Genotypic evaluation of antimicrobial resistance in Staphylococcus spp. isolated from bovine clinical mastitis / [Avaliação genotípica da resistência antimicrobiana em Staphylococcus spp. isolados de mastite clínica bovina]

Bovine clinical mastitis caused by Staphylococcus spp. is a serious and widespread disease in the world of dairy farming. Antimicrobial therapy is of fundamental importance in the prevention and treatment of infectious mastitis, but the indiscriminate use of antimicrobials acts as a determining factor for the spread of the disease. The present study evaluated the resistance profiles of 57 Staphylococcus spp. isolated from bovine clinical mastitis to beta-lactams and gentamicin, relating characteristics of phenotype (in vitro susceptibility tests) and genotype (detection and expression of genes encoding resistance - mecA, mecALGA251, blaZ, femA, femB, and aacA-aphD - using PCR and RT-PCR, respectively). One or more genes coding for resistance to different antimicrobials were detected in 50 Staphylococcus spp. isolates. The femA and femB genes were the most frequent (75.4% for both). The observed expression of the genes was as follows: blaZ (60%), femA (39.5%), aacA-aphD (50%), femB (32.6%), mecA (8.3%), and mecALGA251 (0%). Considering the relevance of the genus Staphylococcus to bovine mastitis, this study aimed to elucidate aspects regarding the genotypic and phenotypic profiles of these microorganisms so as to contribute to the development of effective strategies for mastitis control.(AU)

A mastite clínica bovina causada por Staphylococcus spp. é uma doença grave e generalizada no mundo da pecuária leiteira. A terapia antimicrobiana é de fundamental importância na prevenção e no tratamento da mastite infecciosa, mas o uso indiscriminado de antimicrobianos atua como fator determinante para a disseminação da doença. O presente estudo avaliou os perfis de resistência de 57 Staphylococcus spp. isolados de mastite clínica bovina em relação ao uso de betalactâmicos e gentamicina, relacionando características do fenótipo (testes de suscetibilidade in vitro) e genótipo (detecção e expressão de genes que codificam resistência - mecA, mecALGA251, blaZ, femA, femB, e aacA-aphD - usando PCR e RT-PCR, respectivamente). Um ou mais genes que codificam resistência a diferentes antimicrobianos foram detectados em 50 Staphylococcus spp. isolados. Os genes femA e femB foram os mais frequentes (75,4% para ambos). A expressão observada dos genes foi a seguinte: blaZ (60%), femA (39,5%), aacA-aphD (50%), femB (32,6%), mecA (8,3%) e mecALGA251 (0%). Considerando-se a relevância do gênero Staphylococcus para a mastite bovina, este estudo teve como objetivo elucidar aspectos referentes aos perfis genotípico e fenotípico desses microrganismos, a fim de contribuir para o desenvolvimento de estratégias eficazes para o controle da mastite.(AU)

Virulence, agr groups, antimicrobial resistance and epidemiology of Staphylococcus aureus isolated from bovine subclinical mastitis / Virulência, grupos agr, resistência a antimicrobianos e epidemiologia de isolados de Staphylococcus aureus obtidos de vacas com mastite

Fifty-two Staphylococcus aureus recovered from papillary ostium and milk samples collected from cows with subclinical mastitis and milking environments in three small dairy herds located in southeastern Brazil were subjected to PCR identification based on the thermonuclease (nuc) gene. All the strains were submitted to in vitro antimicrobial susceptibility testing, and we investigated the sequence types (STs), agr groups (I-IV), virulence genes encoding for Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs), biofilm-associated proteins, bi-component toxins, pyrogenic toxin superantigens, and enterotoxins. Screening for oxacillin resistance (2-6 µg/ml oxacillin), beta-lactamase activity assays, and PCR for the mecA/mecC genes detected 26 methicillin-susceptible S. aureus(MSSA) and 26 mec-independent oxacillin-nonsusceptible S. aureus (MIONSA). While MSSA isolates were found to be susceptible to all antimicrobial agents tested, or only resistant to penicillin and ampicillin, MIONSA isolates were multidrug-resistant. ST126-agr group II MSSA isolates were prevalent in milk (n=14) and carried a broad set of virulence genes (clfA, clfB, eno, fnbA, fiB, icaA, icaD, lukED, hla, and hlb), as well as the ST126-agr group II MIONSA isolated from milking liners (n=1), which also carried the eta gene. ST1-agr group III MIONSA isolates (n=4) were found in papillary ostium and milk, but most MIONSA isolates (n=21), which were identified in both papillary ostium and milking liners, were agr-negative and assigned to ST126. The agr-negative and agr group III lineages showed a low potential for virulence. Studies on the characterization of bovine-associated MSSA/MIONSA are essential to reduce S. aureus mastitis to prevent economic losses in dairy production and also to monitor the zoonotic potential of these pathogens associated with invasive infections and treatment failures in healthcare.

Cinquenta e dois isolados de Staphylococcus aureus obtidos de amostras colhidas do óstio papilar, do leite de vacas com mastite subclínica e do ambiente de ordenha em três fazendas de rebanhos leiteiros localizadas no sudeste do Brasil foram identificados por PCR para o gene da termonuclease (nuc). Todos os isolados foram testados para sensibilidade a antimicrobianos e foram investigados os sequence types (STs), grupos agr (I-IV) e genes de virulência que codificam Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs), proteínas associadas a biofilme, toxinas bi-componentes, toxinas pirogênicas com propriedades de superantígenos e enterotoxinas. Triagem para detecção de resistência à oxacilina (2-6 µg/ml oxacilina), ensaios de atividade de enzimas beta-lactamases e PCR para os genes mecA/mecC detectaram 26 estirpes de S. aureus sensíveis à meticilina (methicillin-susceptible S. aureus, MSSA) e 26 estirpes de S. aureus mec-negativas não sensíveis à meticilina (mec-independent oxacillin-nonsusceptible S. aureus, MIONSA). Enquanto os isolados MSSA foram sensíveis a todos os agentes antimicrobianos testados, ou apenas resistentes à penicilina e ampicilina, os isolados MIONSA foram multirresistentes. MSSA ST126-agr grupo II foram prevalentes no leite (n= 14) e apresentaram um amplo conjunto de genes de virulência (clfA, clfB, eno, fnbA, fiB, icaA, icaD, lukED, hla e hlb), assim como o isolado MIONSA ST126-agr grupo II proveniente de um insuflador (n= 1), o qual também apresentou o gene eta. MIONSA ST1-agr grupo III (n= 4) foram identificados no óstio papilar e leite, mas a maioria dos isolados MIONSA (n= 21), encontrados em óstios papilares e insufladores, foram agr-negativos e pertenceram ao ST126. As linhagens agr-negativas e agr grupo III apresentaram baixo potencial de virulência. Estudos sobre a caracterização de MSSA/MIONSA associados a bovinos são essenciais para a redução da mastite causada por S. aureus e de perdas econômicas na produção leiteira e, também, para o monitoramento do potencial zoonótico desses patógenos associados a infecções invasivas e falhas de tratamento em ambientes hospitalares.

