Monitoring the genetic variation of some Escherichia coli strains in wild birds and cattle

To date, there is limited data about the genetic relationship of Escherichia coli between wild birds and cattle because these birds act as silent vectors for many zoonotic bacteria. This study aimed to elucidate the role of rooming wild birds in the vicinity of cattle farm in transmission of the same pathogenic E. coli variants, identifying their virulence, resistance traits and genetic similarities of fimH virulence gene. About 240 faecal/cloacal swabs were collected from both species and examined bacteriologically. Escherichia coli was yielded in 45.8% and 32.5%, respectively, of examined cattle and wild birds. The most prevalent detected E. coli serovar was O26. High tetracycline and chloramphenicol resistance were recorded; however, gentamycin and ciprofloxacin exhibited the highest sensitivity rates. Polymerase chain reaction (PCR) conserved genotypic resistance (tetA and blaCTX-M) and virulence attributes (fimH, stx1, eaeA and ompA) of E. coli isolates were discussed in detail. The fimH gene revealed 100% sequence similarity when comparing with different E. coli isolates globally and locally. Finally, a close genetic association of E. coli with both wild birds and cattle was detected, thus strengthening its role in the dissemination of the infection via environment. Prevention and conservative policy should be carried as E. coli constitute enormous significant zoonotic risks to livestock and animal workers. Also, further studies to the whole genome sequencing of fimH, other virulence and resistance genes of E. coli are recommended trying to limit the possibilities of co-infection and transfer among different species. Contribution The current study recorded updated data about the critical infectious role of wild birds to livestock, including cattle farms in Egypt. It also delivered some recommendations for good hygienic practices in cattle farms which must be implemented for handling animal manure.


Introduction
Wildlife constitutes a magnificent network that might spread a number of potentially significant bacterial diseases with their antimicrobial-resistance traits among livestock animals and infrastructure because of high bird travel and mobilisation in multiple countries during different seasons (Arnold et al. 2016).Migratory and non-migratory wild birds could serve as the main transmitter for the infection with the family Enterobacteriaceae specifically Escherichia coli spp.(Shobrak & Abo-Amer 2014).
Escherichia coli is a Gram-negative environmental bacterium that commensally inhabits the intestinal tract of warm-blooded animals and birds.However, these bacteria could cohabit with other bacteria forming the commensal gut microbiota; some E. coli variants might develop virulent characteristics producing the disease (Umpiérrez et al. 2021).
Hence, the antimicrobial resistance problems could extremely extend to transfer the antibioticresistant genes among different species via plasmid or transposon (Borges et al. 2017).For antimicrobial resistance in the veterinary field, E. coli resistance was consistently the highest (Fahim et al. 2019).Furthermore, E. coli had also been regarded as a reliable indicator bacteria for tracking antibiotic resistance in domestic and wild animals.However, enteropathogenic (EPEC) and Shiga toxigenic E. coli (STEC) are categorised under diarrheagenic E. coli, and the To date, there is limited data about the genetic relationship of Escherichia coli between wild birds and cattle because these birds act as silent vectors for many zoonotic bacteria.This study aimed to elucidate the role of rooming wild birds in the vicinity of cattle farm in transmission of the same pathogenic E. coli variants, identifying their virulence, resistance traits and genetic similarities of fimH virulence gene.About 240 faecal/cloacal swabs were collected from both species and examined bacteriologically.Escherichia coli was yielded in 45.8% and 32.5%, respectively, of examined cattle and wild birds.The most prevalent detected E. coli serovar was O26.High tetracycline and chloramphenicol resistance were recorded; however, gentamycin and ciprofloxacin exhibited the highest sensitivity rates.Polymerase chain reaction (PCR) conserved genotypic resistance (tetA and bla CTX -M) and virulence attributes (fimH, stx1, eaeA and ompA) of E. coli isolates were discussed in detail.The fimH gene revealed 100% sequence similarity when comparing with different E. coli isolates globally and locally.Finally, a close genetic association of E. coli with both wild birds and cattle was detected, thus strengthening its role in the dissemination of the infection via environment.Prevention and conservative policy should be carried as E. coli constitute enormous significant zoonotic risks to livestock and animal workers.Also, further studies to the whole genome sequencing of fimH, other virulence and resistance genes of E. coli are recommended trying to limit the possibilities of co-infection and transfer among different species.

