RESUMO
This study evaluated the genetic diversity and antimicrobial susceptibility of Staphylococcus aureus isolates from dairy cows in Minas Gerais, Brazil. Thirty-seven isolates from five municipalities (8 herds) were genotyped using multi-locus sequence typing (MLST) and susceptibility to 12 antimicrobial agents was tested using the disk diffusion method. High resistance rates for penicillin [75.68% (28/37)], ampicillin [70.27% (26/37)], and tetracycline [70.27% (26/37)] were detected. Multidrug resistance was observed in seven [18.92% (7/37)] isolates, and two were suggestive of Methicillin-resistant Staphylococcus aureus (MRSA). Among the 37 isolates, 33 novel sequence types (ST) and two known STs (ST126 and ST746) were identified in MLST. The clonal complexes more frequently observed were: CC97 [78.38%; (29/37)], CC1 [8.11%; (3/37)] and CC5 [5.40%; (2/37)]. Minimumspanning tree (MST) analysis according to data from municipalities, herds, and resistance patterns for all isolates did not show any clustering pattern. However, the MST comparing all Brazilian S. aureus isolates deposited in the PubMLST database and from this study depicted an association between the genotype and strain origin (clinical sample). Isolates from this study that belong to CC97 were close to database isolates from milk and dairy products, while those that belong to CC1 and CC5 were close to database isolates from human sources and the environment of dairy farms or industries. In conclusion, our results showed a high rate of resistance to penicillins and tetracyclines and great genetic diversity among the S. aureus isolates from bovine mastitis genotyped in the present study.
Este estudo teve como objetivo avaliar a diversidade genética e a suscetibilidade a antimicrobianos de cepas de Staphylococcus aureus isoladas de vacas leiteiras em Minas Gerais, Brasil. Trinta e sete cepas provenientes de cinco municípios (oito rebanhos) foram genotipadas usando a técnica multi-locus sequence typing (MLST) e a suscetibilidade a 12 antimicrobianos foi avaliada pelo método de difusão em disco. Foram detectadas altas taxas de resistência para penicilina [75,68% (28/37)], ampicilina [70,27% (26/37)] e tetraciclina [70,27% (26/37)] entre os isolados. A multirresistência foi observada em sete [18,92% (7/37)] isolados e dois foram classificados como sugestivos de Staphylococcus aureus resistente à meticilina (MRSA). Entre os 37 isolados, 33 novos sequence types (ST) e dois STs conhecidos (ST126 e ST746) foram identificados pelo MLST. Os complexos clonais mais frequentemente observados foram: CC97 [78,38%; (29/37)], CC1 [8,11%; (3/37)] e CC5 [5,40%; (2/37)]. Foi construída uma minimumspanning tree (MSTs) com todos isolados estudados e esta não mostrou padrão de agrupamento quando comparada com dados epidemiológicos como municípios e rebanho, do qual foi isolado, e perfis de resistência a antimicrobianos. Uma segunda MST foi construída comparando os isolados deste estudo e todas as cepas de S. aureus depositadas no banco de dados PubMLST provenientes do Brasil, que mostrou associação entre o genótipo, STs e a origem da cepa. Foi possível observar entre os isolados do estudo que, aqueles que pertenciam ao CC97, eram geneticamente mais próximos das cepas depositadas no PubMLST isoladas de leite e de produtos lácteos, enquanto aqueles que pertenciam aos CC1 e CC5 estavam mais próximos a cepas isoladas de humanos ou do ambiente de fazendas e indústrias de laticínios. Em conclusão, nossos resultados mostraram um alto índice de resistência às penicilinas e tetraciclinas e grande diversidade genética entre as cepas de S. aureus isoladas de casos de mastite bovina.
