Phenotypic and Genotypic Characteristics and Resistance Profile of Staphylococcus spp. from Bovine Mastitis

Background: Bovine mastitis remains one of the health problems that cause the most damage to milk producers. The negative impact of mastitis is due to reduced milk production, early slaughter of females, reduced commercial value of the affected animals, losses in the genetic potential of the herd, expenses with medicines and veterinary medical assistance. Staphylococcus spp. stands out as the cause of this disease and has been able to remain in the mammary gland, becoming resistant to several antimicrobials. The aims of the present study were to characterize the phenotypes, genotypes and resistance profiles of Staphylococcus spp. isolates from bovine mastitis cases in the state of Pernambuco, Brazil.Materials, Methods & Results: These isolates were classified according to biochemical tests and the presence of the nuc gene.  The polymerase chain reaction (PCR) for amplification of the mecA and blaZ genes was used to analyze the genetic potentials of antimicrobial resistance. Isolates were also phenotypically tested for resistance to nine antimicrobials (ampicillin, doxicillin, erythromycin, gentamicin, rifampicin, cephalothin, amoxicillin, nalidixic acid and oxacillin). The genetic potentials for biofilm production were evaluated by the amplifications of the icaD, icaA and bap genes. The phenotypic test of gentian violet was used for biofilm formation analyzes. Ninety-three (93.0%) of the isolates among the Staphylococcus spp. samples were classified as Staphylococcus aureus. The lowest percentage of sensitivity observed was for amoxicillin (28.0%). All of the isolates were sensitive to erythromycin and gentamicin, and 15 (15%) exhibited sensitivity to all of the drugs tested. All of the isolates were negative for the mecA gene, and 36 (36%) were positive for blaZ. In the adhesion microplate tests, 44 (44%) of the isolates were capable of biofilm formation. Of these, seven (15.9%) were strong formers, whereas 16 (36.3%) and 21 (47.8%) were moderate and weak formers, respectively. The icaD gene was confirmed in 89 (89%) of the isolates. The icaA gene was confirmed in 61 (61%) samples, and the bap gene in 52 (52%) samples. One of the samples did not possess icaA, icaD or bap and exhibited moderate biofilm formation according to the microplate adherence test. Sixteen isolates simultaneously exhibited the three genes tested for biofilm production (icaA, icaD and bap) and were negative according to the microplate adherence test.Discussion:  The indiscriminate use of antibiotics to treat mastitis is a common practice in the study area, which may have contributed to the high proportion of herds (88.23%; 15/17) with multi-resistant isolates, constituting a selection factor for the dissemination of resistant bacteria among herds.  The absence of the mecA gene in the present study may be associated with the development of resistant bacteria through another mechanism, such as the overproduction of beta-lactamases. The results demonstrate that antimicrobial resistance occurs in Staphylococcus spp. that cause bovine mastitis in herds of Pernambuco and that these isolates have the a great capacity for biofilm formation. It is necessary to sensitize the professionals involved in the milk production chain of Brazil regarding the importance of the adequate use of antimicrobials for the treatment and control of mastitis, since studies in the country indicate the dissemination of resistant bacterial strains

Virulence Factors in Staphylococcus aureus and Quality of Raw Milk from Dairy Cows in a Semiarid Region of Northeastern Brazil