Levantamento de agentes causadores de mastite e sua resistência a antibióticos / Survey agents of mastitis causes and their resistance to antibiotics

Dentre as enfermidades que acometem o rebanho leiteiro a mastite é a que apresenta o maior impacto econômico, assim com o objetivo de identificar os agentes causadores de mastite e de avaliar a sua resistência a antibióticos, analisou-se 119 amostras de leite, coletadas de 47 unidades produtoras distribuídas em 10 municípios da microrregião de Pelotas no RS. Das amostras analisadas, 76,7% agentes foram classificados como Gram positivos (G+) e 23,3% como Gram negativos (G-) . Das amostras G+ analisados, os que obtiveram maior resistência por parte dos microrganismos isolados, classificados como Gram positivos, foram trimetropin (70% ), seguido da penicilina (65,2%). Já os antimicrobianos com maior resistência por parte dos isolados de G-, foram cefalotina (85,7%), seguido de trimetropin (80,9%). Por fim, as 16 amostras de Staphylococcus coagulase positiva, foram 100% resistentes a estreptomicina. Verifica-se assim a importância de um adequado manejo para o correto uso dessas drogas.

Frequency of microorganisms isolated at different stages of lactation and milk production loss associated with somatic cell count and to mastitis-causing pathogens / Frequência de microrganismos isolados em diferentes fases da lactação e perda de produção de leite associada à contagem de células somáticas e aos patógenos causadores de mastite

The aim of this study was to evaluate the frequency of isolation of agents causing subclinical mastitis in a herd and to estimate production losses associated with SCC> 200,000cells/mL. Three 7-day interval microbiological cultures were performed in all lactating animals from the same farm that was evaluated from June to July. To evaluate the negative and positive isolation frequencies between the lactation phases, a Chi-square test was performed. Simple linear regressions were performed to evaluate the lactation curve of animals grouped by pathogens isolated from negative cows in the microbiological culture and with SCC <200,000cells/mL. To estimate the production losses between the groups, regression coefficients were used. Results found in this experiment were: Culture-negative cows with SCC ≥ 200,000cells/mL, cows testing positive in milk culture, with SCC <200,000cells/mL and cows testing positive in milk culture, with SCC ≥ 200,000cells/mL. Milk production was -3.5; -0.5 and -4.27kg, respectively, when compared to culture-negative cows with SCC <200,000cells/mL. Cows infected with yeast cells, Coagulase-negative staphylococci (CNS), Staphylococcus aureus and environmental streptococci produced -3.42; -0.5; -0.168 and -2.5kg of milk when compared to culture-negative cows with SCC <200,000cells/mL. SCC indicates an inflammatory reaction in the mammary gland and it is directly associated with milk production losses and with presence of microorganisms in the mammary gland.(AU)

O objetivo deste estudo foi avaliar a frequência de isolamento de agentes causadores de mastite subclínica em um rebanho e estimar as perdas de produção associadas com CCS>200.000 cél./mL. Três cultivos microbiológicos intervalados por sete dias foram realizados em todos os animais em lactação da propriedade avaliada, no período de junho a julho. Para avaliar as frequências de isolamento negativo e positivo entre as fases da lactação, foi realizado um teste de qui-quadrado. Foram realizadas regressões lineares simples para avaliar a curva de lactação dos animais agrupados por patógenos isolados em relação a vacas negativas na cultura microbiológica e com CCS < 200.000 cél./mL. Os coeficientes das regressões foram utilizados para estimar as perdas de produção entre os grupos. Vacas com resultado negativo na microbiologia, mas com CCS ≥ 200.000 cél./mL, positivas na microbiologia com CCS < 200.000 cél./mL e positivas com CCS ≥ 200.000 cél./mL, produziram por dia, respectivamente, -3,5; -0,5 e -4,27kg de leite em relação às vacas negativas com CCS < 200.000 cél./mL. Vacas infectadas com células leveduriformes, Staphylococcus coagulase negativa, Staphylococcus aureus e Streptococcus ambientais produziram, respectivamente, -3,42; -0,5; -0,168 e -2,5kg de leite, comparadas a vacas negativas com CCS < 200.000 cél./mL. A CCS, indicativa de reação inflamatória, encontra-se diretamente associada às perdas de produção de leite, assim como a presença do microrganismo na glândula mamária.(AU)

Direct identification of bovine mastitis pathogens by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in pre-incubated milk

ABSTRACT The present study aimed to compare two MALDI-TOF identification methods [(a) direct sample identification after pre-incubation; or (b) use of bacteria isolated on pre-culture)] to standard, traditional bench microbiology. A total of 120 quarter milk samples from 40 Holstein lactating cows were screened based on culture-positive results obtained by microbiological culture (reference method) with the following numbers of quarters positive per cow: 4 cows with 1, 8 cows with 2, 12 cows with 3 and 16 cows with 4 infected quarters per cow. For direct identification method, quarter milk samples (n = 120) were skimmed by centrifugation (10,000 × g/10 min) and pre-incubated at 37 ºC for 12 h. After pre-incubation, quarter milk samples were submitted to total bacterial count by flow cytometry and for a preparation protocol for bacterial ribosomal protein extraction followed by MALDI-TOF MS analysis. The direct MALDI-TOF MS identification method compared to microbiological culture correctly identified isolates of coagulase-negative Staphylococci (27.2%), Streptococcus agalactiae (21.8%), Staphylococcus aureus (14.2%), and Streptococcus uberis (5.2%). The pre-incubation protocol of milk samples, associated to the direct identification method by MALDI-TOF MS, did not increase the identification at species level (score >2.0) of pathogens causing subclinical mastitis in comparison to the method without previous incubation.

