27 Parole chiave Antibiotico-resistenza, Colibacillosi, Escherichia coli patogeno aviario (APEC), Geni di virulenza, PCR. Riassunto La colibacillosi è la malattia batterica che si riscontra più frequentemente nelle specie aviari e i farmaci antimicrobici sono l'arma più utilizzata per ridurre sia l'incidenza che la mortalità ad essa legate. L'uso indiscriminato di antibiotici può, tuttavia, portare al fallimento della terapia e a ingenti perdite economiche da parte dell’allevatore. Questo studio ha l’obiettivo di determinare i tassi di resistenza agli antibiotici da parte di ceppi di Escherichia coli, valutare la possibile correlazione tra l'isolamento di E. coli e le tipologie di allevamento prese in considerazione e rilevare la presenza di E. coli patogeni aviari (APEC) tra i vari E. coli isolati. Mediante l’impiego di 19 agenti antimicrobici, sono stati sottoposti al test di suscettibilità antimicrobica 51 ceppi di E. coli; gli stessi ceppi sono stati analizzati per valutare la presenza di otto geni di virulenza mediante metodica PCR. La resistenza è stata riscontrata più frequentemente verso ampicillina e acido nalidixico, mentre gli isolati di E. coli hanno mostrato una minore resistenza nei confronti delle cefalosporine. Complessivamente, il 40% degli isolati ha mostrato resistenza ad almeno tre o più agenti antimicrobici. Sedici di 51 isolati sono stati definiti ceppi APEC poiché in essi sono stati rilevati almeno cinque degli otto geni di virulenza ricercati. Mentre i geni di virulenza iucD, cvi / cva, irp2 e iss sono stati rilevati in tutti i 16 ceppi APEC, tsh, vat, papC e astA rispettivamente da 11, 7, 5 e 3 ceppi APEC. I nostri risultati dimostrano quanto sia importante approfondire la diffusione del fenomeno dell’antibiotico resistenza e indagare la distribuzione di ceppi APEC in Italia, anche al fine di valutare un’eventuale correlazione tra i due fenomeni, con l’obiettivo di fornire uno strumento preventivo utile. Inoltre, è stato dimostrato come la tipologia di allevamento adottata può influenzare i tassi di antibiotico resistenza. Resistenza antibiotica e geni di virulenza in E. coli patogeni aviari (APEC) isolati in varie tipologie di allevamento Keywords Antibiotic resistance, Avian pathogenic Escherichia coli (APEC), Colibacillosis, PCR, Virulence gene. Summary Colibacillosis is the most frequent bacterial disease in avian species and antimicrobials are the main weapon to reduce incidence and mortality associated to it. However, indiscriminate use of antibiotics may lead to therapy failure and economic losses for the breeder. The aims of this study were to, determine the antibiotic resistance of Escherichia coli isolates, evaluate the correlation between E. coli isolation and systems of breeding included in this study, and identify the avian pathogenic E.coli (APEC) amongst the E. coli strains isolated. A total of 51 E. coli strains were subjected to antimicrobial susceptibility test and they were screened for the presence of virulence genes through PCR. Resistance was most frequently detected against ampicillin and nalidixic acid meanwhile E. coli isolates showed less resistance to the cephalosporins. Overall, 40% of the isolates showed resistance to at least three or more antimicrobials and 16/51 isolates were defined APEC strains. The virulence genes iucD, cvi/ cva, irp2 and iss were detected from all 16 APEC strains. The virulence genes tsh, vat, papC, and astA were detected from 11, 7, 5 and 3 APEC strains, respectively. Results demonstrated the importance of studies on APEC and antibiotic resistance genes in Italy, and it was shown that the systems of breeding might influence the antibiotic resistance. Veterinaria Italiana 2019, 55 (1), 27-33. doi: 10.12834/VetIt.1617.8701.1 Accepted: 19.12.2018 | Available on line: 31.03.2019 Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche ‘Togo Rosati’, Via G. Salvemini 1, 06126 Perugia, Italy #These authors contributed equally to this work. *Corresponding author at: Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche ‘Togo Rosati’ Via G.Salvemini 1, 06126 Perugia, Italy. E‑mail: elisa.sgariglia86@gmail.com. Elisa Sgariglia*#, Nicholas Aconiti Mandolini#, Maira Napoleoni, Laura Medici, Roberta Fraticelli, Michela Conquista, Paola Gianfelici, Monica Staffolani, Stefano Fisichella, Marinella Capuccella, Marta Sargenti and Gianni Perugini Antibiotic resistance pattern and virulence genes in avian pathogenic Escherichia coli (APEC) from different breeding systems 28 Veterinaria Italiana 2019, 55 (1), 27-33. doi: 10.12834/VetIt.1617.8701.1 Antibiotic resistance pattern and virulence genes in APEC Sgariglia et al. Introduction Escherichia coli (E.  coli) is considered a commensal microorganism in people and animals and it is part of normal intestinal microflora in birds (Aarestrup et  al. 2008, De Carli et  al. 2015). Some strains might be pathogenic and cause colibacillosis, an extraintestinal disease characterised by pericarditis, air sacculitis, perihepatitis, peritonitis. Colibacillosis is responsible for high economic losses in chicken industry (Matthijs et al. 2009, Matter et al. 2011, De Carli et  al. 2015). It is the most frequent bacterial disease in avian species and E. coli is considered the first cause of death in poultry sector, even if usually it plays a secondary role during infection (Lutful Kabir 2010). Colibacillosis is caused by avian pathogenic E.  coli (APEC) (Matin et al. 2017) and its pathogenic ability may be localized or systemic. The APEC pathogenic ability is facilitated by broad range of virulence factors which are coded by virulence-associated genes (De Carli et al. 2015). According to molecular criteria, APEC is defined by presence of at least five virulence genes. According to molecular criteria, five genes carried by plasmids were considered as being the most significantly associated with highly pathogenic APEC strains (De Carli et al. 2015). Escherichia coli strains are also considered good indicators of antimicrobial resistance because they are part of the physiological microbiota both in man and animals, and they are also present in the environment (Aarestrup et al. 2008). Antibiotic resistance represents a serious problem to global public health, resulting in a significant impact on animal health and food safety (Aarestrup 2004). The misuse of antimicrobial agents could lead to selection and diffusion of resistant microorganisms with related increase of antibiotic resistance rate (Spellberg 2014). Furthermore, the problem of multi-drug resistance (MDR) can be transmitted and disseminated between animal and human pathogens, leading to treatment problems both animal and human diseases (Collignon et al. 2005). Poultry industries consume wide range of antibiotics, because only few regulations are controlling their use (Hvistendahl 2012). In poultry industries, antibiotics have been used in chicken broilers as growth promoter and disease preventive measures (Bhandari et  al. 2004, Osti et  al. 2017, Shrestha et al. 2017). The main goals of this study were (1) to determine the rates of antibiotic resistance of E.  coli isolated from several avian species, (2) to evaluate possible correlations between E.  coli isolation and the types of breeding and (3) to detect the presence of APEC among E. coli isolates. Table I. Origin of Escherichia coli strains isolated. Progressive number Type of breeding Species and/ or production class Type of samples 1 Industrial breeding Chicken Intestinal swab 2 Industrial breeding Chicken Liver 3 Industrial breeding Chicken Liver 4 Industrial breeding Chicken Intestinal swab 5 Industrial breeding Chicken Liver 6 Industrial breeding Chicken Intestinal swab 7 Industrial breeding Chicken Lung 8 Industrial breeding Chicken Femoral swab 9 Industrial breeding Chicken Spleen 10 Industrial breeding Chicken Liver 11 Industrial breeding Chicken Intestinal swab 12 Industrial breeding Chicken Femoral swab 13 Industrial breeding Chicken Vertebral swab 14 Industrial breeding Chicken Vertebral swab 15 Industrial breeding Chicken Pool organs 16 Industrial breeding Chicken Yolk sac 17 Industrial breeding Hen Liver 18 Industrial breeding Hen Liver 19 Industrial breeding Goose Intestinal swab 20 Industrial breeding Goose Heart 21 Industrial breeding Goose Heart 22 Chick dealer Chicken Liver 23 Chick dealer Chicken Intestinal swab 24 Chick dealer Chicken Liver 25 Chick dealer Chicken Intestinal swab 26 Chick dealer Chicken Liver 27 Chick dealer Chicken Intestinal swab 28 Chick dealer Capon Intestinal swab 29 Chick dealer Hen Intestinal swab 30 Chick dealer Duck Intestinal swab 31 Chick dealer Guinea fowl Intestinal swab 32 Chick dealer Guinea fowl Liver 33 Chick dealer Goose Lung 34 Chick dealer Goose Intestinal swab 35 Chick dealer Goose Lung 36 Chick dealer Goose Intestinal swab 37 Rural breeding Chicken Intestinal swab 38 Rural breeding Chicken Liver 39 Rural breeding Chicken Intestinal