Determinantes genéticos de resistência a antimicrobianos em Staphylococcus spp. e bactérias da família Enterobacteriaceae isolados de leite de vacas e cabras com mastite subclínica em ecossistemas agropecuários do Nordeste brasileiro

Abstract

It was aimed to study the phenotypic and genetic profile of antimicrobial resistance in bacteria of the Staphylococcus genus and Enterobacteriaceae family isolated from raw cow and goat’s milk 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. All milk samples were inoculated into base agar with 5% ovine blood and incubated at 37°C for up to 48 hours. Colonies that presented microscopic morphology of Gram positive cocci were submitted to the catalase test for the identification of Staphylococcus spp. and subsequently submitted to PCR for identification. Gram negative bacteria were identified using biochemical tests. Among bovine and caprine isolates, 16.9% (45/267) S. aureus, 13.1% (35/267) S. epidermidis, 12.7% (34/267) S. haemolyticus and 19.5% (52/267) S. chromogenes were identified, while 37.8% (101/267) were not positive for the tested species. Twenty-six cow milk isolates were identified as Escherichia coli (53.9% [14/26]), Proteus mirabilis (15.4% [4/26]), Klebsiella spp. (26.9% [7/26]) and Citrobacter spp. (3.9% [1/26]). All the isolates were submitted to a screening test to identify BLEE (extended spectrum β-lactamase) -producing bacteria and confirmatory test (Double disc test), using cefotaxime (30 μg), ceftriaxone (30 μg), ceftazidine (30 μg) and amoxicillin with clavulanic acid (30 μg). Of the 26 enterobacteria isolates, 61.5% (16/26) were positive in the BLEE screening test and 12.5% (2/16) were positive in the DDT. Subsequently, all 26 isolates were analyzed for the BLEE-encoding genes, blaSHV and blaTEM. 53.9% (14/26) were positive for one or both genes and 71.4% (10/14) were identified as Escherichia coli. On the other hand, 148 and 119 isolates of Staphylococcus spp. were identified from cows and goats, respectively. for the study of the phenotypic profile of antimicrobial resistance using the technique of diffusion in agar with the discs of antibiotics: penicillin (10 UI), 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 (52.0% [77/148]) was observed, followed by tetracycline (10.1% [15/148]), and none of the isolates was resistant to ceftiofur. Regarding the genetic determinants of resistance, the most observed pattern was the presence of the blaZ gene (40.5% [60/148]) and the tetK gene (22.3% [22/148]). In isolates from goats, the same pattern of resistance was observed as in isolates from cows, with a frequency of resistance to penicillin and tetracycline of 45.0% (53/119) and 30.3% (36/119), respectively. Regarding the resistance genes, a higher frequency of the blaZ gene was observed 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 discs of erythromycin (15 μg) and clindamycin (2 μg). In the isolates of Staphylococcus spp from cows, 0.7% (1/148) were positive D-test, 7.4% (11/148) were resistant to MLS, 1.4% (2/148) were resistant only to to MS (D – negative test) and 2.7% (3/148) were resistant to clindamycin (L+). For isolates from goats, 3.4% (4/119) D-test positive were obtained, 0.8% (1/119) were resistant to MLS and 0.8% (1/119) were L+. These results demonstrate the circulation of antimicrobial resistant strains in the agricultural system of the state of Pernambuco, representing a risk to animal and public health.