Antimicrobial resistance in Enterobacteriaceae from healthy broilers in Egypt: emergence of colistin-resistant and extended-spectrum β-lactamase-producing Escherichia coli

Background Poultry remains one of the most important reservoir for zoonotic multidrug resistant pathogens. The global rise of antimicrobial resistance in Gram-negative bacteria is of reasonable concern and demands intensified surveillance. Methods In 2016, 576 cloacal swabs were collected from 48 broiler farms located in five governorates in northern Egypt. Isolates of Enterobacteriaceae could be cultivated on different media and were identified by MALDI-TOF MS and PCR. Escherichia coli isolates were genotyped by DNA-microarray-based assays. The antimicrobial susceptibility to 14 antibiotics was determined and resistance-associated genes were detected. The VITEK-2 system was applied for phenotypical confirmation of extended-spectrum β-lactamase-producing isolates. The determination of colistin resistance was carried out phenotypically using E-test and genotypically using PCR for detection of the mcr-1 gene. Results Out of 576 samples, 72 representatives of Enterobacteriaceae were isolated and identified as 63 E. coli (87.5%), 5 Enterobacter cloacae (6.9%), 2 Klebsiella pneumoniae (2.8%) and 2 Citrobacter spp. (2.8%). Seven out of 56 cultivated E. coli (12.5%) were confirmed as ESBL-producing E. coli and one isolate (1.8%) as ESBL/carbapenemase-producing E. coli. Five out of 63 E. coli isolates (7.9%) recovered from different poultry flocks were phenotypically resistant to colistin and harboured mcr-1 gene. Conclusions This is the first study reporting colistin resistance and emergence of multidrug resistance in Enterobacteriaceae isolated from healthy broilers in the Nile Delta region, Egypt. Colistin-resistant E. coli in poultry is of public health significance. The global rise of ESBL- and carbapenemase-producing Gram-negative bacteria demands intensified surveillance. ESBL-producing E. coli in poultry farms in Egypt are of major concern that emphasizes the possibility of spread of such strains to humans. The results also reinforce the need to develop strategies and to implement specific control procedures to reduce the use of antibiotics.


Background
Poultry and their products are considered the main vehicle for pathogenic bacteria such as Salmonella (S.) serovars, Escherichia (E.) coli and Klebsiella (K.) spp. that cause foodborne infections in humans [1][2][3].
The prevalence of highly antibiotic-resistant E. coli was recorded in poultry meat more frequently than in all other kinds of meat [4,5].
ESBL-producing Enterobacteriaceae have emerged as pathogens in both poultry and humans [7,11]. Many ESBL-producers are additionally multiresistant to non-βlactam antibiotics, including fluoroquinolones, aminoglycosides, trimethoprim, tetracyclines, sulfonamides and chloramphenicols [12,13]. Resistance to cephalosporins is mediated by ampicillin class C β-lactamases (AmpC β-lactamase) and encoded by bla CMY genes [14,15]. Carbapenems are still the drugs of choice to treat infections with ESBL-producing Enterobacteriaceae in humans [16] and their increasing use reinforces the probability of resistance development to carbapenems among Enterobacteriaceae [17][18][19]. The coexistence of multiple ESBL and carbapenemase genes as well as other antibiotic resistance determinants on mobile elements is of a major concern that might lead to the emergence of organisms with resistance to all antibiotics [6,20,21].
Most ESBLs encoding genes in bacteria of clinical interest are located on plasmids [22]. These plasmids may also carry genes encoding resistance to other drug classes including aminoglycosides and fluoroquinolones [23]. Transmission of ESBLs genes can occur either by emerging bacterial clones or by horizontal gene transfer. In the latter case, plasmids containing resistance genes, spread between bacteria [22]. Colistin recently gained attention as a last-resort antibiotic for treatment of infections caused by multidrug resistant Gram-negative bacteria. In veterinary practice, colistin is a drug of choice for the treatment of frequent digestive tract infections caused by E. coli in food-producing animals [24]. The irrational use of colistin in veterinary practice may be the main cause of the increasing rate of colistin resistance. Recently, the emergence of colistin resistance has caused great concern [25,26] and resistance mediated by the plasmid-borne mcr-1 gene has been detected worldwide in Enterobacteriaceae [27].
In some countries, antimicrobials are used in the poultry industry for treatment of diseased animals, prevention of diseases and promotion of growth [28][29][30]. In Egypt, E. coli infections are considered as one of the most serious diseases leading to economic losses in poultry production [31].
Unfortunately, there are no legislations in Egypt regulating the use of antibiotics. Some of them such as tetracycline, quinolones and beta-lactams are still used for non-therapeutic uses [32]. This improper use of antimicrobials leads to rapid selection of multiresistant strains of Enterobacteriaceae in poultry and plays a key role in the spread of antibiotic-resistant bacteria along the food chain to humans [33][34][35].
In recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been applied as a wide-range technique for bacterial identification [36]. Microarray systems are wellestablished tools for rapid genotypic characterization of bacteria and identification of resistance and virulenceassociated determinants [37]. The data can be obtained in a single experiment with the benefit of saving time and money [38][39][40]. The broth microdilution method proved to be an easy and reliable method for determination of the minimum inhibitory concentration (MIC) of antibiotics and can be used as an alternative technique to agar diffusion test [41][42][43].
The use of a rapid molecular assay as an alternative to phenotypic detection was proved to be a useful option for detection of antibiotic resistance to frequently applied antimicrobial agents in poultry production [43].
The objective of this study was to gain insight into the antimicrobial susceptibility of Enterobacteriaceae, especially E. coli originating from healthy broilers from different districts in northern Egypt and to understand its public health significance. In addition, the prevalence of ESBL/carbapenemase-producing E. coli and colistin resistance were investigated.

