ORIGINAL ARTICLE Virulence-associated genes in faecal and clinical Escherichia coli isolates cultured from broiler chickens in Australia L Awawdeh,a,b* R Forrest,c,†* C Turni,d R Cobbold,b J Henningb and J Gibsonb A healthy chicken’s intestinal flora harbours a rich reservoir of Escherichia coli as part of the commensal microbiota. However, some strains, known as avian pathogenic E. coli (APEC), carry spe- cific virulence genes (VGs) that enable them to invade and cause extraintestinal infections such as avian colibacillosis. Although several VG combinations have been identified, the pathogenic mechanisms associated with APEC are ill-defined. The current study screened a subset of 88 E. coli isolates selected from 237 pre-existing isolates obtained from commercial poultry flocks in Australia. The 88 isolates were selected based on their entero- bacterial repetitive intergenic consensus (ERIC) and antimicrobial resistance (AMR) profiles and included 29 E. coli isolates cultured from chickens with colibacillosis (referred to as clinical E. coli or CEC) and 59 faecal E. coli (FEC) isolates cultured from clinically healthy chickens. The isolates were screened for the presence of 35 previously reported VGs. Of these, 34 were identified, with iucA not being detected. VGs focG, hlyA and sfa/foc were only detected in FEC isolates. Eight VGs had a prevalence of 90% or above in the CEC isolates. Specifically, astA (100%); feoB (96.6%); iutA, iss, ompT, iroN and hlyF (all 93.1%); and vat (89.7%). The prevalence of these were significantly lower in FEC isolates (astA 79.7%, feoB 77.9%, iutA 52.5%, iss 45.8%, ompT 50.9%, iroN 37.3%, hlyF 50.9% and vat 42.4%). The odds ratios that each of these eight VGs were more likely to be asso- ciated with CEC than FEC ranged from 7.8 to 21.9. These eight VGs may be used to better define APEC and diagnostically detect APEC in Australia. Further investigations are needed to identify the roles of these VGs in pathogenicity. Keywords antibiotic resistance profile; avian colibacillosis; avian pathogenic Escherichia coli; enterobacterial repetitive intergenic consensus; extraintestinal pathogenic E. coli; polymerase chain reaction; virulence genes Aust Vet J 2024 doi: 10.1111/avj.13339 Avian colibacillosis is caused by a subgroup of extraintestinal pathogenic Escherichia coli (ExPEC), known as avian pathogenic E. coli (APEC), which have the ability to invade various internal organs and cause systemic disease.1–3 Most E. coli, however, are commensals that coexist in healthy birds’ gut micro- biota, do not cause disease and are known as avian faecal E. coli (AFEC).4–7 A large number of overseas studies have aimed to define and differentiate APEC from AFEC based on whole-genome sequencing (WGS), phylogenetic grouping, virulence genotyping, serotyping, as well as fingerprinting methods such as enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction (PCR), randomly amplified polymorphic DNA (RAPD) and restric- tion fragment length polymorphism (RFLP).4–8 Nevertheless, APEC are still not clearly defined. Several virulence genes (VGs) have been found to be associated with APEC. However, no specific VG, or set of VGs, that contribute entirely to APEC pathogenicity have been identi- fied.9,10 Several overseas studies have differentiated APEC and AFEC based on the presence of five VGs that Johnson et al.6 identified as having a significant association with APEC.4,9,11 These VGs are iss (increase serum survival gene), ompT (outer membrane proteinase gene), hlyF (putative avian hemolysin gene), iroN (salmochelin side- rophore receptor gene) and iutA (aerobactin receptor gene).6 Johnson et al. concluded that E. coli could be considered an APEC if cultured from a lesion in an internal organ of a chicken with colibacillosis and possessed at least four of these five APEC-associated VGs.6 In a previous study, the clonal relatedness between 237 E. coli isolates comprised of 50 clinical isolates cultured from chickens with colibacillosis (CEC) and 187 faecal E. coli (FEC) isolates cultured from healthy chickens in Australia has been determined using ERIC-PCR.12 ERIC-PCR is a molecular typing method that has been employed to study genetic diversity due to its simplicity, enabling the examination of relationships among a large number of isolates.11,12 This method offers advantages such as speed, cost-effectiveness and the ability to categorise E. coli into distinct clonal groups and clusters for epidemio- logical research.13,14 However, it has limitations, including a lack of repeatability and lower discriminatory power compared with other fin- gerprinting methods, such as multi-locus sequence typing (MLST) and pulse-field gel electrophoresis (PFGE).15 In addition, no studies report a clear link between VG and ERIC-PCR profiles. This suggests the existence of distinct VG profiles within clonal groups or clusters, hinting