Frequency of Staphylococcus aureus virulence genes in milk of cows and goats with mastitis / Frequência de genes fatores de virulência de Staphylococcus aureus em leite de vacas e cabras com mastite

The present study determined the frequency of Staphylococcus aureus virulence genes in 2,253 milk samples of cows (n=1000) and goats (n=1253) raised in three different geographical regions of the state Pernambuco, Brazil. The presence of genes of virulence factors associated to adhesion to host cells (fnbA, fnbB, clfA and clfB), toxinosis (sea, seb, sec, sed, seg, seh, sei, tsst, hla and hlb), and capsular polysaccharide (cap5 and cap8) was evaluated by PCR. A total of 123 and 27 S. aureus strains were isolated from cows' and goats' milk, respectively. The sec and tsst genes were detected exclusively in goats' isolates, while the seh gene was only identified in cows' isolates. The number of toxin genes per strain showed that goats' isolates are likely more toxic than bovines' isolates. The cap5 genotype predominated in both host species, especially in strains collected from cows raised in the Agreste region. The cap8 genotype is likely more virulent due to the number of virulence genes per strain. The results of the present study demonstrate that S. aureus may pose a potential threat to human health in Brazil, and, therefore, these results should support actions related to mastitis control programs.(AU)

O presente estudo determinou a frequência de genes de virulência de Staphylococcus aureus em 2253 amostras de leite, sendo de vacas n=1000 e de cabras n=1253, procedentes das três regiões geográficas do estado de Pernambuco, Brasil. A presença de genes de fatores de virulência associados à adesão às células hospedeiras (fnbA, fnbB, clfA e clfB), toxinosis (sea, seb, sec, sed, seg, seh, sei, tsst, hla e hlb) e polissacarídeo capsular (cap5 e cap8) foram avaliadas por PCR. Um total de 123 e 27 cepas de S. aureus foram isoladas do leite de vacas e cabras, respectivamente. Os genes sec e tsst foram detectados exclusivamente em isolados de cabras, enquanto o gene seh foi identificado apenas em isolados de vaca. O número de genes de toxina por cepa mostrou que os isolados de cabras são potencialmente mais tóxicos do que os isolados obtidos de bovinos. O genótipo cap5 predominou em ambas as espécies hospedeiras, especialmente em cepas coletadas de vacas criadas na região Agreste. O genótipo cap8 é potencialmente mais virulento devido ao número de genes de virulência por isolado. Os resultados do presente estudo demonstram que S. aureus pode representar uma ameaça potencial para a saúde humana no Brasil e, portanto, estes resultados devem subsidiar ações relacionadas aos programas de controle de mastite.(AU)

Identification and antimicrobial suceptibility profile of bacteria causing bovine mastitis from dairy farms in Pelotas, Rio Grande do Sul / Identificação e perfil de suscetibilidade antimicrobiana de bactérias causadoras de mastite bovina em propriedades leiteiras de Pelotas, Rio Grande do Sul

Abstract Mastitis is an inflammatory process of the udder tissue caused mainly by the bacteria Staphylococcus aureus. The indiscriminate use of antibiotics fosters conditions that favor the selection of resistant microorganisms, suppressing at the same time susceptible forms, causing a serious problem in dairy cattle. Given the importance in performing an antibiogram to select the most adequate antimicrobial therapy, the aim of this study was to identify bacteria isolated from cow's milk with mastitis, in dairy farms situated in the city of Pelotas, Rio Grande do Sul, and to determinate the susceptibility profile of these isolates against the antibiotics used to treat this illness. A total of 30 isolates of Staphylococcus spp., were selected from milk samples from the udder quarters with subclinical mastitis whose species were identified through the Vitek system. The susceptibility profile was performed by the disk diffusion assay, against: ampicillin, amoxicillin, bacitracin, cephalexin, ceftiofur, enrofloxacin, gentamicin, neomycin, norfloxacin, penicillin G, tetracycline and trimethoprim. In the antibiogram, 100.0% of the isolates were resistant to trimethoprim and 96.7% to tetracycline and neomycin, three strains of Staphylococcus spp., (10.0%) presented resistance to the 12 antibiotics tested and 24 (80.0%) to at least eight. These results showed the difficulty in treating mastitis, due to the pathogens' resistance.

Resumo A mastite se constitui no processo inflamatório da glândula mamária causada principalmente por bactérias Staphylococcus aureus. O uso indiscriminado dos antibióticos promove condições que favorecem a seleção de micro-organismos resistentes e, ao mesmo tempo, suprime formas suscetíveis, causando um grave problema para a bovinocultura leiteira. Tendo em vista a importância da realização do antibiograma para a seleção da terapia antimicrobiana mais adequada, o objetivo deste estudo foi identificar bactérias isoladas de leite de vaca com mastite, oriundas de propriedades leiteiras localizadas na cidade de Pelotas, RS, bem como determinar o perfil de suscetibilidade desses isolados frente a antibióticos usados para o tratamento desta doença. Foram selecionados 30 isolados de Staphylococcus spp. de amostras de leite provenientes de quartos mamários com mastite subclínica, cujas espécies foram identificadas através do sistema Vitek. O perfil de suscetibilidade foi realizado pela técnica de difusão em disco, frente a: ampicilina, amoxicilina, bacitracina, cefalexina, ceftiofur, enrofloxacina, gentamicina, neomicina, norfloxacina, penicilina G, tetraciclina e trimetoprima. No antibiograma, 100,0% dos isolados foram resistentes a trimetoprima e 96,7% a tetraciclina e a neomicina, três cepas (10,0%) foram resistentes aos 12 antibióticos testados e 24 (80,0%) a pelo menos oito. Esses resultados demonstram a dificuldade encontrada no tratamento da mastite devido à resistência dos agentes patológicos.