swab 40 Rural breeding Chicken Liver 41 Rural breeding Chicken Intestinal swab 42 Rural breeding Hen Intestinal swab 43 Rural breeding Hen Lung 44 Rural breeding Hen Heart 45 Rural breeding Hen Liver 46 Rural breeding Hen Spleen 47 Rural breeding Hen Intestinal swab 48 Rural breeding Capon Liver 49 Rural breeding Capon Intestinal swab 50 Rural breeding Pigeon Liver 51 Rural breeding Turkey Intestinal swab 29Veterinaria Italiana 2019, 55 (1), 27-33. doi: 10.12834/VetIt.1617.8701.1 Sgariglia et al. Antibiotic resistance pattern and virulence genes in APEC and 1 minute at 72°C. The amplicons were analyzed by 2% agarose gel electrophoresis (Euroclone®) prepared in 1x TBE buffer (Biorad®). All the PCR products were stained with appropriate intercalating dye and the bands were visualized and photographed under UV light. The amplified product was considered to contain virulence gene if it produced band of the expected size. The amplicon size of the toxin genes of APEC is described on the manufacturer’s instructions. Data related to resistance and virulence genes rates were analyzed by the Fisher exact test. A value of P < 0.05 was considered significant. Results The E.  coli resistant strains found in this study are displayed in Table II. The resistance most frequently observed was against ampicillin and nalidixic acid (23/51, 45%) followed by tetracycline (22/51, 43%), sulphonamide (21/51, 41%), flumequine (17/51, 33%). Escherichia coli isolates exhibited lower resistance to cefotaxime and cefepime (0%) followed by cefoxitin and ceftazidime (1/51, 2%). Overall, 41% (21/51) of the isolates showed resistance to at least three or more antimicrobial agents with a significantly (P  >  0.05) higher number of isolates from animal of industrial breeding (13/21) than isolates obtained from animals of rural breeding (2/21). No significant differences (P > 0.05) were observed between the number of resistent isolates obtained from samples collected from animals of dealer and those from industrial and rural breeding. An E.  coli strain isolated from liver of an animal of industrial breeding was resistant to 11 antimicrobial molecules, including all antimicrobial categories. The rates of resistance according to the type of breeding were illustrated in Figures 1, 2 and 3. Of the 51 E.  coli isolates, 16 (31%) were found to be APEC strains because they contain at least five virulence genes. Out of 16 APEC strains, 7 were isolated from samples collected from animals of industrial breeding (6 chicken and 1 hen), 5  from samples collected from animals of dealer (3 chicken and 2 geese), and 4 from samples collected from animals of rural breeding (2 geese, 1 capon and 1 hen). Seven virulence genes were present in 2 APEC strains, 6 in six strains and 5 in 8 strains. The virulence genes iucD, cvi/cva, irp2 and iss were detected in all 16 APEC strains. The virulence genes tsh, vat, papC, and astA were detected in 11, 7, 5 and 3 APEC strains, respectively. The virulence-associated genes iucD, iss, irp2, and cvi/cva were found in both APEC and non-APEC Materials and methods Fifty-one E.  coli strains from chickens (n  =  27), hens (n  =  9), geese (n  =  7), capons (n  =  3), guinea fowls (n = 2), duck (n = 1), pigeon (n = 1), and turkey (n = 1) originated from chick dealer (n = 15) , rural (n = 15) and industrial (n = 21) breeding were included in the present study. These isolates were from different types of samples (Table I). E.  coli identification was performed with standard microbiological techniques which include studies of colony morphology, Gram staining, and biochemical tests (API Biomerieux®). The E.  coli isolates were tested for antibiotic susceptibility against 19 antimicrobial agents using disk agar diffusion method according to the Clinical Laboratory Standard Institute (CLSI) and EUCAST guidelines, based on available clinical breakpoints. The antibiotics used in this study, belonged to 7 categories of antimicrobial agents, and included ampicillin (AMP 10  µg), amoxicillin/clavulanic acid (AMC 30  µg), cefotaxime (CTX 30  µg), cefepime (FEP 30  µg), cefoxitin (FOX 30  µg), ciprofloxacin (CIP 5  µg), cefazolin (KZ 30  µg), ceftazidime (CAZ 30 µg), chloramphenicol (C 30 µg), enrofloxacin (ENR 5  µg), gentamicin (CN 10  µg), kanamicin (K 30  µg), meropenem (MEM 10 µg), nalidixic acid (NA 30 µg), streptomycin (S 10  µg), compound sulphonamides (S3 300  µg), tetracycline (TE 30  µg), trimethoprim/ sulfamethoxazole (SXT 