at past genetic exchanges between different strains. The current research focused on a subset of the existing isolates obtained from Australian broiler chickens that exhibited diverse *Corresponding author. †Present address: College of Heatlh, Massey University, Palmerston North, New Zealand aSchool of Science, Western Sydney University, Richmond, New South Wales, Australia; l.awawdeh@westernsydney.edu.au bSchool of Veterinary Science, The University of Queensland, Gatton, Queensland, Australia cNursing & Health Science, Te P�ukengajEastern Institute of Technology, Napier, New Zealand; R.Forrest@massey.ac.nz dQueensland Alliance for Agriculture and Food Innovation, Centre for Animal Science, The University of Queensland, Dutton Park, Queensland, Australia Rachel Forrest and Leena Awawdeh have contributed equally to this manuscript and share first authorship. AVIAN AUSTRALIA’S PREMIER VETERINARY SCIENCE TEXT AV IA N © 2024 The Authors. Australian Veterinary Journal published by John Wiley & Sons Australia, Ltd on behalf of Australian Veterinary Association. Australian Veterinary Journal This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 1 https://orcid.org/0000-0001-6236-4772 https://orcid.org/0000-0002-5560-4237 https://orcid.org/0000-0002-0282-1318 mailto:l.awawdeh@westernsydney.edu.au mailto:R.Forrest@massey.ac.nz http://creativecommons.org/licenses/by/4.0/ http://crossmark.crossref.org/dialog/?doi=10.1111%2Favj.13339&domain=pdf&date_stamp=2024-05-09 clonality and were highly antimicrobial resistant.12 This subset of isolates was used to investigate the presence and distribution of 35 VGs reported to be associated with APEC within both CEC and FEC isolates. The 35 VGs investigated included the five APEC- associated VGs that Johnson et al.6 identified and encompassed eight VG categories, including PTJ100 related (cvaC, iroN2, iss2, traT, iutA2, tsh and SitA), adhesins (fimC, papC, papG, papEF, fimH, afa/ dra, focG, sfa/focDE and hlyA), iron acquisition (irp, chuA, fyuA, ireA, feoB, sfaS and IucA3), protectins/serum resistance (iucD, kpsMTII and neuC), invasions (kpsMTK1), toxins (ibeA and Vat), colicins (hlyF, cbi and cma) and miscellaneous (maxI, ompT and astA).13 The aim of the study was to identify an APEC-associated VG profile that distinguished between the CEC and FEC isolate types in Australian broiler chickens. Materials and methods Bacterial isolates and subset selection criteria In total, 237 E. coli isolates (187 FEC and 50 CEC) were available from a previous study.12 For this study, the most resistant isolate from each of the 88 ERIC-PCR profile clusters (see Section 1.2) was selected based on their predetermined AMR profile (see Section 1.3). If more than one isolate from the same cluster had the same AMR profile, then the bird’s health status was used to determine which isolate would be included with isolates cultured from chickens with colibacillosis being selected. Random selec- tions were applied if more than one isolate belonged to the same cluster with the same AMR and health status profile. The subset of E. coli isolates (n = 88) selected for testing in this study are detailed in Table 1. Enterobacterial repetitive intergenic consensus (ERIC)-PCR Clonality between the 237 E. coli isolates had been established by ERIC-PCR.17 The banding patterns were analysed using Gel Com- parII (Applied Maths, Sint-Martens-Latem, Belgium). The similarity was estimated with a Dice Coefficient of 0.1% and a tolerance of 1%, and cluster analysis was performed with Dice Coefficients and an unweighted-pair group method with arithmetic mean (UPGMA). A rooted rendered tree was generated using the UPGMA. Similar to other studies, isolates with a >93% similarity in their ERIC profile were assumed to be closely related (a clonal group).18,19 A cluster was defined as a group of isolates that shared ≥80% similarity in their ERIC-PCR profile patterns. Antimicrobial susceptibility testing of bacterial isolates Antimicrobial susceptibility testing had been performed in a pre- vious study12 using tryptone soy agar disc diffusion as per Clini- cal and Laboratory Standards Institute guidelines (CLSI)20 for 20 antimicrobials of veterinary and/or human health importance. These included amikacin, amoxicillin/clavulanic acid, ampicillin, apramycin, cefoxitin, ceftazidime, ceftiofur, cefovecin, cephalothin, chloramphenicol, ciprofloxacin, florfenicol, gentamicin, imipenem, neomycin, spectinomycin, streptomycin, sulfamethoxazole/tri- methoprim, tetracycline and ticarcillin/clavulanic acid.20 The anti- microbial discs were sourced from Thermo Fisher Scientific Australia Pty Ltd. The quality control organism used was E. coli ATCC 25922. Since there were no specific veterinary breakpoints for poultry, interpretative criteria were extrapolated from the CLSI guidelines for other animal species. For antimicrobials without CLSI