Materials & Methods Objective Extraintestinal pathogenic Escherichia coli in healthy broiler chicken in Italy: a combination of virulence with antibiotic resistance Arash Ghodousi 1,2, Celestino Bonura 2, Laura Saporito 2, Caterina Mammina 2 1.PhD School in Molecular Medicine, University of Palermo, Palermo, Italy 2.Department of Sciences for Health Promotion and Mother-Child Care “G. D’Alessandro”, University of Palermo, Palermo, Italy Abstract (Click on the text to edit) Background Materials & Methods Conclusions References 1.Antimicrobial resistant and extraintestinal pathogenic Escherichia coli in retail foods. J Infect Dis 2.Extraintestinal pathogenic Escherichia coli in poultry meat products on the Finnish retail market. Acta Vet Scand 3.Identification and antimicrobial resistance of extraintestinal pathogenic Escherichia coli from retail meats. J Food Prot 4.Extraintestinal Pathogenic Escherichia coli: A Combination of Virulence with Antibiotic Resistance. Front Microbiol Escherichia coli, a commensal bacterium which is usually found in the intestinal tract of humans and animals, can cause healthcare- and community-acquired infections including urinary tract, post-surgical and systemic infections, based upon their virulence gene content and antibacterial drug resistance. Extraintestinal Pathogenic E. coli (ExPEC), the distinctive E. coli strains that possess the specialized virulence factors (VFs) required for extraintestinal disease, are of particular concern. They can be, indeed, associated with animal and human infections, such as urinary tract and bloodstream infections. Acquired antimicrobial resistance is particularly concerning when it occurs in ExPEC. Antibiotics in livestock production are used for a variety of reasons, such as infectious disease treatment, prevention, control, and growth promotion/feed efficiency. However, any use of antimicrobial drugs, mainly low- dose, prolonged courses among food animals, will select for antibiotic-resistant bacteria, which in turn will spread by direct contact or through food, water, and animal waste application to farm fields. Methods of livestock production differ in antibiotic use influencing the prevalence of antibiotic-resistant bacteria on retail meats. Extended-Spectrum β-Lactamase (ESBL) and AmpC producing E. coli are associated with localized or systemic disease in humans. Resistance genes coding ESBLs are mostly plasmid associated and therefore can easily horizontally spread among E. coli strains. Globally, antimicrobial drug resistant Escherichia coli is amongst the most common etiological agents of invasive disease in humans. In Europe, increasing proportions of infections due to third generation cephalosporins (3GCs) and/or fluoroquinolone resistant extraintestinal pathogenic E. coli (ExPEC) strains are reported. E. coli from poultry are those more closely linked to human E. coli, but lack of reliable data makes it difficult to assess the attributable risk of different food sources. In the present study, our objective was to investigate the antimicrobial resistance profile, phylogenetic background and virulence factors of E. coli isolates from broiler chicken meat sold at retail in Palermo, Italy. Isolation of multidrug resistant (MDR) E. coli was performed during April-December 2013 on a total of 163 chicken meat samples. Susceptibility to a panel of ten antimicrobial agents was determined. PCR assays were carried out to detect extended spectrum β- lactamase (ESBL), plasmid-mediated AmpC β-lactamase and plasmid-mediated quinolone resistance (PMQR) genes, phylogenetic group and ExPEC-associated traits. A single nucleotide polymorphism (SNP) PCR was done to detect E. coli sequence type (ST)131. One hundred thirty-four isolates were MDR. B1 was the most prevalent phylogenetic group (47.8%), followed by groups D (25.4%), A (22.3%) and B2 (4.5%). ESBLs and AmpC β-lactamases were detected by PCR in 132 (98.5%) and 15 (11.2%) isolates. PMQR determinants were detected in 122 (91%) isolates. Twenty-two MDR isolates met the molecular definition of ExPEC. SNP- PCR results confirmed that four B2 isolates were ST131. An alarmingly high prevalence of MDR E. coli from broiler chicken meat is evident in our geographic area. The ongoing use of antimicrobial drugs in livestock should be urgently restricted, particularly in the poultry sector. Isolation of multidrug resistant (MDR) E. coli was performed