25  µg) and flumequine (UB 30  µg). These antibiotics were selected based on World Health Organization and Regional Pharmacovigilance Center recommendations. Furthermore, these antibiotics were a global representation of all antimicrobial classes. Subsequently, E.  coli isolates were investigated for the presence of eight virulence genes with Kylt® APEC ANICON kit. These genes are enteroaggregative toxin (astA), increased serum survival protein (iss), iron-repressible protein (irp2), P fimbrie (papC), aerobactin (iucD), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), and colicin V plasmid operon genes (cvi/ cva) (Exers et al. 2005). For DNA extraction the colony was immersed in 500  µl of DNA Extraction-Mix II and it was resuspended carefully. Consequently, the sample vortexed, incubated for 10-15 minutes at 100  °C  ±  3  °C, vortexed again and centrifuged at 10,000-12,000 g for 5 minutes. The PCR was performed in 20 µl volume containing 18 µl Master-Mix (10 µl 2x PCR-Mix, 2 µl 10x Loading Dye, 6  µl Primer-Mix) and 2  µl DNA template. The cycling conditions were as follows: 94  °C for 3 minutes, 35 cycles of 30 sec at 94 °C, 30 sec at 57 °C, 30 Veterinaria Italiana 2019, 55 (1), 27-33. doi: 10.12834/VetIt.1617.8701.1 Antibiotic resistance pattern and virulence genes in APEC Sgariglia et al. strains but their detection rates were significantly (P < 0.05) higher in APEC isolates. Conversely, the virulence associated gene  papC was found in the APEC isolates only. Discussion Colibacillosis caused by APEC results in huge economic losses in poultry industry throughout the world (Roy et al. 2006, Barnes et al. 2008, Dziva et al. 2008). Antimicrobials are the main weapon to fight both the incidence and the mortality associated with colibacillosis (Harisberger et  al. 2011). Antibiotics were also used as feed additives to improve weight gain (Bower et  al. 1999). However, indiscriminate 0 2 4 6 8 10 12 C h ic ke n Animal species N u m b er C h ic ke n C h ic ke n C h ic ke n C h ic ke n C h ic ke n C h ic ke n C h ic ke n C h ic ke n C h ic ke n C h ic ke n C h ic ke n C h ic ke n C h ic ke n C h ic ke n C h ic ke n H en H en G o o se G o o se G o o se Antimicrobial resistance Antimicrobial category resistance Figure 1. Number of antimicrobials and antimicrobial categories in animals of industrial breeding. Table II. Number of resistant, intermediate and susceptible strains according to the Clinical Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Antimicrobial Antimicrobial acronym Concentration (µg) Resistant (%) Intermediate (%) Susceptible (%) Ampicillin AMP 10 45 0 55 Amoxicillin/clavulanic acid AMC 30 12 0 88 Cefotaxime CTX 30 0 0 100 Cefepime FEP 30 0 0 100 Cefoxitin FOX 30 2 0 98 Ciprofloxacin CIP 5 18 6 76 Cefazolin KZ 30 16 18 66 Ceftazidime CAZ 30 2 2 96 Chloramphenicol C 30 22 0 78 Enrofloxacin ENR 5 18 22 60 Gentamicin CN 10 6 6 88 Kanamicin K 30 6 8 86 Meropenem MEM 10 0 0 100 Nalidixic acid NA 30 45 4 51 Streptomycin S 10 24 12 64 Compound sulphonamides S3 300 41 0 59 Tetracycline TE 30 43 4 53 Trimethoprim/sulphametoxazole SXT 25 25 0 75 Flumequine UB 30 33 12 55 0 2 4 6 8 10 C h ic ke n C h ic ke n C h ic ke n C h ic ke n C h ic ke n C h ic ke n C ap o n H en D u ck G u in ea fo w l G u in ea fo w l G o o se G o o se G o o se G o o se Animal species N u m b er Antimicrobial resistance Antimicrobial category resistance Figure 2. Number of antimicrobials and antimicrobial categories in animals of chick dealer. 0 1 2 3 4 5 6 C h ic ke n C h ic ke n C h ic ke n C h ic ke n C h ic ke n H en H en H en H en H en H en C ap o n C ap o n Pi g eo n Tu rk ey Animal species N u m b er Antimicrobial resistance Antimicrobial category resistance Figure 3. Number of antimicrobials and antimicrobial categories in animals of rural breeding. 