Identification of bovine mastitis pathogens using MALDI-TOF mass spectrometry in Brazil.

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.

Characterization of biofilms and antimicrobial resistance of coagulase-negative Staphylococcus species involved with subclinical mastitis.

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.

Short communication: ß-Lactam resistance and vancomycin heteroresistance in Staphylococcus spp. isolated from bovine subclinical mastitis.

The use of antimicrobial agents has led to the emergence of resistant bacterial strains over a relatively short period. Furthermore, Staphylococcus spp. can produce ß-lactamase, which explains the survival of these strains in a focus of infection despite the use of a ß-lactam antibiotic. The aim of this study was to evaluate the resistance of Staphylococcus spp. isolated from bovine subclinical mastitis to oxacillin and vancomycin (by minimum inhibitory concentration) and to detect vancomycin heteroresistance by a screening method. We also evaluated ß-lactamase production and resistance due to hyperproduction of this enzyme and investigated the mecA and mecC genes and performed staphylococcal cassette chromosome mec typing. For this purpose, 181 Staphylococcus spp. isolated from mastitis subclinical bovine were analyzed. Using the phenotypic method, 33 (18.2%) of Staphylococcus spp. were resistant to oxacillin. In contrast, all isolates were susceptible to vancomycin, and heteroresistance was detected by the screening method in 13 isolates. Production of ß-lactamase was observed in 174 (96%) of the Staphylococcus spp. isolates. The mecA gene was detected in 8 isolates, all of them belonging to the species Staphylococcus epidermidis, and staphylococcal cassette chromosome mec typing revealed the presence of type I and type IV isolates.

Staphylococcus chromogenes, a Coagulase-Negative Staphylococcus Species That Can Clot Plasma.

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.

Presence of mecA-positive multidrug-resistant Staphylococcus epidermidis in bovine milk samples in Brazil.

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.

Microbiological safety of Minas Frescal Cheese (MFC) and tracking the contamination of Escherichia coli and Staphylococcus aureus in MFC processing.

Minas Frescal cheese (MFC) is a traditional food produced and consumed in Brazil, characterized by its soft texture, low sodium, and high moisture content. This study characterized the microbiological contamination by coliforms, Escherichia coli and Staphylococcus aureus, in 99 MFC samples obtained in retail sale and produced by three distinct industrial procedures. Dairy processors were selected to investigate the key points of E. coli and S. aureus contamination during cheese processing. MFC samples produced by the addition of lactic culture presented higher counts of coliforms and E. coli, when compared to other samples (p<0.05). MFC samples produced by the addition of rennet alone presented higher counts of S. aureus when compared to other samples (p<0.05). Fourteen of 19 MFC samples produced by the addition of lactic culture presented E. coli counts higher than 5 × 10(2) colon-forming units/g. The processing steps after pasteurization were identified as the main sources of E. coli and S. aureus contamination of MFC. Based on the results, MFC was characterized as a potential hazard for consumers due to the high frequency of samples contaminated with E. coli and S. aureus counts in noncompliance with Brazilian standards for sanitary quality and safety.

Staphylococcus spp. Isolated from Bovine Subclinical Mastitis in Different Regions of Brazil: Molecular Typing and Biofilm Gene Expression Analysis by RT-qPCR.

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.

Cloxacillin benzathine-loaded polymeric nanocapsules: Physicochemical characterization, cell uptake, and intramammary antimicrobial effect.

The present work shows the development and evaluation of the veterinary antibiotic cloxacillin benzathine (CLOXB) loaded into poly-ε-caprolactone (PCL) nanocapsules (NC), as a potential new treatment strategy to manage bovine intramammary infections, such as mastitis. Staphylococcus aureus-induced mastitis is often a recurrent disease due to the persistence of bacteria within infected cells. CLOXB-PCL NC were prepared by interfacial deposition of preformed biodegradable polymer followed by solvent displacement method. The mean diameter of NC varied from 241 to 428 nm and from 326 to 375 nm, when determined by dynamic light scattering and by atomic force microscopy, respectively. The zeta potential of NC was negative and varied from -28 to -51 mV. In vitro release studies from the NC were performed in two media under sink conditions: PBS with 1% polyethylene glycol or milk. A reversed-phase HPLC method was developed to determine the NC entrapment efficiency and kinetics of CLOXB release from the NC. Free CLOXB dissolution occurred very fast in both media, while drug release from the NC was slower and incomplete (below 50%) after 9 h. CLOXB release kinetics from polymeric NC was fitted with the Korsmeyer-Peppas model indicating that CLOXB release is governed by diffusion following Fick's law. The fluorescence confocal microscopy images of macrophage-like J774A.1 cells reveal NC uptake and internalization in vitro. In addition, antimicrobial effect of the intramammary administration of CLOXB-PCL NC in cows with mastitis resulted in no clinical signs of toxicity and allowed complete pathogen elimination after treatment. The in vivo results obtained in this work suggest that CLOXB-PCL NC could be a promising formulation for the treatment of intramammary infections in cattle, considering their physicochemical properties, release profiles and effects on bovine mastitis control.