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intestinal microbiota of wild birds could include both serovars with their multi-drug resistance attributes (Borges et al. 2017).Moreover, the precise genetic bacterial profile enables more knowledge for the most disseminating genotypes to distinguish its potential links to virulence factors that could infect many animals and free-living birds (Skarżyńska et al. 2021).
As wildlife could play the main role in the microbial spreading in cattle farms, the mechanisms of this bacterial spreading among species in the farm environment are poorly understood (Tormoehlen et al. 2019).The current investigation was conducted to estimate the likelihood of E. coli infections transfer by wild birds that were frequently in contact with cattle and identifying the interrelationship between both species in farms in Egypt.Also, to study the most virulence and antibiotic resistance traits of the E. coli spp.isolates with a special regard to partial-genome resistance of fimH gene.

Bacterial cultivation and identification
All cattle rectal and bird cloacal swabs were enriched overnight onto buffered peptone broth and then incubated at 37 °C under aerobic conditions.Subsequently, the cultures were isolated (on MacConkey, EMB and sheep blood agar plates) and identified using conventional techniques (Quinn et al. 2002).Furthermore, the pathogenicity of each one of E. coli recovered isolates was evaluated separately onto Congo red agar plate medium then they were kept overnight at 37 °C.The cultures were kept at room temperature for 48 h (Singh et al. 2014).After 48 h at room temperature, Congo red-positive pathogenic E. coli isolates appeared with a brick red colour while non-pathogenic ones were colourless (Saha et al. 2020).

Serological identification
The recovered E. coli isolates were serotyped based on their 'O' antigen according to the manual of the Reference Lab for

Antimicrobial susceptibility testing
The purified E. coli isolates were characterised for antibiotic susceptibility on Mueller-Hinton agar plates by the disc diffusion method using 11 different antibiotic discs (tetracycline, chloramphenicol, amoxiclavulinic acid, penicillin, piperacillin, streptomycin, fosfomycin, gentamycin, danofloxacin ciprofloxacin and cefepime), which were selected according to the panel of antibiotics of interest to the dairy industry and public health in our country.The results were interpreted using the standard guidelines of the Clinical and Laboratory Standards Institute (CLSI 2020).
Polymerase chain reaction screening of virulence and resistant traits of Escherichia coli spp.
Following the manufacturer's instructions of QIAamp DNA Mini Kit (QIAGEN, Germany), the genomic deoxyribonucleic acid (DNA) of E. coli bacterial isolates was extracted.The PCR was performed using thermal profiles and oligonucleotides primers (Table 1) and a 25 µL reaction volume containing; 12.5 µL Master Mix (EmeraldAmp Max PCR, Takara, Japan), 1 µL (20 pmol) from each forward and reverse primer (Metabion, Germany), 5.5 µL Dnase free water and 5 µL DNA template.The amplified genes fragments were visualised in ethidium bromide-stained agarose gel 1.5% against 100 base pair (bp) DNA ladder (GeneDirex, Taiwan) in 1× Tris-borate EDTA (TBE) buffer at 100 voltage (V) per 30 min and then recorded using the SynGene Gel Documentation System.All reactions included a negative (non-template) and positive control of reference E. coli strains supplied by AHRI, Dokki, Giza, Egypt.

