35 Parole chiave E. coli fecale aviare, Gruppi filogenetici, Polli, Resistenza antimicrobica, Virulenza. Riassunto L'obiettivo dello studio è stato quello di determinare i caratteri di virulenza e di resistenza antimicrobica di 100 ceppi di E. coli fecali isolati da polli clinicamente sani in Algeria. La maggior parte degli isolati apparteneva ai filogruppi A (45%) e B1 (37%) e mostrava una grande diversità nei profili genetici. Sono stati rilevati i geni fimH, tsh, entB, iutA, irp2, fyuA, iroN, sitA, etsA, etsB, eitA, iss, traT, ompT, hlyF, vat, ibeA, cvaA, cvaB5’, cvaB3’, cvaC, cma e cbi. Le combinazioni tra i geni di virulenza hanno permesso di definire 67 profili di virulenza. Sono stati rilevati alti tassi di resistenza (62‑97%) per amoxicillina, amoxicillina‑acido clavulanico, cefazolina, fluorochinoloni, tetraciclina, trimetoprim, sulfonamidi e sulfametossazolo/ trimetoprim e il 93% dei ceppi presentavano resistenza multipla. Sono stati identificati i geni bla TEM , bla SHV , bla CTX‑M‑1 , tetA, tetB, qnrB, qnrS1, sul1, sul2, sul3, dfrA1, dfrA7, dfrA12 e dfrA14; gli integroni di classe 1 sono stati rilevati nel 49% degli isolati. Una percentuale del 37% dei ceppi era resistente al mercurio, con la presenza del gene merA. Lo studio riporta la presenza, nei ceppi aviari isolati da tamponi fecali, di geni di virulenza di origine plasmidica caratteristici dei ceppi ExPEC associata ad un’elevata resistenza agli antibiotici di prima linea e di integroni di classe 1: un rischio per la salute umana e animale. Geni di virulenza, resistenza antimicrobica e correlazione filogenetica di ceppi di E. coli isolati da allevamenti di broiler in Algeria Keywords Avian fecal E. coli, Broiler chickens, Phylogenetic groups, Virulence, Antimicrobial resistance. Summary The objective of the study was to determine the virulence and antimicrobial resistance traits of 100 fecal E. coli strains isolated from clinically healthy chickens in Algeria. Most of isolates belonged to phylogroups A (45%) and B1 (37%) and showed a great diversity in DNA profiles. The genes fimH, tsh, entB, iutA, irp2, fyuA, iroN, sitA, etsA, etsB, eitA, iss, traT, ompT, hlyF, vat, ibeA, cvaA, cvaB5’, cvaB3’, cvaC, cma and cbi were detected. Combinations of virulence genes defined 67 virulence profiles. High resistance rates (62‑97%) were noted for amoxicillin, amoxicillin‑clavulanic acid, cefazolin, fluoroquinolones, tetracycline, trimethoprim, sulfonamides and sulfamethoxazole/ trimethoprim, and 93% of strains were multidrug‑resistant. Combinations of resistance phenotypes defined 59 resistance patterns. The genes bla TEM , bla SHV , bla CTX‑M‑1 , tetA, tetB, qnrB, qnrS1, sul1, sul2, sul3, dfrA1, dfrA7, dfrA12 and dfrA14 were identified and class 1 integrons were detected in 49% of isolates. A rate of 37% of strains was resistant to mercury, with the presence of merA gene. The study reports the presence in the avian strains isolated from fecal swabs of virulence genes of plasmid origin characteristic of ExPEC strains associated with high resistance to first‑line antibiotics and class 1 integrons, this augurs a risk for human and animal health. Veterinaria Italiana 2019, 55 (1), 35‑46. doi: 10.12834/VetIt.799.3865.2 Accepted: 07.07.2016 | Available on line: 31.03.2019 Laboratory of Cellular and Molecular Biology, Faculty of Biological Sciences, University of Sciences and Technology Houari Boumediene, PB 32 El-Alia, Bab Ezzouar, 16111 Algiers, Algeria *Corresponding author at: Laboratory of Cellular and Molecular Biology, Faculty of Biological Sciences, University of Sciences and Technology Houari Boumediene, PB 32 El-Alia, Bab Ezzouar, 16111 Algiers, Algeria. Tel.: +213 21 24 79 13, fax: +213 21 24 72 17, e-mail: bakourrabah@gmail.com, rbakour@yahoo.fr. Chahinez Messaili, Yamina Messai and Rabah Bakour* Virulence gene profiles, antimicrobial resistance and phylogenetic groups of fecal Escherichia coli strains isolated from broiler chickens in Algeria Nick title First author et al. 36 Veterinaria Italiana 2019, 55 (1), xxx-xxx. doi: 10.12834/VetIt.xxxxx flora in terms of virulence and antimicrobial resistance. In this context, the objective of this study was to assess the prevalence of virulence factors and antimicrobial resistance and to determine the phylogenetic groups in fecal E.  coli strains isolated from clinically healthy broiler chickens. Materials and methods Bacterial strains One hundred non‑repetitive avian fecal E. coli strains (one isolate, one chicken) were recovered from intestines of 45‑47 day old clinically healthy chickens using sterile cotton swabs. Chickens were from five poultry farms in the center of Algeria: Rouiba, Shaoula, Lakhdaria, Tizi‑Ouzou, Bejaia. Strains were identified using API 20E identification system (bioMérieux, France) and by PCR detection of iudA gene (beta‑glucuronidase) (Clermont et al. 2013). Phylogenetic grouping and genotyping of isolates Phylogenetic groups of strains were determined as previously described (Clermont et al. 2013). Molecular typing of strains was performed by enterobacterial repetitive intergenic consensus‑PCR (ERIC‑PCR) using primer ERIC2 (Messai et al. 2008). ERIC profiles are compared visually and those dissimilar by one band or more were considered as different. PCR detection of virulence‑associated gene The DNA template for PCR was extracted using the boiling method (Feria et  al. 