breakpoints, breakpoint information was obtained from the respective manufacturers. In the context of this study, E. coli iso- lates with intermediate susceptibility were considered not resistant, and isolates were labelled as multidrug-resistant (MDR) if they were resistant to one or more antimicrobial agents in three or more antimicrobial classes.21 Case definition All E. coli isolates were categorised as shown in Figure 1. The bird’s health status from which the isolate was obtained was determined independently from the isolate’s VG profile. FEC were further characterised as APEC if sourced from faeces or the Table 1. Sampling details of 29 clinical Escherichia coli (CEC) and 59 faecal E. coli (FEC) isolates obtained from commercial broiler chickens in Australia Type of isolates Number of isolates Sampling site and sample number Location Date of isolation Reference FEC 59 A cloacal swab of healthy chickens Various locations within Southeast Queensland 2013–2014 14 CEC 9 Colibacillosis site in chicken: Liver (n = 2), air sac (n = 1), lung (n = 2), heart (n = 2), spleen (n = 1) and cloacal swabs (n = 1) Three chicken farms within Southeast Queensland 2013 15 CEC 15 Colibacillosis site in chicken: Abdomen (n = 2), air sac (n = 1), heart (n = 1), infraorbital sinus (n = 1), intestine (n = 1), liver (n = 1), lung (n = 3), pericardium (n = 1) peritoneum (n = 1), pleura (n = 1), nasal cavity (n = 1) and trachea (n = 1) Biosecurity sciences laboratory from Queensland 2006–2013 15 CEC 5 Colibacillosis site in chicken: Liver (n = 1), lung (n = 1), pericardium (n = 1), air sac (n = 1) and subcutaneous (n = 1) Australian diagnostic laboratories 2013–2014 16 Australian Veterinary Journal © 2024 The Authors. Australian Veterinary Journal published by John Wiley & Sons Australia, Ltd on behalf of Australian Veterinary Association. 2 AVIAN AV IA N 17510813, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/avj.13339 by M assey U niversity L ibrary, W iley O nline L ibrary on [04/07/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense cloaca and harboured four or more of the APEC-associated VGs (iroN, iutA, iss, hlyF and ompT) defined by Johnson et al6 or AFEC if they did not.6 Upon genotyping, CEC isolates, which harboured four or more of the five APEC-associated VGs, were defined as clinical avian pathogenic E. coli (cAPEC) and those that contained less than four of the selected VGs were identified as potential APEC (pAPEC). Virulence genotyping The selected E. coli isolates (n = 88) had been previously screened for the presence of the five APEC-associated VGs (iroN, iutA, iss, hlyF and ompT).6,15 The isolates were then screened for a further 30 APEC-associated VGs using published single and multiplex PCR assay panels: the first PCR panel targeted astA, irp2, papC, iucD, tsh and vat13; the second panel targeted chuA and traT22; the third panel amplified fyuA; papG and kpsMT k123; the fourth panel screened for fimH, papEF, ireA and ibeA7; the fifth panel targeted sitA and feoB7; sfaS and focG were included in the sixth panel8; the seventh panel amplified cbi; cma and cvaC24; the eighth panel included kpsMTII; hlyA; fimC; neuC; afa/drab, maxI and sfa/foc22 and the ninth panel amplified iucA.24 Statistical analysis Analyses were performed in Stata software (13th edition, Blackburn North Victoria, Australia, www.stata.com). Comparisons of the asso- ciations between each VG for CEC and FEC were carried out using Fisher’s exact test (used instead of the Chi-square in case of small values), and an odds ratio (OR) with their 95% confidence interval (CI) was calculated. A p-value of less than 0.05 was considered significant. Results Isolate case classification Two of the 29 CEC isolates did not harbour any of the 35 APEC- related VGs and were classified as pAPEC, whereas 27 were classified as cAPEC. Nineteen of the FEC were classified as APEC, whereas 40 were AFEC. Virulence genotyping Figure 2 illustrates the 35 APEC-related VG profile for each of the 88 isolates. Table 2 shows the number and prevalence of the 35 APEC-related VGs among the 29 CEC and 59 FEC isolates. None of the CEC or FEC isolates harboured iucA. The APEC-related VGs sfa/foc; focG, and hlyA, were not present in the CEC isolates. Table 2 provides the odds ratio that a particular VG was associated with a CEC rather than an FEC isolate, along with the prevalence of the VG in both the CEC and FEC isolates. The following VGs were identified as being significantly (p-value ≤0.05) associated with CEC isolates: astA (100%); foeB (96.6%); iroN (93.1%); ompT (93.1%); iss (93.1%); iutA (93.1%); iutA (93.1%); vat (89.7%); fimC (86.2%); cvaC (79.3%), tsh (55.2%); ireA (51.7%); papC (44.8%); papEF (41.4%) and ibeA (37.9%). Those with a prevalence of 90% or above (namely, astA, foeB, iroN, omp, iss, iutA, iutA, vat) were associated with odds ratios ranging from 7.8 to 21.9 reflecting that