Gene sequencing of fimH gene
Two retrieved E. coli isolates one from diarrhoeic cattle and the other from Hooded crow were selected for genotyping of the fimH gene.QIAquick ® Gel Extraction Kit (QIAGEN, Germany) was used to purify the PCR products.The sequence reactions were performed using the BigDyeTM Terminator version 3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, United States [US]) and then were analysed using the 3130 Genetic Analyzer (Applied Biosystems™, US).The obtained sequences were trimmed, consensus generated and analysed using the Uniport Ugene software version 43.0 (Okonechnikov et al. 2012).Sequences were investigated using the National Center for Biotechnology Information (NCBI) online basic local alignment tool (BLAST at https://blast.ncbi.nlm.nih.gov/Blast.cgi).For phylogenetic analysis, fimH gene sequences from different sources and origins including Egypt were selected and retrieved from the GenBank in FATSA format.Sequence alignments (MSA) were performed using MUSCLE algorism with the same software Multiple (Okonechnikov et al. 2012).
IQ-TREE was used to construct the phylogenetic tree (Nguyen et al. 2014) using the maximum likelihood method, best model finder and 1000 bootstrap replicates for both nucleotide and protein sequences.The constructed trees were annotated using the Interactive tree of life (iTOL) (Letunic & Bork 2021).

Phenotypic characterisation of the recovered Escherichia coli isolates in cattle and wild birds
The suspected E. coli colonies revealed a pink colouration on MacConkey agar medium, metallic green sheen on EMB and some (enterohaemorrhagic) strains gave haemolysis on blood agar.Microscopically, the isolates appear as Gramnegative rod-shaped motile bacilli.Also, their pathogenicity was confirmed on Congo red medium (gave positive orange or bright red colonies).Biochemically, they all were identified and confirmed as E. coli spp.

Polymerase chain reaction investigations of virulence and resistant attributes of the recovered isolates
Polymerase chain reaction screening of the virulence genes of most 10 multi-drug resistant (MDR) E. coli isolates (Figure 1a-f) revealed that fimH and ompA genes were detected in all isolates 10 of 10 (100%) (Figure 1a and b), while the eaeA gene was detected in 1 of 10 (10%) only of total isolates (Figure 1c), Stx1 gene was demonstrated in 6 of 10 (60%) of MDR isolates (Figure 1d), PCR failed to detect hly and Stx2 genes in the examined E. coli isolates.The presence of tetA and bla CTX -M genes of tetracycline and penicillin β-lactamase inhibitors resistance was confirmed   4.

Phylogenetic analysis of fimH virulence gene
The fimH was detected in all examined isolates with the conventional PCR technique.fimH gene sequences from two selected wild birds (hooded crow) and cattle isolates were examined and then documented in the Gene Bank database and assigned the accession numbers (ON239271 and ON239272), respectively.The phylogenetic analysis of the fimH gene selected from two isolates revealed 99% nucleotides identity between their sequences (cattle and wild bird isolates, Figure 2).Only three single nucleotides polymorphism (SNP) in cattle isolates were shown at positions 192.291,348, but these SNPs did not show any effect at amino acid (a.a) level because they were identical (100% identity, Figure 3).Both cattle and wild bird isolates showed 100% homology with most sequences retrieved from the GenBank from Egypt and geographical locations also from different isolation sources including different animal species, food and environmental source (Table 5).These translated a.a phylogeny results confirmed the high conservation level of the sequenced fimH gene for both retrieved Egyptian E. coli isolates and globally (Figure 4).

