Antimicrobial Resistance Genes of E. coli Isolated from Broiler Chickens in Upper Egypt

The background: E. coli infection is a septicemic disease with an economic importance of poultry causing multiple lesions in broiler chickens. Recent reports described increased resistance of E. coli to many antimicrobial agents. This research work aimed to detect, identify and study antimicrobial resistance genes of E. coli isolated from broiler chickens in Upper Egypt. Methods: Three-hundreds samples, including 200 liver and 100 tracheal swabs, were collected from broiler chicken flocks at different localities in Assiut and El-Minya Governorates. Samples were subjected to isolation and phenotypic identification, serological typing, detection of sensitivity and resistance to antimicrobial drugs and determination of genes responsible for resistance to antimicrobial agents. Results and conclusion: revealed that E. coli could be isolated and phenotypically identified with a percent ratio 70% (210 from 300). Twenty-six out of 30 E. coli isolates were serologically identified. Determination of antimicrobial susceptibility and resistance patterns of E. coli isolates to variable antimicrobial drugs using standard disk diffusion and minimum inhibitory concentration (MIC) methods was done. Detection of antimicrobial resistance genes of 12 serologically identified E. coli isolates showed that ƒloR gene (Florfenicol resistance gene) was detected in 6 isolates and strA-strB gene was detected in 5 isolates, while aadA gene was detected in all 12 isolates. Both strA-strB and aadA are streptomycin resistance genes.


Introduction
Colibacillosis is a systemic disease of poultry caused by avian pathogenic E. coli (APEC) [15] and characterized by septicemia with multiple organ lesions, typically pericarditis, air-sacculitis, perihepatitis, peritonitis, and other extraintestinal lesions. With the increased use of intensive housing, colibacillosis has become the predominant bacterial disease affecting the poultry industry [13], and one of the leading causes of economic loss in the poultry industry worldwide [15].
Approaches to control APEC infections in the poultry industry include improved hygienic methods, vaccination, use of competitive exclusion products, and the introduction of novel immunopotentiators however, each of these practices have had restricted success [55]. This has necessitated the use of antimicrobial chemotherapy to control outbreaks of colibacillosis, but modern reports have described increased resistance to those antimicrobial agents commonly used for treatment [85]. Such overuse or mistreatment leads to spreading of antimicrobial resistance among poultry flocks, and appearance of multiple drug resistance pathogens [65]. Today resistance to antimicrobial agents has become a major concern both for human health and in veterinary medicine [63]. Multi-resistant bacteria are often found in poultry meat [21,34], so that chicken products are suspected to be a source of food borne pathogen and antimicrobial resistance bacteria for humans [9]. E. coli acquire antimicrobial resistance more rapidly than other bacteria. Thus, changes in the resistance of this species may provide a good indicator of resistance in potentially pathogenic bacteria [83]. Multi-drug resistant strains of E. coli are ever-present in both human and animal isolates in different parts of the world [8] and multiple drug resistant, nonpathogenic E. coli found in the intestine is maybe an important reservoir of resistance genes [74] and momentarily drug-resistant E. coli of animal origin may colonize the human intestine [62]. So, E. coli is a commensally intestinal bacterium that usually used to monitor resistance to therapeutically precious antimicrobials in poultry [42].
The aim of the present study is to isolate and identify E. coli from broiler chickens in Upper Egypt, to determine their susceptibility and resistance to antibacterial drugs and to detect antibacterial resistance genes in E. coli isolates.

Samples Collection
A total of 300 samples including, 200 liver samples from freshly dead birds and 100 tracheal swabs from sacrificed diseased broiler chickens, aged from 15 to 45 days old showing clinical signs of colibacillosis, were collected from different broiler flocks at different localities in Assuit and El-Minya Governorates. Samples were collected into sterile tubes containing nutrient broth and were transferred immediately to the laboratory in an icebox, where they subjected to bacteriological examination with special reference for E. coli. Sterilized swabs were used to collect tracheal samples from each bird and then immediately broken off into nutrient broth, 70 tracheal swabs were collected from Assuit and 30 swabs from El-Minya. Liver samples were directly collected into sterile containers, after surface sterilization using heated spatula, a bacteriological loop was inserted into the depth of organ and then inoculated in nutrient broth, 130 samples were collected from Assuit and 70 samples were collected from El-Minya.

