Etiology, antimicrobial susceptibility profile of Staphylococcus spp. and risk factors associated with bovine mastitis in the states of Bahia and Pernambuco

The purpose of this paper was to study the etiology of mastitis, determine the antimicrobial susceptibility profile of Staphylococcus spp. and to identify the risk factors associated with infection in dairy cows in the states of Bahia and Pernambuco, Brazil. From the 2,064 milk samples analyzed, 2.6% were associated with cases of clinical mastitis and 28.2% with subclinical mastitis. In the microbiological culture, Staphylococcus spp. (49.1%) and Corynebacterium spp. (35.3%) were the main agents found, followed by Prototheca spp. (4.6%) and Gram negative bacilli (3.6%). In the antimicrobial susceptibility testing, all 218 Staphylococcus spp. were susceptible to rifampicin and the least effective drug was amoxicillin (32.6%). Multidrug resistance to three or more drugs was observed in 65.6% of Staphylococcus spp. The risk factors identified for mastitis were the extensive production system, not providing feed supplements, teat drying process, not disinfecting the teats before and after milking, and inadequate hygiene habits of the milking workers. The presence of multiresistant isolates in bovine milk demonstrates the importance of the choice and appropriate use of antimicrobial agents. Prophylactic and control measures, including teat antisepsis and best practices for achieving hygienic milking should be established in order to prevent new cases of the disease in herds.

Atividade antimicrobiana do oleorresina de copaíba (Copaifera reticulata) frente a Staphylococcus coagulase positiva isolados de casos de otite em cães

O objetivo do presente trabalho foi investigar o potencial antimicrobiano do oleorresina de Copaifera reticulata Ducke em isolados de Staphylococcus coagulase positiva (SCP) provenientes de casos de otite externa em cães. O método de microdiluição em caldo foi utilizado para determinação da concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) de oleorresina de copaíba. Em adição, foi determinado o perfil de suscetibilidade aos antimicrobianos dos isolados de SCP pelo método de difusão em ágar. Oito classes de antimicrobianos foram usadas para o cálculo de multirresistência antimicrobiana. A determinação da composição química do oleorresina de copaíba foi realizada por cromatografia em fase gasosa acoplada à espectrometria de massas (GC/MS), sendo que β-cariofileno, β-bisaboleno e (E)-α-bergamoteno foram os compostos majoritários. O oleorresina de copaíba demonstrou CIM90 de 0,164mg/mL e CBM90 de 1,31mg/mL. A multirresistência foi verificada em 27% das cepas testadas. Os resultados sugerem que o oleorresina de copaíba exerceu atividade bacteriostática e bactericida mesmo em cepas multirresistentes de Staphylococcus coagulase-positiva.

NaOCl effect on biofilm produced by Staphylococcus aureus isolated from the milking environment and mastitis infected cows

Biofilms constitute a physical barrier, protecting the encased bacteria from detergents and sanitizers. The objective of this work was to analyze the effectiveness of sodium hypochlorite (NaOCl) against strains of Staphylococcus aureus isolated from raw milk of cows with subclinical mastitis and Staphylococcus aureus isolated from the milking environment (blowers and milk conducting tubes). The results revealed that, in the presence of NaOCl (150ppm), the number of adhered cells of the twelve S. aureus strains was significantly reduced. When the same strains were evaluated in biofilm condition, different results were obtained. It was found that, after a contact period of five minutes with NaOCl (150ppm), four strains (two strains from milk , one from the blowers and one from a conductive rubber) were still able to grow. Although with the increasing contact time between the bacteria and the NaOCl (150ppm), no growth was detected for any of the strains. Concerning the efficiency of NaOCl on total biofilm biomass formation by each S. aureus strain, a decrease was observed when these strains were in contact with 150 ppm NaOCl for a total period of 10 minutes. This study highlights the importance of a correct sanitation protocol of all the milk processing units which can indeed significantly reduce the presence of microorganisms, leading to a decrease of cow´s mastitis and milk contamination.

