2.2. Bacterial Isolation and Antimicrobial Susceptibility Testing

Aliquots of the fly homogenates were streaked onto plates containing 5% bovine blood agar, the selective and differential media MacConkey agar, and Phenylethyl Alcohol Blood agar. Plates were incubated at 37 °C for 24 h and individual colonies resembling E. coli, Klebsiella pneumoniae, coagulase-negative Staphylococcus species (CoNS) and S. aureus or S. pseudintermedius were collected and archived for testing.

Gram-negative rods that grew on MacConkey agar as lactose fermenters and were oxidase negative were further speciated as E. coli or Klebsiella pneumoniae by testing for urease and citrate utilization, indole production, and the Voges-Proskauer and Methyl Red tests. Staphylococcus isolates were identified using colony morphology, β-hemolysis on blood agar and conventional biochemical tests, including coagulase, catalase, fermentation of maltose, mannitol, and trehalose and acetoin production.

Antimicrobial susceptibility testing was performed using the Kirby–Bauer agar disk diffusion test with ampicillin, amoxicillin-clavulanic acid, ceftazidime, amikacin, gentamicin, streptomycin, tetracycline, doxycycline, chloramphenicol, ciprofloxacin, and trimethoprim-sulfamethoxazole. Cefoxitin and oxacillin were used to determine methicillin resistance in the Staphylococcus isolates. Multidrug-resistant isolates were classified as having resistance to antimicrobial from three or more drug classes. Resistant to ampicillin in K. pneumoniae was not taken into consideration for their intrinsic resistance to the drug. Extended spectrum beta-lactamase production was identified in isolates resistant to ceftazidime and the reduced susceptibility to amoxicillin/clavulanic acid. For each isolate, an 18–24 h old suspension of bacteria equivalent to a 0.5 McFarland Standard was applied as a uniform lawn onto Mueller Hinton agar and allowed to dry. After the antibiotic impregnated disks were placed onto the agar surface, the plates were incubated at 37 °C for 18–24 h. Zones of inhibition were read and interpretations of susceptible, intermediate and resistant were made using the Clinical and Laboratory Standards Institute Standards [16].