Isolation
Samples were inoculated into nutrient broth then incubated at 37°C for 24 hours. Sub-culturing was carried out on Eosin methylene blue agar and incubation at 37°C for 24 hours. The growth was examined for typical E. coli colonies.

Phenotypic Identification
To confirm the presence of E. coli on suspected isolates, Gram stained films [25], motility test, biochemical reactions, including indole test, methyl red test, Voges-Praskauer test, citrate utilization test, urease test, hydrogen sulphide production test, gelatin hydrolysis test, arginine dehydrolase, nitrate reduction test and fermentation of sugars tests [53] were used.

Serological Identification of Suspected E. coli Isolates
Thirty randomly selected phenotypically identified E. coli isolates were subjected to serological identification using rapid diagnostic E. coli antisera set (DENKA SEIKEN Co., Japan) for diagnosis of the Enteropathogenic types according to Kok et al., (1996) [51].

Determination of Antibacterial Susceptibility and Resistance Patterns of E. coli Isolates
Sensitivity of E. coli isolates to antibacterial agents was determined by two methods as the following:

Standard Disk Diffusion Method
Single colonies that produced on agar media were cultured into broth culture and incubated at 37 •C for 18 hours, about 2 ml of this broth culture were transferred to inoculate the Muller Hinton agar by using sterile pasture pipette, then all excess fluids were removed with pipette and the plate was leaved to be dry. The antibiotic discs were applied at adequate spacing to the surface of plate with sterile forceps and the agar plate was incubated for 24 hours at 37°C. Judgment of the test was done following incubation; the degree of sensitivity was estimated by measuring the visible and clear zone of inhibition produced by diffusion of the used antibacterial agent from the disc into surrounding medium. Interpretation of results was performed according to Quinn et al., (1994) [76] and koneman et al., (1997) [52].  [22,23,70,71].

Determination of Antimicrobial Susceptibility and
Twelve E. coli isolates which were previously phenotypically and serologically identified, were incubated into nutrient broth and cultures were adjusted to be 10 5 CFU/ml [38]. The standard plate count method technique [16] was used to adjust the number of E. coli per milliliter in the inoculated nutrient broth.
The MIC value of colistin-sulphate, doxycycline, gentamicin, streptomycin, norfloxacin, ceftriaxone, florfenicol was determined. All antimicrobial agents were purchased from Sigma (Missouri, USA), except for norfloxacin, which was obtained from Bayer AG (Leverkusen, Germany).

Detection of Antimicrobial Resistance Genes of E. coli Isolates
Total bacterial DNA was extracted from twelve serologically identified E. coli isolates, according to Promega technical manual (Wizard Genomic DNA Purification kit). PCR was used for detection of resistance genes of E. coli isolates including aadA and strA-strB genes for streptomycin and floR gene for florfenicol. The sequences of primers are shown in table 1. The parameters for all reactions were described in the following profile; initial denaturation at 94°C for 4 minutes followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 60°C for streptomycin genes and 58 °C for florfenicol gene for 30 seconds, and extension for 45 seconds at 72°C. The final extension took 10 minutes at 72°C. The PCR product (448bp) was seen by electrophoresis in a 1.5% agarose gel stained with ethidium bromide for visualization performed in a horizontal gel chamber plate. The running buffer was 0.5X TBE (Tris borate EDTA (pH 8.3). The 1 kb plus DNA ladder was used as a reference standard molecular weight marker.

