In vitro assessment of pathogenicity and virulence encoding gene profiles of avian pathogenic Escherichia coli strains associated with colibacillosis in chickens

Abstract

Background:

Avian pathogenic Escherichia coli (APEC) strains have been associated with various disease conditions in avian species due to virulence attributes associated with the organism.

Aims:

This study was carried out to determine the in vitro pathogenic characteristics and virulence encoding genes found in E. coli strains associated with colibacillosis in chickens.

Methods:

Fifty-two stock cultures of E. coli strains isolated from chickens diagnosed of colibacillosis were tested for their ability to produce haemolysis on blood agar and take up Congo red dye. Molecular characterization was carried out by polymerase chain reaction (PCR) amplification of virulence encoding genes associated with APEC.

Results:

Eleven (22%) and 41 (71%) were positive for haemolysis on 5% sheep red blood agar and Congo red agar, respectively. Nine virulence-associated genes were detected as follows: FimH (96%), csgA (52%), iss (48%), iut (33%), tsh (21%), cva (15%), kpsII (10%), pap (2%), and felA (2%).

Conclusion:

The APEC strains exhibited virulence properties and harbored virulence encoding genes which could be a threat to the poultry population and public health. The putative virulence genes were diverse and different in almost all isolate implying that pathogenesis was multi-factorial and the infection was multi-faceted which could be a source of concern in the detection and control of APEC infections.

Introduction

Avian pathogenic Escherichia coli (APEC) strains cause various diseases in chickens and are responsible for large economic losses in the poultry industry worldwide (Zhuang et al., 2014 ▶; Zeinab et al., 2018 ▶). Avian pathogenic Escherichia coli strains are associated with infection of extraintestinal tissues in chickens, turkeys, ducks, and other avian species (Barbieri et al., 2015 ▶). Thus, APEC strains have been implicated in a variety of disease conditions including: coligranuloma, air sac disease, perihepatitis, airsacculitis, pericarditis, egg peritonitis, salpingitis, omphlitis, cellulitis and osteomyelitis or arthritis (Nolan et al., 2013 ▶).

The most important disease syndrome associated with APEC begins as a respiratory tract infection and is often known as airsacculitis or the air sac disease, which in turn can evolve into severe sepsis or systemic infection ultimately leading to the death of the bird. Respiratory lesions observed include airsacculitis with a serous to fibrinous exudates, an initial infiltration with heterophils, and a subsequent predominance of mononuclear phagocytes (Mellata, 2013 ▶). Sites of entry into the bloodstream are presumed to be the gas exchange region of the lung and the air sacs, which are relatively vulnerable to colonization and invasion by bacteria due to lack of resident macrophages (Guabirabaand and Schouler, 2015 ▶).

Avian pathogenic E. coli isolates possess several potential virulence factors related to colonization, temperature-sensitive haemagglutinin, complement resistance and increased serum survival (Mainil, 2013 ▶). Knowledge of virulence factors associated with APEC is necessary in explaining the pathogenesis of these organisms which could be helpful in management and control of the disease. These virulence factors are usually encoded or mediated by genes which often are transferable from pathogenic to non-pathogenic strains and vice versa, in a multi-cultural environment like the gastrointestinal tract (GIT) (Ogura et al., 2009 ▶).

The presence of certain virulence-associated genes among APEC strains as well as similar disease patterns and phylogenetic background is an indication of a significant zoonotic risk of avian-derived E. coli infections (Bauchart et al., 2010 ▶). Escherichia coli isolated from healthy chickens have been reported to contain extraintestinal pathogenic Escherichia coli (ExPEC)-associated gene and can cause ExPEC-associated infections in animal models and thus may pose a health threat to the host, including humans (Stromberg et al., 2017 ▶). Since chickens are usually in close contact with humans in the poultry industry value chain, APEC could also be of high public health risk.

Many studies across the globe including Nigeria have shown the prevalence of APEC strains among chickens diagnosed of colibacillosis (Olarinmoye et al., 2013 ▶; Barbieri et al., 2015 ▶; Ali et al., 2019 ▶). Different virulence-associated genes of APEC have also been documented worldwide (Wang et al., 2015 ▶; Sarowska et al., 2019 ▶). The purpose of this study was to determine the virulence-associated genes among E. coli strains isolated from chickens with colibacillosis in Enugu State, Nigeria.