these VGs were much more likely to be associated with CEC than FEC (Table 2). For the following VGs, a significant difference between the CEC and FEC prevalence was not detected: afa-drab, cbi, chuA, cma, fimH, fyuA, irp2, iucD, kpsMT11, kpsMTK1, maxI, neuC, papG, sfaS, sitA and traT (Table 2). Seven per cent, 51.7% and 41.4% of the CEC harboured 10, 20 and 30 VGs in comparison with 22%, 71.2% and 6.7% of the FEC. Figure 1. Flow chart detailing the case classification applied to all Escherichia coli isolates (n = 88) included in the current study. The five selected VGs were iss; iutA; iroN; ompT and hlyF.6 © 2024 The Authors. Australian Veterinary Journal published by John Wiley & Sons Australia, Ltd on behalf of Australian Veterinary Association. Australian Veterinary Journal 3 AVIAN AV IA N 17510813, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/avj.13339 by M assey U niversity L ibrary, W iley O nline L ibrary on [04/07/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense http://www.stata.com Figure 2. Rooted rendered virulence gene profiles of 29 clinical E. coli (CEC) and 59 faecal E. coli (FEC) isolates obtained from broiler chickens in Australia. Virulence genes screened (n = 35): afa/draB, afimbrial/Dr antigen-specific adhesin; astA, EAST1 (heat-stable cytotoxin associated with enteroaggregative E. coli); cbi, corresponding immunity; cma, Colicin M-resembles B-lactam; chuA, heme receptor gene (heme uptake); cvi/cva, structural genes of colicin V operon (microcin ColV); feoB, ferrous iron transport protein B fimC, type 1 fimbriae (d-mannose-specific adhesin); fimH, adhesive subunit of type 1 fimbriae; fyuA, ferric yersiniabactin uptake (yersiniabactin receptor); hlyA, haemolysin A; ibeA, invasion of brain endothelium; ireA, //�responsive element (putative catecholate siderophore receptor); iroN, catecholate siderophore (salmochelin) receptor; irp2, iron-repressible protein (yersiniabactin synthesis); iss, increased serum survival; iucD, aerobactin synthesis; iutA, ferric aerobactin receptor (iron uptake/transport); kpsMTI, group I capsule antigens; kpsMT K1, group I capsule antigens; kpsMT II, group II capsule antigens; maxI, pathogenicity-associated island marker; neuC, K1 capsular polysaccharide; ompT, outer membrane protease; papC, P-fimbriae; sitA, putative iron transport gene; sfa/focDE, sfa (S fimbriae) and foc (F1C fimbriae; traT, surface exclusion protein (serum survival factor); tsh, temperature-sensitive haemagglutinin; vat, vacuolating autotransporter toxin. Australian Veterinary Journal © 2024 The Authors. Australian Veterinary Journal published by John Wiley & Sons Australia, Ltd on behalf of Australian Veterinary Association. 4 AVIAN AV IA N 17510813, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/avj.13339 by M assey U niversity L ibrary, W iley O nline L ibrary on [04/07/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense Discussion Globally, colibacillosis or APEC infection is considered a significant problem affecting the poultry industry and translates into multimillion-dollar losses annually.4,6,25 Interestingly, although vac- cines have been developed to alleviate this problem, none have effec- tively controlled this infection or disease.26,27 Despite the intensive research globally, the genetic diversity of APEC makes it hard to reach a consensus on the definition of APEC.6,7,26 In the current study, the E. coli isolates (29 CEC and 59 FEC) carried an assortment of APEC-associated VGs, some significantly associated with CEC. The varying prevalence of the VGs reported in this study is compa- rable with several overseas studies, which have reported variations in VG frequency and combinations in CEC and FEC isolates.6,7,27–29 The genetic diversity and relatedness of isolates used in the current study was previously determined using ERIC-PCR20 as it is a simpler when compared with other molecular typing methods and allows the processing of a large number of isolates.30,31 It has the benefits of being rapid, cost-effective and able to classify E. coli into different clonal groups or clusters for epidemiological studies.30–35 The lack of repeatability and discriminatory power and lower typing ability com- pared with other fingerprinting methods, such as MLST and PFGE, are some of the disadvantages of this protocol.30 However, Ewers et al.36 and Knobl et al.37 have reported that none of the molecular typing methods have the ability to differentiate between CEC and FEC isolates. Consistent with this, the ERIC-PCR profiles did not differentiate between CEC and FEC in the current study, and there was no clear association between VG pattern and ERIC-PCR profile. This may suggest the presence of distinct VG profile patterns within clonal groups or clusters and past genetic exchanges between various strains. Phylogenetic analysis using WGS is recommended to provide rapid, accurate and