Incidence of E. coli
Examination of broiler chickens in different localities of Assiut and El-Minya Governorates showed typical clinical signs and post mortem lesions of colibacillosis. The results revealed that over all isolation incidences were 210 suspected E. coli isolates out of 300 examined samples with a percentage 70%. The incidence of the suspected E. coli isolates from liver in freshly dead birds was 150 out of 200 examined samples with percentage 75%. The examination of living diseased birds resulted in isolation of 40 E. coli suspected samples of tracheal swabs out of 100 examined one with a percentage 40%. Generally, it was observed that there was higher incidence rate obtained from liver samples collected from freshly dead birds than that collected from trachea of scarified diseased birds in both Governorates as showed in table 2 and figure 1.  Table 3. Biochemical characters of the suspected E. coli isolates.

Biochemical test Result
The growth of suspected E. coli on eosin methylene blue (EMB) agar, appeared as dark green-black colonies with metallic sheen. Gram stained slides of suspected colonies showed gram negative medium sized bacilli (single short to long slender rods). Single suspected E. coli colonies were inoculated into semi-solid media, and results revealed that the appearance of inverted tree at site of stabbing (motile bacteria). The suspected E. coli colonies were biochemically identified as described in table 3.

Serological Identification of E. coli Suspected Isolates
As summarized in

Determination of Antimicrobial Susceptibility and Resistance Patterns of E. coli Isolates Using Standard Disk Diffusion Method
Results revealed that all isolates were sensitive to doxycycline then to gentamycin and norfloxacin with different percentages, while the highest percentage of resistance to cefradine, spectinomycin, amoxicillin, colistin sulfate and streptomycin respectively, results were summarized in table 5.

Determination of Antimicrobial Susceptibility and Resistance Patterns of E. coli Isolates Using MIC
Results of antibacterial susceptibility and resistance patterns for 7 different antibacterial drugs using MIC were summarized in tables 6 and 7. Results showed that all isolates were sensitive to gentamycin, 11 out of 12 isolates were sensitive to doxycycline, norfloxacin, Ceftriaxone. While the highest rate of resistance was to florfenicol where the number of resistant isolates was 11 isolates out of total of 12 tested E. coli isolates, also number of resistant strains to streptomycin was 10 isolates out of total number of isolates.

Detection of Antimicrobial Resistance Genes of E. coli Isolates
Detection of antibacterial resistance genes in 12 serologically identified E. coli isolates showed that ƒloR gene (Florfenicol resistance gene) was detected in 6 isolates and strA-strB gene was detected in 5 isolates, while aadA gene was detected in all 12 isolates. both strA-strB and aadA are streptomycin resistance gene. Results were summarized in tables 8 and figures 2, 3, 4.