Materials and Methods

Escherichia coli strains

Fifty-two stock cultures of E. coli strains isolated from the liver (20 strains), spleen (10 strains), heart blood (18 strains), and oviduct (4 strains) of broilers and layers diagnosed with colibacillosis in Nsukka, Nigeria were screened for the presence of virulence encoding genes. The stock cultures were inoculated onto nutrient broth and incubated for 24 h at 37°C. An aliquot of the broth culture was sub-cultured onto MacConkey agar (MCA) and confirmed by biochemical tests.

In vitro pathogenicity testing

Haemolytic activity

Escherichia coli isolates were streaked on blood agar plates. The inoculated blood agar plates were incubated at 37°C for 24 h and colonies producing clear zones of haemolysis were then recorded as hemolytic strains (Fakruddin et al., 2012 ▶). A known haemolytic Staphylococcus spp. and non-haemolytic Klebsiella pneumoniae isolates were used as positive and negative controls, respectively.

Congo red uptake

Each isolate was inoculated on a separate Congo red agar plate and incubated at 37°C for 24 h. After 24 h incubation, the cultures were left at room temperature for 48 h to facilitate annotation of results (Osman et al., 2012 ▶). Congo red uptake was indicated by the appearance of red colonies on the Congo red agar while colourless colonies indicated an inability to take up Congo red. A known Congo red positive E. coli and Congo red negative Salmonella isolates were used as positive and negative controls, respectively.

DNA extraction

DNA extraction was done following the standard phenol-chloroform method described by Sharpe (2005) ▶. Each E. coli strain was inoculated into 10 ml nutrient broth (HiMedia, India) and incubated at 37°C for 24 h. One ml of the culture was centrifuged at 12,000 g for 2 min. The cell pellet was then re-suspended in 200 µL of Tris-EDTA buffer (pH = 7.2) and 30 µL of lysozyme (2000U/µL). The mixture was incubated at 37°C for 1 h. It was then mixed with 33 µL of 10% sodium dodecyl sulphate (v/v) and incubated at 62°C for 30 min. Three-hundred microlitres of phenol: chloroform: isoamyl alcohol (25:24:1) were added to the mixture and vortex for 10 s followed by centrifugation at 12,000 g for 1 min. The top aqueous phase was collected into a new centrifuge tube and added to 1/10 volume of 3 M sodium acetate and mixed by inversion. It was then mixed with 2 volumes of 100% ethanol and incubated on ice for 5 min. The samples were then centrifuged at 12,000 g for 5 min and the supernatant removed. The DNA pellet was washed with 1 ml 70% ethanol and centrifuged at 12,000 g for 1 min and air-dried for 10 min. The DNA was re-suspended in 100 µL of Tris-EDTA buffer (pH = 8.0).

Detection of virulence-associated genes

The APEC strains were investigated for the presence of virulence-associated genes by polymerase chain reaction (PCR) following the procedure described by Rocha et al. (2008) ▶. The genes investigated were FimH, pap, felA, sfa, fac, csgA, tsh, cvaC, kpsII, iss, iutA, and cnf. The sequences of the primers used and PCR conditions are presented in Table 1.

Table 1.

Sequence of PCR primers, product size, annealing temperature and cycles (Rocha et al., 2008)

Gene	Primer sequence (5´-3´)	Product size (bp)	Annealing temp (°C)	cycles
fimH	CGA GTT ATT ACC CTG TTT GCT G (F)	878	55	35
	ACG CCA ATA ATC GAT TGC AC (R)
papC	GAC GGC TGT ACT GCA GGG TGT GGC G	328	63	30
	ATA TCC TTT CTG CAG GGA TGC AAT A
felA	GGC AGT GGT GTC TTT TGG TG	270	63	35
	GGC CCA GTA AAA GAT AAT TGA ACC
Sfa	CTC CGG AGA ACT GGG TGC ATC TTA C	410	55	35
	CGG AGG AGT AAT TAC AAA CCT GGC A
Fac	GGT GGA ACC GCA GAA AAT AC	388	58	35
	GAA CTG TTG GGG AAA GAG TG
csgA	ATC AGT ACG GTG GTG GTA ACT C	103	64	40
	CCA ACA TCT GCA CCG TTA CCA C
Tsh	GGT GGT GCA CTG GAG TGG	620	55	30
	AGT CCA GCG TGA TAG TGG
Cva	CAC ACA CAA ACG GGA GCT GTT	680	63	30
	CTT CCC GCA GCA TAG TTC CAT
Kpsll	GCG CAT TTG CTG ATA CTG TTG	272	65	30
	CAT CCA GAC GAT AAG CAT GAG CA
Iss	GTG GCG AAA ACT AGT AAA ACA GC	760	61	30
	CGC CTC GGG GTG GAT AA
iutA	GGC TGG ACA TCA TGG GAA CTG G	300	63	35
	CGT CGG GAA CGG GTA GAA TCG
cnf	CTG GAC TCG AGG TGG TGG	533	55	30
	GAA CTT ATT AAG GAT AGT