Isolation and characterization of bacterial strains
During 2016, 576 cloacal swabs were randomly collected from apparently healthy broilers housed in 48 farms located in five governorates, namely: Dakahlia, Kafr El-Sheikh, Damietta, Gharbiya and Sharkiya, in the Nile Delta region, Egypt. An overview about investigated poultry farms, the number of birds and the number of collected samples are given in Table 1

Identification by MALDI-TOF MS
Isolates were identified using MALDI-TOF MS [44]. Interpretation of results was performed according to the manufacturer's recommendation: score of ≥ 2.3 represented reliable species level identification; score 2.0-2.29, probable species level identification; score 1.7-1.9, probable genus level identification, and score ≤ 1.7 was considered an unreliable identification [45].

DNA extraction and purification
Genomic DNA was extracted from bacterial cultures using High Pure PCR Template Purification Kit (Roche Diagnostics, Mannheim, Germany) according to the instructions of the manufacturer.

Identification of E. coli isolates using PCR
The identified E. coli isolates were confirmed at species level using a specific PCR assay targeting 16S rRNA genes with primers ECO-1 (5′-GAC CTC GGT TTA GTT CAC AGA-3′) and ECO-2 (5′-CAC ACG CTG ACG CTG ACC A-3′) which geared from previous study by Seidavi et al. [46]. The PCR reaction was carried out with the following amplification conditions: An initial denaturation step at 96 °C for 60 s was followed by 35 cycles of denaturation (96 °C for 15 s), annealing (58 °C for 60 s) and extension (72 °C at 45 s) with a final extension at 72 °C for 60 s. PCR resulted in 585 bp amplicons. PCR products were analyzed on a 1.5% agarose gel, stained with ethidium bromide and visualized under UV light.

Genoserotyping of E. coli isolates using microarray assay
The serotypes of E. coli isolates were determined using the E. coli SeroGenoTyping AS-1 Kit (Alere Technologies GmbH, Jena, Germany). Five microliters of extracted RNA-free DNA (with a concentration of at least 100 ng/ μl) were biotin-labeled by a primer extension amplification using E. coli SeroGenoTyping AS-1 Kit according to manufacturer's instructions. The procedures for multiplex labelling, hybridization and data analysis were carried out as described in a previous study [47].

Phenotypic antibiotic susceptibility testing
The antibiotic susceptibility testing of all isolates was performed with the MICRONAUT system using commercial 96-well microtiter plates (Merlin, Bornheim, Germany) as recommended by the manufacturer. This system allowed the determination of minimum inhibitory concentrations (MICs) of 14 antimicrobial agents (Tables 2, 3) in serial dilutions of the antibiotics. Overnight grown bacteria were suspended in NaCl solution (0.9%) to obtain a turbidity corresponding to a McFarland standard of 0.5 (Dr. Lange, CADAS photometer 30, Berlin, Germany). One hundred microliters of the suspension were diluted with 10 ml of Mueller-Hinton broth (Oxoid GmbH) resulting in a concentration of approximately 10 6 -10 7 colony forming units (cfu)/ ml. In total, 100 µl of the inoculum were given in each well of the plate. After sealing the plates, they were incubated for 18 h to 24 h at 37 °C. Reading of plates was done with a photometer (Merlin) at a wavelength of 620 nm. An optical density of > 0.1 was interpreted as an indication of growth. MICs were interpreted with the advanced expert system MCN-6 (Merlin) using the guidelines of the German Institute for Standardization (Deutsches Institut für Normung, Berlin, Germany). E. coli ATCC 25922, E. coli ATCC 35218 and K. pneumoniae ATCC 700603 were used as quality controls.