Relationship between virulence factor genes in coagulase-negative Staphylococcus spp. and failure of antimicrobial treatment of subclinical mastitis in sheep / Relação entre genes de fatores de virulência em Staphylococcus spp. coagulase-negativos e a falha do tratamento antimicrobiano da mastite subclínica ovina

Coagulase-negative Staphylococcus spp. (CNS) are the main microorganisms involved in ovine mastitis. Treatment at the end of lactation can contribute towards cure and prevention of subclinical cases during the subsequent lactation. However, virulence factors and resistance mechanisms presented by CNS can decrease cure rates. The aims of the study were to identify the species of CNS in milk of mastitic ewes with and without antimicrobial treatment, and to investigate the presence of genes relating to resistance of β-lactam antimicrobials, formation of biofilms, production of enterotoxins and production of the toxic shock syndrome toxin. Cases of failure in the treatment were related with the presence/absence of the respective genes. Sixty sheep were divided into three groups: G1, without treatment; G2, animals treated via the intramammary route with 100mg of cloxacillin during drying off; and G3, sheep treated via the intramammary route with 50 mg of nanoparticulate cloxacillin. Milk samples were gathered during drying off and 15 and 30 days after the parturition of the subsequent lactation. The analyses to identify the species of CNS were carried out by means of the internal transcribe spacer technique and the investigation of the genes responsible for the virulence factors and resistance to oxacillin was performed using the polymerase chain reaction (PCR) technique. No sample was positive for the mecA gene. The only gene relating to production of enterotoxins was sec. Among the genes relating to production of biofilm, icaD was the only one identified in the three experimental groups. Staphylococcus warneri was the main species of CNS isolated during the pre and post-partum periods of the sheep. The species carrying genes relating to production of enterotoxins and biofilms were present in uncured sheep.(AU)

Staphylococus spp. coagulase-negativos (SCN) estão entre os principais micro-organismos envolvidos na mastite ovina. O tratamento ao final da lactação pode contribuir com a cura e a prevenção de casos subclínicos durante a lactação seguinte. Todavia, fatores de virulência e mecanismos de resistência apresentados por SCN podem reduzir as taxas de cura. Os objetivos desse estudo foram identificar as espécies de SCN no leite de ovelhas com mastite com e sem tratamento antimicrobiano e investigar a presença de genes relacionados com resistência a antibióticos beta lactâmicos, formação de biofilmes, produção de enterotoxinas e produção da toxina da síndrome do choque tóxico. Casos de falhas no tratamento foram relacionados com a presença/ausência dos respectivos genes. Sessenta ovelhas foram divididas em três grupos: G1, sem tratamento; G2, animais tratados via intramamária com 100mg de cloxacilina antes da secagem; e G3, ovelhas tratadas via intramamária com 50 mg de cloxacilina nanoparticulada. Amostras de leite foram obtidas durante a secagem e 15 e 30 dias depois do parto na lactação seguinte. As análises para identificar as espécies de SCN foram conduzidas por meio da técnica de Internal transcribe spacer e a investigação dos genes responsáveis pelos fatores de virulência e resistência à oxacilina foi realizada usando a técnica reação em cadeia da polimerase. Nenhuma amostra foi positiva para o gene mecA. O único gene relacionado com a produção de enterotoxinas foi o sec. Dentre os genes relacionados com a produção de biofilme, icaD foi o único identificado nos três grupos experimentais. Staphylococcus warneri foi a principal espécie de SCN isolada durante o pré e pós-parto. As espécies que apresentaram genes relacionados com a produção de enterotoxinas e biofilmes estavam presentes nas ovelhas não curadas.(AU)

Antifungal susceptibility profile of diferent yeasts isolates from wild animals, cow's milk with subclinical mastitis and hospital environment / Perfil de suscetibilidade antifúngica de diferentes leveduras isoladas da microbiota de animais silvestres, leite com mastite subclínica e ambiente hospitalar

Abstract Yeast infections have acquired great importance due to increasing frequency in immunocompromised patients or patients undergoing invasive diagnostic and therapeutic techniques, and also because of its high morbidity and mortality. At the same time, it has been seen an increase in the emergence of new pathogenic species difficult to diagnose and treat. The aim of this study was to determine the in vitro susceptibility of 89 yeasts from different sources against the antifungals amphotericin B, voriconazole, fluconazole and flucytosine, using the VITEK® 2 Compact system. The antifungal susceptibility was performed automatically by the Vitek® 2 Compact system. The origin of the yeasts was: Group 1 - microbiota of wild animals (W) (26/89), 2 - cow's milk with subclinical mastitis (M) (27/89) and 3 - hospital enviorment (H) (36/89). Of the 89 yeasts submitted to the Vitek® 2 test, 25 (20.9%) were resistant to fluconazole, 11 (12.36%) to amphotericin B, 3 (3.37%) to voriconazole, and no sample was resistant to flucytosine. Regarding the minimum inhibitory concentration (MIC), fluconazole showed an MIC between 1 and 64 mg/mL for the three groups, voriconazole had an MIC between 0.12 and 8 mg/mL, amphotericin B had an MIC between 0.25 and 4 mg/mL for group H and group W respectively, between 0.25 and 16 mg/mL for group M and flucytosine had an MIC equal to 1μg/mL for all groups. The yeasts isolated from the H group showed the highest resistance to fluconazole 12/89 (13.49%), followed by group W (7.87%) and group M (5.62%). The more resistant group to voriconazole was followed by the M and H groups, the W group showed no resistance to this antifungal. Group H was the least resistant (2.25%) to amphotericin.

Resumo As infecções por leveduras têm adquirido grande importância, devido ao aumento da sua frequência em pacientes imunocomprometidos ou pacientes submetidos a técnicas diagnosticas e terapêuticas agressivas, e devido sua alta morbidade e mortalidade. Paralelamente tem-se observado um incremento na aparição de novas espécies patógenas difíceis de diagnosticar e tratar. O objetivo desse estudo foi avaliar a suscetibilidade in vitro de 89 leveduras de diferentes origens frente aos antifúngicos Anfotericina B, Voriconazol, Fluconazol e Fluocitocina pelo Sistema Vitek® 2. O antifungigrama foi realizado automaticamente pelo Vitek® 2 Compact. A origem das leveduras foi: Grupo 1- Microbiota de Animais Silvestres (S) (26/89), 2- Leite com mastite bovina subclínica (L) (27/89) e 3- Ambiente Hospitalar (H) (36/89). Das 89 leveduras submetidas à carta Vitek®, 25 (20.09%) foram resistentes ao fluconazol, oito (8.99%) à anfotericina B, três (3.37%) ao voriconazol, e nenhuma amostra mostrou-se resistente a fluocitosina. O grupo três (H) foi mais resistente ao fluconazol que os demais, já o dois (L) foi mais resistente ao voriconazol e a anfotericina B que os outros dois. O fluconazol pode ter apresentado maior número de resistências devido ser um fármaco comumente usado principalmente em humanos. As leveduras isoladas de humanos apresentaram maior número de resistências aos fármacos testados do que as leveduras isoladas de animais silvestres. O que pode ocorrer devido a uma maior exposição dos humanos aos fármacos em relação aos animais que vivem isolados em ambientes selvagens e na maioria dos casos nunca teve contato com fármacos de qualquer origem.