PCR: Polymerase chain reaction

The PCR assay was performed in a total volume of 25 µL of a mixture containing 3 µL of DNA template, 1 µL of each primer (IDT-Integrated DNA Technologies, Singapore), 1 × Taq buffer (10 mM Tris-HCl, pH = 8.8, 50 mM KCl), 1.0 mM MgCl₂, 0.2 mM of dNTPs and 1.25U Taq DNA polymerase (Promega, USA). The PCR condition was as follows: initial denaturation at 94°C for 5 min; followed by repeated cycle of denaturation at 94°C for 1 min, annealing for 30 s, and extension at 72°C for 30 s, and a final extension at 72°C for 7 min. Reaction products were separated by agarose gel electrophoresis by adding 1 μL of EZ-Vision DNA dye (Amresco, USA) to 5 μL of PCR product onto a 1% agarose gel (Vivantis, Malaysia). The buffer in the electrophoresis chamber and the agarose gel was 1 × Tris-acetate-EDTA (TAE) buffer. One-hundred volts and 400 mA were applied across the gel for 30 min. DNA in the gel was visualized under ultraviolet light (UV) using UVItec Gel Documentation System (USA). A 1 kb molecular weight marker (Promega, USA) was used.

Statistical analysis

Data were presented in the form of percentages, tables and images. Chi-square test was used to determine the association between virulence genes and chicken type and source of infection. Significance was accepted at 5% probability level.

Results

In vitro pathogenicity test

Out of fifty-two E. coli isolates used in this study, 11 (19%) of them were haemolytic on 5% sheep blood agar while 41 (71%) of the isolates were Congo red positive. Five of the haemolytic strains were isolated from broilers while six were gotten from layers. Out of the forty-one strains that bound to Congo red dye, 18 were from layers while 23 were from broilers chickens.

Occurrence and distribution of virulence-associated genes of APEC from chickens

The APEC strains harbored 9 out of the 12 genes investigated. FimH (878 bp) gene (Fig. 1) has the highest occurrence (96.2%) while pap and felA genes (Figs. 2 and 3, respectively) had the least occurrence (2%). None of the strains was positive for sfa, fac, and cnf genes (Table 2). Out of the 50 FimH positive strains, 14 (27.5%) were from broilers while 37 (72.5%) were from layers (Table 3).

Fig. 1.

Representative gel of FimH gene in APEC strains. Positive strains produced 878 bp band. First Lane on the right is 1.5 kb DNA ladder, Lanes 1-3, and 5-13: Positive, and Lane 4: Negative

Fig. 2.

Representative gel of papC gene detection in APEC strains. Positive strains produced 328 bp band. First and last Lanes are 1.5 kb DNA ladder, Lane 20: Positive and the other Lanes: Negative

Fig. 3.

Representative gel of felA gene detection in APEC strains. Positive strain produced 270 bp band. First and last Lanes are 1.5 kb DNA ladder, Lane 20: Positive and the other Lanes: Negative

Table 2.

Percentage distribution of the virulence associated genes detected in APEC strains in Enugu State

Genes	fimH	pap	felA	sfa	fac	csgA	tsh	cvaC	kpsII	iss	iut	cnf
No. of positive (%)	50 (96.2)	1 (1.9)	1 (1.9)	0 (0.0)	0 (0.0)	27 (51.9)	11 (21.2)	8 (15.4)	5 (9.6)	25 (48.1)	17 (32.7)	0 (0.0)

Table 3.