2002). All isolates were screened for the presence of 31 virulence associated genes (VAGs) by multiplex and simplex PCR (Table I). E.  coli EC7372, ECOR66 and CFT073 were used as control strains. Biofilm formation assay Biofilm formation (BF) ability was assessed as previously described (Naves et  al. 2008). Briefly, a 100 fold dilution in LB medium of an overnight bacterial culture was distributed in 96‑well polystyrene microplate. After incubation at 37  °C for 24 h, the optical density (OD) of bacterial growth was measured at 630  nm. Plate was then emptied, washed with sterile distilled water, air dried for 20 min and stained with 130 µl of 1% crystal violet for 5 min. Dye was discarded, and plate was washed five times to remove excess dye and air dried for 1 hour. The biofilm‑bound dye was eluted with 130 µl of 95% ethanol and the absorbance was measured at 570 Introduction Most Escherichia coli are commensal bacteria present in the gut of humans and warm‑blooded animals. However, some strains can harbor virulence genes and might be associated with various intestinal (InPEC strains) or extraintestinal (ExPEC strains) infections in humans and animals (Bélanger et  al. 2011). Different combinations of virulence factors define pathotypes, which are specific of a type of infection. The ExPEC group includes UPEC (uropathogenic), NMEC (newborn meningitic), SePEC (septicaemia associated) and APEC (avian pathogenic) strains (Bélanger et al. 2011). Eight phylogenetic groups are now recognized in E. coli: seven (A, B1, B2, C, D, E, F) belong to E.  coli sensu stricto, whereas the eighth is the Escherichia cryptic clade I (Clermont et al. 2013). A and B1 groups generally include commensal strains and certain intestinal pathogens while B2 and to a lesser extent D groups are characteristic of extraintestinal pathogens (Ewers et al. 2007, Mellata 2013). Avian colibacillosis caused by APEC strains is one of the major causes of economic losses in the poultry industry in Algeria and throughout the world; it manifests by various injuries as airsacculitis, peritonitis, polyserositis and sepsis. APEC strains are characterized by the expression of various virulence factors including adhesins, toxins, iron uptake systems and serum resistance (Johnson et  al. 2008, Bélanger et  al. 2011, Mellata 2013). There are genotypic similarities between APEC and human ExPEC, mainly UPEC and NMEC, suggesting zoonotic potential among APEC strains (Bélanger et al. 2011, Mellata 2013). In addition to the virulence factors, antibiotic resistance of bacteria determines their impact in infectious diseases. The increase of antibiotic resistance has worldwide reached alarming proportions; it is inherent in large part to the widespread use of antibiotics in intensive livestock, especially poultry (Mellata 2013). Healthy poultry is considered the main reservoir of virulence genes and antibiotic resistance (Rodriguez‑Sieck et al. 2005, Ewers et al. 2009). Pathogenic strains result generally from commensals by the acquisition of infectious capacity through horizontal transfer of virulence genes (Johnson and Nolan 2009, Mellata et al. 2010). In addition to inter‑animal transfer, virulent and/or resistant bacteria and genes can even reach humans via contaminated environment and food chain (Graham et  al. 2008, Vincent et  al. 2010). In order to better understand and monitor the emergence of pathogens and antibiotic‑resistant strains from healthy animal reservoir, it is necessary to know the presence and prevalence of virulence factors and antibiotic resistance. These investigations are very important in Algeria, because of the lack of data on this issue in this country, especially since veterinary practices, farming conditions and environment have a considerable impact on the evolution of intestinal First author et al. Nick title Veterinaria Italiana 2019, 55 (1), xxx-xxx. doi: 10.12834/VetIt.xxxxx 37 used (µg/ disk): amoxicillin (AMX) (25 µg), amoxicillin/ clavulanic acid (AMC) (20  µg/10  µg), cefazolin (CZ) (30 μg), cefotaxime (CTX) (30 μg), ceftazidime (CAZ) (30 μg), nalidixic acid (NA) (30  µg), ciprofloxacin (CIP) (5 µg), pefloxacin (PEF) (5  µg), ofloxacin (OFX) (5  µg), tetracycline (TE) (30  µg), kanamycin (K) (30  µg), netilmicin (NET) (30 µg), gentamicin (GM) (15 µg), sulfonamides (SSS) (200  µg), trimethoprim (TMP) (5  µg), sulfamethoxazole/trimethoprim (SXT) (1.25  µg/23.75  µg) and colistin (CS) (50  µg). E.  coli ATCC 25922 was used as a control strain. Multiple antibiotic resistance index (MAR) was used to check the antibiotic resistance. MAR is calculated as a ratio a/b, ‘a’ is the number of antibiotics to which the isolate is resistant and ‘b’ is the total number of antibiotics to which it is exposed. nm. The Specific Biofilm Formation index (SBF) was determined as follow: SBF = T ‑ C/G. T is OD 570 value of wells containing strains to be tested; C is OD 570 of control wells containing bacteria‑free medium and G is OD 630 of bacterial growth. Strains were classified as weak biofilm producer (SBF ≤ 1) or strong biofilm producer (SBF > 1). Antimicrobial susceptibility testing Antibiotic susceptibility was tested by the disk diffusion method according to guidelines of antibiogram committee of the French Society for Microbiology (CA‑SFM 2013) (www. sfm‑microbiologie.org). The following disks of antibiotics (Bio‑Rad, Marnes la