Assuntos
Animais , Bovinos , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/genética , Variação Genética , Doenças dos Bovinos , Mastite Bovina , Anti-InfecciososRESUMO
ABSTRACT: This study evaluated the genetic diversity and antimicrobial susceptibility of Staphylococcus aureus isolates from dairy cows in Minas Gerais, Brazil. Thirty-seven isolates from five municipalities (8 herds) were genotyped using multi-locus sequence typing (MLST) and susceptibility to 12 antimicrobial agents was tested using the disk diffusion method. High resistance rates for penicillin [75.68% (28/37)], ampicillin [70.27% (26/37)], and tetracycline [70.27% (26/37)] were detected. Multidrug resistance was observed in seven [18.92% (7/37)] isolates, and two were suggestive of Methicillin-resistant Staphylococcus aureus (MRSA). Among the 37 isolates, 33 novel sequence types (ST) and two known STs (ST126 and ST746) were identified in MLST. The clonal complexes more frequently observed were: CC97 [78.38%; (29/37)], CC1 [8.11%; (3/37)] and CC5 [5.40%; (2/37)]. Minimum‐spanning tree (MST) analysis according to data from municipalities, herds, and resistance patterns for all isolates did not show any clustering pattern. However, the MST comparing all Brazilian S. aureus isolates deposited in the PubMLST database and from this study depicted an association between the genotype and strain origin (clinical sample). Isolates from this study that belong to CC97 were close to database isolates from milk and dairy products, while those that belong to CC1 and CC5 were close to database isolates from human sources and the environment of dairy farms or industries. In conclusion, our results showed a high rate of resistance to penicillins and tetracyclines and great genetic diversity among the S. aureus isolates from bovine mastitis genotyped in the present study.
RESUMO: Este estudo teve como objetivo avaliar a diversidade genética e a suscetibilidade a antimicrobianos de cepas de Staphylococcus aureus isoladas de vacas leiteiras em Minas Gerais, Brasil. Trinta e sete cepas provenientes de cinco municípios (oito rebanhos) foram genotipadas usando a técnica multi-locus sequence typing (MLST) e a suscetibilidade a 12 antimicrobianos foi avaliada pelo método de difusão em disco. Foram detectadas altas taxas de resistência para penicilina [75,68% (28/37)], ampicilina [70,27% (26/37)] e tetraciclina [70,27% (26/37)] entre os isolados. A multirresistência foi observada em sete [18,92% (7/37)] isolados e dois foram classificados como sugestivos de Staphylococcus aureus resistente à meticilina (MRSA). Entre os 37 isolados, 33 novos sequence types (ST) e dois STs conhecidos (ST126 e ST746) foram identificados pelo MLST. Os complexos clonais mais frequentemente observados foram: CC97 [78,38%; (29/37)], CC1 [8,11%; (3/37)] e CC5 [5,40%; (2/37)]. Foi construída uma minimum‐spanning tree (MSTs) com todos isolados estudados e esta não mostrou padrão de agrupamento quando comparada com dados epidemiológicos como municípios e rebanho, do qual foi isolado, e perfis de resistência a antimicrobianos. Uma segunda MST foi construída comparando os isolados deste estudo e todas as cepas de S. aureus depositadas no banco de dados PubMLST provenientes do Brasil, que mostrou associação entre o genótipo, STs e a origem da cepa. Foi possível observar entre os isolados do estudo que, aqueles que pertenciam ao CC97, eram geneticamente mais próximos das cepas depositadas no PubMLST isoladas de leite e de produtos lácteos, enquanto aqueles que pertenciam aos CC1 e CC5 estavam mais próximos a cepas isoladas de humanos ou do ambiente de fazendas e indústrias de laticínios. Em conclusão, nossos resultados mostraram um alto índice de resistência às penicilinas e tetraciclinas e grande diversidade genética entre as cepas de S. aureus isoladas de casos de mastite bovina.