valuable genetic data.38–40 Unlike ERIC-PCR which only amplifies a small portion of the genome and can only be used to distinguish between strain of bacteria, WGS can be used to not only differentiated between strains of bacteria but also to identify genetic variations between strains and identify genes associated with specific traits. These data have the discriminatory power for future APEC pathogenesis studies and epidemiological investigations to improve the bird’s welfare and help develop prevention and control measures against avian colibacillosis.40 The ability of WGS to com- pare and link with international bacterial typing databases is consid- ered one of the advantages in comparison with other genetic typing methods, such as ERIC, where data are not standardised and cannot be compared between laboratories.41 The frequency of APEC-related VGs among the E. coli isolates varied as reported in Table 2. VG profiles may be influenced by several fac- tors such as geographical locations, season, the bird’s immune status, the sampling sites and different husbandry and vaccination proto- cols.6,42,43 Therefore, APEC-associated VG markers specific to Australian conditions need to be determined to better define and identify APEC in order to reduce the economic losses associated with the disease in the poultry industry. The positive association between avian colibacillosis and the presence of APEC-associated VGs, either individually or in different combinations, has been previ- ously documented in other parts of the world.6,7,44–47 In the current study, 15 VGs were found to be significantly associated with CEC when compared with FEC: cvi/cvaC; iroN; iss; iutA; tsh; fimC; papC; papEF; ireA; feoB; ibeA; vat; hlyF; ompT and astA (Table 2). The odds ratios for those VGs that had a CEC prevalence of 90% or above (astA, foeB, iroN, ompT, iss, iutA, hlyF and vat) ranged from 7.8 to 21.9 (Table 2). Thus, using these eight VGs in combination has a very high likelihood of predicting CEC isolates from FEC iso- lates and therefore can be used to define APEC in Australian broiler chickens. Not surprisingly, the five VGs (iutA, iss, ompT, iroN and hlyF) identified by Johnson et al.6 were among these eight high prev- alence APEC-associated VGs. Studies in Brazil (82%–95%) and Egypt (90%–94%) have reported a similar frequency of these five VGs among APEC isolates cultured from lesions of birds with colibacillosis.9,11 Ewers et al.13 found that the five VGs described by Johnson et al.,6 as well as iucD, irp2 and astA could identify and dif- ferentiate APEC from AFEC, of which astA was among the eight high prevalence VGs identified in this study (Table 2). Interestingly, iucD and irp2 were not found to be significantly associated with CEC in Australian broiler chickens which supports the notion that there is variation in the genes underlying the pathogenicity of E. coli strains making it difficult to identify a comprehensive VG profile that defines APEC globally. The present study investigated 35 APEC-associated VGs to also facil- itate a better understanding of the pathogenic mechanisms associ- ated with APEC. Each of the APEC-associated VGs investigated serve distinct roles in the extraintestinal pathogenicity of APEC and can be categorised into different groups according to their functions and contribution to the APEC pathogenicity mechanisms.4,48 At dif- ferent stages of infection, alternative VGs could be involved in the pathogenicity mechanism of APEC; therefore, for functions where multiple VGs have been identified such as colonisation (fimH, fimC, papC, papEF and tsh), invasion (ibeA, vat), iron acquisition (iutA, iroN, IreA, feoB), serum complement resistance (iss) and putative iron transport (sitA), their prevalence may vary between APEC strains.7 Consistent with this notion are the findings with regard to iss, which encodes for a lipoprotein of the bacterial outer membrane that facilitates resistance to serum complement.4,7,10,49–52 In the cur- rent study, the prevalence of iss in APEC was 93.1% but this varies in other countries such as the USA (80.5%), Germany (82.7%), Spain (91%), Egypt (72%) and Brazil (51%).6,9,36,53,54 This indicates that APEC strains exhibit genetic diversity underpinning their pathogenic mechanisms, highlighting the needs for additional research to eluci- date the prevalence of APEC-associated VGs in different countries. The adhesin genes, fimC and papC genes, were significantly associ- ated with CEC in the isolates obtained from Australian broiler chickens. These genes mediate the adherence of E. coli to host epi- thelial cells to enable colonisation.55 The prevalence of fimC in this study was reported as 86.2% (Table 2), which is comparable with Brazil (86%) but lower than that reported for Germany (92%) and Korea (90.1%) for E. coli obtained from chickens with colibacillosis.36,37,47 The positive association