Beta-lactamase detection in Staphylococcus aureus and coagulase-negative Staphylococcus isolated from bovine mastitis

The objectives of the study were to evaluate the presence/production of beta-lactamases by both phenotypic and genotypic methods, verify whether results are dependent of bacteria type (Staphylococcus aureus versus coagulase-negative Staphylococcus - CNS) and verify the agreement between tests. A total of 200 bacteria samples from 21 different herds were enrolled, being 100 CNS and 100 S. aureus. Beta-lactamase presence/detection was performed by different tests (PCR, clover leaf test - CLT, Nitrocefin disk, and in vitro resistance to penicillin). Results of all tests were not dependent of bacteria type (CNS or S. aureus). Several S. aureus beta-lactamase producing isolates were from the same herd. Phenotypic tests excluding in vitro resistance to penicillin showed a strong association measured by the kappa coefficient for both bacteria species. Nitrocefin and CLT are more reliable tests for detecting beta-lactamase production in staphylococci.

Suscetibilidade antimicrobiana de Staphylococcus spp. isolados de cães com pioderma superficial

Foram obtidas 154 amostras de lesões cutâneas de cães com pioderma superficial atendidos no Serviço de Dermatologia Veterinária do Hospital Veterinário Universitário (HVU) da Universidade Federal de Santa Maria (UFSM), com o objetivo de determinar a suscetibilidade antimicrobiana de isolados de Staphylococcus spp. e avaliar a presença de multirresistência. Após isolamento e identificação, as cepas foram submetidas ao teste de sensibilidade aos antimicrobianos, cujos resultados evidenciaram menores percentuais de resistência à associação amoxicilina e ácido clavulânico (1,9%), cefadroxil (1,9%), cefalexina (1,9%) e vancomicina (0,6%). Os maiores percentuais de resistência foram frente à amoxicilina (60,4%) e penicilina G (60,4%). A multirresistência foi detectada em 23,4% e a resistência à meticilina em 5,8% das amostras. Pode-se concluir que os isolados de Staphylococcus spp. apresentam elevada suscetibilidade aos antimicrobianos comumente utilizados no tratamento dos piodermas superficiais em cães atendidos no HVU-UFSM, como a cefalexina e a amoxicilina associada ao ácido clavulânico, confirmando a eleição desses fármacos para o tratamento de cães com esta afecção. A suscetibilidade diminuída das cepas frente às fluoroquinolonas, também recomendadas pela literatura para o tratamento de pioderma, permite sugerir que estes fármacos não devem mais ser considerados na seleção empírica. A identificação de Staphylococcus spp. multirresistentes na população canina estudada justifica análises bacteriológicas periódicas e regionais de lesões cutâneas de cães com pioderma superficial, a fim de minimizar a seleção de bactérias resistentes, possíveis falhas terapêuticas e também motiva a antimicrobianoterapia prudente.

Atividade antibacteriana do extrato hidroalcoólico de Punica granatum Linn. sobre Staphylococcus spp. isolados de leite bovino

Mastite bovina é caraterizada por inflamação da glândula mamária, geralmente em resposta à infecção bacteriana, compromete quali-quantitativamente a produção leiteira. Este estudo objetivou verificar a atividade antibacteriana in vitro do extrato hidroalcoólico da casca da romã sobre bactérias isoladas de leite bovino. As colônias de Staphylococcus spp. foram ressuspendidas a escala 6 de MacFarland e ajustada a sua concentração por espectrofotometria UV visível na concentração de 10 mL-1. Os extratos foram avaliados em quintuplicata, em sete concentrações: de 4mg mL-1 até 0,0625 mg.mL-1. A sensibilidade dos isolados microbianos foi determinada utilizando o teste de difusão em disco e os resultados que apresentaram zonas de inibição correspondentes a valores a partir de 15 mm, foram considerados sensíveis. Os resultados foram avaliados pelo método ANOVA, teste de Tukey 5%, utilizando o SISVAR 5.3 -DEX/UFLA. Adicionalmente o extrato foi avaliado quanto à atividade antioxidante, teores de fenóis e flavonoides totais. Para tanto o extrato foi diluído em sete concentrações: de 25 a 1000µg.mL-1, e avaliado em triplicata. O crescimento bacteriano foi inibido a partir da concentração de 4mg.mL-1 e a ação antioxidante foi verificada a partir de 50µg.mL-1, com valores correspondentes a 4.62%, atingindo platô de 64,90% na concentração de 500µg.mL-1. Na avaliação da atividade captadora de radicais, empregando o radical livre DPPH, o extrato demonstrou atividade antioxidante (IC50%= 378,80µg/mL). Porém, não foi possível correlacionar a atividade antioxidante aos teores de fenóis e flavonoides. Talvez a presença de outras substâncias alcaloides e taninos presentes no extrato, possam ter sido as responsáveis pela atividade antioxidante encontrada. Conclui-se que o extrato hidroalcoólico de Punica granatum Linn. apresenta atividade antimicrobiana contra Staphylococcus spp., demonstrando potencial benefício para o controle da mastite bovina.