Discussion
The current study aimed to detect the occurrence of E. coli from different localities of Upper Egypt as well as to investigate antibiotic resistance, that was done by collection of liver and tracheal swabs from diseased broiler chicken flocks, all flocks were suspected to be infected with colibacillosis and the main clinical manifestations were depression, ruffled feathers, huddling together, loss of appetite, retardation of growth, respiratory distress, white pasty diarrhea, these finding were also recorded by several authors; Kumar et al., 1988 [54] and Moltarana et al., 1993 [68]. The main post-mortem findings were pericarditis, peritonitis, perihepatitis, enteritis, congestion in internal organs, petechial hemorrhages on parenchymatous organs, and air sacs varied from slight turbidity and thickness to severe muco-or fibrinopurulent inflammation, air-sacculitis and omphalitis, similar post mortem findings were described by Sojka, 1965 [80]; Awaad, 1972 [10]; Awad 1973 [11]; Bahy, 1985 [12] and Barnes and Gross, 1997 [14].
By studying isolated E. coli strains, the micro-organism was Gram negative rods, lactose fermenter on Eosin -methylene Blue (EMB) agar. The biochemical identification of isolates showed that all isolates were catalase positive, oxidase negative, also they were positive to indole, methyl red tests and negative for Voges Proskauer, urease, H 2 S production and citrate utilization. They ferment glucose, lactose, sorbitol and mannitol, but sucrose fermentation was variable. These characters were in conformity with Cruickshank et al., 1975 [25]; Ewing, 1986 [30] and MacFaddin, 2000 [59].
Our results revealed that over all isolation incidences according to morphology and biochemical characteristics was 210 suspected E. coli isolates out of 300 examined samples with a percentage 70%. The incidence of E. coli isolates from liver in dead birds was 150 suspected E. coli samples out of 200 examined samples. The examination of tracheal swabs from living diseased birds resulted in isolation of 40 suspected E. coli samples out of 100 examined one with a percentage 40%. Isolation of E. coli from the liver had been reported by Modestas et al. [67], 2010. It was found that E. coli caused respiratory infections in poultry and could be recovered from the trachea [40]. Serotyping is the basic method for typing of E. coli strains. In the present study the results showed that, twenty-six typeable E. coli isolates [86.6% (26/30)] and four isolates were negative [13.3% (4/30)] from a total thirty isolates that have been serotyped. Among the typeable isolates, 23% of isolates belonged to the O78 serogroup, 15.38% of isolates belonged to O2 serogroup, 11.5% of isolates belonged to the O91 serotype, also 11.5% of isolates belonged to O1 serotype and 7.69% of isolates belonged to serotypes O127, O146, while the percentage of isolation was 3.84% for each of O114, O121, O44, O163, O26, O124 serotypes, and our results were consistent with a study in which serotypes O1, O2 and O78 were present in 15% to 61% of colibacillosis clinical cases [26]. Other studies reported that these serogroups are commonly associated with avian colibacillosis on a worldwide scale and confirmed their role as particularly adapted pathogens that permit involvement in extra intestinal infections [29,46,56,64,75].
Presently, antimicrobial therapy is one of the primary control measures for reducing morbidity and mortality due to APEC associated avian colibacillosis [7,26,35,84]. The majority of E. coli isolates tested in this study for detection of phenotypic pattern of sensitivity and resistance by standard disk diffusion method, demonstrating 66% resistance to cefradine, 58% to spectinomycin, 57% to amoxicillin, 51% to streptomycin, and 50% to colistin-sulfate, nearly similar phenotypic resistance have been detected in APEC strains isolated from diseased chickens with colibacillosis in United States [44,89], Korea [47,50], Iran [86], China [85,87], United Kingdom [77], Australia [72], Egypt [4], while 76% of the isolates were sensitive to gentamycin and this finding is in agreement with a study, in which the prevalence of antimicrobial sensitivity for gentamicin was 71.2% in E. coli isolates from broilers with colibacillosis in Morocco [41], but this percentage is lower than a percentage reported in a study, which found that 97% of the environmental strains of E. coli were sensitive to gentamicin [6], and higher than the sensitivity rates that detected in other studies carried out and reported that sensitivity rates of APEC to gentamicin were 60% [1] and 64% [19]. Resistance to gentamicin was recorded among fifty-five E. coli isolates from poultry meat in Tunisia and results revealed that with 2% of isolates were resistant [81]. All isolates in our study were sensitive to doxycycline by disk diffusion method with percentage 100%, while results in the studies carried out by other authors, showed high rates of sensitivity to doxycycline by percentage of 52% [19], 53.75% [39], 58% [78] and 70.12% [88], indicating that this drug didn't used widely in the localities of samples collection, also this may reflect different patterns of antimicrobial use in different regions. Also, 67% of our isolates were sensitive to norfloxacin this result nearly similar to that detected by a study in which sensitivity to norfloxacin Chansiripornchai et al., 1995, which was 70.65% [19].