Coquette, France) were Table I. All primer sequences used in PCR for detecting E. coli virulence associate genes. — cont’d Gene Primer sequence (5’-3’) Annealing temp. (C°) Expected size (bp) Reference Adhesion papA atggcagtggtgtcttttggtg cgtcccaccatacgtgctcttc 63° 720 Johnson and Stell 2000 fimH tgcagaacggataagccgtgg gcagtcacctgccctccggta 63° 508 Johnson and Stell 2000 afa/draBC ggcagagggccggcaacaggc cccgtaacgcgccagcatctc 63° 559 Johnson and Stell 2000 bmaE atggcgctaacttgccatgctg agggggacatatagcccccttc 63° 507 Johnson and Stell 2000 sfa/focDE ctccggagaactgggtgcatcttac cggaggagtaattacaaacctggca 63° 410 Johnson and Stell 2000 tsh gggaaatgacctgaatgctgg ccgctcatcagtcagtaccac 60° 420 Johnson et al. 2006a Protectins kpsMTII gcgcatttgctgatactgttg catccagacgataagcatgagca 63° 272 Johnson and Stell 2000 kpsMTIII tcctcttgctactattccccct aggcgtatccatccctcctaac 63° 392 Johnson and Stell 2000 Serum resistance iss cagcaacccgaaccacttgatg agcattgccagagcggcagaa 63° 323 Johnson et al. 2008 traT ggtgtggtgcgatgagcacag cacggttcagccatccctgag 63° 290 Johnson et al. 2008 Iron-Related entB atttcctcaacttctggggc agcatcggtggcggtggtca 57° 371 El Fertas-Aissani et al. 2013 iroN aagtcaaagcaggggttgcccg gacgccgacattaagacgcag 63° 667 Johnson et al. 2006a fyuA gcgacgggaagcgatgattta taaatgccaggtcaggtcact 56° 547 El Fertas-Aissani et al. 2013 irp2 aaggattcgctgttaccggac tcgtcgggcagcgtttcttct 57° 287 El Fertas-Aissani et al. 2013 iutA ggctggacatcatgggaactgg cgtcgggaacgggtagaatcg 63° 300 Johnson and Stell 2000 sitA agggggcacaactgattctcg taccgggccgttttctgtgc 59° 608 Johnson et al. 2006a eitA acgccgggttaatagttgggagatag atcgatagcgtcagcccggaagttag 60° 450 Johnson et al. 2006a etsA caactgggcgggaacgaaatcagga tcagttccgcgctggcaacaacctac 60° 284 Johnson et al. 2006a etsB cagcagcgcttcggacaaaatctcct ttccccaccactctccgttctcaaac 60° 380 Johnson et al. 2006a continued Nick title First author et al. 38 Veterinaria Italiana 2019, 55 (1), xxx-xxx. doi: 10.12834/VetIt.xxxxx (Šeputienė et  al. 2010); mercuric reductase merA (Bass et al. 1999). Class 1 integrons were searched by multiplex PCR targeting int1, sul1 and qacEΔ1 genes (Messai et al. 2008). PCR products of positive reactions for qnrB, qnrS, bla CTX‑M , dfrA1, dfrA5, dfrA7 and dfrA12 were sequenced and analyzed with the BLAST and FASTA programs of the National Center of Biotechnology Information (www.ncbi.nlm.nhi.gov). Conjugation assay and plasmid analysis Conjugation assay was performed for the cefotaxime resistant strain using sodium azide resistant E.  coli BM21 as recipient. Exponential culture of donor isolate (1 volume) and recipient (2 volumes) were inoculated as a spot on Brain Heart Infusion Agar (BHIA). After overnight incubation at 37 ºC, transconjugants were selected on Mueller Hinton agar supplemented with cefotaxime (4 μg/ml) and sodium azide (300 μg/ml). Plasmid DNA was extracted by alcalin lysis method (Kado and Liu 1981) and analyzed by electrophoresis on 0.7% (wt/vol) agarose gels at 5 volts/cm. Plasmid size was estimated by using reference plasmids, pRK2013 (48 kb) and pIP173 (126.8 kb). Plasmid incompatibility group was determined by a PCR‑based replicon typing method (Carattoli et al. 2005). The screening of isolates for ESBL production was performed by the Double‑Disc Synergy Test (DDST) (Messai et al. 2008). Heavy metal susceptibility was assessed by the agar‑dilution method on Mueller Hinton medium, the following heavy metal concentrations were tested: HgCl 2 : 2.7, 13.57, 27.15 and 54.3 µg/ml; CuCl 2 and ZnCl 2 : 100, 200, 400, 800, 1600 and 3,200 µg/ ml; Pb(NO3) 2 : 400, 800, 1,600, 2,400 and 3,200 μg/ ml; Cd(NO 3 ) 2 . 4H 2 O: 7, 12.5, 25, 50, 100, 200, 400 and 800 μg/ml. MIC values indicative of metal resistance were: 200 μg/ml for Cd2+, 1,600 µg/ml for Zn2+, 3,200 µg/ml for Cu2+ and Pb2+ (Calormiris et al. 1984) and 27.15 µg/ml for Hg2+ (Edlund et al. 1996). Detection of antibiotic resistance genes and integrons Simplex and multiplex PCR were used for the detection of following resistance genes as previously described: β‑lactamases bla TEM , bla SHV and bla CTX‑M (Messai et  al. 2008); plasmid mediated quinolone resistance (PMQR) genes qnrA, qnrB, qnrS, and qepA (Figueira et  al. 2011); aminoglycoside‑modifying enzyme aac(6’)-Ib (Figueira et al. 2011); tetracycline efflux pumps tetA and tetB (Guardabassi et  al. 2000); dihydropteroate synthases sul1, sul2 and sul3 (Frank et  al. 2007, Messai et  al. 2008); dihydofolate reductase dfrA1, dfrA5, dfrA7, dfrA8 and dfrA12 Table I. Table I. All primer sequences used in PCR for detecting E. coli virulence associate genes. — cont’d Gene Primer sequence (5’-3’) Annealing temp. (C°) Expected size (bp) Reference Toxins vat aacggttggtggcaacaatcc agccctgtagaatggcgagta 58° 420 Restieri et al. 2007 hlyA aacaaggataagcactgttctggct accatataagcggtcattcccgtca 63° 1177 Johnson and Stell 2000 hlyF ggccacagtcgtttagggtgcttacc ggcggtttaggcattccgatactcag 63° 450 Johnson et al. 2008 cnf1 aagatggagtttcctatgcaggag cattcagagtcctgccctcattatt 63° 498 Johnson and Stell 2000 Colicins cvaA atccgggcgttgtctgacgggaaagttg accagggaacagaggcacccggcgtatt 63° 319 Johnson et al. 2006a cvaB5’ tggccacccgggctctttcactggagtt atgcgggtctgcagggtttccgactgga 63° 550 Johnson et al. 2006a cvaB3’ ggcccgtgccgcctcctatttta tcccgcaccggaagcaccagttat 63° 247 Johnson et al. 2006a cvaC cacacacaaacgggagctgtt cttcccgcagcatagttccat 63° 679 Johnson and Stell 2000 cbi acaagacagcaccagttatgggtatt