Discussion
Many studies implicated the crucial role of wild birds in the pathogenesis of E. coli spp. in livestock animals (Fadel, Afifi & Al-Qabili 2017;Fahim et al. 2019).However, its pathogenesis in these birds was still unclear.Diarrhoeagenic strains of E. coli were isolated from different wild bird species including migratory and non-migratory (Ahmed et al. 2019), Passeriformes, Columbiformes and Pelecaniformes (Sanches et al. 2017).Escherichia coli also was isolated from birds of prey, waterfowls and passerines.
Farm animals could infect wild birds or vice versa (Fadel et al. 2017).They infiltrated animal enclosures in search of water and food hence infecting them with different pathogens or even acquiring the infection from these animals.Moreover, the feeding practice of cattle in open yards could result in the accumulation of their manure and so the attraction of these wild birds to those farms (Medhanie et al. 2015).
The present article stated that E. coli was detected higher (45.8%) from all examined apparently healthy and diarrhoeic cattle than from different wild birds (32.5%).For apparently healthy cattle, it was detected in 17 of 60 (28.3%); however, it was isolated from 38 of 60 (63.3%) of diarrhoeic animals.However, from different wild bird species, 18 of 21 isolates were obtained from the hooded crow (85.7%), 9 of
In the same way, recent studies discussed the propagation rate of E. coli in wild birds, cattle and their environment in which it was found in a range of 17% -47% in the faeces samples of wild birds (house sparrows, red-winged blackbirds, European starling and brown-headed cowbirds) despite its percentage was recorded higher in cattle farms (Tormoehlen et al. 2019).Also, 478 positive E. coli samples of migratory birds were reported in China from a total of 1387 (34.7%) faecal, cloacal and throat samples (Yuan et al. 2021).Ahmed et al. ( 2019) isolated E. coli from 60% and 45% of examined hooded crows and cattle egrets in Egypt, respectively.However, a higher rate of E. coli was recovered from the faeces of wild birds (70%) than migratory waterfowls (33.3%) (Fahim et al. 2019).From a different point of view, a large number of E. coli were isolated from egret wild birds than from cattle in the same study (Fashae et al. 2021) This variation in the isolation rate in different studies might be because of either insensitivity testing method or other anonymous agents (Ballem et al. 2021).
Moreover, E. coli isolates were recorded also in the United States (21% of beef cattle and 13% of dairy cattle) (Venegas-Vargas et al. 2016).Also, 112 of 409 positive E. coli isolates were retrieved from cattle in Portugal with a prevalence of 27.4%, and 133 STEC isolates were identified (Ballem et al. 2021).In addition, 106 and 29 E. coli isolates were yielded from 77 diarrheic and in-contact calves (Awad et al. 2020).
Antibiotic-resistant bacteria could pose a rising hazard to global public health and accompanied environmental contamination problems (WHO 2017   animal production.As a result of the diversity in ecological niches, the migratory birds act as reservoirs and transporters of antibiotic-resistant bacteria and consequently play a significant epidemiological role in the dissemination of antibiotic-resistant genes (ARGs) (Cao et al. 2020).These birds could carry ARGs during migration leading to the dissemination of MDR bacteria and ARGs through the environment (Yuan et al. 2021).
The presented information in this study displayed MDR phenomena of the yielded isolates because they showed high antimicrobial resistances against tetracycline and chloramphenicol with prevalence rates of 95.7% and 93.6%, respectively, followed by piperacillin, penicillin and streptomycin (90.4%, 88.3% and 88%), respectively.Meanwhile, gentamycin, cefepime, ciprofloxacin and danofloxacin were highly sensitive where the lowest resistance was recorded (9.6% and 10.6%, 12.8% and 26.6%), respectively, as shown in Table 3.These results might be of good importance in management routines for cattle farms to control the spread of antimicrobial resistance.
Similar to this, the wild bird E. coli isolates exhibited bacterial resistance in many studies in the last years.Escherichia coli isolates showed great resistance to penicillin G, piperacillin, tetracycline, cotrimoxazole, ampicillin and nitrofurantoin (Shinde et al. 2020).Also, 376 recovered E. coli isolates from Hooded and White-naped cranes in Japan were found resistant to oxytetracycline, ampicillin and nalidixic acid antibiotics.A high resistance level was also recorded against tetracycline followed by sulfamethoxazole, ampicillin, trimethoprim and ciprofloxacin in most E. coli isolates (Suenaga et al. 2019).Furthermore, 87 of 88 egret's and 53 of 55 cattle E. coli isolates were found to have MDR against more than one antimicrobial.Tetracycline resistance was highest in E. coli isolates from egret birds (n = 85/87), further followed by streptomycin (n = 69/87) and ciprofloxacin resistance (n = 38/87).