RESUMO
Staphylococcus is one of the most frequent etiological agents of bovine mastitis, causing economic losses to milk farming. This study aimed to describe the milk production and management practices in 15 herds located in three microregions of the State of Rondônia and to identify the antimicrobial resistance profile of 97 isolates of the genus Staphylococcus from these herds. The isolates were subjected to antimicrobial susceptibility tests using the agar diffusion method. Twenty-nine S. aureus isolates were selected to determine their minimum inhibitory concentration (MIC). In the farms studied, the breeding system was semi-intensive, with the Girolando breed being predominant. Milking was performed predominantly using a mechanical milking system (86.7%), twice a day (66.7%), in the presence of the calf (53.8%). The average number of lactating cows in the farms was 24, with an average milk production of 204.9 L d-1 and a milk productivity of 10.2 L animal-1 d-1. The use of antimicrobials for the treatment and prevention of bovine mastitis was reported for all properties, and therapy for dry cows was adopted in 80% of the herds. The percent susceptibility to antimicrobials ranged from 85.5% to 100% for S. aureus, 22.2% to 88.9% for coagulase-positive staphylococci (CPS), and 69.2% to 100% for coagulase-negative staphylococci (CNS), with the highest resistance frequencies for penicillin, ampicillin, and tetracycline. Among the S. aureus isolates, 100% susceptibility to oxacillin, cefalexin, and gentamicin was observed, and among the CNS isolates, 100% susceptibility to gentamicin was observed. None of the antimicrobials tested showed 100% in vitro effectiveness for CPS. CPS and CNS presented lower percentages of susceptibility to penicillin, ampicillin, and tetracycline than the S. aureus isolates. Twelve resistance patterns were detected, six of which were multiresistance patterns. The most prevalent resistance patterns were penicillin and ampicillin (PEN-AMP) and penicillin, ampicillin, and tetracycline (PEN-AMP-TET). Results of the MIC assay revealed that all 29 S. aureus isolates were susceptible to cephalothin, cefoxitin, tetracycline, and erythromycin, whereas 25 (86.2%) were susceptible to penicillin.(AU)
O gênero Staphylococcus destaca-se como um dos agentes etiológicos mais frequentes da mastite bovina, causando prejuízos econômicos à pecuária de leite. Este estudo teve o objetivo de descrever a produção de leite e práticas de manejo de 15 rebanhos localizados em três microrregiões do Estado de Rondônia, e identificar o perfil de resistência aos antimicrobianos de 97 isolados do gênero Staphylococcus provenientes destes rebanhos. Os isolados foram submetidos a testes de suscetibilidade a antimicrobianos, utilizandose a técnica de difusão em ágar. Foram selecionados 29 isolados de Staphylococcus aureus (S. aureus) para determinação da concentração inibitória mínima (CIM). Nas propriedades estudadas, o sistema de criação era semi-intensivo, com predomínio da raça Girolando. A ordenha era realizada predominantemente em sistema de ordenha mecânica (86,7%), duas vezes ao dia (66,7%) e com a presença do bezerro (53,8%). A média do número de vacas em lactação das propriedades era de 24 animais, com média de produção de leite de 204,9 L d-1 e produtividade de leite de 10,2 L animal-1 d-1. O uso de antimicrobianos para o tratamento e prevenção da mastite bovina foi relatado em todas as propriedades, sendo adotada a terapia da vaca seca em 80% dos rebanhos. A percentagem de susceptibilidade aos antimicrobianos variou de 85,5 a 100% para S. aureus, 22,2 a 88,9% para Staphylococcus coagulase positivos (SCP) e 69,2 a 100% para Staphylococcus coagulase negativos (SCN), sendo as maiores frequências de resistência para penicilina, ampicilina e tetraciclina. Entre os isolados de S. aureus foi observada 100% de susceptibilidade aos antimicrobianos oxacilina, cefalexina e gentamicina, e entre os isolados de SCN, 100% de susceptibilidade à gentamicina. Nenhum dos antimicrobianos testados apresentou 100% de efetividade in vitro para SCP. SCP e SCN apresentaram menores percentagens de susceptibilidade à penicilina, ampicilina e tetraciclina do que os isolados de S. aureus. Foram observados 12 padrões de resistência, sendo seis padrões de multirresistência. Os padrões de resistência mais prevalentes foram resistência à penicilina e ampicilina (PEN-AMP) e à penicilina, ampicilina e tetraciclina (PEN-AMP-TET). A determinação da CIM dos 29 isolados de S. aureus mostraram que todos foram susceptíveis à cefalotina, cefoxitina, tetraciclina e eritromicina, e 25 (86,2%) foram susceptíveis à penicilina.(AU)
Assuntos
Animais , Masculino , Bovinos , Staphylococcus , Testes de Sensibilidade Microbiana , Coagulase , Mastite Bovina , Anti-Infecciosos , ÁgarRESUMO
In this Research Communication we evaluate the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify 380 bacteria isolated from cases of bovine mastitis in Brazil. MALDI-TOF MS identifications were compared to previous identifications by biochemical tests and 16S rRNA sequencing. MALDI-TOF MS achieved a typeability of 95.5%. The accuracy of MALDI-TOF MS for the identification of Staphylococcus isolates was 93.2%. The agreement between MALDI-TOF MS and biochemical identification of Streptococcus agalactiae was 96%, however, the agreement between these techniques for identifying other catalase-negative, Gram-positive cocci was lower. Agreement in identifying Gram-negative bacteria at the genus level was 90.5%. Our findings corroborate that MALDI-TOF MS is an accurate, rapid and simple technique for identifying bovine mastitis pathogens. The availability of this methodology in some research institutions would represent a significant step toward increasing the diagnosis and epidemiological studies of bovine mastitis and other animal infectious diseases in Brazil.