According to results of MIC test, 84% of E. coli isolates tested in this study were resistant to streptomycin, this resistance levels is nearly like many previous studies [2,27,31,33,72,89]. In our research, results of MIC indicated that 91.6% of E. coli isolates were resistant to florfenicol while, another study in china reported 29% resistance in E. coli isolates from chicken [57]. In the United States, it was demonstrated that a low percentage (4%) of avian E. coli isolates already display resistance to florfenicol, although the drug is not used therapeutically in chickens [48]. E. coli isolated from the faeces of intensively farmed and free-range poultry in Australia, in which small number of isolates were resistant to florfenicol by a percentage 2%, as florfenicol is not registered for use in poultry in Australia [72]. Although our study revealed high level of phenotypic resistance to streptomycin and florfenicol using MIC test, but also it revealed high level of sensitivity to gentamicin, doxycycline, norfloxacin, Ceftriaxone and Colistin sulphate, with sensitivity rates of 100%, 91.67%, 91%, 91%, and 75% respectively, these results nearly similar to a study in Germany, in which the antimicrobial resistance of E. coli strains isolated during 1999-2001 from cattle, swine and poultry was detected [37]. It was detected that the percentage of resistance of APEC isolates to Ceftriaxone was 3% [89]. It was revealed that resistance of E. coli isolates to Ceftriaxone and gentamicin, was 0% for both antibiotics [73]. The resistance of E. coli isolates from animal origin to colistin was detected, and results reported that 9.6% of isolates were resistant [18], while in another study, resistance of E. coli isolates to colistin was 6.3% [69]. The low level of resistance to these classes of antibiotics in broilers was not surprising possibly due to different antibiotic practices in the different countries. The results obtained in this study confirm that the disk diffusion method is not the recommended test to monitor resistance as the meaning of MIC value itself is interpreted against published standards yielding a qualitative result of resistant, intermediate, or sensitive as well as giving the treating physician and/or veterinarian quantitative data on the degree of resistance or sensitivity allowing them to optimize therapy. In contrast, the disc diffusion method is a strictly qualitative method and does not provide quantitative data [45,61].
Variation in results of antibacterial susceptibility and resistance patterns of E. coli may due to several factors, from which method used for determination, types of antibacterial drugs used, and types and doses of prophylactic antibacterial drug used in poultry farms.
The results of genotypic resistance for detection of resistance genes of streptomycin and florfenicol demonstrated that the two genes responsible for resistance of streptomycin (aadA gene and strA-strB gene) were presented by rate of 100% and 41.66% respectively in E. coli isolates, and this genotypic pattern is similar to the observed pattern of phenotypic resistance of E. coli isolates detected with MIC, and these results were consistent with many other authors [2,5,36,50,58,72,87]. The floR gene that responsible for resistance of florfenicol presented at 54.5% (6/11) of the isolates, while the observed pattern of phenotypic resistance, of E. coli isolates detected with MIC to florfenicol was 91.66%. Florfenicol resistance gene (floR) could be detected in 17.8% of the tested APEC isolates [5]. the floR gene was previously identified in 4.5% of APEC isolates from broiler chickens in Canada [17]. The variation between the phenotypic and genotypic results of florfenicol is due to several causes, from which; The floR gene is located either in the chromosomal DNA [24,28,32] or on a plasmid [48,49,84]; two types of mobile elements, both of which are widespread among Enterobacteriaceae, may be considered to carry the floR gene; gene cassettes/integrons or transposons. Although the floR gene is located between two integrons structures in the chromosomal multi-resistance gene clusters present in several Salmonella serovars [66], analysis of the sequences flanking floR in all floR-associated database entries did not reveal any sequences that suggested direct involvement of gene cassettes/integrons in the spread of floR. Therefore, the location of this gene on a transposable element appeared to be a likely explanation for its various positions on plasmids or in the chromosomal DNA.
It was concluded that E. coli isolated from broiler chickens in different localities of upper Egypt were resistant in high rates to streptomycin and florfenicol. MIC were more recommended than disk diffusion method in mentoring antibacterial drug sensitivity and resistance patterns. Streptomycin and florfenicol resistance genes were detected in E. coli isolates from broiler chickens.