For cattle E. coli isolates multiple authors reported MDR patterns of E. coli isolates (Iweriebor et al. 2015, Mahmoud et al. 2020).The MDR phenomena are of great concern because the resistant strain could be transmitted to humans by consumption of either milk or food carrying antibioticresistant bacteria, which could lead to the acquisition of antibiotic-resistant infections (Geletu, Usmael & Ibrahim 2022), and also could be transmitted to accompanying animals and their offspring (Roca-Saavedra et al. 2018).It was previously documented that resistant strains selected during an antimicrobial treatment last for a long time in the intestinal tract when this treatment ceases.In addition, these resistant strains could modify animal health.
The results in this study were consistent with Geletu et al. (2022) who revealed that tetracycline (80%) was the drug that most E. coli isolates from dairy cattle were extremely resistant to, followed by ceftriaxone and vancomycin (83%).However, gentamycin (90%) and nitrofurantoin (70%) were the most sensitive drugs, respectively.Additionally, tetracycline resistance (n = 47/53) was the most often seen phenotype in cow cefotaxime-resistant E. coli, followed by streptomycin (n = 46/53) and ciprofloxacin (n = 17/53) resistance (Fashae et al. 2021).Moreover, Mahmoud et al. (2015) recorded the E. coli.resistance against oxytetracycline and ampicillin in cattle samples.Moreover, the most responsive medications, nevertheless, were ceftiofur (40%) and lincospectine (56.6%), followed by danofloxacin (56.6%), enrofloxacin (40%) and danofloxacine (56.6%).Furthermore, sulfamethoxazole, ampicillin, trimethoprim and ciprofloxacin were the antibiotics with the highest rates of resistance in E. coli isolates, followed by tetracycline (Hang et al. 2019).In the present study, the highest resistance levels of E. coli isolates might be because of the non-judicious use of antibiotics on a cattle farm.Also, this high antimicrobial resistance of E. coli isolates in cattle might confer a selective advantage towards intestinal colonisation, which might itself increase the faecal shedding of antimicrobial-resistant E. coli (Harkins, McAllister & Reynolds 2020).
Studying the genotypic virulence attributes of the isolated bacterial species was applied by conventional PCR technique.Our results indicated that the fimH gene, one of the virulence genes involved in bacterial adhesion, was found to be present in 10 of 10 (100%) of the tested E. coli.Also, identical findings were recorded by Nüesch-Inderbinen (et al. 2018) who discovered that the fimH gene was present in all (100%) of their isolates.In the present study, eaeA (attaching and effacing virulence factor) was detected in 1 of 10 (10%) of tested E. coli isolates, despite this finding complied with a study by Sanches et al. (2017) in which eaeA gene was found in a rate of 5.74%.Moreover, Mohamed and Sayed (2017) implied that eaeA gene was exhibited in 43.75% of yielded E. coli isolates.These results disagreed with the finding by Nüesch-Inderbinen et al. ( 2018) who recorded that eaeA gene was not present in any of the studied E. coli isolates.
Depending on the retrieved data in this study, PCR confirmed the positivity of the ompA gene (outer membrane protein A) virulence gene in 10 of 10 (100%) of E. coli isolates.The frequency of detected ompA gene in our study was much higher than another study in which this gene was detected in (82%) of E. coli isolates (Ammar et al. 2015).
While the presence of virulence factors such as Shiga toxin (Stx1 and Stx2) and α-haemolysin (hly) of E. coli is pivotal for suggesting the increased pathogenicity of these strains, serogroups are still crucial for identifying potential diseases.In this study, the prevalence of Stx1 virulence gene was 6 of 10 (60%), while (Stx2 and hly) genes failed to be detected.In accordance with the recorded result, Nasef, El Oksh and Ibrahim (2017) detected Stx1, Stx2 and hly (45%, 65% and 80%), respectively.
Studying the antimicrobial genotypic attributes of the isolates was applied by PCR to investigate the presence of tetracycline and penicillin β-lactams resistance gene (tetA and bla CTX -M), the results indicated positive detection in all 10 of 10 (100% for each) of the tested E. coli.Similar outcomes were reported by (Fashae et al. 2021) who detected the bla CTX -M gene in 83.3% of MDR E. coli isolates.Furthermore, according to Gholami-Ahangaran et al. (2021), all E. coli isolates from faecal samples of pet birds included the tetA gene.
The phylogenetic analysis of the fimH gene of two selected E. coli isolates from both resident free-living wild birds and cattle, which was in contact, demonstrated a high conservation level of the gene at (a.a) level as previously proved by Vandemaele, Hensen and Goddeeris (2004).The homology was 100% with gene sequence from different resources including avian, cattle, birds, pig and from food sources such as milk and ice cream and also from environmental sources such as water, sewage and farm soil prove the potential role of wild birds as a reservoir for E. coli having MDR genes.This was concordant with results mentioned by Nabil et al. (2020).However, the detected SNPs between both examined samples represented no change at protein level proves the common source nature of pathogens suggesting the possible role of wild birds to contaminate water sources and disseminating the infection to cattle farms (Fahim et al. 2019).Also, our findings could highlight the public health concern of presence of wild birds carrying E. coli with MDR genes in contact with dairy cattle farms and its surrounding environment that could transmit infection to human through the food chain.