Identification of coagulase-negative staphylococci from bovine mastitis using RFLP-PCR of the groEL gene.

Coagulase-negative staphylococci (CNS) have become the predominant pathogens causing bovine mastitis in many countries. CNS infections are associated with damage to milk secretory tissue of the mammary gland by increased connective tissue stroma, moderate increases of somatic cells count in milk and significant production decreases. These consequences impose serious economic losses for the farmers and the dairy industry. Routine veterinary laboratories do not usually identify CNS at the species level. Thereby, the aims of this study were to identify the most common staphylococcal pathogens involved in bovine mastitis using PCR-restriction fragment length polymorphism (RFLP) analysis of a partial groEL gene sequence and to compare our results with the identification carried out by the conventional method. A total of 54 isolates of Staphylococcus, involved in bovine mastitis, were analyzed by this method. The size and number of the fragments obtained by either AluI or HindIII/PvuII digestions made possible to form clear patterns differentiating, among the isolates, 11 of the most common species of animal staphylococcal pathogens. Most of the isolates clustered together with the reference strain of Staphylococcus chromogenes (28) and the type strain of Staphylococcus epidermidis (8). Besides, some isolates clustered together with the type strain of Staphylococcus aureus (5). All patterns were confirmed by the conventional biochemical method, showing concordant results. Thus, the PCR-RFLP of the groEL gene constitutes a reliable and reproducible molecular method for identification of CNS species responsible for bovine mastitis.

Genetic diversity and antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Minas Gerais, Brazil / Diversidade genética e susceptibilidade a antimicrobianos em Staphylococcus aureus isoladas de mastite bovina em Minas Gerais, Brasil

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.

Genetic diversity and antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Minas Gerais, Brazil / Diversidade genética e susceptibilidade a antimicrobianos em Staphylococcus aureus isoladas de mastite bovina em Minas Gerais, Brasil

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.

Antimicrobial resistance profile of Staphylococcus spp. isolates in cattle herds from Western Amazon / Perfil de resistência antimicrobiana de Staphylococcus spp. isolados em rebanhos leiteiros da Amazônia Ocidental

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)

Accurate identification of atypical Staphylococcus chromogenes plasma-clotting strains causing bovine mastitis / Identificação acurada de cepas atípicas de Staphylococcus chromogenes que coagulam o plasma envolvidas com mastite bovina

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.

Accurate identification of atypical Staphylococcus chromogenes plasma-clotting strains causing bovine mastitis / Identificação acurada de cepas atípicas de Staphylococcus chromogenes que coagulam o plasma envolvidas com mastite bovina

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)

Antimicrobial activity of marine sponges against coagulase-negative staphylococci isolated from bovine mastitis.

Bovine mastitis remains worldwide a major challenge for the dairy industry despite the widespread implementation of control strategies. The increasing number of coagulase-negative staphylococci (CNS) causing mastitis and of bacteria resistant to conventional antibiotics has become a serious problem in recent years. Marine sponges are a rich source of bioactive compounds, and many species can be useful for the development of new antimicrobial drugs. In the present study, 49 CNS strains were isolated from bovine mastitis cases from 21 different dairy herds kept at farms in Southeast Brazil. Strains were analyzed for antimicrobial susceptibility and mecA gene detection. Fifty-nine percent of the CNS strains were resistant to at least one of the drugs tested and 12.2% were classified as multiresistant. Three strains carried the mecA gene, confering resistance to the beta-lactamic antibiotics. In addition, the CNS strains were submitted to in vitro screening for antimicrobial activities of extracts from marine sponges. Extracts from the sponge species Cinachyrella sp., Haliclona sp. and Petromica citrina showed antibacterial activity against 61% of the CNS strains, including strains resistant to conventional antibiotics. Extracts from P. citrina showed the largest spectrum of inhibitory activity. The aqueous extract inhibited 51% of the CNS strains and presented a bactericidal effect over susceptible and multiresistant-bacteria at a minimal inhibitory concentration of 1.024µg/ml. This study shows the potential of marine sponges as new sources of antibiotics and disinfectants for the control of CNS involved in bovine mastitis.