Occurrence of enterotoxin-encoding genes in Staphylococcus aureus causing mastitis in lactating goats

Staphylococcal enterotoxins are the leading cause of human food poisoning worldwide. Staphylococcus spp. are the main mastitis-causing agents in goats and frequently found in high counts in goat milk. This study aimed to investigate the occurrence of enterotoxin-encoding genes in Staphylococcus aureus associated with mastitis in lactating goats in Paraiba State, Brazil. Milk samples (n=2024) were collected from 393 farms. Staphylococcus aureus was isolated in 55 milk samples. Classical (sea, seb, sec, sed, see) and novel (seg, seh, sei) enterotoxin-encoding genes were investigated by means of polymerase chain reaction (PCR). From thirty-six tested isolates, enterotoxin-encoding genes were detected in 7 (19.5%) S. aureus. The gene encoding enterotoxin C (seC) was identified in six isolates, while seiwas observed in only one isolate. The genes sea, seb, sed, see, seg and seh were not observed amongst the S. aureus investigated in this study. In summary, S. aureus causing mastitis in goats can harbor enterotoxin-encoding genes and seC was the most frequent gene observed amongst the investigated isolates. This finding is important for surveillance purposes, since enterotoxin C should be investigated in human staphylococcal food poisoning outbreaks caused by consumption of goat milk and dairy products.

Risk factors associated with the antimicrobial resistance of Staphylococcus aureus isolated from bovine mastitis

The objective of this study was to evaluate herd management practices and mastitis treatment procedures as risk factors associated with Staphylococcus aureus antimicrobial resistance. For this study, 13 herds were selected to participate in the study to evaluate the association between their management practices and mastitis treatment procedures and in vitro antimicrobial susceptibility. A total of 1069 composite milk samples were collected aseptically from the selected cows in four different periods over two years. The samples were used for microbiological culturing of S. aureus isolates and evaluation of their antimicrobial susceptibility. A total of 756 samples (70.7%) were culture-positive, and S. aureus comprised 27.77% (n=210) of the isolates. The S. aureus isolates were tested using the disk-diffusion susceptibility assay with the following antimicrobials: ampicillin 10mg; clindamycin 2μg; penicillin 1mg; ceftiofur 30μg; gentamicin 10mg; sulfa-trimethoprim 25μg; enrofloxacin 5μg; sulfonamide 300μg; tetracycline 30μg; oxacillin 1mg; cephalothin 30μg and erythromycin 5μg. The variables that were significantly associated with S. aureus resistance were as follows: the treatment of clinical mastitis for ampicillin (OR=2.18), dry cow treatment for enrofloxacin (OR=2.11) and not sending milk samples for microbiological culture and susceptibility tests, for ampicillin (OR=2.57) and penicillin (OR=4.69). In conclusion, the identification of risk factors for S. aureus resistance against various mastitis antimicrobials is an important information that may help in practical recommendations for prudent use of antimicrobial in milk production.

Ação dos desinfetantes sobre a adesão e biofilme consolidado de Staphylococcus spp.

Staphylococcus spp. são reconhecidos como importantes causadores de mastites em rebanhos leiteiros. Esses micro-organismos têm a capacidade de produzir uma estrutura denominada biofilme, que é responsável pela sobrevivência e muitas vezes pela resistência a ação de produtos desinfetantes e as demais condições adversas. Neste trabalho avaliou-se a ação de dois produtos pós-dipping a base de iodo (0,7%) e clorexidine (2,0%) sobre a adesão de Staphylococcus coagulase positiva (SCP) e Staphy-lococcus coagulase negativa (SCN) isolados de casos de mastite subclínica, e também sobre biofilmes pré formados a partir destes isolados. Os produtos testados apresentaram uma alta redução na taxa de adesão de todos os isolados. No entanto, a ação sobre os biofilmes consolidados só foi estatisticamente significativa sobre os SCN. Assim, ressalta-se a importância dos programas sanitários a fim de prevenir a formação de biofilmes e diminuir as fontes de contaminação da glândula mamaria em sistemas de produção leiteira.