Assuntos
Mastite Bovina/microbiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/veterinária , Animais , Brasil , Bovinos , Feminino , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/classificação , Bactérias Gram-Positivas/genética , Leite/microbiologia , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Análise de Sequência de RNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Staphylococcus/genética , Streptococcus agalactiae/genéticaRESUMO
Biofilm formation is a central feature to guarantee staphylococcal persistence in hosts and is associated with several diseases that are difficult to treat. In this research paper, biofilm formation and antimicrobial susceptibility were investigated in staphylococcal strains belonging to several species. These strains were isolated from the milk of cows with subclinical mastitis and most of them were coagulase-negative, with the prevalence of Staphylococcus chromogenes. High genetic diversity was observed among the strains by pulsed field gel electrophoresis. Antimicrobial resistance was assessed by disk diffusion and more than 50% of the strains were resistant to ampicillin and penicillin G, with multi-resistance profiles (13.6%) also being observed. Most strains (65.9%) formed biofilms when cultivated in BHI supplemented with 1% glucose. Most strains (72.7%) carried the intercellular adhesion gene (icaA), while less than half (36.3%) carried the biofilm-associated protein gene (bap). Concentrations of up to 10xMIC of erythromycin and tetracycline were not sufficient to suppress cell viability in preformed biofilms. Our results revealed that a genetically diverse group of biofilm-forming Staphylococcus species can be involved in subclinical mastitis. Since high antimicrobial concentrations cannot eradicate biofilm cells in vitro, their use in dairy animals may be ineffective in controlling infections, while supporting selection of resistant microorganisms. These data reinforce the need for alternative therapies aiming at disrupting biofilms for effective disease control.
Assuntos
Biofilmes/crescimento & desenvolvimento , Farmacorresistência Bacteriana/fisiologia , Mastite Bovina/microbiologia , Infecções Estafilocócicas/veterinária , Staphylococcus/efeitos dos fármacos , Staphylococcus/fisiologia , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Bovinos , Coagulase/análise , Farmacorresistência Bacteriana/genética , Feminino , Variação Genética , Mastite Bovina/tratamento farmacológico , Testes de Sensibilidade Microbiana/veterinária , Infecções Estafilocócicas/microbiologia , Staphylococcus/genéticaRESUMO
Bovine mastitis is mainly caused by bacteria of the genus Staphylococcus spp., which possess different virulence factors, including the capacity for biofilm formation that provides enhanced protection against the action of immune system components and serves as a barrier against the penetration of antimicrobial agents. This study aimed to characterize 181 Staphylococcus spp. Strains-including Staphylococcusaureus and coagulase-negative staphylococci (CoNS) isolated from bovine subclinical mastitis in six Brazilian states-by molecular methods. RT-qPCR was used to verify the expression of genes of the ica operon-mainly responsible for biofilm formation-as well as bap and bhp. Chromosome similarity among the isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The icaA gene was detected in 79 (43.6%) isolates, icaB in 24 (13.2%), icaC in 57 (31.4%), and icaD in 127 (70.1%). The bap gene was identified in 66 (36.4%) isolates, while the bhp gene was found in nine (4.9%). RT-qPCR confirmed the expression of the icaA gene in 60 (75.9%) isolates, of icaB in six (25%), of icaC in 26 (45.6%), and of icaD in 80 (63%). Clonal typing of the isolates by PFGE permitted the identification of eight Staphylococcusaureus clusters that simultaneously included ≥3 strains, with a similarity of ≥80%. Regarding the other species studied, three clusters were observed for Staphylococcuschromogenes and four clusters for Staphylococcusepidermidis. Only one cluster each was identified for Staphylococcussaprophyticus and Staphylococcussimulans, while the other species did not form any cluster. With respect to MLST, ST126 and ST1 were the prevalent sequence types in S. aureus, while in S.epidermidis all sequence types were different. These results reveal strains with the same evolutionary origin as other isolates, which might cause infections in humans and animals, suggesting their ability to spread between these species.