Distribution of E. coli strains positive for virulence gene from different tissues and bird type

Bird type	Tissue collected	No. of positive (%)
Laying bird (layers)	Heart	13 (35.1)
	Liver	12 (32.4)
	Spleen	8 (21.6)
	Oviduct	4 (10.8)
	Sub total	37 (72.5)
Broilers	Heart	4 (28.6)
	Liver	8 (57.1)
	Spleen	2 (25)
	Sub total	14 (27.5)
	Grand total	51 (100)

The patterns of occurrence of the virulence-associated gene detected from the APEC strains are presented in Table 4. A total of 22 patterns were observed, with fimH-csgA-iss being the predominant combination. The number of virulence genes per strain ranged from 1 to 8, with the majority (35.3%) of the strains harboring three virulence genes.

Table 4.

Patterns of occurrence of virulence associated genes in APEC strains from chicken samples

S/No.	Virulence gene pattern	No. of positive (%)
1	fimH	11 (21.6)
2	fimH-csgA	5 (9.8)
3	fimH-iss	3 (5.9)
4	fimH-tsh	1 (2.0)
5	fimH-iut	1 (2.0)
6	fimH-csgA-iss	9 (17.6)
7	fimH-tsh-kpsII	1 (2.0)
8	fimH-tsh-cvaC	1 (2.0)
9	fimH-csgA-iut	2 (3.9)
10	fimH-cvaC-iut	3 (5.9)
11	fimH-iss-iut	1 (2.0)
12	csgA-tsh-iss	1 (2.0)
13	fimH-csgA-tsh-iss	1 (2.0)
14	fimH-csgA-kpsII-iss	2 (3.9)
15	fimH-csgA-iss-iut	2 (3.9)
16	fimH-tsh-cvaC-iut	1 (2.0)
17	fimH-csgA-kpsII-iss-iut	1 (2.0)
18	fimH-csgA-tsh-iss-iut	1 (2.0)
19	fimH-tsh-cvaC-iss-iut	1 (2.0)
20	fimH-csgA-tsh-kpsII-iss-iut	1 (2.0)
21	fimH-csgA-tsh-cvaC-iss-iut	1 (2.0)
22	fimH-pap-felA-csgA-tsh-cvaC-iss-iut	1 (2.0)
Total		51 (100)

S/No.: Serial number

The association between virulence gene and bird type and source of isolation is shown in Table 5. There was no significant association between virulence gene and bird type (P>0.05) but there was a significant association between the virulence genes and the tissue of isolation (P<0.05).

Table 5.

Association between virulence genes and type of bird and tissue samples

Variables		Virulence genes
		fimH	pap	felA	csgA	tsh	cvaC	kpsII	iss	iut
Type of bird	Broilers	14	0	0	7	3	1	0	9	3
	Layers	37	1	1	20	9	7	5	15	12
Organ	Liver	20*	0	0	10	6	2	2	9	4
	Spleen	10*	0	0	5	1	3	1	4	6
	Heart	17*	1	1	10	5	3	2	10	4
	Oviduct	4*	0	0	2	0	0	0	1	1

^* Significant association (P<0.05)

Discussion

Haemolysis is usually associated with pathogenicity of E. coli, especially the more severe forms of infection and is usually seen in E. coli strains isolated from blood (Daga et al., 2019 ▶). In this study, 11% of the E. coli strains were haemolytic which is higher than 1.5% haemolytic strains reported by Shankar et al. (2010) ▶ among APEC strains isolated from colisepticeamic chickens lower than 37.03% of APEC strains isolated from broiler chickens reported by AL-Saiedi and Al-Mayah (2014) ▶. Although it was reported that haemolytic activity is one of the important factors of pathogenicity in APEC strains, Sharada et al. (1999) ▶ stated that avian E. coli must not be haemolytic before they can be classified as pathogenic. Al-Arfaj et al. (2016) ▶ recognised haemolysis, Congo red uptake among others as phenotypic markers of virulence among E. coli strains associated with colibacillosis in chicken. Avian E. coli strains isolated from blood usually record high heamolytic activity but since these strains were isolated from other organs, it is only probable that haemolysis may not be the major pathway to their pathogenesis.

Congo red uptake by E. coli is a marker for differentiation of colisepticaemic (invasive) strains from non-coliseptecaemic E. coli in poultry (Al-Arfaj et al., 2016 ▶). An increase in virulence of bacteria strains has also been reported in bacteria that bind to Congo red dye (Ambalam et al., 2012 ▶). Among the 57 APEC strains isolated from chickens in this study, 41 (71%) bound to Congo red dye (positive), suggesting that most of the APEC strains studied were invasive and therefore pathogenic. This result was higher than the findings who reported that 40% of E. coli isolates from clinical cases of colibacillosis in the northern part of Nigeria were Congo red positive. This result was also higher than the 60% reported by AL-Saiedi and Al-Mayah (2014) ▶.