gttgttggttttgttggcgtagttat 63° 430 Johnson et al. 2006b cma cagcgccattaccccataaatagtga ggttcgttcgccggtgtaagcgttag 63° 498 Johnson et al. 2006b Miscellaneous ompT tcatcccggaagcctccctcactactat tagcgtttgctgcactggcttctgatac 63° 496 Johnson et al. 2008 ibeA aggcaggtgtgcgccgcgtac tggtgctccggcaaaccatgc 63° 170 Johnson and Stell 2000 First author et al. Nick title Veterinaria Italiana 2019, 55 (1), xxx-xxx. doi: 10.12834/VetIt.xxxxx 39 iron acquisition and transport, entB, iutA, irp2, fyuA, iroN, sitA, etsA, etsB and eitA were found in 77%, 17%, 14%, 13%, 19%, 35%, 4%, 6% and 16% of strains, respectively. Serum resistance‑associated genes, iss and traT, were present at rates of 20% and 58%, respectively. Capsular genes kpsMTII and kpsMTIII were not found. A rate of 27% of strains harbored ompT and avian hemolysin hlyF genes. The vat gene was present in 5% of strains and, hemolysin A and cytotoxic necrotizing factor 1 genes, hlyA and cnf1, were absent. The invasion gene ibeA was detected in a single strain (1 %). The colicin V, B and M operon genes, cvaA, cvaB5’, cvaB3', cvaC, cma and cbi, were present in 12%, 14%, 10%, 2%, 10% and 10% of strains, respectively, revealing a total of 24% of strains harboring a presumptive plasmid ColV and/or ColBM (ColV: 14%, ColBM: 6%, ColV+ColBM: 4%). All strains had a biofilm formation (BF) ability, of which 20% were strong biofilm producers. The combinations of the different virulence factors allowed to distinguish 67 virulence profiles including 0 to 16 virulence genes, and based on the gene combination “iutA, hlyF, iss, iroN, ompT”, 5% of strains could be assigned to APEC pathotype (Johnson et  al. 2008) (Table II). The Fisher’s exact test showed significant association between colV operon and phylogenetic group B1, with a rate of 29.7% in B1 strains versus 11.1% in the non‑B1 strains (P = 0.021). Antibiotic susceptibility testing showed high level of resistance to amoxicillin (97%), amoxicillin‑clavulanic acid (72%), cefazolin (73%), nalidixic acid (97%), ofloxacin (78%), ciprofloxacin (62%), pefloxacin (68%), tetracycline (90%), trimethoprim (75%), sulfonamides (75%) and sulfamethoxazole/ trimethoprim (69%). Resistance rates of 53%, 6% and 5% were observed Statistical analysis For comparison of rates, Fisher’s exact test was used, p < 0.05 was considered significant. Results Phylogenetic analysis allowed to assign isolates to phylogroups A (45%), B1 (37%), B2 (3%), C (1%), D (3%), E (4%), F (4%), I (1%) and unknown (2%). Molecular typing of strains by ERIC‑PCR showed 80 different genetic profiles. The phenotypic and genotypic screening for 31 virulence‑associated genes (Figure 1) showed the presence of fimH gene in almost all strains (91%), while the other adhesin genes (papA, bmaE, sfa/focDE, afa/drABC) were absent. The temperature‑sensitive hemagglutinin gene tsh was detected in 2% of strains. Genes of 0 10 20 30 40 50 60 70 80 90 100 �m H pa pA ts h bm aE sf a/ fo cD E af a/ dr aB C kp sM T II kp sM T III en tB iu tA fy uA irp 2 iro N si tA et sA et sB ei tA tr aT is s om pT hl yA hl yF va t cn f1 ib eA cv aA cv aB 5’ cv aB 3’ cv aC cb i cm a P re va le n ce % Phylogenetic groups A B1 Other: B2, D, C, F, E, I, UK Figure 1. Prevalence of virulence-associated genes and their distribution according to phylogenetic groups. Table II. Virulence and antimicrobial resistance patterns of some representative E. coli strains. — cont’d Phylo-group DNA profile Strain Virulence gene profile Antimicrobial resistance pattern Resistance and integron genes A E40 S67 fimH entB traT hlyF colV AMX AMC CZ NA CIP PEF OFX TE SSS TMP SXT blaTEM tetA E73 S111 fimH entB traT eit vat BF AMX AMC CZ NA CIP PEF TE K SSS TMP SXT blaTEM tetA sul2 sul3 E38 S65 fimH entB yers hlyF colV AMX NA CIP PEF OFX TE K SSS tetA tetB sul2 E53 S84 fimH entB sitA iss ompT hlyF AMX AMC CZ NA CIP PEF OFX TE SSS TMP SSS TMP SXT blaTEM blaSHV E80 S118 fimH iroN sitA iss ompT hlyF AMX AMC CZ NA CIP PEFOFX bla TEM E22 S37 fimH entB yers sitA traT ompT hlyF AMX AMC CZ NA CIP PEF OFX TE K SSS TMP SXT Hg tetA sul1 sul2 dfrA12 qacE∆1 merA E39 S66 fimH entB iroN sitA iss hlyF colBM AMX CZ NA CIP PEF bla TEM E8 S14 fimH entB iutA sitA ets traT iss colBM colV AMX AMC CZ NA CIP PEF OFX TE K SSS Hg blaTEM blaSHV tetB sul2 int1 E78 S116 fimH entB iroN sitA eit traT iss ompT hlyF AMX AMC CZ NA CIP PEF OFX TE SSS TMP SXT blaTEM blaSHV tetA sul2 qacE∆1 int1 E45 S72 fimH tsh entB iroN iutA sitA ets eit traT iss ompT hlyF colV AMX AMC CZ NA CIP PEF OFX TE K SSS TMP SXT bla TEM bla SHV tetB sul1 sul2 dfrA7 int1 qacE∆1 continued Nick title First author et al. 40 Veterinaria Italiana 2019, 55 (1), xxx-xxx. doi: 10.12834/VetIt.xxxxx by conjugation in association with an IncI1 plasmid of about 118 kb. Tetracycline resistance genes tetA and tetB were detected in 74% and 12% of the strains. Plasmid mediated quinolone resistance determinants qnrB and qnrS1 were present in 12% and 1% of strains. Sulfonamide resistance genes sul1, sul2 and sul3 were identified in 14%, 53% and 10% of strains and trimethoprim resistance genes dfrA1, dfrA7, dfrA12 and dfrA14 were present in 5%, 7%, 13% and 43% of strains, respectively. The combinations of resistance phenotypes allowed distinguish 59 antibiotic resistance patterns for kanamycin, netilmicin and gentamicin. One strain (1%) was resistant to cefotaxime and positive for DDST. All strains were susceptible to