Background: Milk quality is considered unsatisfactory in Brazil due to factors of a social, economic, cultural, and climatic nature. Mastitis is the main disease that affects dairy herds. Microorganisms of the genus Staphylococcus are the most frequently isolated pathogens in cases of mastitis in bovines. Staphylococcus aureus requires more attention because they have the ability to develop resistance to antimicrobials used in the treatment of mastitis. Thus, the aim of the present study was to evaluate virulence factors in isolates of S. aureus as well as analyze the hygienic-sanitary quality of raw milk produced on dairy farms in a semiarid region of northeastern Brazil.Material, Methods & Results: Samples were taken from milk containers at 44 properties. Fifty-eight isolates of Staphylococcus spp. were analyzed and genotypically identified as S. aureus. In the phenotypic characterization, 56.8% (33/58) of the isolates demonstrated moderate biofilm production. In the genotypic characterization (icaA, icaD and bap genes), icaA was the most representative among the isolates. No resistance to cephalothin or oxacillin was found, but 62% (36/58) of the isolates exhibited resistance to ampicillin, amoxicillin, and penicillin. In the genotypic evaluation in response to β-lactam antibiotics, 50% (29/58) of the isolates exhibited the blaZ gene. Total bacterial counts were determined based on the standard plate count and flow cytometry, for which a significant positive correlation was found (r = 0.61, P < 0.01). The somatic cell count was also performed using flow cytometry and demonstrated that 6.8% (3/44) of the properties had values above one million cells per mL. The analysis of Staphylococcus spp. revealed that 20% (9/44) of the properties had significant counts for the production of staphylococcal enterotoxins. Regarding total coliforms, 56.8% (25/44) of the milk analyzed had counts above 1.0 x 103 CFUs/mL. There were no cases of the isolation of Pseudomonas spp. Streptococcus spp. was isolated on 6.8% (3/44) of the properties. The results of the sequencing revealed one isolate to be Lactococcus lactis and two to be Enterococcus spp.Discussion: None of the 58 isolates of S. aureus was classified as negative in the phenotypic characterization of biofilmforming capacity, which suggests the presence of genes involved in this process. Studies have shown that the molecular techniques used to identify ica genes encoding biofilm synthesis are very important for the identification of virulent strains. The isolates demonstrated resistance to β-lactam antibiotics of the class of penicillins, which are the most widely employed antimicrobial agents for the treatment of diseases in dairy herds. The presence of the blaZ gene in 50% of the isolates is in agreement with data reported in other studies developed in northeastern Brazil. In 50% (29/58) of the isolates, the presence of the blaZ gene was not observed. Of these, (8/29) were resistant to ampicillin, amoxicillin and penicillin. This is due to the existence of other bacterial mechanisms. Research has shown that some isolates possessed three mechanisms (blaZ, mecA and msrA) of antimicrobial resistance simultaneously. The results of the quality analyses demonstrate that some properties produce milk of low quality, which underscores the need for management strategies directed at reducing the contamination of the product

Formigas (Hymenoptera: Formicidae) como vetores na transmissão de Staphylococcus aureus em um hospital público

Objetivo: analisar o potencial de formigas como veículos mecânicos de bactérias do gênero Staphylococcus aureus no centro cirúrgico e unidade de terapia intensiva de um hospital público. Metodologia: trata-se de um estudo descritivo. As formigas foram coletadas utilizando-se iscas atrativas que ficavam expostas por três horas sendo, após a exposição, incubadas por 24h a 35ºC. A susceptibilidade aos antimicrobianos foi analisada através da técnica de disco-difusão. Os isolados de Cocos gram-positivos foram submetidos a Reação em Cadeia da Polimerase e observado os genes nuc, mecA blaZ e icaAD. Resultados: foram coletadas 592 formigas pertencentes à espécie Tapinoma melanocephalum e Pheidole sp. Todos os isolados apresentaram o gene mecA, 93,7% apresentaram resultado positivo para o gene blaZ e 12,5% para o gene icaA e icaD. Sete isolados apresentaram multirresistência. Conclusão: os resultados sugerem que as formigas podem agir como veiculadoras de bactérias multirresistentes no ambiente hospitalar. Abstract Objective: to analyze the potential of ants as mechanical vehicles of bacteria of the genus Staphylococcus Aureus in the surgical center and intensive care unit of a public hospital. Methodology: this was a descriptive study. The ants were collected using attractive baits that were exposed for three hours and, after exposure, incubated for 24 hours at 35ºC. The susceptibility to antimicrobials was analyzed using the disk-diffusion technique. Gram-positive Cocos isolates were subjected to Polymerase Chain Reaction and the nuc, mecA blaZ and icaAD genes were observed. Results: 592 ants belonging to the species Tapinoma melanocephalum and Pheidole sp. All isolates showed the mecA gene, 93.7% were positive for the blaZ gene and 12.5% for the icaA and icaD gene. Seven isolates showed multidrug resistance. Conclusion: the results suggest that ants can act as carriers of multidrug-resistant bacteria in the hospital environment

Molecular characterization of virulence factors in Aeromonas hydrophila obtained from fish

Multiple factors can be involved in the virulence processes of Aeromonas hydrophila. The objective of the present paper was to verify the presence of aerolysin, hidrolipase, elastase and lipase virulence genes through the polymerase chain reaction (PCR) in A. hydrophila isolates obtained from fish of the São Francisco River Valley, and to evaluate virulence according to the presence of these genes in Nile tilapia fingerlings. One hundred and fourteen isolates from the bacteria were used. DNA was heat extracted and PCR undertaken using specific primers described in the literature. For in vivo tests Nile tilapia fingerlings were used. From the PCR tests, negative isolates for all genes tested were selected, positive isolates for two genes (aerolysin and elastase) and positive for the four genes tested. These were inoculated at a concentration of 10(8) UFC/ml into the tilapias, considered as treatments; another group of animals was used as control (with inoculation of saline solution). In all, 12 distinct standards regarding the presence of virulence factors in isolates from A. hydrophila, were observed. Of the 114 isolates analyzed, 100 (87.72%) presented at least one of the virulence factors under study. The virulence factors were widely distributed among the A. hydrophila isolates. Aerolysin was the most frequent virulence factor present in the isolates analyzed. A. hydrophila led to the mortality of the Nile tilapia fingerlings, regardless of the absence or quantity of virulence genes tested