of papC as well as iss, tsh, cva/cvi and vat with APEC were found in E. coli isolates sourced from chickens affected with colibacillosis in Germany.22 The preva- lence of papC, which enables the adherence and the survival of E. coli in the internal organs,55 was 44.8% in the current study. A lower prevalence in E. coli isolates from broiler chickens with colibacillosis was also observed in the United States of America © 2024 The Authors. Australian Veterinary Journal published by John Wiley & Sons Australia, Ltd on behalf of Australian Veterinary Association. Australian Veterinary Journal 5 AVIAN AV IA N 17510813, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/avj.13339 by M assey U niversity L ibrary, W iley O nline L ibrary on [04/07/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense Table 2. Number (N), prevalence (%) and odds ratio (OR) with 95% confidence interval (CI) for 35 avian pathogenic Escherichia coli (APEC)- associated virulence genes among 29 clinical E. coli (CEC) and 59 faecal E. coli (FEC) from broiler chickens in Australia Putative gene function Virulence genea,b ORc (95% CI) CEC N (%) FEC N (%) p-value PTJ100 related genes cvaC 3.42 (1.14, 11.8) 23 (79.3) 31 (52.5) 0.026 iroN2 21.9 (4.76, 208) 27 (93.1) 22 (37.3) <0.001 iss2 15.6 (3.39, 147) 27 (93.1) 27 (45.8) <0.001 traT 1.45 (0.50, 4.45) 22 (72.4) 38 (64.4) 0.616 iutA2 11.9 (2.59, 112) 27 (93.1) 31 (52.5) 0.002 tsh 4.27 (1.51, 12.6) 16 (55.2) 13 (22.1) 0.005 SitA 4.38 (0.94, 43.9) 27 (93.1) 44 (74.6) 0.064 Adhesins fimC 7.24 (2.13, 32.2) 25 (86.2) 27 (45.8) 0.004 papC 6.98 (2.08, 26.3) 13 (44.8) 6 (10.2) 0.001 papG 2.88 (0.89, 9.46) 10 (34.5) 9 (15.3) 0.079 papEF 3.85 (1.24, 12.4) 12 (41.4) 9 (15.3) 0.017 fimH 1.55 (0.58, 4.22) 16 (55.2) 26 (44.1) 0.451 afa/dra 1.16 (0.27, 4.36) 5 (17.3) 9 (15.3) 1.000 focG - 2 (3.4) sfa/foc - 2 (3.4) hlyA - 1 (1.7) Iron acquisition irp2 0.48 (0.05, 2.62) 2 (6.9) 8 (13.6) 0.591 chuA 2.10 (0.69, 7.33) 23 (79.3) 38 (64.4) 0.242 fyuA 1.27 (0.47, 3.41) 15 (51.7) 27 (45.8) 0.761 ireA 2.84 (1.03, 8.04) 15 (51.7) 16 (27.1) 0.043 feoB 7.80 (1.06, 348) 28 (96.6) 46 (77.9) 0.041 sfaS 1.38 (0.11, 12.8) 2 (6.9) 3 (5.08) 1.000 IucA - - Protectins/serum resistance iucD 1.61 (0.54, 5.22) 22 (75.9) 39 (66.1) 0.496 kpsMTII 2.27 (0.73, 7.05) 10 (34.5) 11 (18.6) 0.173 neuC 0.82 (0.28, 2.29) 9 (31.1) 21 (35.6) 0.86 Invasions kpsMTK1 2.27 (0.73, 7.05) 10 (34.5) 11 (18.6) 0.173 Toxins ibeA 3.34 (1.06, 10.8) 11 (37.9) 9 (15.3) 0.038 vat 11.5 (3.02, 65.7) 26 (89.7) 25 (42.4) <0.001 Colicin genes hlyF2 12.7 (2.78, 120) 27 (93.1) 30 (50.9) <0.001 cbi 2.41 (0.68, 8.46) 8 (27.6) 8 (13.6) 0.139 cma 2.09 (0.61, 7.11) 8 (27.6) 9 (15.3) 0.276 Miscellaneous maxI 0.71 (0.24, 1.97) 9 (31.1) 23 (38.9) 0.627 ompT2 12.7 (2.78, 120) 27 (93.1) 30 (50.9) <0.001 astA 9.99 (1.55, ∞) 29 (100) 47 (79.7) 0.011 a afa/draB, afimbrial/Dr antigen-specific adhesin; astA, EAST1 (heat-stable cytotoxin associated with enteroaggregative E. coli); cbi, corresponding immunity; cma, Colicin M-resembles B-lactam; chuA, heme receptor gene (heme uptake); cvi/cva, structural genes of colicin V operon (microcin ColV); feoB, ferrous iron transport protein B fimC, type 1 fimbriae (d-mannose-specific adhesin); fimH, adhesive subunit of type 1 fimbriae; fyuA, ferric yersiniabactin uptake (yersiniabactin receptor); hlyA, haemolysin A; ibeA, invasion of brain endothelium; ireA, //-responsive element (putative catecholate siderophore receptor); iroN, catecholate siderophore (salmochelin) receptor; irp2, iron-repressible protein (yersiniabactin synthesis); iss, increased serum survival; iucD, aerobactin synthesis; iutA, ferric aerobactin receptor (iron uptake/trans- port); kpsMTI, group I capsule antigens; kpsMT K1, group I capsule antigens; kpsMT II, group II capsule antigens; maxI, pathogenicity-associated island marker; neuC, K1 capsular polysaccharide; ompT, outer membrane protease; papC, P-fimbriae; sitA, putative iron transport gene; sfa/focDE, sfa (S fimbriae) and foc (F1C) fimbriae; traT, surface exclusion protein (serum survival factor); tsh, temperature-sensitive haemagglutinin; vat, vacuolating autotransporter toxin. b The PCR for these genes was performed in previous study.15 c Virulence gene iucA was not detected in CEC and FEC, whereas hlyA, focG and sfa/foc were not detected in CEC; therefore, ORs for these could not be calculated. Bolded p values are significant using the α = 0.05 level. Australian Veterinary Journal © 2024 The Authors. Australian Veterinary Journal published by John Wiley & Sons Australia, Ltd on behalf of Australian Veterinary Association. 