Pathogenicity and Antimicrobial resistance of E. Coli

Escherichia coli (E. coli), is responsible for different pathologies in birds, the most outstanding ones are: respiratory chronic disease, onfalitis, salpingitis, septicemia, peritonitis, syndrome of the swollen head, enteritis and cellulites (Gross, 1994). Cellulitis is an acute purulent inflammation affecting subcutaneous tissue (Norton, 1997). The frequent isolation of E. coli from cellulitis and the further experimental reproduction of such a pathology, as of the inoculation of E. coli samples, prove that this microorganism is the responsible one for this type of lesion (Messier et al., 1993). The objective of this work, is that of evaluating antimicrobial resistance, as well as the pathogenicity of E. coli samples, isolated from cellulites lesions in chicken.

Forty eight samples of E. coli isolated from cellulitis lesions in broilers, from Parana, Brazil, were used. Antimicrobial resistance tests were done using the diffusion technique of antibiotic-impregnated filter paper disks. Three colonies were isolated by TSA in a container with 3 mL of nourishing broth, at 37º C during 2 hours. Culture was diluted at 1:100 in sterile physiologic saline and seeded by means of a swab in Mueller-Hinton agar, with the purpose of achieving a confluent growth. After drying the plates, the following antimicrobial disks were used: nalidixic acid (30 mcg), ampiciline (10 mcg), ceftiofur (30 mcg), ciprofloxacine (30 mcg), chloramphenicol (30 mcg), doxycycline (10 mcg), enrofloxacine (5 mcg), florfenicol (30 mcg), gentamicine (10 mcg), linocmicine+espectinomicine (50+100 mcg), neomicine (30 mcg), norfloxacine (10 mcg), sulfa+trimetoprim (25 mcg), sulfonamides (300 mcg) and tetracyclines (30 mcg). Plates were incubated at 37º C during 24 hours. Previously, ATCC 25922 disks sensitive to all antimicrobial drugs were tested. Results were determined by measuring the growth inhibition halos and by comparing them with the values presented in the standard tables. The antimicrobial multiple resistance index (IRMA) for each sample, was estimated according to Kaspar & Burgess (1990), (IRMA= number of resistant antibiotics / total number of tested antibiotics).

Pathogenicity of E. coli strains was evaluated in 500 Cobb chicken, one day old, divided into 50 groups of 10 birds each. Each group was inoculated with a different sample of E. coli. Two groups were inoculated with a sterile  saline solution, just and only. Birds received 0.1 mL of the inoculant subcutaneously, in the cervical region and were daily evaluated during 7 days, observing mortality (Brito et al., 2003). According to the mortality rate (MR), samples were classified as: high pathogenicity (MR > 80%), intermediate pathogenicity (MR 50% and 80%), low pathogenicity (MR < 50%) and non pathogenic (MR = 0%).

The evaluation of the sensitivity of E. coli samples, isolated from the lesions of cellulitis in broilers at slaughter, was done by means of an antibiogram, using 16 commercial drugs. The most efficient drugs tested were ceftiofur and florfenicol. Tested samples presented 100 % of sensitivity to such antibiotics. The following sensitivity percentages were verified for the other drugs: neomicine (98%), lincomicine + espectinomicine (94%), chloramphenicol (90%), ampiciline (75%), norfloxacine (75%), gentamicine (71%), ciprofloxacine (67%), enrofloxacine (62%), sulfa+trimetoprim (62%), nalidixic acid (50%), sulfonamides (46%), tetracycline (21%) and doxicycline (4%). In several works, high percentages of tetracycline resistance were observed (Cloud et al., 1985; Peighambari et al., 1995). All samples presented resistance to at least one of the drugs they were subject to. Of the 48 analyzed samples, eight were resistant to one drug and eleven to four of the drugs tested. The average IRMA of the forty eight samples was of 0.323.

Regarding the classification of the pathogenicity of E. coli, we can stress that of the forty eight analyzed samples of E. coli, 71% was classified as of high pathogenicity and 29% of the samples presented intermediate pathogenicity, proving that all of the samples were pathogenic and that none of them proved to be non pathogenic, or of low pathogenicity in in vivo trials.

The variation in the results of antimicrobial resistance may be explained because of endless factors. The occurrence of antimicrobial resistance may be determined by a chromosome mutation, or transmitted by plasmids. We cannot stress that more pathogenic samples have a higher resistance, but there may exist a plasmid transference, reaching a higher resistance to one or more  antimicrobials, an elevation in the pathogenicity of the bacteria (Gross, 1994).

Escherichia coli samples proved to be pathogenic for birds, presenting high sensitivity to florfenicol and ceftiofur. Such bacteria present high resistance to tetracycline y doxicycline.

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Messier S, Quessy S, Robinson Y, Devriese LA, Hommez J, Fairbrother JM. 1993. Focal dermatitis and celullitis in broiler chickens. Avian Dis. 37:839-844.

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Apoio: Conselho Nacional Desenvolvimento Científico e Tecnológico - CNPq - Programa RHAE.