Celularidade do leite e Unidades Formadoras de Colônias nas mastites causadas por Staphylococcus coagulase positiva e coagulase negativa

Resumo: A mastite é a principal afecção do gado destinado à produção leiteira, que impacta significativamente a cadeia produtiva do leite, com reflexos ainda para a saúde pública. Estudou-se aspectos relacionados à etiologia, celularidade e de contagem bacteriana em 10 propriedades leiteiras, localizadas no Estado de São Paulo. Foram examinadas 1148 vacas em lactação, totalizando 4584 glândulas mamárias. Foram considerados os casos, em que houve isolamento de estafilococos coagulase positiva (SCP) e estafilococos coagulase negativa (SCN). Os resultados revelaram microbiota com vários patógenos e diferentes espécies de SCN (128 casos) e SCP (45), Staphylococcus aureus(90), Streptococcus agalactiae(70), Streptococcus dysgalactiae (69), Streptococcus uberis(29), Corynebacteriumspp. (230), Klebsiella pneumoniae(28), Klebsiella oxytoca(2), Escherichia coli(15), Enterobactersp. (3). Os resultados de contagem de células somáticas (CCS) relacionados aos SCP e SCN não mostraram diferenças entre as propriedades avaliadas, entretanto com diferenças significantes ao se avaliar a CCS entre os dois grupos de estafilococos, como pode ser evidenciado ao comparar SCN Discreto e SCP exuberante (P<0,01), SCP Discreto e SCP exuberante (P<0,001) e SCN moderado e SCP exuberante (P<0,01). A avaliação da CCS relacionada à intensidade da infecção, considerando-se como crescimento discreto o isolamento de até nove colônias, moderado de dez a 29 colônias e exuberante, com 30 ou mais colônias, revelou para ambos os grupos de estafilococos que quanto maior o número de unidades formadoras de colônias (UFC), a CCS é mais elevada, sendo sempre maior nos casos de SCP. Conclui-se que quando há maior número de UFC, há concomitantemente maior CCS/mL de leite, no caso dos SCP e SCN, o que mostra relação direta da intensidade do processo infeccioso com a resposta da celularidade do leite, bem como pela relevância desses na etiologia das mastites e dos aspectos negativos tanto para a produção, quanto na qualidade do leite produzido nas propriedades.

PCR-based ribotyping of Staphylococcus aureus

Genotyping techniques are valuable tools for the epidemiologic study of Staphylococcus aureus infections in the hospital setting. Pulsed-field gel electrophoresis (PFGE) is the current method of choice for S. aureus strain typing. However, the method is laborious and requires expensive equipment. In the present study, we evaluated the natural polymorphism of the genomic 16S-23S rRNA region for genotyping purpose, by PCR-based ribotyping. Three primer pairs were tested to determine the size of amplicons produced and to obtain better discrimination with agar gel electrophoresis and ethidium bromide staining. The resolution of the typing system was determined using sets of bacteria obtained from clinical specimens from a large tertiary care hospital. These included DNA from three samples obtained from a bacteremic patient, six strains with known and diverse PGFE patterns, and 88 strains collected over a 3-month period in the same hospital. Amplification patterns obtained from samples from the same patient were identical, and PFGE from samples known to be different produced three genotypes. Amplification of DNA from 61 methicillin-resistant isolates produced only one pattern. Methicillin-sensitive strains yielded a diversity of patterns, pointing to a true polyclonal distribution throughout the hospital (22 unique patterns from 27 strains). Computer-based software can be used to differentiate among identifiable strains, given the low number of bands and good characterization of PCR products. PCR-based ribotyping can be a useful technique for genotyping methicillin-sensitive S. aureus strains, but is of limited value for methicillin-resistant strains.