Factors associated with microbiological and clinical cure of mastitis in dairy cows / Fatores associados à cura microbiológica e clínica da mastite em vacas leiteiras

709 clinical mastitis cases were analyzed and treated with antimicrobial combination cephalexin-neomycin and the anti-inflammatory prednisolone. A sample of milk was collected to perform a microbiological culture before starting the treatment and 14 days later. Somatic cell count (SCC) was obtained from samples collected on the day of the clinical case (D0), 14 days after (D14) and 28 days after (D28). Of the total, 435 (61.4%) at the D0 exhibited growth of microorganisms. Of the isolated agents, 365 (84%) were Gram-positive, and 66 (16%) were Gram-negative. A clinical cure was achieved in 63% of cases. Bacteriological cure occurred in 75% of cases. Only at D28 after the clinical case a significant SCC reduction was verified. The logistic regression for clinical cure showed significant effects for days in milk and parity (P< 0.05). For bacteriological cure, there were significant effects of Log (SCC) D0; clinical cure and quarter affected (P< 0.05). In the principal component analysis, the Temperature-Humidity Index was associated with reduced clinical cure of clinical mastitis cases.(AU)

Setecentos e nove casos clínicos de mastite foram analisados e tratados com combinação antimicrobiana à base de cefalexina-neomicina e o anti-inflamatório prednisolona. Uma amostra de leite foi coletada para realização de cultura microbiológica antes do início do tratamento e 14 dias depois. A contagem de células somáticas (SCC) foi obtida de amostras coletadas no dia do caso clínico (D0), 14 dias após (D14) e 28 dias após (D28). Do total, 435 (61,4%) no D0 apresentaram crescimento de microrganismos, enquanto em 274 (38,6%) não houve crescimento. Dos agentes isolados, 365 (84%) eram Gram-positivos e 66 (16%) eram Gram-negativos. A cura clínica foi alcançada em 63% dos casos. A cura bacteriológica ocorreu em 75% dos casos. Apenas no D28 verificou-se uma redução significativa na SCC. A regressão logística para a cura clínica mostrou efeitos significativos para dias em lactação e paridade (P<0,05). Para a cura bacteriológica, houve efeitos significativos de Log (SCC) D0; cura clínica e quarto afetado (P<0,05). Na análise do componente principal, o índice de temperatura-umidade foi associado com a redução da cura clínica dos casos clínicos da mastite.(AU)

Produção de biofilme por Staphylococcus chromogenes isolados de amostras de leite provenientes de rebanhos bovinos com mastite / Biofilme production by Staphylococcus chromogenes isolated from milk samples from bovine with mastitis

Foram estudadas 135 vacas mestiças, provenientes de 10 rebanhos leiteiros no estado do Acre. O objetivo foi identificar espécies de Staphylococcus isoladas dos quartos mamários de vacas com mastite e, posteriormente, avaliar a capacidade de produção de biofilme pela espécie Staphylococcus chromogenes. A caracterização dos isolados presentes nas amostras encontradas, correspondentes a Staphylococcus sp., foi realizada utilizando-se a técnica do MALDI TOF MS (Matrix Associated Laser Desorption-Ionization - Time of Flight - Mass Spectrometry). Foram identificados: S. chromogenes (36), Staphylococcus saprophyticus (5), S. chromogenes ou Staphylococcus hycus (5), Staphylococcus haemolyticus (4), Staphylococcus epidermidis (3), Staphylococcus hycus (3), Staphylococcus aureus (1), Staphylococcus auriculares (1), Staphylococcus kloosii (1) e Staphylococcus xylosus (1). A espécie S. chromogenes correspondeu a 60% dos isolados do gênero (17 isolados coagularam o plasma de coelho no teste da coagulase em tubo), sendo 83,3% dos isolados (30/36) produtores de biofilme, não estando esse fator de virulência associado ao fenótipo de coagulação do plasma. A identificação desses microrganismos é importante para a elucidação da etiologia da mastite bovina. O alto percentual de S. chromogenes, produtores de biofilme, isolados de vacas com mastite é um achado relevante e pode revelar uma mudança de perfil na colonização de agentes etiológicos causadores dessa enfermidade.(AU)

A total of 135 crossbred cows was studied, from ten dairy herds on the state of Acre. The purpose was to identify species of Staphylococcus isolated from the mammary quarters of cows with mastitis. Additionally, the capacity of biofilm production by the species Staphylococcus chromogenes was evaluated. The sample characterization of the isolates was performed using the MALDI TOF MS (Matrix Associated Laser Desorption-Ionization - Time of Flight - Mass Spectrometry). The following species were identified by MALDI TOFF MS: S. chromogenes (36), Staphylococcus saprophyticus (5), S. chromogenes or Staphylococcus hycus (5), Staphylococcus haemolyticus (4), Staphylococcus epidermidis (3), Staphylococcus hycus (3), Staphylococcus aureus (1), Staphylococcus auriculares (1), Staphylococcus kloosii (1) and Staphylococcus xylosus (1). S. chromogenes corresponded to 60% of the Staphylococcus isolates (17 isolates positive on tube coagulase test). From those, 83.33% (30/36) of them were biofilm producers. This virulence factor had no association with the plasma coagulation phenotype. The identification of these microorganisms is important for the elucidation of the bovine mastitis etiology. The high percentage of S. chromogenes, biofilm producers, isolated from cows with mastitis is an important finding. This may reveal a profile change on the colonization of etiologic agents that cause this disease.(AU)

Streptococcus uberis: In vitro biofilm production in response to carbohydrates and skim milk / Streptococcus uberis: producción in vitro de biofilm en respuesta a carbohidratos y leche descremada