Quality and safety of milk and dairy products / ptos

Milk quality is defined on the basis of its composition and hygiene. The somatic cell count (SCC) is the main indicator of milk quality and the limit of 400,000 cells per milliliter of milk is adopted in the international market. The SCC indicates the mastitis situation in herds, making control of this disease a major challenge for producers. High somatic cell and bacteria counts in raw milk adversely affect cheese yield and the quality, shelf life and sensory characteristics of processed milk and dairy products. Food pathogens can be found in raw milk, and in processed milk and dairy products as a result of post-processing contamination. The presence of chemical residues, notably antibiotics, may affect the manufacture of fermented products and the health of consumers. Prudent use of these drugs is an issue for dairy farmers. It is recommended, therefore, the adoption of good dairy practice programs, both in primary production and in processing plants. Consumers are aware of all these aspects. Ignoring these facts could jeopardize the sectors survival.

A qualidade do leite é definida com base na sua composição e higiene. A contagem de células somáticas (CCS) é o principal indicador de qualidade e o limite usado no mercado internacional é de 400.000 células por mililitro de leite. A CCS indica a situação de mastite nos rebanhos. O controle dessa doença é um dos maiores desafios para os produtores de leite. Alta CCS e alta contagem de bactérias no leite cru interferem com o rendimento de queijos e a qualidade, a vida de prateleira e as características sensoriais do leite e seus derivados. Patógenos alimentares podem contaminar o leite cru e até o leite industrializado, como consequência da contaminação durante e após o processamento. A presença de resíduos de substâncias químics, notadamente os antimicrobianos, pode afetar a manufatura de produtos fermentados e a saúde dos consumidores. Isso torna imperativo o uso prudente dessas drogas pelos produtores de leite. É importante, portanto, a adoção de programas de boas práticas, tanto na produção primária quanto na indústria. Os consumidores estão atentos para todos esses aspectos. Ignorar esses fatos pode colocar em risco a sobrevivência do setor.

Quality and safety of milk and dairy products / ptos

Milk quality is defined on the basis of its composition and hygiene. The somatic cell count (SCC) is the main indicator of milk quality and the limit of 400,000 cells per milliliter of milk is adopted in the international market. The SCC indicates the mastitis situation in herds, making control of this disease a major challenge for producers. High somatic cell and bacteria counts in raw milk adversely affect cheese yield and the quality, shelf life and sensory characteristics of processed milk and dairy products. Food pathogens can be found in raw milk, and in processed milk and dairy products as a result of post-processing contamination. The presence of chemical residues, notably antibiotics, may affect the manufacture of fermented products and the health of consumers. Prudent use of these drugs is an issue for dairy farmers. It is recommended, therefore, the adoption of good dairy practice programs, both in primary production and in processing plants. Consumers are aware of all these aspects. Ignoring these facts could jeopardize the sectors survival.(AU)

A qualidade do leite é definida com base na sua composição e higiene. A contagem de células somáticas (CCS) é o principal indicador de qualidade e o limite usado no mercado internacional é de 400.000 células por mililitro de leite. A CCS indica a situação de mastite nos rebanhos. O controle dessa doença é um dos maiores desafios para os produtores de leite. Alta CCS e alta contagem de bactérias no leite cru interferem com o rendimento de queijos e a qualidade, a vida de prateleira e as características sensoriais do leite e seus derivados. Patógenos alimentares podem contaminar o leite cru e até o leite industrializado, como consequência da contaminação durante e após o processamento. A presença de resíduos de substâncias químics, notadamente os antimicrobianos, pode afetar a manufatura de produtos fermentados e a saúde dos consumidores. Isso torna imperativo o uso prudente dessas drogas pelos produtores de leite. É importante, portanto, a adoção de programas de boas práticas, tanto na produção primária quanto na indústria. Os consumidores estão atentos para todos esses aspectos. Ignorar esses fatos pode colocar em risco a sobrevivência do setor.(AU)

Relationship between total bacteria counts and somatic cell counts from mammary quarters infected by mastitis pathogens / Relação entre contagem total de bactérias e contagem de células somáticas de quartos mamários infectados por patógenos da mastite

