Investigation of susceptibility of Staphylococcus species to some antibacterial drugs by disk diffusion and broth microdilution

Abstract

The objective of this work was to identify isolated Staphylococcus species and to investigate their sensitivity to some antibacterial drugs. The material used for these investigations were Staphylococcus isolates originating from milk samples. A total of 25 strains of Staphylococcus isolates were examined, including 24 from milk samples from cows with mastitis, and one strain was isolated from a milk sample from a cow following treatment for mastitis. For primary identification, catalase and oxidase tests were used, as well as the free coagulase test. Following the preliminary tests, the isolated strains were identified using commercial systems ID32 STAPH (bioMérieux, France) and the BBL Crystal Gram-Positive ID Kit (Becton Dickinson, USA) according to the enclosed instructions. The Staphylococcus isolates were examined for sensitivity to the following: oxacillin, penicillin, cefoxitin, gentamicin, erythromycin, chloramphenicol, tetracycline, ciprofloxacin, sulfametoxazol/trimetoprim, and vacomycin using the disk diffusion method and the broth microdilution method as recommended by the Clinical and Laboratory Strandards Institute - CLSI(2003), and the results were interpreted according to CLSI recommendations from 2008 and 2010. Antibiogram disks manufactured by Becton Dickinson (USA) were used, and the broth microdilution method was applied using pure antibiotic substances from different manufacturers: erythromycin, chloramphenicol, cefoxitin, gentamicin, oxacillin, tetracycline (Sigma Aldrich, USA), sulfametoxazol (Fluka, USA), penicillin (Calbiochem, Germany), vancomycin (Abbott laboratories, USA), ciprofloxacin and trimetoprim (Zdravlje A.D., Serbia). All 25 strains were catalase positive and oxidase negative. Of the 25 strains, 19 were coagulase positive and 6 were coagulase negative.With the implementation of the disk diffusion method on 19 strains of S. aureus, 17 were established to be resistant to penicillin (89.5%), and 2 strains to gentamicin (10.5%). The investigation of 3 strains of S. xylosus using the disk diffusion method showed that one strain was resistant to tetracycline (33.3%) and to oxacillin (33.3%), while another strain was found to be resistant to penicillin (33.3%). The third strain of S. xylosus was sensitive to all the examined antibiotics. Two strains of S. simulans and one strain of S. haemolyticus were not found to be resistant to any of the examined antibiotics using the disk diffusion method. The implementation of the broth microdilution method yielded in 13 strains of S. aureus resistance to penicillin (68.4%) with MIC values from 0.5 to 4 μg/m, in 2 strains to gentamicin (10.5%) with MIC values of 32 μg/ml, and intermediary sensitivity to chloramphenicol was established in 9 strains of S. aureus (47.4%) with MIC values of 16 μg/ml and to vancomycin in 1 strain of S. aureus (5.3%) with MIC values of 4 μg/ml.

Cilj ovog rada je bila identifikacija izolovanih vrsta stafilokoka i ispitivanje njihove osetljivosti na neke antibakterijske lekove. Kao materijal u ovom ispitivanju korišćeni su izolati stafilokoka poreklom iz uzoraka mleka. Ukupno je ispitano 25 sojeva izolovanih stafilokoka od kojih su 24 poticala iz uzoraka mleka krava sa mastitisom, a jedan soj je izolovan iz uzorka mleka krave nakon lečenja mastitisa. U primarnoj identifikaciji su korišćeni katalaza i oksidaza testovi, kao i test prisustva slobodne koagulaze. Nakon izvođenja preliminarnih testova, vršena je identifikacija izolovanih sojeva, primenom komercijalnih sistema ID32 STAPH (bioMérieux, Francuska) i BBL Crystal Gram-Positive ID Kit (Becton Dickinson, SAD) prema uputstvima proizvođača. Osetljivost izolovanih sojeva stafilokoka ispitivana je na: oksacilin, penicilin, cefoksitin, gentamicin, eritromicin, hloramfenikol, tetraciklin, ciprofloksacin, sulfametoksazol/trimetoprim i vankomicin primenom disk difuzione metode i mikrodilucione metode u bujonu prema preporukama Instituta za kliničke i laboratorijske standarde (Clinical and Laboratory Strandards Institute - CLSI (2003), a tumačenje rezultata je vršeno prema preporukama CLSI iz 2008. i 2010. godine. Korišćeni su antibiogram diskovi proizvođača Becton Dickinson (SAD), a za mikrodilucionu metodu u bujonu korišćene su čiste supstance antibakterijskih lekova različitih proizvođača: eritromicin, hloramfenikol, cefoksitin, gentamicin, oksacilin, tetraciklin (Sigma Aldrich, SAD), sulfametoksazol (Fluka, SAD), penicilin (Calbiochem, Nemačka), vankomicin (Abbott laboratories, SAD), ciprofloksacin i trimetoprim (Zdravlje A.D., Srbija). Svih 25 sojeva je bilo katalaza pozitivno i oksidaza negativno. Od 25 sojeva, 19 je bilo koagulaza-pozitivno, a 6 koagulaza-negativno. Primenom disk difuzione metode od 19 sojeva S. aureus kod 17 je utvrđena rezistencija na penicilin (89,5%), a kod 2 soja na gentamicin (10,5%). Od 3 soja S. xylosus, kod jednog je primenom disk difuzione metode utvrđena rezistencija na tetraciklin (33,3%) i na oksacilin (33,3%), dok je kod drugog soja utvrđena rezistencija na penicilin (33,3%). Treći soj S. xylosus je bio osetljiv na sve ispitivane antibiotike. Kod dva soja S. simulans i jednog soja S. haemolyticus nije utvrđena rezistencija ni na jedan od ispitivanih antibiotika primenom disk difuzione metode. Primenom mikrodilucione metode u bujonu kod 13 sojeva S. aureus je utvrđena rezistencija na penicilin (68,4%) sa vrednostima minimalne inhibitorne koncentracije (MIC) od 0,5 do 4 μg/m, kod 2 soja na gentamicin (10,5%) sa vrednostima MIC od 32 μg/ml, a intermedijarna osetljivost na hloramfenikol utvrđena je kod 9 sojeva S. aureus (47,4%) sa vrednostima MIC od 16 μg/ml i na vankomicin kod jednog soja S. aureus (5,3%) čija je vrednost MIC iznosila 4 μg/ml.