CONICET | Buscador de Institutos y Recursos Humanos

Introduction. Resistance to extended spectrum beta-lactams, especially third- and fourth-generation cephalosporins and penems, is of special interest for human and veterinary medicine. Salmonella spp. are important zoonotic pathogens in humans and animals. Increasing rates of antimicrobial resistance in Salmonella isolates have been reported from a number of developing and developed countries. The purpose of this study was to carry out a preliminary screening to determine the occurrence of antibiotic resistance of Salmonella enterica isolates recovered in the context of a surveillance program among poultry farms from Argentina. Material and Methods. A selected group of 27 strains of Salmonella enterica recovered from broilers and laying hens farms from Entre Ríos and Buenos Aires provinces over the period 2010-2012 was examined for resistance to 22 antibiotics. Organisms were obtained from pools of poultry droppings or by fecal boot swab samples. Each isolate represented a contaminated farm. Identification of serovars was performed by the agglutination technique using the antisera produced by the Instituto Nacional de Producción de Reactivos y Biológicos -ANLIS, Dr. Carlos G Malbràn. To investigate the antimicrobial susceptibilities of the isolates, the Kirby-Bauer disk diffusion susceptibility test was performed using Mueller-Hinton Agar (MHA) plates according to the Clinical Laboratory Standards Institute (CLSI) using the following antimicrobials: ampicillin (AMP, 10 μg), cephalotin (CEP, 30 μg), cefuroxime (CXM, 30 μg), cefotaxime (CTX, 30 μg), ceftazidime (CAZ, 30 μg), cefoxitin (FOX, 30 μg), ceftriaxone (CRO, 30 μg), ceftiofur (CFT, 30 μg), cefepime (FEP, 30 μg), aztreonam (AZT, 30 μg), kanamycin (KA, 30 μg), gentamicin (GEN, 10 μg), streptomycin (ST, 10 μg), tetracycline (TET, 30 μg), chloramphenicol (CLR, 30 μg), trimethoprim?sulfamethoxazole (SxT, 1.25/ 23.75 μg), nalidixic acid (NAL, 30 μg), ciprofloxacin (CIP, 5 μg), amikacin (AMI, 30 μg), imipenem (IMI, 10 μg), meropenem (MEM, 10 μg) and enrofloxacin (ENR, 5 μg). Third- generation cephalosporins resistant strains were characterized as extended spectrum beta-lactamase (ESBL) producers by using the CLSI-recommended phenotypic confirmatory test (PCT) or AmpC for boronic acid synergy test, respectively. The presence of beta-lactamase genes was detected by PCR using plasmidic DNA purified according to the Kado and Liu method. Briefly, isolates were analysed using a multiplex PCR for the detection of family-specific plasmid-mediated AmpC β-lactamase genes (ACC, CIT, DHA, EBC, FOX and MOX) and by simple PCR with primers for blaCMY-2. The presence of blaCTX-M genes was investigated by a screening PCR with universal primers for blaCTX-M-like genes. The positive isolates detected were further analysed by simple PCRs using specific primers for the blaCTX-M groups M-1, M-2, M-8, M-9 and M-25. Results. The 27 Salmonella isolates belonged to 20 serovars. Four isolates (S. Heidelberg Nº2, S. Havanna, S, Oranienburg and S. Thyphimurium) showed resistance to ceftiofur. S. Heidelberg Nº2 was also resistant to ampicillin, cefalotin, cefuroxime, cefotaxime, ceftazidime, ceftriaxone, cefepime and aztreonam, whereas S. Havanna, S, Oranienburg and S. Thyphimurium were resistant to ampicillin, cefalotin, cefuroxime, cefotaxime, ceftazidime, ceftriaxone and cefoxitin. On the basis of their antibiotic susceptibility patterns, and presence of genes detected by PCR, S. Heidelberg Nº2 was found to carry CTX-M-2 group determinants. On the other hand, an AmpC beta-lactamase CMY-2-like was identified in S. Havanna, S. Oranienburg and S. Thyphimurium, respectively. Resistance to streptomycin (70%), tetracycline (59 %), nalidixic acid (55%), ciprofloxacin (40%) and enrofloxacin (37%) were highest, whereas resistance to chloramphenicol (18.5 %), kanamycin (15%) and gentamycin (3.7 %) were low. All strains were susceptible to amikacin, meropenem, imipenem and trimethoprim/sulfamethoxazole. Discussion. Resistance to ceftiofur and ceftriaxone, two third generation cephalosporins, has significant clinical implications for both human and veterinary medicine. Ceftriaxone is the drug of choice for treatment of severe salmonellosis in humans, especially in children. Ceftiofur is a cephalosporin whose use has been increased in the last years in the treatment of bacterial infections in food animals. Poultry production is relevant as both direct and indirect source of human salmonellosis, and antimicrobial resistance is an emerging problem of public health concern. S. Oranienburg producing beta-lactamase of the CTX-M-2 group was reported in a pediatric hospital in Tucumán, Argentina. Recently, a CMY-2 plasmid mediated β-lactamase in S. Heidelberg was detected for first time in Argentina and South America in an input patient. These results emphasize the need of implementing an Argentinean Integrated Program for Antimicrobial Resistance Surveillance.

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