ABSTRACT
Antimicrobial resistance (AMR) is a global issue, posing a grave threat to the public, animal, and environmental health. The AMR surveillance at the level of the hatchery is crucial to develop an AMR control strategy in the poultry industry. The objective of this study was to investigate the AMR profiles of bacteria isolated from yolk material of non-viable broiler chicken embryos at hatch from commercial hatcheries in western Canada. Antimicrobial susceptibility testing was done using the Kirby–Bauer disk diffusion method focusing on Escherichia coli (n = 170) and Enterococcus (n = 256) species, which are commonly used as indicators of AMR evolution. E. coli isolates were resistant to tetracycline, ampicillin, amoxycillin-clavulanic acid, triple sulpha, ceftiofur, gentamycin, and spectinomycin at the rate of 52.9%, 50.6%, 40.0% 31.8%, 29.4%, 29.4%, 21.8% respectively. Among those, 37.1% of E. coli were multidrug resistant. The descending order of antimicrobial resistance of E. faecalis was; tetracycline (61.9%), ceftiofur (46.2%), bacitracin (43.9%), erythromycin (31.4%) and tylosin (27.4%). Multidrug resistance was detected in 40.4% of E. faecalis isolates, and 85.7% of E. faecium isolates. To the best of our knowledge, this is the first report on AMR surveillance of non-viable chicken embryos. Overall, the present study revealed that non-viable chicken embryos, an overlooked niche for AMR surveillance, harbour multidrug-resistant E. coli, and enterococci that can be a substantial source of superbugs in the environment. Our data also highlight the urgency of including non-viable chicken embryos in AMR surveillance programme to understand AMR dissemination and its control.
KEYWORDS: Chicken embryos, AMR, multi-drug resistance, antibiotics, hatchery
1. Introduction
Antimicrobial resistance (AMR) has become a serious threat to public, animal and environmental health [1,2]. AMR control is a global priority and the World Health Organization (WHO) has initiated a global action plan to mitigate the emergence and dissemination of AMR [1,2].The emergence of AMR is multifactorial and may include indiscriminate antimicrobial use and resistance gene transfer from one organism to another. The inappropriate and excessive antimicrobial use in farm animals has been suggested as one of the major causes of the emergence of multidrug-resistant superbugs [3]. Consumer awareness about the antimicrobial use in farm-animals and the potential of AMR development is dictating a trend of an increased market demand for organic and antibiotics-free animal products [4].
The European Union banned the vancomycin analogue, avoparcin, in 1997 and bacitracin, spiramycin, tylosin, and virginiamycin in 1999 for the purpose of prophylactic antimicrobial use in farm animals including poultry feed [5]. Although a reduction of vancomycin resistant enterococci (VRE) was observed in poultry products in the European Union following the ban on avoparcin since 1997, there has been no reduction of VRE observed in humans [5]. Moreover, the fluoroquinolone ban in the USA since 2006 as therapeutic use in the poultry industry, did not result in the reduction of ciprofloxacin resistant Campylobacter in poultry products [6]. Because of these complexities and poor understanding of AMR, concerted efforts are required to identify the potential sources of AMR in a variety of agricultural settings to develop an appropriate control measures [7].
Although, there is no direct evidence available, however literatures suggest that poultry is a potential source of AMR transmission to humans [8]. In commercial poultry production, AMR development and dissemination can occur at several stages of production, such as, at breeder level, at hatchery and at the production farm level. Most of the data on AMR in poultry were generated from the production farms [9] or from the retail poultry meat [10]. In the poultry industry, commercial hatcheries act as a link between breeder farms and the production farms. Recent studies suggest that the hatchery is a potential reservoir for antimicrobial resistant bacteria [2] and day-old chicks are a potential source of AMR in chicken farms [11]. The comparison of AMR data generated from hatchery samples versus AMR data obtained from poultry farms at the end of production cycle may provide important clue regarding AMR development and its dissemination in the poultry industry [12]. The bacterial contamination of hatching eggs can occur at breeder farm level, egg transport and storage, and at hatchery level [13]. Bacterial contamination of developing chicken embryos in hatcheries occurs in many possible ways including contamination of egg shells and penetration of bacteria via cracks in the egg shell, or due to thin egg shells [2,14]. Transmission of bacteria from hatching eggs to their progeny has been demonstrated for bacterial species such as Campylobacter and Salmonella [15,16]. Most of the studies related to AMR surveillance at the hatchery level have profiled fluff-derived bacteria [17] or day-old chicks [12]. Given that contaminated eggs explode during incubation [18], which may facilitate dissemination of AMR from dead embryos to healthy live embryos and ultimately reaching to humans through contaminated poultry. The contaminated non-viable chicken embryos have been an overlooked niche for AMR surveillance.