Vitek-2 system
All suspected ESBL isolates were subsequently confirmed using an automated microdilution system (VITEK-2, bioMérieux Deutschland GmbH, Nürtingen, Germany) according to the instructions of the manufacturer. For this study, the test card AST-N289 was used that included the following antibiotics

Detection of antibiotic resistance and virulence-associated genes of E. coli isolates by microarray analysis
Antimicrobial resistance (AMR) genotypes and other resistance genes were ascertained using the CarbDetect AS-2 Kit and E. coli PanType AS-2 Kit, respectively (Alere Technologies GmbH). The data were automatically summarized by the "result collector", a software tool provided by Alere Technologies GmbH. An antibiotic resistance genotype was assigned to be a carrier of a group of genes which have been described to confer resistance to a family of antibiotics (e.g., the genotype "bla CTX-M1/15 , bla TEM " conferring resistance to 3rd generation cephalosporins).
The detection of virulence-associated genes was performed using E. coli PanType AS-2 Kit (Alere Technologies GmbH). Twenty-eight different combinations of genes encoding virulence factors associated with adhesion, fimbriae production, secretion systems, SPATE   (serine protease auto-transporters), toxins and miscellaneous genes were detected. The genes were detected and analysed by the "result collector", a software tool provided by Alere Technologies GmbH.
Seven out of 63 E. coli isolates could not be re-cultivated for testing of antibiotic resistance after applying MALDI-TOF MS (11.1%), while DNA was extracted from preserving solution for further identification.

Antimicrobial susceptibility testing
The results of phenotypic antibiotic susceptibility testing of 56 re-cultivated E. coli isolates were given in Table 2 (Tables 2 and 6). Fifty-five out of 56 E. coli isolates (98.2%) were resistant to antibiotics of at least three different classes of antimicrobial agents and thus they were defined as multidrug resistant isolates ( Table 2).
The antimicrobial susceptibility profiles for other species of Enterobacteriaceae were presented in Table 3. All 5 Enterobacter cloacae isolates were resistant to penicillin, erythromycin and rifampicin. Two Citrobacter spp. isolates were resistant to penicillin, rifampicin, streptomycin and ceftazidime. Two K. pneumoniae strains were sensitive to amikacin and imipenem but resistant to the rest of the antibiotics tested.

Detection of antibiotic resistance determinants in E. coli by microarray analysis
Several resistance genes were identified in 15 phenotypically resistant E. coli using microarray analysis ( Table 4). The isolates were originated from four districts located in four provinces, namely Dakahliya (n = 7), Damietta (n = 3), Gharbiya (n = 3) and Kafr El-Sheikh (n = 4). Frequently identified resistance genes were aadA1 associated with resistance to aminoglycosides (n = 12), sul2 responsible for sulphonamide resistance (n = 10) and floR connected with resistance to chloramphenicol (n = 10).
In this study, five E. coli isolates harboured qnrS gene while one isolate possessed qnrB gene associated with quinolone resistance. In two phenotypically ciprofloxacin-resistant E. coli, qnrS gene was detected ( Table 4).
The sul and dfrA genes associated with sulphonamides/ trimethoprim resistance were detected in 16 and 13 E. coli isolates, respectively (Table 4). Meanwhile, sul3 gene corresponding to sulphonamide resistance was amplified in two susceptible E. coli to sulphonamide/trimethoprim. Eleven E. coli phenotypically resistant to tetracycline were harboured one or more tet genes (tetA, tetB or tetC) ( Table 4). Chloramphenicol resistance-associated genes catA1, catB3, cmlA1 and floR were detected in 13 E. coli isolates. Out of 13 chloramphenicol resistant isolates, 10 isolates harboured one or more resistance genes. The cmlA1 gene was detected once in an E. coli strain that was phenotypically susceptible to chloramphenicol.
Ten different genes (aac6, aac6Ib, aadA1, aadA2, aadA4, aadB, ant2, aphA, strA and strB) associated with aminoglycoside resistance were detected in 14 out of phenotypically tested E. coli isolates ( Table 4). All isolates harbouring at least one of described genes were phenotypically resistant to streptomycin, but four of them were susceptible to gentamicin (Tables 3, 4). All isolates with aminoglycoside resistance-associated genes were susceptible to amikacin.