Streptococcus uberis has become one of the most important environmental pathogens associated with clinical and subclinical bovine mastitis. Biofilm confers to bacteria more resistance to physical and chemical agents as well as to different mechanisms of the innate immune system. The aim of this work was to evaluate the ability of in vitro biofilm production in 32 S. uberis isolates from bovine mastitis and identified by biochemical tests and milk subsequently confirmed by the amplification of the pauA gene. The isolates were cultivated in TMP broth and TMP broth with the addition of 0.5% glucose, 1% sucrose, 1% lactose or 0.5% skim milk in microtiter plates stained with crystal violet. We demonstrated that S. uberis isolated from bovine mastitis are able to produce biofilms in TMP broth and, also that biofilm formation by S. uberis can be significantly enhanced by the addition of 0.5% glucose or 1% sucrose to TMP broth. This may suggest that the carbohydrates in milk or within the ruminant gut might affect the growth mode of S. uberis. In addition, our results showed that in vitro biofilm production under different conditions of supplementation displays variation among the isolates and that each isolate shows a particular profile of biofilm production. This phenotypic heterogeneity in biofilm production exhibited by S. uberis could at least partly explain why this bacterium has the ability to adapt to different niches facilitating survival to diverse and stressful conditions.

Streptococcus uberis es uno de los más importantes patógenos medioambientales asociados a la mastitis bovina clínica y subclínica. El biofilm confiere a las bacterias resistencia a agentes físicos y químicos, como así también a diferentes mecanismos del sistema inmune innato. El objetivo del presente estudio fue evaluar la habilidad de producción de biofilm in vitro de 32 aislamientos de S. uberis recuperados de mastitis bovina, previamente identificados por pruebas bioquímicas y confirmados por la amplificación del gen pauA. Los aislamientos fueron cultivados en caldo TMP sin carbohidratos, y además en caldo TMP con la adición de 0,5% de glucosa, 1% de sacarosa, 1% de lactosa o 0,5% de leche descremada, en placas de microtitulación tenidas con cristal violeta. Se demostró que dichos aislamientos son capaces de producir biofilm en caldo TMP, y además se observó un incremento significativo en la producción de biofilm en caldo TMP suplementado con 0,5% de glucosa o con 1% de sacarosa. Así, los carbohidratos de la leche o los presentes dentro del intestino de los rumiantes podrían afectar el modo de crecimiento de S. uberis. Además, nuestros hallazgos mostraron que la producción de biofilm in vitro en diferentes condiciones de suplementación presenta variabilidad entre los aislamientos de S. uberis y que cada aislamiento muestra un perfil particular de producción de biofilm. Esta heterogeneidad fenotípica en la producción de biofilm de S. uberis podría explicar, al menos en parte, por qué esta bacteria tiene la habilidad de adaptarse a diferentes nichos, lo que le facilita la supervivencia frente a condiciones diversas y estresantes.

Molecular epidemiology of Streptococcus agalactiae isolated from mastitis in Brazilian dairy herds

Abstract Streptococcus agalactiae is one of the most common pathogens leading to mastitis in dairy herds worldwide; consequently, the pathogen causes major economic losses for affected farmers. In this study, multilocus sequence typing (MLST), genotypic capsular typing by multiplex polymerase chain reaction (PCR), and virulence gene detection were performed to address the molecular epidemiology of 59 bovine (mastitis) S. agalactiae isolates from 36 dairy farms located in the largest milk-producing mesoregions in Brazil (Minas Gerais, São Paulo, Paraná, and Pernambuco). We screened for the virulence genes bac, bca, bibA, cfb, hylB, fbsA, fbsB, PI-1, PI-2a, and PI-2b, which are associated with adhesion, invasion, tissue damage, and/or immune evasion. Furthermore, five capsular types were identified (Ia, Ib, II, III, and IV), and a few isolates were classified as non-typeable (NT). MLST revealed the following eight sequence types (STs): ST-61, ST-67, ST-103, ST-146, ST-226, ST-314, and ST-570, which were clustered in five clonal complexes (CC64, CC67, CC103, CC17, and CC314), and one singleton, ST-91. Among the virulence genes screened in this study, PI-2b, fbsB, cfb, and hylB appear to be the most important during mastitis development in cattle. Collectively, these results establish the molecular epidemiology of S. agalactiae isolated from cows in Brazilian herds. We believe that the data presented here provide a foundation for future research aimed at developing and implementing new preventative and treatment options for mastitis caused by S. agalactiae.

Species identification and antimicrobial susceptibility profile of bacteria causing subclinical mastitis in buffalo / Identificação de espécies e perfil de susceptibilidade antimicrobiano de bactérias causadoras de mastite subclínica em búfalos

Microorganisms causing subclinical mastitis in water buffalo were isolated from 20 buffalo milk samples at four dairy farms located in central region of São Paulo State, Brazil, through testing of somatic cell count (SCC), standard plate count (SPC), biochemical, PCR assays and antimicrobial profile. The SCC showed average of 721,000 cells/mL in the milk, indicating the presence of subclinical mastitis. The overall average for SPC was 1.8 x 104 CFU/mL. The microorganism most frequently isolation according to biochemical tests were: Staphylococcus epidermidis (17%), Staphylococcus aureus (15%), Bacillus spp. (14%), Acinetobacter spp. (12.5%); with intermediate frequency: Pseudomonas aeruginosa (9.5%); Shigella flexneri (7.0%), Streptococcus spp. (5.5%), Corynebacterium spp. (5.0%), Escherichia coli (4.5%), Serratia marcescens (4.0%), Stenotrophomonas maltophilia (4.0%), and low incidence: Klebsiella rhinoscleromatis (0.5%), Klebsiella ozaenae (0.5%), Tatumella ptyseos (0.5%), Enterobacter cloacae (0.5%). The molecular analysis indicated that samples positive by culture method of the genera Staphylococcus, Streptococcus and E. coli were positive by PCR. Para S. aureus and S. epidermidis the highest percentages of observed sensitivity were gentamicin (100%) and vancomycin (100%); for the genus Streptococcus to gentamicin and oxacillin and E. coli to Ampicilin. These findings may help in the control and treatment of subclinical mastitis in buffaloes and contribute to improving the efficiency and quality of the milk produced.(AU)