ceftazidime, amikacin and colistin (Figure 2). Molecular identification by PCR and sequencing of resistance genes (Figure  3) revealed the presence of broad‑spectrum beta‑lactamase (BSBL) genes bla TEM , bla SHV and extended‑beta‑lactamase (ESBL) bla CTX‑M‑1 allele in 70%, 50% and 1% of isolates, respectively. ESBL allele bla CTX‑M‑1 and, amoxicilline, cefazolin and cefotaxime resistance phenotypes of the cefotaxime‑resistant strain were transferable Table II. Virulence and antimicrobial resistance patterns of some representative E. coli strains. — cont’d Phylo-group DNA profile Strain Virulence gene profile Antimicrobial resistance pattern Resistance and integron genes B1 E47 S75 fimH entB iutA sitA traT AMX AMC NA CIP PEF OFX TE K SSS bla TEM tetB sul2 E8 S12 fimH entB iroN ompT hlyF BF AMX AMC CZ NA CIP PEF OFX TE K SSS TMP SXT blaTEM tetA dfrA5 int1 E29 S53 fimH sitA traT iss ompT colV AMX AMC NA CIP PEF OFX TE K SSS TMP SXT Hg blaTEM tetA sul2 dfrA5 merA int1 E37 S63 fimH entB iroN sitA traT iss ompT AMX AMC CZ NA CIP PEF OFX TE SSS TMP SXT Hg bla TEM bla SHV tetA sul2 dfrA1 dfrA5 merA int1 E69 S103 fimH entB iroN sitA iss ompT hlyF AMX CZ NA CIP PEF OFX TE bla TEM bla SHV tetA E59 S93 fimH entB sitA eit iss ompT hlyF colV AMX AMC CZ NA CIP PEF OFX TE K SSS TMP SXT blaTEM tetA sul2 dfrA5 merA int1 E31 S55 fimH entB iroN sitA traT ompT colBM colV AMX AMC NA TE K blaTEM blaSHV tetA E64 S98 fimH entB iroN sitA traT iss ompT hlyF colV AMX AMC CZ NA CIP PEF TE SSS TMP SXT Hg blaTEM blaSHV tetA sul2 dfrA5 merA int1 E52 S83 fimH iroN iutA sitA eit iss ompT hlyF colV AMX AMC CZ NA OFX TE K blaTEM tetA dfrA5 int1 E63 S97 fimH entB iutA sitA traT iss ompT hlyF colV AMX AMC CZ NA CIP PEF TE K SSS TMP SXT blaTEM blaSHV tetA tetB sul2 dfrA7 int1 E19 S31 fimH entB iroN sitA traT iss ompT hlyF colBM AMX AMC NA CIP PEF OFX TE K SSS TMP SXT Hg tetA sul1 dfrA12 qacE∆1 E50 S81 fimH entB iroN iutA sitA traT ompT hlyF colV AMX AMC CZ NA CIP PEF OFX TE K SSS TMP SXT blaTEM blaSHV tetA sul2 dfrA5 merA int1 E16 S27 fimH tsh entB iroN iutA sitA traT iss colBM colV AMX AMC CZ NA CIP PEF OFX TE K SSS TMP SXT blaTEM tetB sul1 sul2 sul3 dfrA7 qacE∆1 E51 S82 fimH iroN iutA sitA eitA traT iss ompT hlyF colV AMX AMC CZ NA CIP PEF OFX TE SSS TMP SXT blaTEM blaSHV tetA sul2 dfrA5 int1 E72 S110 fimH entB iroN fyuA irp2 iutA sitA eit traT iss ompT hlyF colV BF AMX AMC CZ NA CIP PEF OFX TE SSS TMP SXT Hg tetB sul2 dfrA5 merA int1 D E12 S19 fimH IutA sitA ets ompT hlyF colBM AMX NA CIP PEF TE SSS TMP SXT bla SHV tetA sul2 B2 E33 S58 fimH entB AMX AMC OFX qnrB E20 S35 fimH entB traT AMX AMC CZ NA CIP PEF OFX TE SSS bla TEM tetA E37 S64 fimH entB ompT AMX AMC CZ NA CIP PEF OFX TE K SSS TMP SXT blaSHV tetA sul3 dfrA5 int1 F E54 S86 iutA ibeA sitA colV BF AMX AMC CZ NA CIP PEF OFX TE K NET SSS TMP SXT blaTEM tetB sul1 sul2 dfrA7 qacE∆ int1 E61 S95 fimH entB iutA sitA ets traT ompT hlyF BF AMX AMC CZ NA CIP PEF OFX TE K SSS TMP SXT blaTEM tetA sul2 dfrA5 int1 E60 S94 fimH entB iutA sitA ets eit traT ompT hlyF colV AMX CZ NA CIP PEF OFX TE K SSS TMP SXT blaTEM tetA sul2 dfrA5 int1 E30 S54 fimH entB iutA fyuA sitA ets traT ompT hlyF vat colBM AMX AMC CZ NA CIP PEF OFX TE K SSS TMP SXT Hg blaTEM blaSHV tetA sul2 dfrA5 merA int1 E E9 S15 fimH iroN IutA sitA traT iss ompT hlyF colBM colV AMX AMC CZ CTX NA CIP PEF TE K SSS TMP SXT Hg bla TEM bla SHV bla CTX-M-1 tetA sul1 sul2 sul3 dfrA12 qacE∆1 merA C E5 S6 fimH entB fyuA, irp2 iutA traT iss ompT hlyF colBM AMX AMC NA OFX TE Hg blaSHV tetA First author et al. Nick title Veterinaria Italiana 2019, 55 (1), xxx-xxx. doi: 10.12834/VetIt.xxxxx 41 Agar dilution MICs of Cd2+, Zn2+, Cu2+, Pb2+ and Hg2+ were, respectively, from 25 µg/ml to 50 µg/ml, 100 µg/ml to 400 µg/ml, 800 µg/ml to 1,600 µg/ml, 3,200 µg/ml and 2.7 µg/ml to >  54.3 µg/ml. A total of 37 strains were resistant to mercury, of which 26 (70.2%) harbored merA gene, and 69.2% of merA were associated with class 1 integrons. All strains were susceptible to the other heavy metals tested. There were significant associations between antimicrobial resistance and virulence factors. (resistance to 1‑7 antibiotic families); 93% of strains had a multidrug‑resistance (MDR) phenotype, they resisted to at least three antibiotic classes, and 95% had a MAR index from 0.27 to 0.72. No statistically significant association was found between antibiotic resistance and phylogenetic groups. Class 1 integrons were detected in 49% of strains, fourty five (91.8%) of them lacked the 3’‑conserved sequence (3’‑CS) that contains qacEΔ1 and sul1 and one lacked only sul1. In these cases, sulfonamides resistance was conferred by sul2 and/or sul3. Furthermore, 10% of strains contain qacEΔ1 gene in absence of integrons. P re va le n ce % 0 10 20 30 40 50 60 70 80 90 100 A M X A M C C Z C TX C A Z N A O FX PE F C IP TE K N ET G M A N SS S TM P SX T C S H g Phylogenetic groups A B1 Other: B2, D, C, F, E, I, UK Figure 2. Prevalence of E. coli antimicrobial resistance phenotypes and their distribution according to phylogenetic groups. P re va le n ce % Phylogenetic groups A B1 Other: B2, D, C, F, E, I, UK 0 10 20 30 40 50 60 70 80 bl a T EM bl a S H V bl a C TX -M -1 te tA te tB qn rB qn rS 1 su l1 su l2 su l3 df rA 1 df rA 14 df rA 7 df rA 12 qa cE ∆ 1 m er A in t1 Figure 3. Prevalence of E. coli antimicrobial resistance genes and their distribution according to phylogenetic