Our recent study revealed that the majority of non-viable broiler chicken embryos examined in western Canadian hatcheries were co-infected with Enterococcus species and Escherichia coli [19]. Enterococcus species and E. coli colonizing the gut of animals are used as bacterial indicators to monitor the prevalence and dissemination of AMR between food animal species and humans [20]. Moreover, E. coli and Enterococcus species can cause significant economic loses to the poultry industry [21]. Hence, present study was designed to fill the knowledge gap by investigating AMR of non-viable chicken embryo using clinical microbiology technique [22]. To the best of our knowledge, this is the first report on AMR surveillance on non-viable chicken embryos in hatcheries.
2. Materials and methods
2.1. Bacterial isolates
E. coli (n = 170) and Enterococcus (n = 256) isolates i.e. [E. faecalis (n = 223), E. faecium (n = 21), Enterococcus avium (n = 5), Enterococcus gallinarum (n = 5) and Enterococcus casseliflavus (n = 2)] were recovered from yolk material of non-viable broiler chicken embryos at hatch (21 days of incubation), from three commercial broiler hatcheries in western Canada during 2013 and 2014 [19]. Bacterial swabs were cultured on 5% Columbia sheep blood agar (BA) (Oxoid Company, Napean, ON) and bacterial identification was done by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonics, Milton, ON) as previously described [23]. Bacterial isolates were stored in brain heart infusion (BHI) broth (DIFCO®, Detroit, MI) containing 20% glycerol (Thermo Fisher Scientific, Waltham, MA) at −80 C for further studies.
2.2. Antimicrobial susceptibility testing
Each bacterial isolate was streaked on 5% Columbia sheep BA and incubated at 37 C overnight and tested for antimicrobial susceptibility testing using the standard Kirby–Bauer disk diffusion method. Selection of disk concentration, test method and interpretation of zone diameter were done as recommended by the Clinical Laboratory Standards Institute (CLSI) [24,25]. E. coli (ATCC 25,922) and Staphylococcus aureus (ATCC 25,923) were used as reference strains for E. coli and Enterococcus species respectively. The following antimicrobial agents and disk potency were used: amoxycillin-clavulanic acid (AUG,30 μg), ampicillin (AMP,10 μg), apramycin (APR,15 μg), bacitracin (BAC, 10 IU), ceftiofur (CEF, 30 μg), chloramphenicol (CHL, 30 μg), ciprofloxacin (CIP, 5 μg), enrofloxacin (ENR, 5 μg), erythromycin (ERY, 15 μg), florfenicol (FLO, 30 μg), gentamicin (GEN, 10 μg), gentamicin [(120 μg, to determine high level resistance to aminoglycosides in Enterococcus species)], lincomycin (LIN, 2 μg), neomycin (NEO, 30 μg), penicillin G (PEN, 10 units), spectinomycin (SPE, 100 μg), tetracycline (TET, 30 μg), trimethoprim-sulphonamide (SXT, 1.25 μg), triple sulpha (SSS, 0.25 mg) and tylosin (TYL, 60 μg), vancomycin (VAN, 30 μg). The antimicrobials used in this study represented 10 classes; namely β-lactams (AUG, AMP, CEF, PEN), aminoglycosides (GEN, NEO, SPE), cyclic polypeptides (APR, BAC), phenicols (CHL, FLO), fluoroquinolones (CIP, ENR), lincosamides (LIN), macrolides (ERY, TYL), tetracyclines (TET), glycopeptides (VAN) and folate pathways inhibitors (SSS, SXT). The inhibition zone diameter of each antimicrobial agent was measured using the BIOMIC V3 − 2014-Microbiology Digital Image Analysis system (Giles Scientific Inc, Santa Barbara, California, USA). Inhibition zone diameters were used to categorize antimicrobial susceptibility of the isolate as susceptible, intermediate and resistant according to the CLSI recommendations except for sulphonamides, where the European Committee on Antimicrobial Susceptibility Testing (EUCAST) version 4.0 interpretive criteria were used [26]. Multidrug resistance was enumerated as acquired non-susceptibility to at least one agent in three or more antimicrobial classes [27]. Intrinsic AMR was disregarded in this enumeration.