This study was conducted to establish the relationship between somatic cell count (SCC) and bacterial shedding from mammary quarters according to mastitis pathogens. Milk samples from 638 mammary quarters were examined for mastitis pathogens, SCC and total bacterial count (TBC). The raw data of SCC and TBC were used to perform descriptive statistics. The significance of the arithmetic mean differences between SCC and TBC according to bacteriological examination results was determined by a two-tailed unpaired t-test. Pearson and Spearman´s correlations were done with logarithmic data and linear regression analyses. The geometric means of the bacteriological examination results were (cells mL-1; CFU mL-1): no growth (52,000; 12,000), coagulase-negative staphylococci (85,000; 17,000), Staphylococcus aureus (587,000; 77,000); other streptococci (432,000; 108,000) and Streptococcus agalactiae (1,572,000; 333,000). The Pearson and Spearman's correlations between SCC and TBC were higher than 0.60 for all mastitis pathogens. The regression analyses slopes showed different increase in TBC with the same increase in SCC according to mastitis pathogens. The slope for S. agalactiae (0.542) was higher than that for other mastitis pathogens. The results suggest that the intensity of inflammatory process was associated with number of mastitis pathogens shedding from the mammary gland.

Este estudo foi realizado com objetivo de estabelecer a relação entre contagem de células somáticas (CCS) e a liberação de bactérias de quartos mamários de acordo com os patógenos da mastite. Amostras de leite de 638 quartos mamários foram examinadas para identificação dos patógenos da mastite, CCS e contagem total de bactérias (CTB). Estatísticas descritivas foram utilizadas para avaliar os dados brutos de CCS e CTB. A diferença entre médias para CCS e CTB de acordo com os resultados dos exames bacteriológicos foi avaliada pelo teste T para amostras independentes. Foram realizadas a correlação de Pearson, de Spearman e regressão linear com os dados transformados. As médias geométricas de acordo com os resultados dos exames bacteriológicos foram (células mL-1; UFC mL-1): sem crescimento (52.000; 12.000), estafilococos coagulase negativo (85.000; 17.000), Staphylococcus aureus (587.000; 77000); outros estreptococus (432.000; 108.000) e Streptococcus agalactiae (1.572.000; 333.000). A correlação de Pearson e Spearman entre CCS e CTB foi maior que 0,60 para todos os patógenos da mastite. O coeficiente angular das regressões lineares mostrou diferentes aumentos da CTB como o mesmo aumento da CCS de acordo com os patógenos da mastite. O coeficiente angular para o S. agalactiae (0.542) foi maior em relação aos outros patógenos da mastite. Os resultados sugerem que a intensidade do processo inflamatório foi associada com a quantidade de bactérias da mastite liberada pela glândula mamária.

Composition and bulk tank somatic cell counts of milk from dairy goat herds in Southeastern Brazil / Composição e contagem de células somáticas no leite de rebanhos caprinos do Sudeste do Brasil

The milk composition and somatic cell count (SCC) are requirements for assessment milk quality and mastitis in goat herds. Studies conducted with dairy goat herds indicated that the milk composition differed among them to due to factors such as genetic, feeding, system of production, stage of lactation, year and year-season. The objective of this study was to assess SCC and other milk quality indicators (fat, protein, lactose, and total solids) for goat milk bulk tank. The influence of the herd and year-season on the milk composition as well as herd, milking system and year-season on SCC was also evaluated. Thirteen Brazilian dairy goat herds with about 1,400 dairy goats were included in the study. Six herds were milked by hand and in the remaining seven machine milking was used. Herds were sampled at weekly intervals during two lactations. A total of 913 bulk milk samples were analysed using a automated equipment. The general average percent values for fat, protein, lactose and total solids were, respectively,3.44, 2.95, 4.45 and 11.69. The effect of herd and season was significant for all milk components and bulk milk goat somatic cell count (SCC).The SCC average of all 13 herds was 779,000 cells/ml. The average SCC values of herds milked by hand and by machine were 1,121,000and 848,000 cells/ml respectively. In both groups, the SCC was lowerin the winter and higher in the autumn. Herd characteristics were responsible for higher variability on components and SCC in goat herds.(AU)

