The investigation of new forms of resistance to some antibiotics in E coli strains isolated from piglets

Abstract

The resistance of bacteria poses a significant problem everywhere in the world, and consequently in our country as well. The non-critical use of antibacterial medicines in human and veterinary medicine has contributed to the spreading of this resistance. Due to the topical importance of this problem, large numbers of states in the world are financing projects of which the objective is to follow and monitor bacterial resistance. The objective of this investigation was to isolate and identify pathogenic strains of E. coli from piglets with clinically manifest diarrhea and to examine the sensitivity of the isolated strains to a certain number of selected antibiotics. The material for these investigations were parts of intestines (jejunum, ileum) from piglets that died, rectal smears and feces of diseased piglets sampled pig farms in the vicinity of Belgrade. Conventional methods of microbiological diagnostics were used for isolation, and conventional and commercial tests API 20E (bio Merieux, France) were used for identification. Following biochemical identification using hyper immune serums for certain group (O) antigens: (O8, O138, O139, O147, O149, and O157), the serological typization of the strains was carried out. Commercial antiserums: T K88 (F4), K99 (F5), and 987P (F6), Toxigenic E. coli pili antisera, Denka Seiken Co. Ltd. Tokyo, Japan) were used to establish the presence and to identify fimbrial antigens-adhesions through slide agglutination reaction. The sensitivity of the antibiotics to the isolated strains of E. coli was examined using the disc diffusion method according to Kirby Bauer and the microdillution method in bouillon according to CLSI recommendations (2008). Examinations using the microdillution method in bouillon were performed with pure active antibiotic substances: ampicillin, apramycin, gentamicin, kanamycin, tetracycline, ceftriaxone, and ciprofloxacin (Sigma, USA). A total of 400 E. coli strains were isolated, including 48 E. coli strains that are within the category of pathogenic strains. Out of the total number of pathogenic strains of E. coli, 32 (66.67%) were found to be multi resistant to 3 or more than 3 antibiotics, and 16 (33.33%) pathogenic strains of E. coli were resistant to 2 or 1 of the examined antibiotics. Resistance to tetracycline and ampicillin was established in 89.58% isolated strains of E. coli, and to ciprofloxacin in 37.5% strains. The lowest percentage of resistance to ceftriaxone was established in 4.17% strains of E. coli. The isolated strains of E. coli were also found to be resistant to certain amino glycosides antibiotics. Thus, resistance to apramycin was established in 18.75% strains whose MIC values were higher than 128 µg/mL. Resistance to kanamycin was established in 52.08% strains of E. coli, and in these, 96.00% strains showed MIC kanamycin values of over 256µg/mL. Resistance to the third amino glycosides antibiotic, gentamicin, was established in 33.33% E. coli strains. All the E. coli strains that led to diarrhea in piglets were resistant to at least two antibiotics, and more than 50% strains were found to be resistant to more than 3 antibiotics. It is significant to stress that all isolated strains of E. coli covered by these investigations were sensitive only to amikacyn. .

Rezistencija bakterija predstavlja značajan problem svuda u svetu, pa i kod nas. Širenju rezistencije doprinela je nekritička upotreba antibakterijskih lekova u humanoj i veterinarskoj medicini. Zbog aktuelnosti problema veliki broj država finansira projekte čiji je cilj praćenje i nadzor nad rezistencijom bakterija. Cilj ovog istraživanja je bio izolacija i identifikacija patogenih sojeva E. coli kod prasadi sa klinički manifestnom dijarejom i ispitivanje osetljivosti izolovanih sojeva na određeni broj odabranih antibiotika. Materijal za ovo ispitivanje predstavljali su delovi creva (jejunum, ileum) uginule prasadi, rektalni brisevi i feces obolele prasadi uzorkovani na farmama svinja iz okoline Beograda. Za izolaciju su korišćene klasične metode mikrobiološke dijagnostike, a za identifikaciju klasični i komercijalni testovi API 20E (bioMerieux, Francuska). Nakon biohemijske identifikacije pomoću hiperimunih seruma za određene grupne (O) antigene (O8, O138, O139, O147, O149 i O157) vršena je serološka tipizacija sojeva. Za utvrđivanje prisustva i identifikaciju fimbrijalnih antigena - adhezina primenom reakcije aglutinacije na pločici su korišćeni komercijalni antiserumi: K88 (F4), K99 (F5) i 987P (F6), Toxigenic E. coli pili antisera, Denka Seiken Co. Ltd. Tokyo, Japan. Osetljivost na antibiotike izolovanih sojeva E. coli ispitivana je primenom disk difuzione metode po Kirby Baueru i mikrodilucione metode u bujonu prema preporukama CLSI (2008). Ispitivanje mikrodilucionom metodom u bujonu je vršeno sa čistim aktivnim supstancama antibiotika: ampicilina, apramicina, gentamicina, kanamicina, tetraciklina, ceftriaksona i ciprofloksacina (Sigma, SAD). Ukupno je izolovano 400 sojeva E. coli, od kojih je 48 svrstano u kategoriju patogenih sojeva. Od navedenog broja patogenih sojeva E. coli kod 32 (66,67%) je ustanovljena multirezistencija na 3 i više od 3 antibiotika, a kod 16 sojeva (33,33%) rezistencija na 2 ili 1 ispitivani antibiotik. Rezistencija na tetraciklin i ampicilin ustanovljena je kod 89,58% izolovanih sojeva E. coli, a na ciprofloksacin kod 37,5% sojeva. Najniži procenat rezistencije na ceftriakson ustanovljen je kod 4,17% sojeva E. coli. Prisustvo rezistencije kod izolovanih sojeva E. coli ustanovljeno je i na neke aminoglikozidne antibiotike. Tako je rezistencija ustanovljena kod 18,75% sojeva kod kojih su vrednosti MIC-a bile veće od 128 μg/mL. Kod 52,08% sojeva E. coli otkrivena je rezistencija na kanamicin, a od navedenog procenta kod 96,00% sojeva vrednosti MIC-a kanamicina iznosile su preko 256 μg/mL. Takođe je kod 33,33% sojeva E. coli ustanovljena rezistencija i na treći aminoglikozidni antibiotik, gentamicin. Svi sojevi E. coli koji su uzrokovali dijareju kod prasadi bili su rezistentni na najmanje dva antibiotika, a kod preko 50% sojeva otkriveno je prisustvo rezistencije na više od 3 antibiotika. Značajno je naglasiti da su svi izolovani sojevi E. coli obuhvaćeni ispitivanjem bili osetljivi samo na amikacin.