Microrganismos causadores de mastites subclínicas em búfalas foram isolados desde 20 amostras de leite de búfalos de quatro granjas leiteiras localizadas na região central do Estado de São Paulo, Brasil, através dos testes contagem de células somáticas (CCS), contagem padrão em placas (CPP), provas bioquímicas, reações de PCR e perfil antimicrobiano. A CCS apresentou uma mediana de 721.000 cel/mL no leite, indicando presença de mastite subclínica. A média geral de CPP foi de 1,8x104 UFC/mL. Os microrganismos com maior frequência de isolamento segundo os testes bioquímicos foram: Staphylococcus epidermidis (17%), Staphylococcus aureus (15%), Bacillus spp. (14%), Acinetobacter spp. (12,5%); frequência intermediaria: Pseudomonas aeruginosa (9,5%); Shigella flexneri (7,0%), Streptococcus spp. (5,5%), Corynebacterium spp. (5,0%), Escherichia coli (4,5%), Serratia marcescens (4,0%), Stenotrophomonas maltophilia (4,0%), e baixa incidência: Klebsiella rhinoscleromatis (0,5%), Klebsiella ozaenae (0,5%), Tatumella ptyseos (0,5%), Enterobacter cloacae (0,5%). A análise molecular indicou que as amostras positivas pelo método de cultura dos gêneros Staphylococcus, Streptococcus e Escherichia coli foram positivas por PCR. Para S. aureus e S. epidermidis os maiores percentuais de sensibilidade observados foram gentamicina (100%) e vancomicina (100%); para o gênero Streptococcus à gentamicina e oxacilina e para E. coli à ampicilina. Este resultados podem ajudar no controle e tratamento da mastite subclínica em búfalos e contribuir para melhorar a eficiência e qualidade do leite produzido.(AU)

Biofilm production and beta-lactamic resistance in Brazilian Staphylococcus aureus isolates from bovine mastitis

Abstract Staphylococcus spp. play an important role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most relevant species due to the production of virulence factors such as slime, which is required for biofilm formation. This study aimed to evaluate biofilm production and its possible relation to beta-lactamic resistance in 20 S. aureus isolates from bovine mastitic milk. The isolates were characterized by pheno-genotypic and MALDI TOF-MS assays and tested for genes such as icaA, icaD, bap, agr RNAIII, agr I, agr II, agr III, and agr IV, which are related to slime production and its regulation. Biofilm production in microplates was evaluated considering the intervals determined along the bacterial growth curve. In addition, to determine the most suitable time interval for biofilm analysis, scanning electron microscopy was performed. Furthermore, genes such as mecA and blaZ that are related to beta-lactamic resistance and oxacillin susceptibility were tested. All the studied isolates were biofilm producers and mostly presented icaA and icaD. The Agr type II genes were significantly prevalent. According to the SEM, gradual changes in the bacterial arrangement were observed during biofilm formation along the growth curve phases, and the peak was reached at the stationary phase. In this study, the penicillin resistance was related to the production of beta-lactamase, and the high minimal bactericidal concentration for cefoxitin was possibly associated with biofilm protection. Therefore, further studies are warranted to better understand biofilm formation, possibly contributing to our knowledge about bacterial resistance in vivo.

The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) identification versus biochemical tests: a study with enterobacteria from a dairy cattle environment

Abstract Mastitis adversely affects milk production and in general cows do not regain their full production levels post recovery, leading to considerable economic losses. Moreover the percentage decrease in milk production depends on the specific pathogen that caused the infection and enterobacteria are responsible for this greater reduction. Phenotypic tests are among the currently available methods used worldwide to identify enterobacteria; however they tend to misdiagnose the species despite the multiple tests carried out. On the other hand The Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) technique has been attracting attention for its precise identification of several microorganisms at species level. In the current study, 183 enterobacteria were detected in milk (n = 47) and fecal samples (n = 94) from cows, and samples from water (n = 23) and milk lines (n = 19). All these samples were collected from a farm in Rio de Janeiro with the specific purpose of presenting the MALDI-TOF MS technique as an efficient methodology to identify Enterobacteriaceae from bovine environments. The MALDI-TOF MS technique results matched the biochemical test results in 92.9% (170/183) of the enterobacteria species and the gyrB sequencing confirmed 100% of the proteomic technique results. The amino acid decarboxylation test made the most misidentifications and Enterobacter spp. was the most misidentified genus (76.9%, 10/13). These results aim to clarify the current biochemical errors in enterobacteria identification, considering isolates from a bovine environment, and show the importance for more careful readings of phenotypic tests which are often used in veterinary microbiology laboratories.

Expression of CD14 and toll-like receptors 2 and 4 by milk neutrophils in bovine mammary glands infected with Corynebacterium bovis / Expressão de CD14 e dos receptores do tipo toll 2 e 4 por neutrófilos lácteos provenientes de glândulas infectadas por Corynebacterium bovis

This study evaluated the expression of CD14, toll-like receptor (TLR) 2 and TLR4 on the surface of milk neutrophils in bovine mammary glands infected with Corynebacterium bovis. Here, we used 23 culture-negative control quarters with no abnormal secretion on the strip cup test and milk somatic cell count lower than 1x105 cells/mL, and 14 C. bovis infected quarters. The identification of neutrophils, as well as, the percentage of neutrophils that expressed CD14, TLR2 and TLR4 were analyzed by flow cytometry using monoclonal antibodies. The present study encountered no significant difference in the percentages of milk neutrophils that expressed TLR2 and TLR4 or in the expression of TLR4 by milk neutrophils. Conversely, a lower median fluorescence intensity of TLR2 in milk neutrophils was observed in C. bovis-infected quarters. The percentage of neutrophils that expressed CD14 and the median fluorescence intensity of CD14 in milk neutrophils was also lower in C. bovis-infected quarters...