31Veterinaria Italiana 2019, 55 (1), 27-33. doi: 10.12834/VetIt.1617.8701.1 Sgariglia et al. Antibiotic resistance pattern and virulence genes in APEC iucD, both related to iron acquisition system, tend to be present on the same strain; in this study all the APEC strains contained either iron acquisition systems. Although at lower frequency, the astA gene was also detected in both the APEC and non-APEC isolates. In contrast, the virulence gene papC was detected in APEC strains only. This finding confirms what reported in other similar studies and supports the hypothesis that it is an important virulent gene. The vat and tsh genes were distribuited in both the APEC and non-APEC strains. In this study, the frequency of virulence genes iucD, cvi/cva, irp2 and iss observed in APEC strains was higher than that found by Subedi and colleagues (Subedi et  al. 2018) which reported frequency of irp2 and cvi/cva of 73.3% and 57.8%, respectively. In other studies the frequency of irp2 observed (59.3% and 67%) was also much lower (Sadeghi Bonjar et  al. 2017, Kwon et  al. 2008). Furthemore, Kwon and colleagues reported 16% frequency of cvi/cva virulence gene (Kwon et  al. 2008). The frequency of tsh virulence gene (69%) was comparable with literature data (Subedi et al. 2018). Lower frequencies were found for vat, papC and astA virulence genes (44%, 31% and 19%, respectively). Four APEC strains from rural breeding showed no resistance (2 strains) or only resistance to nalidixic acid (other 2 strains). However, it should be highlighted that 3/7 isolates from industrial breeding resulted susceptible to all drugs. Out of 9 resistant APEC strains, 6 showed multidrug resistance to at least three or more antimicrobials agents (4 from industrial breeding and 2 from dealer). Although four strains from rural breeding were identified as APEC, they did not show high level resistance. Conversely, the strains from industrial breeding showed multidrug resistance suggesting a possible excessive use of antibiotics in poultry industries (Subedi et al. 2018). These results demonstrate the importance of studies on APEC, antibiotic resistance rates in our country and its correlation with poultry breeding, aiming to acquire preventive measures to minimize losses due to APEC and multidrug-resistance that plays an important role and have high significance to public health. Acknowledgements The authors would like to thank Valentina Stefanetti for her valuable technical assistance and Andrea Felici for his help in statistical analysis. use of antibiotics has provided selective pressure for the emergence of drug resistance strains which may lead to therapy failure and potential economic losses for breeders (Scioli et al. 1983, Quednau et al. 1998, Bower et al. 1999, Oosterik et al. 2014). Of the nineteen antibiotics tested in this study, only two showed 100% efficacy against all E. coli strains. The highest rates of resistance (45%) were found with ampicillin and nalidixic acid. Other studies have also shown similar resistance but with higher rates (Yang et  al. 2004, Li et  al. 2010, Shrestha et  al. 2011, Yassin et  al. 2017, Matin et  al. 2017, Shrestha et  al. 2017, Bakhshi et  al. 2017, Subedi et  al. 2018). Similarly, for quinolones such as ciprofloxacin and enrofloxacin which in this study showed moderate resistance (18%) in other studies resistance rates higher than 50% were reported (Yassin et  al. 2017, Subedi et al. 2018). The resistance rates of the E. coli strains included in this study to ceftazidime and cefoxitin were very low. In contrast with a previous study of Younis and colleagues (Younis et  al. 2017), that found 95.8%, 90.4% and 76.7% resistance rates against cefepime, cefoxitin and cefotaxime, respectively, all the E.  coli isolates included in this study were susceptible to cefotaxine and cefepime.. Tetracycline resistance in our isolates was quite high (43%) which is consistent with what found by other authors (Vandemaele et  al. 2002, Smet et  al. 2008, Salehi et al. 2010, Persoons et al. 2012, Younis et al. 2017). The high levels of resistance to sulfonamides (41%) revealed in this study, was not unexpected. Indeed sulfonamides were widely and continuously used for a long time and resistance was already described before 1950 (Yassin et al. 2017). The resistance to aminoglycosides was quite low in our study (gentamicin and kanamicin 6%, streptomycin 24%), that is in disagreement with other authors which reported higher levels of resistance (Yassin et al. 2017, Yousin et al. 2017). In this study, the frequency of eight virulence genes and their correlation with phenotypic antibiotic resistance were evaluated. E. coli strains are defined APEC when at least five virulence genes are detected. The virulence genes iucD, iss, irp2, and cvi/cva were found in both, APEC and non-APEC strains; the presence of these genes in both groups of strains might indicate that they are not associated with virulence. However, iss gene has been described as a virulence gene of recognised importance in E. coli of chickens (Ellis et  al. 1988). 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