during April-December 2014 on a total of 163 chicken meat samples. Susceptibility to a panel of ten antimicrobial agents was determined. PCR assays were carried out to detect extended spectrum β-lactamase (ESBL), plasmid-mediated AmpC β-lactamase and plasmid-mediated quinolone resistance (PMQR) genes, phylogenetic group and ExPEC-associated traits. A single nucleotide polymorphism (SNP) PCR was done to detect E. coli sequence type (ST)131. In summary, our results show that in Sicily a large proportion of retail chicken meat samples carry cephalosporin and ciprofloxacin resistant E. coli. A high prevalence of ESBL and PMQR determinants was also found in these isolates, which raises a serious public health concern. A prudent use of antimicrobial agents is urgent in veterinary as well as in human medicine to minimize the selection and spread of antibiotic resistant strains and their resistance genetic determinants. In this study, our objective was to investigate phylogenetic background, antimicrobial resistance profile, virulence factors and their association in E. coli isolates recovered from retail broiler chicken meat on sale in Sicily, Italy. Fig. 1. Prevalence of resistance to the antibacterial drugs under study among the 134 MDR E. coli isolates by phylogenetic group. Fig. 2. Distribution of the resistance genetic determinants under study by phylogroup Results & Discussion All 163 chicken meat samples under investigation grew E. coli of which 136 proved to be MDR. The results of antibiotic susceptibility testing can be found in Figure 1. Phylogenetic group B1 was the most prevalent (64 isolates, 47.8%) followed by groups D (34 isolates, 25.4%), A (30 isolates, 22.3%) and B2 (eight isolates, 4.5%). ESBLs and AmpC β-lactamases were detected by PCR in 132 (98.5%) and 15 (11.2%) isolates, respectively, whilst PMQR determinants were detected in 122 (91%) of isolates (Figure 2). Based on the molecular definition of ExPEC, 24 isolates out of 40 belonging to phylogroups B2 and D were attributed with the status of ExPEC (all the B2 isolates and 16 out of 34 [47.1%] D isolates). The most prevalent virulence factors were kpsMT II and iutA. SNP-PCR results confirmed that 4 isolates of phylogroup B2 were ST131. These four isolates were positive for both ESBL and PMQR determinants. In this study, MDR E. coli were recovered from 66.9% of poultry meat samples on sale in Palermo, Italy. These results appear to be consistent with findings from other European and non-European countries. However, results from different studies are difficult to compare because of different settings and sample processing methods. Our findings also can be cautiously compared with those of previous studies because of our MDR E. coli detection approach. The predominant ESBL gene families among our E. coli isolates were bla SHV, bla TEM, and bla CTX-M-1 group, which is comparable with previous data from European countries. TEM, SHV and CTX-M-1 group are reported as the most prevalent β-lactam resistance determinants in the food animal reservoir. The prevalence of plasmid-mediated quinolone resistance was very high among our isolates which highlights a serious clinical and public health issue of concern. qnrA was the only PMQR determinant detected in all ciprofloxacin resistant isolates accounting for a prevalence of about 90%, substantially higher than any other previous report. The majority of the isolates in our study belonged to the phylogenetic group B1, followed by groups D, A and B2. These results are consistent with previously reports from both European and non European countries Within our collection, 22 (16.4%) E. coli isolates could be classified as ExPEC. Johnson et al. 1 found 21% of 110 E. coli isolates from chicken products to meet the criteria for ExPEC identification. In a further study by Lyhs et al. 2 22% of E. coli strains from chicken samples could be classified as ExPEC. Xia et al. 3 among different retail meats found the highest rate of ExPEC strains in ground turkey (23.5%) and chicken breasts (20.2%). Of interest, our D isolates were more frequently antibiotic resistant, ESBL and AmpC carrying compared with those belonging to phylogroups A and B1, which is consistent with literature reporting a close combination of antibiotic resistance and virulence traits 4. B2 phylogroup isolates behaved similarly but their limited number does not allow to draw meaningful conclusions.