O presente estudo objetivou avaliar alterações na expressão de CD14, e dos receptores do tipo toll (TLR) 2 e 4 na superfície de neutrófilos lácteos provenientes de glândulas mamárias infectadas por Corynebacterium bovis. O presente estudo utilizou 23 quartos negativos no exame bacteriológico, sem alterações na prova de fundo escuro e com contagem automática de células somáticas menor que 1 x105 células/mL, e 14 quartos mamários infectados por C. bovis A identificação de neutrófilos, assim como a porcentagem de neutrófilos lácteos que expressaram CD14, TLR2 e 4 foram avaliadas por citometria de fluxo utilizando anticorpos monoclonais. A porcentagem de neutrófilos que expressaram TLR2 e TLR4 nos quartos mamários infectados por C. bovis não diferiu dos quartos mamários sadios, assim como na expressão de TLR4. No entanto, a intensidade de fluorescência do TLR2 na superfície dos neutrófilos foi menor nos quartos mamários infectados por C. bovis. A porcentagem de neutrófilos que expressaram CD14 e a intensidade de fluorescência da molécula de CD14 foi menor na superfície dos neutrófilos lácteos dos quartos infectados por C. bovis...

Factores de virulencia de Staphylococcus aureus asociados con infecciones mamarias en bovinos: relevancia y rol como agentes inmunógenos / [Virulence factors of Staphylococcus aureus associated with intramammary infections in cows: relevance and role as immunogens].

Staphylococcus aureus is the most prevalent mastitis pathogen in Argentina and worldwide. Lack of effectiveness of traditional control measures based on milking hygiene and antibiotic therapy against this organism has led to the development of alternatives directed to prevent the disease. Among them, the manipulation of host immune mechanisms through vaccination has been explored. The identification of virulence factors able to stimulate host immune defenses is key to developing a rational vaccine. S. aureus has multiple virulence factors that interact with the host at different stages of an intramammary infection. The use of some of these factors as immunogens has been shown to elicit protective responses in the host. The structure, function, and use as immunogens of S. aureus virulence factors considered to be relevant at different stages of intrammamary infections caused by this organism are reviewed in this article.

Prevalence and antibiotic resistance of mastitis pathogens isolated from dairy herds transitioning to organic management

Changes in udder health and antibiotic resistance of mastitis pathogens isolated from dairies upon conversion from conventional to organic management over a 3-year period was studied. Coagulase-negative staphylococci (CNS) were the most prevalent mastitis pathogens isolated. CNS were significantly less resistant to beta-lactam antibiotics when isolated from milk after the herd transitioned to organic management. Cessation of the use of antimicrobial therapies in dairies in combination with organic management could lead to a reduction in the antimicrobial resistance of mastitis pathogens.

Breed and season influence on milk quality parameters and in mastitis occurrence / Influência da raça e estação em aspectos de qualidade do leite e na ocorrência de mastites

The aims of the present study were to evaluate the performance of Jersey and Holstein cows under different rainfall conditions (dry and rainy seasons) by monitoring aspects related to subclinical mastitis (somatic cell count, microbiological isolation, type of isolated pathogen), milk quality (lactose, protein, fat, total solids) and production (mean milk production) of both breeds. The study was carried out in a dairy farm located in the state of São Paulo, Brazil. Eight visitations were done to the farm, four in a period of high rainfall and four in a period of low rainfall. Milk samples were collected from 79 Holstein cows and 37 Jersey cows for electronic somatic cell count and determination of the main milk components (protein, fat, total solids, lactose). Milk fat, protein, total solids and production were influenced by breed and the season, with similar tendencies for both breeds in both seasons. Somatic cell count (SCC) showed similar results for both breeds. Holstein cows with intramammary infections (IMI) presented a higher increase in SCC when compared to Jersey cows (P<0.001). In the dry season, 53 animals had IMI in at least one month during the study, which 32 were Holstein and 21 were Jersey cows. In the rainy season, 65 animals had intramammary infection, being 43 Holstein and 22 Jersey cows. The frequency of IMI cases was larger in the rainy season than in the dry season. Jersey cows had a lower chance of showing IMI signs and symptoms than Holstein cows in the rainy season (odds ratio=0.52). The larger number of IMI cases in the rainy season may have led to a lower milk lactose rate for both breeds, thus milk lactose rate can be considered an indicator of IMI status. There was prevalence of contagious pathogens overall in the study. The applied model showed that environmental pathogens were more frequently isolated from the breed Jersey, regardless of the study season. There seems to be differences in the immune response of Jersey and Holstein breeds.

Os objetivos do presente estudo foram avaliar a performance das raças Jersey e Holandesa em diferentes condições de pluviosidade (estações seca e chuvosa) através do monitoramento de aspectos relacionados com a mastite subclínica (contagem de células somáticas, isolamento microbiológico, tipo de patógeno isolado), qualidade do leite (lactose, proteína, gordura e sólidos totais) e produção (media da produção de leite) de ambas as raças. O estudo foi conduzido em propriedade leiteira localizada no estado de São Paulo, Brasil. Oito visitas foram realizadas a propriedade, quatro em período de alta pluviosidade e outro em período de baixa pluviosidade. Amostras de leite foram colhidas de todos os animais para contagem eletrônica de células somáticas e determinação dos principais componentes do leite (proteína, gordura, sólidos totais, lactose). Gordura, proteína, sólidos totais e produção leiteira foram influenciadas tanto pela raça como pela estação, apresentando tendências similares para ambas raças em ambas estações. A contagem de células somáticas (CCS) apresentou resultados similares para ambas as raças. Vacas holandesas com infecção intramamária (IIM) apresentaram um maior aumento na CCS quando comparadas as vacas Jersey (P<0.001). Na estação seca, 53 animais tiveram IIM em pelo menos um mês do estudo. Destes, 32 eram da raça Holandesa e 21 da raça Jersey. Na estação chuvosa, 65 animais possuíram infecção intramamária, dos quais 43 eram da raça Holandesa e 22 da raça Jersey. A frequência de casos de IIM foi maior na estação chuvosa em comparação à estação seca. Vacas Jersey apresentaram uma menor chance de desenvolver sinais e sintomas de IIM em relação às vacas holandesas na estação chuvosa (razão de chances=0.52). O maior numero de casos de IIM na estação chuvosa pode ter contribuído para uma menor taxa de lactose no leite para ambas as raças, sendo que esta pode ser considerada como um indicador do status de IIM. Existiu uma prevalência de patógenos contagiosos durante todo o experimento. O modelo estatístico aplicado mostrou que patógenos ambientais foram mais frequentemente isolados da raça Jersey, independentemente do período do estudo. Aparentemente existem diferenças na resposta imune das raças Jersey e Holandesa.