Detection of new antibiotic resistance gene profile in Escherichia coli
associated with avian leukosis virus infection from broiler chickens

The Escherichia coli (E. coli) is prevailing worldwide, but the epidemiology of E. coli infections feature regional distribution characteristics to some extent. E. coli, as a zoonotic pathogen, can be transferred from animals to humans through food chain or via contact with wounds, causing a public health risk. We reported the swelling of proventriculus and tracheal bleeding following the death in two broiler chickens (Gallus gallus domesticus) from Beijing, China. To investigate whether a virus was involved in the infection, Madin Darby Bovine Kidney (MDCK) cells were co-cultured with supernatants of proventriculus, trachea and spleen homogenates. The avian leucosis virus was detected in the samples of proventriculus and trachea, but the avian influenza virus, the Newcastle disease virus and the avian infectious laryngotracheitis virus were not detected. E. coli isolates were resistant to almost all the antimicrobial as tested except for the combinations of amoxicillin/clavulanic acid and sulfamethoxazole/trimethoprim. PCR tests demonstrated the presence of antibiotic resistance genes in these E. coli isolates and further research revealed a novel gene profile with the presence of CTX-M-1, gyrA, gyrB, oqxA, oqxB, parC and Sul2 antibiotic resistance genes in a strain isolated from a proventriculus sample. These results demonstrated that the presence of antibiotic resistant E. coli would not necessarily cause outbreak of large-scale disease. However, when the bacteria carrying new antibiotic resistance genes enter the environment, it may result in the development of more virulent strains which will potentially impact human and animal health.


Introduction
The bacterium E. coli can be found in many kinds of livestock such as poultry, farmed animals, other land animals and some aquatic animals, among which, pigs and chickens are the mostly susceptible of infection. E. coli is an important opportunistic zoonotic pathogen, belonging to the family Enterobacteriaceae, but it is usually considered as a commensal bacterium in humans and animals, and used as an indicator of enteric pathogens and environmental contamination by feces (Jiang et al., 2014). E. coli is mainly found in the feces of patients with gastrointestinal infections and can be mainly transmitted among people through fecal and oral routes. Our study detected new antibiotic resistance gene profiles in E. coli and further research indicated avian leukosis virus infection in dead broiler chickens that were the sources of these isolates. Studies suggest that ESBL/ AmpC-producing E. coli are isolated with increasing frequency from animals, food, environmental sources and humans (Dorado-Garcia et al., 2018). To date, several different fluoroquinolone resistance mechanisms have been proposed, such as the aminoglycoside acetyltransferase AAC (6′)-Ib-cr, which acetylates several fluoroquinolones with piperazinyl substitutions; the qnr families (qnrA, qnrB,qnrS, qnrC, and qnrD) which protect DNA gyrase and topoisomerase IV from quinolone inhibition; and the active multidrug efflux pump qepA and oqxA/B, which can remove fluoroquinolones from the bacterial cells (Poirel et al., 2012;Ruiz et al., 2012;. Recent experiments found acquisition of mcr-1 and co-carriage of virulence genes in avian pathogenic E. coli isolated from municipal wastewater influents in Japan (Hayashi et al., 2019). E. coli pathotypes have been identified to be largely responsible for moderating severe diarrhea transmitted from contaminated human or animal feces to susceptible hosts via environmental reservoirs such as hands, water and soil . The soil may represent a direct source or act as an intermediary matrix for the transmission of antibiotic resistant pathogenic E. coli strains, particularly in rural settings, and the presence of E. coli in soil is of public health significance . These studies show the significance of, and the need for urgent measures for reducing environmental contamination by bacteria carrying antibiotic resistance genes possibly resulting from contamination in the course of human activities such as random disposal of untreated water, soil and waste, etc.
The antibiotic resistant isolates and resistance genes of E. coli can be transferred to humans and animals through food, water, poor hygiene, improper cooking, logistics, storage and wound contact, causing potential harms to the public health (Ryu et al., 2012). Avian pathogenic E. coli is the etiologic agent of avian colibacillosis, which is the most common disease causing chicken morbidity (Mohamed et al., 2018). However, human activities influence the epidemiology of transmission among human, environment and animals. This study aimed at investigating the antibiotic susceptibility pattern and resistance gene types of E. coli through biochemical and gene sequencing methods to reveal valuable information for preventing bacterial diseases.

Materials and Methods
Samples collection and testing for microorganisms Two dead broiler chickens were taken from a chicken farm on the outskirts of Beijing, China, and transferred to the laboratory at National Research Center for Wildlife-Borne Diseases. Six isolates of E. coli were obtained from proventriculus and trachea of the broiler chickens. Proventriculus samples were obtained in a sterile environment by first cutting the skin and muscle tissue of the broiler with sterile scissors, and then cutting the proventriculus with another pair of sterile scissors. The inner surface of the proventriculus was sampled by using a sterile inoculation loop, and it was then removed with sterile scissors. The proventriculus was fixed in 10% neutral buffered formalin and embedded in paraffin before the tissue sections were stained with hematoxylin and eosin for histological examination. To detect the viral infections, a cell culture approach was used. Briefly, a Madin-Darby canine kidney cells (MDCK) line was cultured in high-glucose Dulbecco's modified Eagle's medium (Gibco) with 10% fetal bovine serum (Gibco) and incubated for 5 days with supernatants of homogenate of proventriculus, trachea and spleen from the dead broiler chickens .