Conclusion
This study reported updated data about the critical infectious role of faecal matter of wild birds to cattle farm.Highly virulent and resistant pathogenic serovar of E. coli could be disseminated towards different animal species triggering several diseases, threatening their health and impairing the animal farm economy.Hence, strict recommendations for animal manure with good hygienic practices in cattle farms should be implicated.Also, one health approach should be implicated to inherit the dispersion of multiple antimicrobial resistance phenomena.Furthermore, more advanced sequencing approaches should be studied on the whole genome level for such bacteria to indicate the interrelationship of virulent and resistant genes in different animal, human and wild bird species.

FIGURE 2 :
FIGURE 2:The maximum likelihood (ML) phylogenetic tree for partially nucleotide sequenced FimH gene of E. coli isolated from wild bird and cattle.The tree was constructed in IQ-TREE with Model Finders.The accession number and a brief GenBank ID are assigned to each retrieved sequence from different l isolation sources and origins.The two isolates are highlighted in red with a green background, various clades are designated with different colours, and the numbers above the branches are the branch length.The bootstrap values were computed from 1000 bootstrap repeats and branch length as visualised by iTOL.The tree's roots are in the middle.

FIGURE 4 :
FIGURE 4: Rooted maximum likelihood phylogenetic tree for a translated protein of fimH gene Escherichia coli sequences obtained from wild bird and cattle isolates.The branch colour represents the homology between protein sequences.Rings from inner to outer: the first ring (origin) represents the geographical location of selected isolates, the second ring (source) is for the sample source of isolates, the branch length, and bootstrap values computed from 1000 bootstrap repeats are also visualised.

TABLE 1 :
Primer sequences and amplicon sizes of target genes.
Source: Please see the full reference list of the article for more information bp, base pair.

TABLE 2 :
The prevalence of different Escherichia coli serotypes in cattle and wild birds.

TABLE 3 :
Phenotypic characterisation of antibiotic resistance profile of Escherichia coli isolates.
Note: Lane (L), DNA marker; lane (P), positive control; lane (N), negative control in all figures.bp, base pair

TABLE 4 :
Polymerase chain reaction amplification results of different virulence and resistant genes of isolates.

TABLE 5 :
Source modifier tabulates for fimH gene isolates and strains sequences retrieved from GenBank for alignment, phylogenetic analysis, and tree construction for isolates from wilds birds and cattle in Egypt.

Table 5
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TABLE 5 (
Continues...): Source modifier tabulates for fimH gene isolates and strains sequences retrieved from GenBank for alignment, phylogenetic analysis, and tree construction for isolates from wilds birds and cattle in Egypt.