Use of molecular epidemiology to monitor the nosocomial dissemination of methicillin-resistant Staphylococcus aureus in a University Hospital from 1991 to 2001

Methicillin-resistant Staphylococcus aureus (MRSA) has been the cause of major outbreaks and epidemics among hospitalized patients, with high mortality and morbidity rates. We studied the genomic diversity of MRSA strains isolated from patients with nosocomial infection in a University Hospital from 1991 to 2001. The study consisted of two periods: period I, from 1991 to 1993 and period II from 1995 to 2001. DNA was typed by pulsed-field gel electrophoresis and the similarity among the MRSA strains was determined by cluster analysis. During period I, 73 strains presented five distinctive DNA profiles: A, B, C, D, and E. Profile A was the most frequent DNA pattern and was identified in 55 (75.3%) strains; three closely related and four possibly related profiles were also identified. During period II, 80 (68.8%) of 117 strains showed the same endemic profile A identified during period I, 18 (13.7%) closely related profiles and 18 (13.7%) possibly related profiles and, only one strain presented an unrelated profile. Cluster analysis showed a 96% coefficient of similarity between profile A from period I and profile A from period II, which were considered to be from the same clone. The molecular monitoring of MRSA strains permitted the determination of the clonal dissemination and the maintenance of a dominant endemic strain during a 10-year period and the presence of closely and possibly related patterns for endemic profile A. However, further studies are necessary to improve the understanding of the dissemination of the endemic profile in this hospital.

Inactivation of Staphylococcus aureus and Salmonella enteritidis in tryptic soy broth and caviar samples by high pressure processing

We studied the action of high pressure processing on the inactivation of two foodborne pathogens, Staphylococcus aureus ATCC 6538 and Salmonella enteritidis ATCC 13076, suspended in a culture medium and inoculated into caviar samples. The baroresistance of the two pathogens in a tryptic soy broth suspension at a concentration of 10(8)-10(9) colony-forming units/ml was tested for continuous and cycled pressurization in the 150- to 550-MPa range and for 15-min treatments at room temperature. The increase of cycle number permitted the reduction of the pressure level able to totally inactivate both microorganisms in the tryptic soy broth suspension, whereas the effect of different procedure times on complete inactivation of the microorganisms inoculated into caviar was similar.

Hospital strain colonization by Staphylococcus epidermidis

The skin and mucous membranes of healthy subjects are colonized by strains of Staphylococcus epidermidis showing a high diversity of genomic DNA polymorphisms. Prolonged hospitalization and the use of invasive procedures promote changes in the microbiota with subsequent colonization by hospital strains. We report here a patient with prolonged hospitalization due to chronic pancreatitis who was treated with multiple antibiotics, invasive procedures and abdominal surgery. We studied the dynamics of skin colonization by S. epidermidis leading to the development of catheter-related infections and compared the genotypic profile of clinical and microbiota strains by pulsed field gel electrophoresis. During hospitalization, the normal S. epidermidis skin microbiota exhibiting a polymorphic genomic DNA profile was replaced with a hospital-acquired biofilm-producer S. epidermidis strain that subsequently caused repetitive catheter-related infections.

The first report in Brazil of severe infection caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA)

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emergent pathogen in Brazil. However, there are no data on the prevalence of CA-MRSA. We report here the first well-characterized case of severe life-threatening CA-MRSA infection in a child living in Rio de Janeiro city. The patient had many complications including hematogenous osteomyelitis and involvement of multiple sites requiring drainage of soft-tissue abscess, and pleural and pericardial empyema. The MRSA isolates recovered were genotyped using PFGE, SCCmec typing and multilocus sequence typing. Disk diffusion tests were performed following Clinical and Laboratory Standards Institute recommendations. In addition, the presence of Panton-Valentine leukocidin (PVL) was assessed by PCR amplification, using specific primers for lukF-pv (encoding for the F subunit of the PVL). The bacterial isolates were related to the ST30-SCCmecIV lineage (Oceania Southwest Pacific clone), a PVL producer CA-MRSA previously detected in Porto Alegre, RS, Brazil. Also, the isolates analyzed were susceptible to all non-β-lactam antibiotics tested. The present report demonstrates that disseminated CA-MRSA disease is also occurring in Rio de Janeiro. Thus, the empirical treatment of moderate or severe infections suspected of being associated with CA-MRSA needs to be reviewed in order to allow prompt initiation of an effective therapy that also covers these microorganisms.