A composição do leite bem como a contagem de células somáticas (CCS) são requisitos para avaliar a qualidade do leite e mastite em rebanhos caprinos. Estudos conduzidos indicaram que a composição do leite varia entre os rebanhos devido a fatores genéticos, alimentação, sistema de produção, estágio de lactação, ano e estação do ano. O objetivo do estudo foi avaliar a CCS e outros indicadores de qualidade(gordura, proteína, lactose e sólidos totais) no leite de rebanhos caprinos. A influência do rebanho e estação do ano sobre a composição bem como a influência de rebanho, tipo de ordenha e estação do ano sobre a CCS também foi avaliado. Treze rebanhos caprinos localizados no Brasil, com aproximadamente 1.400 matrizes foram incluídos no estudo, Sendo ordenhados manualmente e os outros sete com equipamento de ordenha. As amostras de leite dos rebanhos foram coletadas semanalmente durante duas lactações. O total de 913 amostras foi analisado no equipamento automatizado. A média dos valores percentuais para gordura, proteína, lactose e sólidos totais foram, respectivamente; 3,44; 2,95; 4,45 e 11,69. O efeito de rebanho e estação do ano foi significante para todos os componentes do leite e CCS. A média para CCS de todos 13 rebanhos foi 779.000 células/ml. As médias para CCS dos rebanhos ordenhados manualmente e mecanicamente foram, respectivamente, 1.121.000 e 848.000 células/ml. Em ambos grupos, a CCS foi menor no inverno e maior no outono. As características de rebanho foram responsáveis pela maior variação dos componentes e CCS no leite de rebanhos caprinos.(AU)

Composition and bulk tank somatic cell counts of milk from dairy goat herds in Southeastern Brazil / Composição e contagem de células somáticas no leite de rebanhos caprinos do Sudeste do Brasil

The milk composition and somatic cell count (SCC) are requirements for assessment milk quality and mastitis in goat herds. Studies conducted with dairy goat herds indicated that the milk composition differed among them to due to factors such as genetic, feeding, system of production, stage of lactation, year and year-season. The objective of this study was to assess SCC and other milk quality indicators (fat, protein, lactose, and total solids) for goat milk bulk tank. The influence of the herd and year-season on the milk composition as well as herd, milking system and year-season on SCC was also evaluated. Thirteen Brazilian dairy goat herds with about 1,400 dairy goats were included in the study. Six herds were milked by hand and in the remaining seven machine milking was used. Herds were sampled at weekly intervals during two lactations. A total of 913 bulk milk samples were analysed using a automated equipment. The general average percent values for fat, protein, lactose and total solids were, respectively,3.44, 2.95, 4.45 and 11.69. The effect of herd and season was significant for all milk components and bulk milk goat somatic cell count (SCC).The SCC average of all 13 herds was 779,000 cells/ml. The average SCC values of herds milked by hand and by machine were 1,121,000and 848,000 cells/ml respectively. In both groups, the SCC was lowerin the winter and higher in the autumn. Herd characteristics were responsible for higher variability on components and SCC in

A composição do leite bem como a contagem de células somáticas (CCS) são requisitos para avaliar a qualidade do leite e mastite em rebanhos caprinos. Estudos conduzidos indicaram que a composição do leite varia entre os rebanhos devido a fatores genéticos, alimentação, sistema de produção, estágio de lactação, ano e estação do ano. O objetivo do estudo foi avaliar a CCS e outros indicadores de qualidade(gordura, proteína, lactose e sólidos totais) no leite de rebanhos caprinos. A influência do rebanho e estação do ano sobre a composição bem como a influência de rebanho, tipo de ordenha e estação do ano sobre a CCS também foi avaliado. Treze rebanhos caprinos localizados no Brasil, com aproximadamente 1.400 matrizes foram incluídos no estudo, Sendo ordenhados manualmente e os outros sete com equipamento de ordenha. As amostras de leite dos rebanhos foram coletadas semanalmente durante duas lactações. O total de 913 amostras foi analisado no equipamento automatizado. A média dos valores percentuais para gordura, proteína, lactose e sólidos totais foram, respectivamente; 3,44; 2,95; 4,45 e 11,69. O efeito de rebanho e estação do ano foi significante para todos os componentes do leite e CCS. A média para CCS de todos 13 rebanhos foi 779.000 células/ml. As médias para CCS dos rebanhos ordenhados manualmente e mecanicamente foram, respectivamente, 1.121.000 e 848.000 células/ml. Em ambos grupos, a CCS foi menor no inverno e maior no outono. As características de rebanho foram responsáveis pela maior variação dos componentes e CCS no leite de rebanhos caprinos