Aeromonas hydrophila in tilapia (Oreochromis niloticus) after the intake of aflatoxins

The objectives of this study were to assess the effect of the intake of aflatoxin on the development of tilapia and to evaluate the impact of inoculation with Aeromonas hydrophila on performance parameters, so these two individual tests were performed. One hundred and twenty fingerlings aged 35 days old, with mean weight of 1.55 ± 0.005 g and mean length of 5 cm were used in each test, distributed in 20 tanks. Each experimental unit consisted of a 60 L tank with six fingerlings. In the first experiment, increasing levels of aflatoxin (0.350, 0.757, 1.177 mg.kg feed-1) were used as treatments and, for the control group, a diet without aflatoxin was used in a completely randomized design with four treatments and five replicates. In the second experiment, a control group of Nile tilapia fingerlings was used and received a diet without aflatoxin, inoculated with saline solution (group 1) and Aeromonas hydrophila (group 2), as well as groups of animals fed on diets containing 0.350 mg.kg feed-1of aflatoxin (group 3), 0.757 mg.kg feed-1of aflatoxin (group 4) and 1.177 mg.kg feed-1of aflatoxin (group 5), and these groups were inoculated with Aeromonas hydrophila. The survival rate and the total length of fingerlings were influenced by the treatments (p < 0.05). The synergistic action of aflatoxins and Aeromonas hydrophila was effective and caused the death of experimental fish, thus affecting feed conversion and length

Molecular screening for the mutation associated with canine degenerative myelopathy (SOD1:c.118G > A) in German Shepherd dogs in Brazil.

Canine Degenerative Myelopathy is a late onset recessive autosomal disease characterized by a progressive ascending degeneration of the spinal cord. Two causal mutations are associated with this disease: a transition (c.118G>A) in exon 2 of the SOD1 that was described in several breeds and a transversion (c.52A>T) in exon 1 of the same gene described in Bernese Mountain dogs. The aim of this study was to understand the impact of the SOD1:c.118G > A mutation by genotyping a population of German Shepherd dogs in Brazil. A PCR-RFLP approach was used to genotype 97 healthy individuals belonging from the Northeast (Bahia and Pernambuco states) and South (Santa Catarina state) regions of Brazil. A total of 95 individuals were successfully genotyped resulting in an observed genotype frequency (with 95% confidence interval) of: 0.758 (0.672-0.844), 0.242 (0.156-0.328) and 0.000 (0.000-0.000) for "GG", "AG" and "AA" genotypes, respectively. To our knowledge, this is the first attempt to describe the presence of the "A" allele associated with CDM (SOD1:c.118G > A) in German Shepherd dogs in Brazil and, as such, these results contribute toward important epidemiological data in this country and to the knowledge of the distribution of the aforementioned mutation worldwide

MICROBIOTA BACTERIANA COM POTENCIAL PATOGÊNICO EM PACAMÃ E PERFIL DE SENSIBILIDADE A ANTIMICROBIANO

In aquaculture, infections caused by bacteria are factors that cause damage. The purpose of this study was to identify bacteria with pathogenic potential in pacamã (Lophiosilurus alexandri) of São Francisco Valley. One hundred and fifteen animals were used for sampling from gills, kidneys, external lesions and eggs. The samples were streaked in Trypticase soy agar. The biochemical and antimicrobial susceptibility profiles were determined. The bacterial species identified were A.hydrophila, A. salmonicida, A. media, Acinetobacter spp., P. alcaligenes, E. aerogenes, E. agglomerans, K. oxytoca, K. pneumoniae, P.shigelloides, V. parahaemo- lyticus, V. metschnikovii and S. sannei. The sensitivity to antimicrobial was 85% to norfloxacin, 79% to ceftri- axone, 78% to enrofloxacin, 68% to sulfazothrim, 60% to nitrofurantoin, 59% to tetracycline, 55% to nalidixic acid, 49% to streptomycin, 45% to erythromycin, 32% to neomycin, 7% to ampicillin and 3% to lincomycin. Multiple resistance was observed to all isolates analyzed. Considering the occurrence of pathogenic bacteria in pacamã and its resistance to antimicrobial drugs, security measures should be adopted during antimicrobialther- apy, with the use of antibiotics that did not show resistance strains or trying natural products as an alternative to antibiotics, ensuring protection of human and animal health, as well as the environment