6 AVIAN AV IA N 17510813, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/avj.13339 by M assey U niversity L ibrary, W iley O nline L ibrary on [04/07/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense (USA) (24.8%) and Germany (22.7%).6,36 Consistent with a role in virulence, the prevalence of papC in E. coli collected from healthy chickens was very low, with rates of (0%) in the USA and (7.13%) in Japan.56,57 The APEC-associated VG tsh contributes mainly to the respiratory form of avian colibacillosis.58–61 In this study, 55% of CEC isolates carried this gene; however, respiratory tract sample sites only accounted for 11 of the 29 isolates (38%, Table 1) suggesting that in Australian broiler chickens tsh may also contribute to non- respiratory forms of colibacillosis. Other studies have reported the association between tsh and CEC,45,62 though these studies reported a prevalence of tsh as low as 19%63 and as high as 85.3%,45,62 which may indicate the dominant form of avian colibacillosis (non- respiratory versus respiratory, respectively) in these studies. The APEC-associated VG vat encodes for the Vat protein with cyto- toxic activity for the chicken embryo fibroblast and kidney cells.64 Several studies have reported that the vat gene is encoded in the APEC pathogenicity island (PAI) and occurs in a higher frequency among APEC compared with AFEC.13,36,65 Consistent with this, vat had a prevalence of 89.7% in CEC isolates compared with 42.4% in FEC isolates in this study. The current study also found that seven pTJ100-plasmids-related genes (iroN, traT, iutA, sitA, iss, cvaC and tsh) were more prevalent among CEC isolates than FEC isolates (Table 2) which is consistent with previous studies.6,7,66 This finding reflects the role of plasmids in the pathogenicity and the transfer of certain VGs among the path- ogenic as well as commensal E. coli isolates. This highlights the need for further molecular studies to identify the role of plasmids and other forms of horizontal gene transfer in APEC pathogenicity.66,67 The prevalence of the following VGs: papG; irp2; chuA; kpsMT11; kpsMT K1; cbi; cma; traT; fimH; afa-drab; fyuA; sfaS; iucD; neuC and maxI was not found to be different between CEC and FEC iso- lates (Table 2). The presence of APEC-associated VGs in FEC isolates indicates that AFEC may act as a reservoir for some VGs.1,10,68 In the current study, iucA which is involved in one of the iron acqui- sition systems from both CEC and FEC isolates, was not detected. This differs from a previous study by Tivendale et al.51 who identi- fied the co-presence of iss, tsh and iucA in plasmids found in E. coli strains isolated from birds with severe colibacillosis in Australia. This difference in the detection of iucA between the studies could be cor- related with variation in the levels of virulence of the E. coli isolates in the Tivendale et al.51 study compared with the current study. Tivendale et al.51 demonstrated the ability of the three most virulent APEC strains to amplify the iucA gene compared with less virulent APEC strains. Other studies have also suggested that iucA plays a vital role in the pathogenicity of APEC.69,70 Collectively the result from this study indicates that a VG profile consisting of eight genes, namely, astA, foeB, iroN, ompT, iss, iutA, hlyF and vat could offer a highly sensitive approach for detecting APEC strains in Australian broiler chickens. Despite this finding, it is essential to recognise the existing limitations within this methodol- ogy. The fact that three PCRs would be needed to investigate the presence of the VGs to determine APEC status is not a practical approach and highlights the need to use faster and more accurate methods to screen for the presence of the APEC-associated VGs. A number of methods may be suitable for multiplex analysis in one assay, such as Luminex, which allows multiplexing of up to 100 analytes in a single well of a microtiter plate.71 In addition to PCR and genotyping methods, DNA microarrays are able to char- acterise bacteria by detecting large numbers of VGs and antimicro- bial resistance genes.72 Furthermore, microarrays can identify genomic variations between different strains of bacteria. With the advances seen in recent years, whole-genome sequencing will become feasible for practical routine screening to rapidly characterise and identify large numbers of virulence genes.73,74 Although the current study identified a set of APEC-associated VGs (iroN, iss, iutA, tsh, fimC, papC, papEF, vat, hlyF, astA, ibeA, feoB, ireA, cvi/cvaC and ompT) that were more likely associated with CEC the pathogenicity of these VGs still has to be explored. Furthermore, whereas this study provides important information about APEC- associated VG profiles for isolates obtained between 2006 and 2014, it needs to be acknowledged that there is the potential for shifts in E. coli strains and, therefore VG profiles, over time. Further research using more recent data is needed to ascertain the current prevalence of APEC in broiler chickens in Australia along with their VG pro- files. Comparing more recent data with that of this study will allow a better understanding of