Occurrence of diverse Extended-spectrum beta-lactamase (ESBL)- and AmpC beta-lactamase-encoding genes in Avian Pathogenic Escherichia coli from poultry in Italy

Avian pathogenic Escherichìa coli (APEC) cause infections with high morbidity and mortality in poultry flocks. Beta-lactams like orai penicillins, ampicillin and amoxicillin, are among the most used antimicrobials in poultry but they can be inactivated by enzymes including extendedspectrum |3-lactamases (ESBLs)- and AmpC P-lactamases. Information on occurrence and mechanisms of beta-lactam resistance in APEC is limited, although highly relevant for animai welfare and economy within the poultry industry. This study aimed at investigating the prevalence and diversity of ESBL- and AmpC P-lactamase-encoding genes in APEC from chickens, turkeys and layer hens collected from industriai poultry farms across Italy between 2008 and 2012. A total of 226 non-repetitive clinical E. coli isolates from turkeys (n=104), broilers (n=99) and layer hens (n=23) was screened for ESBL/AmpC production by phenotypic tests according to Clinical and Laboratory Standards Institute (CESI) guidelines. Isolates resistant to at least one thirdgeneration cephalosporin were tested for presence of TEM-, SHV-, CTX-Mand CMY-2-encoding genes and mutations in the chromosomal AmpC promoter by PCR and sequencing. A total of 31 (14%) isolates from ali sources displayed resistance to at least one third-generation cephalosporin and 26 (84%) of those isolates were confìrmed as ESBLs producers. The ESBL-encoding genes è/aSHV-12, è/aCTX-M-1, blaCTX-M-2 and è/aCTX-M-14 were detected in one, twelve, one and two isolates, respectively. The AmpC P-lactamase-encoding gene è/aCMY-2 was detected in two isolates, and ten isolates presented mutations in the chromosomal AmpC promoter compatible with AmpC overproduction. è/aTEM-1 was detected in fifteen isolates associateci with 6/aCTX-M-l (n=8), WaCTX-M-14 (n=l), WaCMY-2 (n=2), and AmpC promoter mutations (n=I). In three isolates, the molecular bases for ESBL phenotype were not elucidated and are currently under investigation. This is the first study characterizing ESBL/AmpC-encoding genes in APEC in poultry in Italy. A broad diversity of ESBL/AmpC-encoding genes was detected, including variants not previously reported in Italian poultry (6/aCTX-M-14). This suggests that APEC acquired such genes on multiple occasions, and emphasizes the need of studies identifying the causes for occurrence of ESBL/AmpC-encoding genes in poultry flocks