Literature review of reported infections with S. warneri include catheter related or unrelated bacteremias with or without immunosuppression, endocarditis (Cimiotti et al. 2007), neonatal infections (Buttery et al. 1997). Considering the animal health, besides the relevance of S. epidermidis and S. warneri on bovine mastitis etiology (Siqueira 2011, Guimarães et al. 2013), S. warneri was accountable for bovine abortion (Barigye et al. 2007) and canine meningoencephalitis (Espino et al. 2006). Conclusion Microbiology laboratories require rapid, sensitive and specific techniques to perform identification tests. In the last decades, new molecular methods have been developed and introduced in clinical laboratories of microbiology to improve the credibility of these tests. 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