Ninety-eight percent of the 52 E. coli strains screened were positive for at least one of the 12 virulence genes studied which are usually implicated in the pathogenicity

of APEC strains (Nakazato et al., 2009 ▶). This indicates that the E. coli strains are APEC strains since they were isolated from confirmed cases of colibacillosis in chicken. This finding is in agreement with the findings of Mbanga and Nyararai (2015) ▶ and Mohamed et al. (2018) ▶ who found 93% and 98% of E. coli strains from chicken in Zimbabwe and Algeria, respectively, haboring at least one virulence-associated gene.

The type I fimbrial adhesion gene (FimH) was the most prevalent gene detected in this study. FimH encodes for type 1 pili (Ionica et al., 2012 ▶) and plays a role in extraintestinal E. coli translocation through the intestinal epithelium and invasion (Poole et al., 2017 ▶). FimH is also the gene responsible for the mannose-specific or receptor-specific adhesin encoding the synthesis of type 1 fimbriae (Ionica et al., 2012 ▶). FimH in the form of Fim DsG complex is a relevant target for the development of anti-adhesive drugs (Sauer et al., 2016 ▶). The high binding ability of FimH could result in increased bacterial binding to target cells and increased pathogenicity of E. coli; thus, FimH could be used to design vaccine for the prevention of E. coli infections by blocking the bacterial attachment and colonization (Hojati et al., 2015 ▶). Previously, authors like Rodriguez-Siek et al. (2005) ▶ in USA and Wu et al. (2012) ▶ in the UK had reported similar occurrence of 98.1% and 100% of the FimH gene among APEC strains, respectively. However, a lower prevalence rate of 33.3% of FimH was recorded in APEC strains in Zimbabwe (Mbanga and Nyararai, 2015 ▶).

From the present study, 2% of the APEC strains harboured the papC gene. The pap operon which encodes for P fimbriae, is involved in bacterial colonization in respiratory epithelium which directly affects the intensity of infection (Melican et al., 2011 ▶). The pap gene has been reported to play a significant role during septicaemic infection as it was observed to be associated more with pathogenic E. coli isolates from septicaemic chickens than from healthy chickens (Subashchandrabose and Mobley, 2015 ▶). This gene is often present in urinary tract infections in humans and chickens (Rahdar et al., 2015 ▶), thereby making the strains a potential zoonotic danger. This finding is lower than that of Samah et al. (2015) ▶ and Hasani et al. (2017) ▶ who reported 8.3% and 20% prevalence, respectively of papC gene in E. coli strains tested. The felA is the operon that codifies a serological variant of P fimbriae (F11) (Tseng et al., 2018 ▶). In this study, 2% of the strains were also positive for felA gene. Different results have been obtained by researchers in different regions. For instances, Rocha et al. (2008) ▶ reported 38.8% in Brazil and Rodriguez-Siek et al. (2005) ▶ reported a higher prevalence rate of 78% in the United Kingdom. The variation in these reports could be attributed to regional differences and sample size investigated.

The csgA gene had a prevalence rate of 51.9% in the present study. Curli fibers encoded by csgA gene have been reported to be essential for the internalization of bacteria causing avian septicaemia in vitro (Van Gerven et al., 2018 ▶). This finding was lower than what Dho-Moulin and Fairbrother (1999) ▶ reported (99%) in E. coli from diseased chicken. csgA gene has been linked to biofilm formation in rats and increased invasion (Oppong et al., 2015 ▶) which could be a source of concern in antimicrobial resistance.

The cvaC gene encoding for Colicin V is involved in extra-intestinal infections affecting humans and animals by interfering with membrane formation and inhibiting bacterial growth (Gérard et al., 2005 ▶) thereby reducing bacterial population and competition. Expression of numerous virulence genes including cvaC, iss, and iutA were associated with the pathogenesis of colibacillosis in boiler chickens with gross and histopathological lesions (Sharif et al., 2018 ▶). In this study, 15.38% of the strains were positive for cvaC gene. This finding was similar to the findings of Ghafoor et al. (2017) ▶ who reported 10.52% in their studies. Our finding was lower than the findings of Kumar et al. (2013) ▶ who reported a higher prevalence of 35%.