the epidemiology associated with colibacillosis so that the Australian poultry industry is better equipped to track and combat this disease. Conclusions The current study identified 15 specific VGs (iroN, iss, iutA, tsh, fimC, papC, papEF, vat, hlyF, astA, ibeA, feoB, ireA, cvi/cvaC and ompT) that had a significantly higher prevalence in CEC than in FEC isolates; however, they were present in both indicating that AFEC may act as a reservoir for VGs. These results confirm that the set of five VGs (iroN, iutA, iss, hlyF and ompT) previously used to define APEC in other countries can also be applied in Australian broiler chickens as they had a significantly higher prevalence in CEC compared with FEC isolates. An additional ten APEC-associated VGs were also identified (tsh, fimC, papC, papEF, vat, astA, ibeA, feoB, ireA and cvi/cvaC) that were significantly more likely to be found in the avian E. coli strains sourced from chickens with colibacillosis in Australia. Of these, eight VGs (astA, feoB, iutA, iss, ompT, iroN, hlyF and vat) have been identified as a VG profile for the identification of APEC in Australian broiler chickens. VGs are often located on plasmids, such as PTJ100-related genes iroN, traT, iutA, sitA, iss, cvaC and tsh highlighting the association between avian pathogenicity E. coli strains and the possession of mobile genetic elements. Given iroN, iutA and iss were identified as APEC- associated VGs in Australian broiler chickens, further studies are required to investigate the possibility of using these VGs in the field and diagnostic laboratories to identify avian colibacillosis in Australia. Rapid detection of avian colibacillosis could minimise the economic losses and welfare effects associated with the disease. A study to investigate the role of these VGs and their contribution to the pathogenicity of avian pathogenic E. coli is also recommended. © 2024 The Authors. Australian Veterinary Journal published by John Wiley & Sons Australia, Ltd on behalf of Australian Veterinary Association. Australian Veterinary Journal 7 AVIAN AV IA N 17510813, 0, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/avj.13339 by M assey U niversity L ibrary, W iley O nline L ibrary on [04/07/2024]. 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See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense https://doi.org/10.4137/bbi.s12462 https://doi.org/10.1016/j.ijfoodmicro.2014.07.002 https://doi.org/10.1186/s40793-015-0126-6 https://doi.org/10.1186/s42522-020-0010-1 https://doi.org/10.1186/s42522-020-0010-1 https://doi.org/10.3389/fmicb.2013.00188 https://doi.org/10.1080/00071668.2014.935998 https://doi.org/10.1080/00071668.2014.935998 https://doi.org/10.1080/00071668.2012.711904 https://doi.org/10.1016/s0378-1135(03)00076-2 https://doi.org/10.4142/jvs.2012.13.2.145 https://doi.org/10.1080/03079450802216652 https://doi.org/10.1016/J.VETMIC.2005.08.001 https://doi.org/10.1016/J.VETMIC.2005.08.001 https://doi.org/10.1128/IAI.71.6.3285-3293.2003 https://doi.org/10.1128/iai.72.11.6554-6560.2004 https://doi.org/10.1128/IAI.71.1.536-540.2003 https://doi.org/10.3382/ps/pev256 https://doi.org/10.1371/journal.pone.0143191 https://doi.org/10.1371/journal.pone.0143191 https://doi.org/10.1006/mpat.1996.0116 https://doi.org/10.1006/mpat.1996.0116 https://doi.org/10.1371/journal.pone.0180599 https://doi.org/10.1371/journal.pone.0180599 https://doi.org/10.1016/j.psj.2022.102007 https://doi.org/10.1016/j.psj.2022.102007 https://doi.org/10.1128/cmr.18.2.264-292.2005 https://doi.org/10.1016/s0378-1135(03)00125-1 https://doi.org/10.1016/s0378-1135(03)00125-1 https://doi.org/10.1128/IAI.68.7.4145-4154.2000 https://doi.org/10.1128/IAI.68.7.4145-4154.2000 https://doi.org/10.2307/1592582 https://doi.org/10.2307/1592582 https://doi.org/10.1016/j.vetmic.2005.01.021 https://doi.org/10.1073/pnas.232686799 https://doi.org/10.1073/pnas.232686799 https://doi.org/10.1128/IAI.71.9.5087-5096.2003 https://doi.org/10.1128/IAI.71.9.5087-5096.2003 https://doi.org/10.1099/mic.0.024869-0 https://doi.org/10.1099/mic.0.024869-0 https://doi.org/10.2141/jpsa.46.260 https://doi.org/10.1078/1438-4221-00143 https://doi.org/10.1371/journal.pone.0057794 https://doi.org/10.1016/j.vetmic.2012.04.024 https://doi.org/10.1128/aem.00419-07 https://doi.org/10.3389/fmicb.2016.00644 https://doi.org/10.3389/fmicb.2016.00644 https://doi.org/10.1111/1751-7915.12389 Virulence-associated genes in faecal and clinical Escherichia coli isolates cultured from broiler chickens in Australia Materials and methods Bacterial isolates and subset selection criteria Enterobacterial repetitive intergenic consensus (ERIC)-PCR Antimicrobial susceptibility testing of bacterial isolates Case definition Virulence genotyping Statistical analysis Results Isolate case classification Virulence genotyping Discussion Conclusions Acknowledgments Conflicts of interest and sources of funding References