RESUMO
ABSTRACT: 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.
RESUMO: 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.
RESUMO
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.(AU)
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.(AU)
Assuntos
Animais , Bovinos , Staphylococcus , Mastite Bovina/diagnóstico , Mastite Bovina/sangue , Testes de Coagulação SanguíneaRESUMO
Staphylococcus chromogenes is one of the main coagulase-negative staphylococci isolated from mastitis of dairy cows. We describe S. chromogenes isolates that can clot plasma. Since the main pathogen causing mastitis is coagulase-positive Staphylococcus aureus, the coagulase-positive phenotype of S. chromogenes described here can easily lead to misidentification.
Assuntos
Coagulação Sanguínea , Coagulase/deficiência , Plasma/metabolismo , Staphylococcus/metabolismo , Animais , Bovinos , Mastite Bovina/diagnóstico , Mastite Bovina/microbiologia , Staphylococcus/classificação , Staphylococcus/isolamento & purificaçãoRESUMO
Bacteria of the genus Staphylococcus are one of the major pathogens causing bovine mastitis. In recent decades, resistance of this genus to oxacillin (methicillin) has been a matter of concern due to the possibility of reducing the effectiveness of mastitis treatments and the transfer of resistance determinants to other bacteria. Oxacillin resistance was studied in 170 staphylococci from bovine milk samples, including 79 Staphylococcus aureus and 91 coagulase-negative staphylococci (CNS). The susceptibility profile of 10 antimicrobial agents used in veterinary practice was determined by the Etest method. In addition to the Etest, the phenotypic characterization of oxacillin resistance was tested using the cefoxitin disk diffusion test. All isolates were screened by PCR to detect the mecA gene in 2 different regions of the gene. The isolates with an oxacillin minimum inhibitory concentration ≥0.5 µg/mL or resistant to cefoxitin were identified by sequencing a 536-bp fragment of the 16S rRNA gene. This group of isolates was also evaluated for the presence of blaZ and mecC genes. Molecular analysis of the mecA gene was carried out by typing of the staphylococcal cassette chromosome mec (SCCmec). The relatedness of the mecA-positive isolates was evaluated by macrorestriction of chromosomal DNA followed by pulsed-field gel electrophoresis. With the exception of penicillin and oxacillin, 86% of the isolates showed susceptibility to cephalothin, gentamicin, erythromycin, sulfonamide, trimethoprim-sulfamethoxazole, and tetracycline. All S. aureus isolates were susceptible to oxacillin, whereas 47% (n=43) of the CNS isolates were resistant. The CNS isolates showed a higher resistance to cephalothin, erythromycin, tetracycline, and gentamicin in comparison with S. aureus. The mecA gene was only detected in 10 CNS isolates, identified as Staphylococcus epidermidis, and classified into 3 pulsotypes (A, B, and C) and 4 subtypes (A1, B1, B2, and B3). Among the isolates with an oxacillin resistance phenotype, 12 were positive for the blaZ gene, and 9 of them were mecA-positive. Two of the oxacillin-resistant isolates amplified the mecA homolog gene of Staphylococcus sciuri and none amplified mecC. Three SCCmec types, I, IV, and V, were found. Our results suggest that Staphylococcus epidermidis can be a reservoir for mecA for other Staphylococcus species. Studies investigating the molecular and phenotypic profile of antimicrobial resistance in staphylococcal species should be performed for controlling the spread of resistance and the selection of appropriate therapeutic measures.