groups. Table III. Association between E. coli virulence genes and antimicrobial susceptibility phenotypes (P < 0.05). % of virulence gene among resistance strains - % of virulence gene among susceptible strains P Virulence gene (%) Antimicrobials (% resistance , % susceptibility) AMC (72, 28) CZ (73, 27) CIP (62, 38) PEF (68, 32) OFX (78, 22) TE (90, 10) K (53, 47) SSS (75, 25) TMP (75, 25) SXT (69, 31) Hg (37, 63) iroN (19) 22.22-10.71 20.53-14.81 25.8-7.89 0.035* 18.60-9.37 19.23-18.18 18.89-20 18.87-19.15 18.67-20 18.67-20 20.29-16.13 18.91-19.05 fyuA (13) 12.5-14.28 8.21-25.92 0.039** 11.29-15.79 10.29-18.75 8.97-27.27 0.034** 14.44-0 9.43-17.02 8-28 0.016** 10.66-20 7.25-25.80 24.32-6.35 0.014* irp2 (14) 11.11-21.43 13.51-33.33 0.001** 9.68-21.05 10.29-21.05 10.26-27.27 15.55-0 9.43-19.15 8-32 0.005** 13.33-16 7.24-29.03 27.03-6.35 0.006* iutA (17) 20.83-7.14 19.18-11.11 24.19-5.26 0.014* 22.06-6.25 17.95-13.64 18.89-0 24.53-8.51 20-8 17.33-16 18.84-12.90 13.51-19.05 sitA (35) 40.28-21.43 36.99-29.63 48.39-13.15 0.0004* 44.11-15.62 0.006* 37.18-27.27 36.67-20 43.39-25.53 40-20 37.33-28 40.57-22.58 35.13-34.92 traT (58) 58.33-57.14 56.16-62.96 58.06-57.89 52.94-68.75 53.85-72.73 63.33-10 0.001* 67.92-46.81 0.043* 62.66-44 64-40 62.32-48.39 67.57-52.38 iss (20) 23.61-10.71 21.91-14.81 29.03-5.26 0.004* 26.47-6.25 0.017* 20.51-18.18 20-20 18.87-21.28 20-20 18.67-24 20.29-19.35 21.62-19.05 ompT (27) 31.94-14.28 28.77-22.22 38.70-7.89 0.0009* 35.29-9.37 0.007* 26.92-27.27 28.89-10 32.07-21.27 29.33-20 0.001* 29.33-20 0.001* 31.88-16.13 0.001* 27.03-26.98 hlyF (27) 27.78-25 28.77-22.22 37.09-10.53 0.004* 33.82-12.5 0.030* 24.36-36.36 28.89-10 26.41-27.66 20.67-28 26.67-28 27.54-25.81 24.32-28.57 cvaA (12) 15.28-3.57 15.07-3.70 17.74-2.63 0.027* 16.18-3.12 12.82-9.09 13.33-0 16.98-6.38 14.67-4 12-12 13.04-9.68 10.81-12.69 cvaB5’(14) 19.44-0 0.004* 16.44-7.40 19.35-5.26 17.65-6.25 14.10-13.63 15.55-0 22.64-4.25 0.009* 16-8 14.67-12 15.94-9.68 13.52-14.28 Positive* and negative** significant association between virulence gene and antimicrobial resistance (P < 0.05). Nick title First author et al. 42 Veterinaria Italiana 2019, 55 (1), xxx-xxx. doi: 10.12834/VetIt.xxxxx and Bonnet and colleagues (32%) (Bonnet et  al. 2009). The gene sitA belongs to sitABCD operon encoding an iron and manganese ABC transport system, whose role in virulence and resistance to oxidative stress of APEC was demonstrated (Sabri et  al. 2008). The gene sitA was mostly associated with pathogenic strains than fecal ones; however, its rate in our strains (35%) was higher than those reported in fecal strains (27%, 19%) (Amabile de Campos et al. 2008, Kemmett et al. 2013). Genes eitA and etsA/etsB are located in the eitABCD and etsABC operons encoding putative iron ABC transporters identified in high pathogenic APEC and induced in vivo during infection in chickens (Johnson et al. 2008, Tuntufye et al. 2012); their prevalence (16%, 4%, 6%) was relatively lower than that reported by Johnson and colleagues in commensal strains (43%, 43%, 44%) (Johnson et  al. 2008). Serum resistance is one of the pathogenicity mechanisms of APEC strains, there is a correlation between serum resistance and the ability of bacteria to persist in body fluids and internal organs (Mellata et  al. 2003). Genes iss and traT involved at least in part in serum resistance, were detected at rates below (20%, 58%) those reported in fecal E. coli by Bonnet and colleagues (traT, 86.3%) (Bonnet et al. 2009) and Johnson and colleagues (iss, 60%) (Johnson et al. 2008), consistent with results of Hiki and colleagues (iss, 20.5%) (Hiki et al. 2014) and higher than in Kemmett and colleagues (iss, 10%) (Kemmett et al. 2013). The avian hemolysin gene hlyF, epidemiologically associated to the most virulent APEC, was found at prevalence (27%) close to that of Hiki and colleagues (28.2%) (Hiki et  al. 2014). The serine protease gene ompT, involved in providing defense against cationic antimicrobial peptides secreted by the epithelial cells and macrophages, was present at rate (27%) close to result of Hiki and colleagues (29.5%) (Hiki et  al. 2014), but under those recorded by Rodriguez‑Sieck and colleagues (45.2%) (Rodriguez‑Sieck et  al. 2005), Johnson and colleagues (42%, 47%) (Johnson et  al. 2008) and Bonnet and colleagues (46.7%) (Bonnet et  al. 2009). The invasion‑related gene ibeA was present in our strains at low rate (1%) compared to that previously reported in fecal strains (7.7%, 16%) (Rodriguez‑Sieck et  al. 2005, Kemmett et  al. 2013). The prevalence of vat gene (5%) matches that reported by Kemmett and colleagues (6%) (Kemmett et  al. 2013) in fecal strains. Biofilm is a form of bacterial resistance to antimicrobials, opsonization and phagocytosis. Rate of strong biofilm producers among our strains was lower (20%) than the 30% reported by Skyberg and colleagues (Skyberg et al. 2007). Most of the detected genes (iutA, fyuA, irp2, iroN, fimH, cvaC, traT, iss, sitA, ompT, hlyF, cvaA, etsA, etsB, eitA, tsh) have been described in pathogenicity islands associated with virulence plasmids in APEC, of which ColV and ColBM plasmids as pAPEC‑O1, Resistance to ciprofloxacin was distinguished by its association to 7 virulence genes (iutA, iroN, sitA, iss, ompT, hlyF, cvaA) (Table III). Discussion Most of our isolates (82%) belonged to A and B1 phylogenetic groups in accordance with