Banca de QUALIFICAÇÃO: TANIA ALEXANDRA ORTEGA SIERRA

Genetic determinants of antimicrobial resistance in Staphylococcus spp. and bacteria of the Enterobacteriaceae family isolated from milk from cows and goats in agricultural ecosystems in Northeast Brazil

antimicrobial resistance, Staphylococcus spp., Enterobacteriaceae family, BLEE, MLS, MRS.

The objective was to study the phenotypic and genetic profile of antimicrobial resistance in bacteria of the genus Staphylococcus and the family Enterobacteriaceae isolated from milk from cows and goats with mastitis in the state of Pernambuco. Milk samples were collected from 426 cows and 323 goats, in 6 and 17 bovine and goat dairy farms, respectively. Milk samples from cows were seeded on sheep blood agar and incubated at 37°C for up to 48 hours. Colonies that presented as Gram positive cocci were submitted to the catalase test for the identification of Staphylococcus spp. and subsequently identified. S. aureus, S. epidermidis, S haemolyticus and S. chromogenes were identified using molecular techniques. Gram negative bacteria were identified using biochemical tests (glucose fermentation, lactose fermentation, motility, citrate utilization, lysine decarboxylation, H2S production, gas production, indole production, urease production and phenylalanine deaminase production). Twenty six isolates from cow's milk were identified as Escherichia coli (53.85% [14/26]), Proteus mirabilis (15.38% [4/26]), Klebsiella spp. (26.92% [7/26]) and Citrobacter spp. (3.85% [1/26]). These bacteria were submitted to a screening test to identify bacteria that produce ESBLE (spectrum β-lactamase). These bacteria were subjected to a screening test to identify bacteria producing ESBLE (extended spectrum β-lactamase) and confirmatory testing (Double disc test), using cefotaxime (30 µg), ceftriaxone (30 µg), ceftazidine (30 µg). µg) and amoxicillin with clavulanic acid (30 µg). Of the 26 enterobacterial isolates, 61.54% (16/26) were positive in the screening test for ESBLE and 12.5% (2/16) were positive in the DDT. Subsequently, all 26 isolates were analyzed for the genes encoding ESBL, blaSHV and blaTEM, and 53.85% (14/26) were positive for one or both genes and 71.43% (10/14) were identified. such as Escherichia coli. On the other hand, 148 and 119 isolates of Staphylococcus spp. of cows and goats, respectively, to study the phenotypic profile of resistance in the Muller Hinton agar diffusion technique with antibiotic discs: penicillin (10 IU), tetracycline (30 µg), cefoxitin (30 µg), erythromycin (15 µg ), Clindamycin (2 µg) and ceftiofur (30 µg). In bovine isolates, a significant frequency of resistance to penicillin (52.03% [77/148]) was observed, followed by tetracycline (10.14% [15/148]), and no isolate was resistant to ceftiofur. Regarding the genetic determinants of resistance, the most observed pattern was the presence of the blaZ gene (40.54% [60/148]) and the tetK gene (22.3% [22/148]). The same pattern of resistance was observed in the goat isolates as in the cow isolates, with a frequency of resistance to penicillin and tetracycline of 45.0% (53/119) and 30.3% (36/119), respectively. Regarding resistance genes, there was a higher frequency of the blaZ gene with 50.4% (60/119) followed by tetK with 45.0% (53/119). In Staphylococcus spp. of both species, the D – test was performed to identify strains capable of inducing resistance to lincosamides and streptogramin, using disks of erythromycin (15 µg) and clindamycin (2 µg) placed at a distance of 20 mm between them on Muller agar Hinton. In Staphylococcus spp. of cows, 0.7% (1/148) of D – positive test was obtained, 7.4% (11/148) were resistant to MLS, 1.4% (2/148) were resistant to MS only (D – negative test) and 2.7% (3/148) were L+. for the goat isolates, 3.4% (4/119) D - positive test was obtained, 0.8% (1/119) were resistant to MLS and 0.8% (1/119) were resistant to clindamycin (L+) . The results obtained demonstrate the circulation of antimicrobial-resistant strains in the agricultural system of the state of Pernambuco, representing a risk to animal and public health.