Twelve isolates (21.15%) haboured temperature-sensitive hemagglutinin (tsh) gene which encodes for autotransporter protein which is frequently found in APEC (Sarowska et al., 2019 ▶). Hasani et al. (2017) ▶ reported prevalence rates of 49.3% of the tsh gene in 71 APEC strains studied in Iran which is slightly higher than what was found in this study. Similarly, Won (2009) ▶ reported 55% of tsh gene among 118 APEC strains studied in Korea. The low prevalence rate may be connected with a relatively low prevalence of cvaC which has been reported to be associated with tsh. Paixao et al. (2016) ▶ have reported its role during pathogenesis of APEC infections in high-lethality E. coli isolates and its link to colicin V genes when they were present on the same plasmid. It contributes to the development of lesions in the air sac and is associated with high virulence among APEC strains.

The iutA gene is one of genes that encodes for siderophores (aerobactin operon). Aerobactin is produced more especially by invasive E. coli. The aerobactin system enables microorganisms to grow in iron-free media at low concentration (Garénaux et al., 2011 ▶). The aerobaction system plays a role in the persistence and generation of lesions in APEC infected chicken (Mbanga and Nyararai, 2015 ▶). The iutA prevalence of 32.7% recorded in this study is lower than that reported by Sharif et al. (2005) ▶ and Mbanga and Nyararai (2015) ▶ who found 96% and 80% prevalence of iutA gene, respectively. However, our finding was similar to Wu et al. (2012) ▶ who reported prevalence rate of 50% in their studies.

The iss gene was prevalent in 48.08% of the APEC strains. Increased serum survival (iss) gene is known to be associated with serum resistance (Barbieri et al., 2013 ▶). It is considered the most significantly associated gene with APEC strains (Dissanayake et al., 2014 ▶). The iss gene has been detected at a higher percentage in extraintestinal strains of the diseased birds that reached 72.2% when compared to no detection in the intestinal strains and this gives insight to the importance of its pathogenicity (Mohamed et al., 2014 ▶). This finding was lower than Dissanayake et al. (2014) ▶ who reported that 80.5% of APEC isolates were positive for the iss gene in the United States of America. Samples analysed in this study showed 9.6% positive for kpsII gene. The K1 and K5 antigens are codified by kps genes (Wijetunge et al., 2015 ▶). The K1 antigen is thought to be an important virulence factor of E. coli while K5 antigen occurs frequently amongst E. coli strains isolated from extra-intestinal infections (Sarowska et al., 2019 ▶). This finding was lower than the 18% reported by Rocha et al. (2008) ▶ in Brazil.

The virulence genes investigated in this study, occurred in various combinations with fimH-csgA-iss being the most predominant. Avian pathogenic E. coli strains possess virulence traits that make them live extraintestinally and each strain has several virulence factors with several combinations of genes (Circella et al., 2012 ▶). In this study, 12 (23%) strains had 4 or more genes while 18 (35.3%) revealed 3 of the virulence-associated genes. Possessing iutA, tsh and cva/cvi, colicin V plasmids have been considered to be a defining feature of the APEC strains (Borzi et al., 2018 ▶). The diversity of genes associated with pathogenicity detected among the E. coli strains tested in this study and other studies especially among APEC strains may indicate an interaction among these virulence traits.

There was no significant association (P>0.05) between the virulence genes and the chicken type and source of isolation but there was a significant association (P<0.05) between fimH gene and the organ of isolation. This was in disagreement with the findings of Vandamaele et al. (2005) who did not find an association between the occurrence of fimH gene and chicken type and organ of isolation.

The E. coli strains showed multiple pathways to virulence which highlights the danger imposed by these organisms to their hosts. Because these genes may be carried by mobile genetic elements, the spread of virulence genes among E. coli strains could be a huge risk. This is more dangerous since some E. coli strains are usually commensal and could acquire virulence attributes thereby becoming pathogenic especially in immune-compromised hosts. The public health implication of this is also enormous as there is always a continual exposure of humans to chicken and poultry manure. Escherichia coli from chicken in the study area had been reported to be multi-drug resistant (Ugwu et al., 2017 ▶) and following the report of Stella et al. (2016) ▶ that virulence genes are common in E. coli strains resistant to one or more antimicrobials, then there is a possibility of animal and human health being hypothetically in danger. A difficult to treat, highly virulent E. coli strains could be a problem to the poultry industry and have the potential to be a major public health hazard.

Conflict of interest

The authors declare that they do not have any conflict of interest.