many previous studies (Johnson et  al. 2008, Bonnet et  al. 2009, Hiki et  al. 2014). The clonal relationship investigated by ERIC‑PCR showed a large genetic diversity among strains. Almost all of our isolates (99%) possessed at least one of the examined virulence genes. Type 1 fimbriae are ubiquitous among E.  coli strains including APEC, they are involved in the initiation of the colonization of respiratory tract epithelium (Wooley et  al. 1998). According to our result (2%), tsh was reported at low frequency (10%‑11.2%) in avian fecal E.  coli, while it was described at higher rates (49.7%‑97.7%) in APEC strains; it would have a role in the colonization of tracheal mucosa and in the development of lesions in the air sacs, it was proposed as a marker of APEC (Dozois et  al. 2000, Dozois et  al. 2003, Amabile de Campos et  al. 2005, Bonnet et  al. 2009). Seven iron‑related genes were detected; enterobactin was reported among pathogens and commensals; however, it was found in our study at a rate higher (77%) than in commensals (3%‑13.2%) and close to those in APEC (40%‑75%) (Amabile de Campos et  al. 2008, Bonnet et  al. 2009). The transformation of enterobactin to salmochelin (C‑glucosylation) mediated by iroBCDEN gene cluster can prevent siderocalin binding (Dozois et  al. 2003, Garénaux et  al. 2013). Gene iroN representative of this gene cluster was detected at rate (19%) close to that reported in fecal strains by Johnson and colleagues (21%) (Johnson et al. 2008), but lower than in study of Bonnet and colleagues (62.4%) (Bonnet et  al. 2009). Aerobactin and Yersiniabactin were reported at high frequency in pathogenic strains (Amabile de Campos et al. 2008, Bonnet et al. 2009), their significant role in APEC virulence was demonstrated (Dozois et al. 2003, Tuntufye et al. 2012). Yersiniabactin allowed evasion of siderocalin and prevents reactive oxygen species production by innate immune cells. The rate of aerobactin was close to those of fecal E. coli in certain studies (12.2%‑15%) (Amabile de Campos et  al. 2008, Bonnet et  al. 2009, Kemmett et  al. 2013) but lower than in others (25.9%, 35.5%) (Rodriguez‑Sieck et  al. 2005, Johnson et  al. 2008). The prevalence of yersiniabactin (14%) is consistent with that reported in fecal E. coli by Amabile de Campos and colleagues (13%) (Amabile de Campos et al. 2008) and Kemmett and colleagues (11%) (Kemmett et al. 2013), whereas it was lower than in studies of Rodriguez‑Sieck and colleagues (30.1%) (Rodriguez‑Sieck et  al. 2005), Johnson and colleagues (30%) (Johnson et  al. 2008) First author et al. Nick title Veterinaria Italiana 2019, 55 (1), xxx-xxx. doi: 10.12834/VetIt.xxxxx 43 Links between antimicrobial resistance and ExPEC strains in animal food products, specifically chicken meat, and human infections were observed (Vincent et al. 2010). BSBL genes bla TEM and bla SHV were detected in a large number of strains, as already reported (Gyles 2008, Bonnet et  al. 2009, Wang et  al. 2013), they would be the cause of resistance to amoxicillin, amoxicillin‑clavulanic acid and cefazolin in our strains. Conversely, ESBL bla CTX‑M‑1 , located in transferable IncI1plasmid, was found in the single cefotaxime‑resistant strain (1%). The prevalence of broad cephalosporin resistance and ESBLs varies by country, our results were in accordance with those of studies from Europe and China (de Jong et  al. 2012, Wang et  al. 2013). CTX‑M‑1 ESBL and IncI1 plasmids are among the most widespread, particularly in animal strains including poultry (Caratolli 2009, Johnson and Nolan 2009); however, it should be noted that CTX‑M‑1 type is reported for the first time in Algeria in this study. Plasmid mediated quinolone resistance genes qnrB (12%) and qnrS1 (1%) were detected, their presence among avian E.  coli strains was reported with varying frequency by country. Their transfer from animal to human was reported, they contribute to the emergence of highly quinolone‑resistant bacteria mainly due to mutations in the DNA gyrase and topoisomerase IV genes (Szmolka and Nagy 2013). A rate of 92% of tetracycline‑resistant strains had tetA and/or tetB, they are the most frequently involved among avian strains with a predominance of tetA (Guerra et  al. 2003, Bonnet et  al. 2009, Johnson et  al. 2012). The three dihydropteroate synthase genes sul1, sul2 and sul3 were detected in 72.3% of sulfonamides‑resistant strains; they play an important role in sulfonamide resistance and are significantly related to integrons and transposons. Consistent with our result, sul2 gene was the most widely distributed in avian E.  coli (Guerra et  al. 2003, Wang et  al. 2013). Genes dfrA1, dfrA7, dfrA12 and dfrA14 were detected alone or in combination in 77.3% of trimethoprim‑resistant strains; dfrA1, dfrA12, dfrA14 and dfrA17 were the most commonly identified, inside integrons (Guerra et  al. 2003, Machado et  al. 2008). Integrons are important contributors in the emergence and dissemination of antimicrobial resistance, half of our strains carried class 1 integrons, the most frequently detected in avian E. coli. Our prevalence is equal to that reported in Greece (49.6%) (Vasilakopoulou et  al. 2009) and higher than those recorded in Portugal (22.5%) (Machado et  al. 2008) and Germany (36%) (Guerra et  al. 2003). The majority of detected integrons lacked sul1 and qacEΔ1, this truncated structure was