Table 4 Genoserotypes and resistance profiles of Escherichia coli isolates possessed resistance genes from different farms in four districts in Egypt
The correlation between the genotypic and phenotypic antimicrobial resistance of E. coli was demonstrated in Table 4.
Thirteen out of 15 isolates harbouring bla genes were analyzed using the VITEK-2 ( Table 5). Two samples could not be tested, as they could not be re-cultivated. All isolates possessing beta-lactam resistance genes were resistant to piperacillin, while one isolate was susceptible to moxifloxacin. All isolates were susceptible to fosfomycin. The narrow-spectrum beta-lactamase gene bla OXA-1 was detected once in one ESBL isolates originated from poultry farm in Damietta.

Genotypic and phenotypic identification of resistance to colistin
Plasmid-mediated colistin resistance gene mcr-1 was detected in 5 out of 63 E. coli (8.0%) using a PCR assay. All of them were phenotypically confirmed as resistant to colistin using E-test (Table 6). All colistin resistant E. coli isolates were phenotypically resistant to rifampicin, penicillin and erythromycin but were susceptible to carbapenems. The colistin-resistant isolates originated from different poultry flocks in Dakahliya, Kafr El-Sheikh, Damietta and Gharbiya (Table 6).

Microarray analysis concerning virulence-associated genes
The virulence genes detected by microarray were differently distributed all over the isolated E. coli. One isolate 16CS0740 isolated from poultry farm in Dakahliya harboured 7 genes of virulence-associated secretion system: cif, espA, espF_O103H2, espJ, nleA, nleB O157:H7 and tccP.
eae and iha genes, involved in adhesion, were identified in 16CS0740 and 16CS0752, repectively. Two isolates harboured serine protease autotransporter genes. 16CS0774 carried tsh while 16CS0775 had pic and vat genes.
The distribution of virulence-associated genes in E. coli isolates possessed antimicrobial resistance-associated genes was demonstrated in Table 4.

Discussion
Escherichia coli is a commensal pathogen of the intestinal tract of young and adult poultry [49]. Among healthy chickens, 10 to 15% of intestinal coliform bacteria may belong to potentially pathogenic serotypes of E. coli [50].
The identification of bacterial foodborne pathogens of zoonotic significance using rapid, accurate and reliable Table 5 Results of antimicrobial resistance testing using VITEK-2 system PIP piperacillin, TZP piperacillin/tazobactam, CTX cefotaxime, CAZ ceftazidime, FEB cefepime, ATM aztreonam, IMP imipenem, MEM meropenem, AMK amikacin, GEN gentamicin, TOB tobramycin, CIP ciprofloxacin, MXF moxifloxacin, TGC tigecycline, FOS fosfomycin, CT colistin, T/S trimethoprim/sulfamethoxazole   [44,51]. In 2016, 576 cloacal swabs were collected from 48 poultry farms located in 5 governorates in northern Egypt. The samples were screened for multidrug resistant bacteria and investigated for the antimicrobial resistance of E. coli. Seven out of 56 E. coli isolates (12.5%) were producing ESBLs. To analyze the underlying molecular antimicrobial resistance mechanism, all E. coli isolates were genotyped using the multiplex microarray technique.