Results
Clinical isolates and their identification PCR amplified fragment was detected by 1% agarose gel electrophoresis, and the results indicated that the amplified products were 1500 bp in length with good specificity. The sequence and biochemical tests identified six isolates (E1, E2, E3, E4, E5 and E6) as E. coli. Our study showed that the cell morphology of the proventriculus samples was the same as that of the control (Fig. 1). Histopathological biopsy of proventriculus samples revealed cells with diffuse hemorrhage, cell gap enlargement and an increase in lymphocytes (Fig. 2). Further, evidences were found that ALV co-infection with E. coli was available in the proventriculus and trachea samples. BLAST analysis showed that ALV DNA sequence of the ALV subgroup E (KC610515) was present, while AIV, DNV and AILTV were not detected (Fig. 3).
Antibiotic resistance testing of bacterial isolates Except for amoxicillin/clavulanic acid and sulfamethoxazole/ trimethoprim, our study showed that all six isolates were multi drug resistant (MDR) to almost all antibiotics, including beta-lactams (cefazolin, cephalothin, cefotaxime, ampicillin and piperacillin), aminoglycosides (gentamicin,
tobramycin and amikacin) and quinolones (ciprofloxacin, levofloxacin and ofloxacin). Additionally, these MDR isolates were resistant to aztreonam and chloramphenicol. While the 16S rDNA gene sequences showed a closer similarity of isolates to E. coli (Fig. 4), several MDR isolates (E3, E4, E5 and E6) were susceptible to nitrofurantoin, although this was not found in isolates E1 and E2. The antibiotic resistance patterns of the six MDR isolates were presented in Tab. 1.
Detection of antibiotic resistance genes AAC (3)-II was detected in isolates E1, E3, E5 and E6; CTX-M-1, gyrA, gyrB, parC and Sul2 were detected in all six isolates; oqxA and oqxB were found in E2; and qnrB testing produced a weak band in E2 and E3. However, cmlA, blaKPC, NDM-1, OXA, qepA, qnrA, qnrC, qnrD and qnrS antibiotic resistance genes were not amplified in any isolate (Tab. 2 and Appendix Fig. 1). The RT-PCR results revealed the novel coexistence of CTX-M-1, oqxA, oqxB, parC and Sul2 resistance genes in an isolate (E2) from a proventriculus sample that was also positive for ALV. Our study also found the combination of antibiotic resistance   ALV was found from proventriculus and trachea, but AIV, DNV and AILTV were not detected in this study. E1-4 from proventriculus, E5 and E6 were found from trachea, N: negative control.

Discussion
Our results found that E. coli isolated from the broiler chickens showed different antibiotic resistance genes. The problem of antibiotic resistance has been concerned for many years, and the increase in antibiotic resistance is difficult to control the given population growth and the climate change. At present, the use of antibiotics is the main approach to prevent and treat diseases in the poultry industry, but the abuse of antibiotics has resulted in the emergence of antibiotic resistance in E. coli in the global area. Disease caused by this organism is one of the common disease agents infected by bacteria in poultry, causing vitelline peritonitis, salpingitis, septicemia, airsacculitis, perihepatitis and pericarditis (Wang et al., 2013). The antibiotic resistance genes such as AAC(6')-Ib-cr and qepA have been identified in many clinical isolates with high-level antibiotic resistance (Cavaco et al., 2009;Zhao et al., 2010).
In this study, we identified a novel isolate possessing CTX-M-1, oqxA, oqxB, parC and Sul2 resistance genes, which is, to our knowledge, the first report of such combination. Extended-spectrum β-lactamases (ESBLs), such as CTX-M-1, CTX-M-2, CTX-M-14, CTX-M-15 and CTX-M-55, have been found in many European countries and China, and are associated with E. coli isolates from poultry and other animals (Rao et al., 2014). E. coli is ubiquitous in warm blooded animals and usually will not produce human diseases; but the variations within E. coli, especially new combinations of antibiotic resistance genes, may result in new manifestations of human diseases in the future. For example, the plasmid mediated quinolone resistance (PMQR) genes oqxA and oqxB, expressed in a novel E. coli isolate, may result in occurrence of a complex antibiotic resistance mechanism (Santos et al., 2014). To our knowledge, few papers have been published regarding the coexistence of PMQR and ESBL encoding genes in E. coli isolated from proventriculus (Ammar et al., 2016;Gouvêa et al., 2015). The industry is faced with a dilemma because on one hand, antibiotics are required to combat the high incidence of poultry disease; on the other hand, their use is the ultimate selection pressure that may promote widespread bacterial resistance. Our investigation showed that E. coli isolate E2 was not distinct in terms of antibiotic susceptibility when compared with other E. coli isolates although we cannot exclude the possibility that E. coli E2 will not have novel susceptibilities to other antibiotics. We usually only consider the presence of antibiotic resistant bacteria themselves, but our experiments showed their coexistence with ALV subgroup E in the broiler chickens. This result suggests that it will be interesting to study further whether the presence of pathogenic microorganisms other than E. coli can promote antibiotic resistance in bacteria.
Our study indicates that the antibiotic resistant E. coli is commonly seen in poultry and also reveals a novel antibiotic resistance gene pattern in E. coli isolated from the proventriculus. These data may help clinical veterinarians to better understand the antibiotic resistance present in relevant pathogenic E. coli. In addition, as an important foodborne pathogen, the antibiotic resistance gene spectrum of E. coli in poultry may provide an early warning of emerging antibiotic resistance patterns for the sake of human health and surveillance.

Conflicts of Interest:
The authors declare no conflict to interest.