already described, it generally contains sul3 at the 3'‑end and is linked to IS26, which probably is the cause of the 3'‑CS deletion (Dawes et al. 2010, Sáenz pAPEC‑O2‑ColV and pAPEC‑O1‑ColBM (Johnson and Nolan 2009, Mellata et  al. 2010). The simultaneous presence of several of these plasmid‑borne virulence‑associated genes and, operon ColV (cvaA, cvaB, cvaC) and/or colB/M (cbi, cma) genes in 24% of our strains augurs that the latter harbored ColV and/or ColBM plasmids. These virulence plasmids have an important role in pathogenicity, evolution from commensal to pathogenic state and zoonotic risk (Johnson and Nolan 2009, Mellata et  al. 2010). In addition to plasmid genes, some of our isolates possessed certain chromosomal genes (fyuA, vat, ibeA), which characterize APEC strains (Johnson et al. 2008). The phylogroup B2 strains were characterized by the presence of little virulence genes, mainly chromosomal and ubiquitous (entB, fimH), compared to other groups and the majority of strains carrying presumptive ColV plasmids belonged to phylogroup B1 (61.1%), this finding is not in accordance with previous reported data (Johnson et  al. 2008). No statistical association with phylogenetic groups was noted for the remaining virulence factors. The combination of virulence factors showed a diversity in virulence profiles (n = 67). Five percent of our isolates possessed the gene combination “iutA, hlyF, iss, iroN, ompT” defined as the most significantly genes associated with highly pathogenic APEC strains (Johnson et  al. 2008), and 12% harbored at least 4 genes of this combination. These findings were in agreement with results from Hiki and colleagues (Hiki et al. 2014). The prevalence of virulence factors found among our strains differ for some of them from those reported in other countries, environmental conditions (feed, production systems, veterinary practices) can modulate the distribution of virulence determinants (Amabile de Campos et al. 2008, Bonnet et al. 2009, Kemmet et al. 2013) High resistance rates were observed for amoxicillin, amoxicillin‑clavulanic acid, cefazolin, fluoroquinolones, tetracycline, trimethoprim, sulfonamides and sulfamethoxazole/ trimethoprim. These results reflect the general trend worldwide both for fecal and APEC strains; however, resistance rates vary by country. In comparison, our rates are significantly higher than those recorded in the USA (Johnson et  al. 2012), Europe (de Jong et  al. 2012), Canada (Bonnet et al. 2009), Japan (Hiki et al. 2014), and lower than those from Egypt (Mohamed et  al. 2014), China (Wang et  al. 2013). The range of antibiotics used in Algeria for prophylaxis, therapy and growth promotion covers various families; this can directly affect antimicrobial resistance of endogenous bacteria. Furthermore, the environment can also be a source of resistant organisms and resistance genes for animals (Bélanger et  al. 2011). Nick title First author et al. 44 Veterinaria Italiana 2019, 55 (1), xxx-xxx. doi: 10.12834/VetIt.xxxxx resistance was associated with fewer virulence genes in comparison to ciprofloxacin‑susceptible strains (Graziani et  al. 2009). The molecular mechanisms underlying association between resistance and virulence remains to understand. This study, the first in Algeria devoted to virulence and antimicrobial resistance of fecal strains from healthy broiler chickens, reported the presence of ExPEC virulence genes typically found in pathogenicity islands located on plasmids, particularly ColV and ColBM plasmids. High prevalence of MDR phenotype was observed, with resistance to first line antibiotics including amoxicillin‑clavulanate, fluoroquinolones and trimethoprim‑sulfamethoxazole, as well as various plasmidic resistance genes and class1 integrons. Intensive chicken farming in the current conditions in Algeria really constitutes a source of virulence and antimicrobial resistance genes that may spread and exacerbate virulence and resistance of animal and human pathogenic strains. This situation should incite to take measures at the level of farming conditions and veterinary practices. et al. 2010). The presence of the gene qacEΔ1 in the absence of integrons would probably be the result of a selection pressure by quaternary ammonium compounds which are, among disinfectants, the most used in the poultry industry. The mercury resistance (37%) can result from an anthropogenic selection pressure or co‑selection by antibiotics. The detection of merA gene associated in the majority of cases to class 1 integrons is indicative of the presence of the transposon Tn21 which carries mercury resistance operon (mer) and an integron (In2). This transposon allows co‑selection by antibiotics and mercury. This finding is in agreement with that already reported among avian strains (Bass et al. 1999, Johnson et al. 2012). Many associations between antimicrobial resistance and virulence factors were noted, the most remarkable was resistance to ciprofloxacin that was statistically associated with seven virulence genes. The combination of ExPEC virulence factors and antibiotic resistance was reported (Pitout 2012); however, in contrast to our results for ciprofloxacin, previous studies demonstrated that ciprofloxacin Amabile de Campos T., Stehling E.G., Ferreira A., Castro A.F.P., Brocchi M. & Silveira W.D. 2005. 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