Isolate PIP TZP CTX CAZ FEB ATM IMP MEM AMK GEN TOB CIP MXF TGC FOS CT T/S
The results of this study were in accordance with previous reports which demonstrated a high prevalence of E. coli in poultry farms and their environment in Egypt [34,[52][53][54].
In previous studies on broiler chickens in Egypt, high phenotypic resistance rates were found to penicillin, rifampicin, erythromycin, trimethoprim/sulphamethoxazole, streptomycin and tetracycline [53]. Antimicrobial resistance rates in this study for amoxicillin (26.8%), gentamicin (19.6%) and imipenem (1.8%) were lower than those of E. coli isolates from poultry reported in Egypt [53], in China [55], in United States [56], in Korea [57], in United Kingdom [58], in Australia [59] and in Portugal [60]. In the present investigation, the most striking finding was that E. coli isolates showed a low resistance rate to fluoroquinolones (ciprofloxacin (21.4%) and levofloxacin (14.3%)). Cephalosporins are the first-line antimicrobials for treating human bacterial infections [61]. In addition, a considerable resistance to ceftazidime was detected among E. coli isolates from healthy broilers (41.1%).
In this study, one carbapenem-resistant isolate (1.8%) was found within all E. coli isolates. A higher rate was determined with retail chicken meat, 11.3% carbapenemase-producing Enterobacteriaceae including E. coli in Egypt in a previous study [62].
Few reports discussed prevalence of ESBL-producing E. coli isolated from healthy birds in Egypt. Here, ESBL and/or AmpC β-lactamase-producing isolates were detected in seven out of tested 56 E. coli (12.5%) isolated from healthy broilers. Two ESBL-producing strains were isolated from one farm in Gharbiya governorate (Farm 4) while five other isolates could be recovered from different farms in Dakahliya and Kafr El-Sheikh governorates. Two of the ESBL-producing isolates 16CS0740 and 16CS0747 from Dakahliya and Gharbiya, respectively were additionally carrying bla OXA-7 gene characteristic for β-lactamase-producing bacteria (Table 4).
The prevalence of ESBLs has been found to be variable worldwide with Asian countries having the highest rates [67].
The resistance-associated genes bla TEM , bla SHV , and bla CMY were previously reported in Enterobacteriaceae isolated from septicaemic broilers [68] and from humans [69,70] in Egypt.
In this study, bla TEM resistance gene was detected in 20.6% of E. coli isolates. This result was in accordance with previous reports in China [71,72]. bla CMY was detected in 3.5% of 56 E. coli isolates while the prevalence of bla CMY-2 amongst E. coli isolates from broilers in Japan was 69.5% [73]. In Belgium, 49.0% of ceftiofur-resistant E. coli isolates derived from five broiler farms carried bla CMY-2 [74]. Moreover, 12.1% of avian pathogenic E. coli strains and 9.5% of strains recovered from meat were found positive as carriers of bla CTX-M in Palestine [75].
qnrB and qnrS genes associated with quinolone resistance were detected in one and five isolates, respectively, which is lower than described previously in E. coli isolated from chickens in China [72,76]. On the other hand, qnrA, qnrB, and qnrS genes were detected in 0.75, 3.9 and 5.1%, respectively of E. coli from chicken samples in China [77].
Many studies found similarities between virulenceassociated genes in human and avian E. coli isolates including iss, fliC, iha and ireA genes [78].
In a previous study, the virulence genes iroN, ompT, iss, iutA, and hlyF were detected in 80.2% of isolated E. coli [68]. In this study, only 9 (16.7%) of the 56 E. coli isolates carried 2 genes (iroN, iss) together characteristic for avian pathogenic E. coli.
The mcr-1 gene is now reported all over the world in Enterobacteriaceae from animals, food and humans [79]. In 2015, first time mcr-1 gene was detected in livestock and raw meat samples in addition to human beings in China [27]. In this study, five E. coli isolates (8.9%) were phenotypically resistant to colistin and harboured mcr-1 gene associated with colistin resistance. This result was higher than reported in E. coli isolates from pigs, poultry and turkey in France with 0.5, 1.8 and 5.9%, respectively [80] and 5.6% of E. coli isolates from broilers in Germany [81], while it was lower than in E. coli isolates from poultry in China [27].
In previous studies conducted in China and Austria, the majority of phenotypically colistin-resistant E. coli isolates carried the mcr-1 gene [82,83].

Conclusion
To the best of our knowledge, this study is the first report discussing the antibiotic susceptibility profiles of Enterobacteriaceae and ESBL-producing E. coli isolated from healthy broilers in the Nile Delta in Egypt. The emergence of colistin-resistant E. coli isolates in poultry is of public health significance and considered as potential source of transmission of plasmid-mediated mcr-1 to humans. It was shown that molecular biological methods such as microarray investigation are reliable and fast tools for detection of geno-serotypes, resistance-and virulence-associated determinants.
The results reinforce the need to develop surveillance strategies and to implement specific control procedures to reduce the use of antibiotics and subsequently the development of antimicrobial resistance by over-/misuse of antibiotic agents.