3. Results
3.1. Antimicrobial resistance of E. coli
E. coli isolates were resistant to TET, AMP, AUG, SSS, CEF, GEN and SPE at the rate of 52.9%, 50.6%, 40.0%, 31.8%, 29.4%, 29.4% and 21.8% respectively. The descending order of AMR to the remainder of the antimicrobials were CIP (7.1%), NEO (7.1%), ENR (6.5%), APR (5.3%), FLO (3.5%), SXT (3.5%) and CHL (2.9%) (Figure 1). Multidrug resistance was seen in 63 of 170 (37.1%) E. coli isolates of which 17.1% (n = 29) of E. coli were resistant to three classes of antimicrobials, 15.9% (n = 27) of E. coli were resistant to four classes of antimicrobials and 4.1% (n = 7) of E. coli were resistant to five classes of antimicrobials (Figure 2). The intrinsic resistance of E. coli was noted for BAC (99.4%), LIN (99.4%), TYL (98.2%), VAN (97.7%), PEN (97.1%) and ERY (91.2%). The AMR profile of all E. coli isolates are shown in Tables 1 and 2. AMR phenotypes of E. coli, in descending order, were TET (23/170), AUG (R) + AMP(R) + CEF(R) + GEN(R) + SPE(R) + TET(R) + SSS (R) (9/170), AUG (R) + AMP (R) + CEF (R) + CIP (R) + ENR (R) + TET (R) + SSS (R) (8/170) and AUG (R) + AMP (R) + CEF (R) (8/170). Pan-resistance was not observed for E. coli but pan-susceptibility was observed in 18.82% isolates.
Figure 1.
Antimicrobial resistance profile of E. coli.
Figure 2.
(Panel A) Antimicrobial resistance profile of E. coli to each drug class and (panel B) indicates the multidrug resistance profile of E. coli.
Table 1.
Antimicrobial resistance profile of E. coli.
| Drug class | Drug | Disk potency | Resistance percentage (n = 170) |
|---|---|---|---|
| β-lactam | AUG | 30 μg | 40.0 |
| AMP | 10 μg | 50.6 | |
| CEF | 30 μg | 29.4 | |
| Phenicols | CHL | 30 μg | 2.9 |
| FLO | 30 μg | 3.5 | |
| Fluoroquinolones | ENR | 5 μg | 6.5 |
| CIP | 5 μg | 7.1 | |
| Aminoglycosides | GEN | 10 μg | 29.4 |
| NEO | 30 μg | 7.1 | |
| SPE | 100 μg | 21.8 | |
| Tetracyclines | TET | 30 μg | 52.9 |
| Cyclic polypeptides | APR | 15 μg | 5.3 |
| Folate pathways inhibitors | SSS | 31.58 μg | 31.8 |
| SXT | 1.25–23.75 μg | 3.5 |
Table 2.
Summary of resistance profiles of E. coli (n = 170)
| Resistance profile | Number of isolates | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| AUG | AMP | CEF | CIP | ENR | GEN | SPE | TET | SXT | SSS | 2 |
| AUG | AMP | CEF | CHL | FLO | GEN | NEO | TET | SSS | 1 | |
| AUG | AMP | CEF | CHL | FLO | GEN | SPE | TET | SSS | 1 | |
| AUG | AMP | CEF | CIP | GEN | SPE | TET | SSS | 1 | ||
| AUG | AMP | CHL | FLO | GEN | SPE | TET | SSS | 1 | ||
| AUG | CEF | CHL | FLO | GEN | SPE | TET | SSS | 1 | ||
| AUG | AMP | CEF | GEN | SPE | TET | SSS | 9 | |||
| AUG | AMP | CEF | CIP | ENR | TET | SSS | 8 | |||
| AUG | AMP | CEF | CHL | FLO | TET | SSS | 1 | |||
| AUG | AMP | GEN | NEO | TET | SXT | SSS | 1 | |||
| AUG | AMP | APR | CEF | GEN | NEO | TET | 1 | |||
| AUG | AMP | CEF | GEN | SPE | SSS | 3 | ||||
| AUG | AMP | GEN | SPE | TET | SSS | 3 | ||||
| AUG | AMP | CEF | GEN | TET | SSS | 1 | ||||
| AUG | AMP | CEF | CIP | ENR | SSS | 1 | ||||
| AUG | AMP | CEF | FLO | TET | SSS | 1 | ||||
| AUG | AMP | APR | CEF | GEN | NEO | 1 | ||||
| AUG | AMP | APR | CEF | NEO | 2 | |||||
| AUG | AMP | CEF | GEN | SSS | 1 | |||||
| AUG | AMP | GEN | NEO | TET | 1 | |||||
| AUG | AMP | GEN | TET | SSS | 1 | |||||
| AUG | AMP | TET | SXT | SSS | 1 | |||||
| AUG | AMP | CEF | TET | 4 | ||||||
| AUG | AMP | CEF | GEN | 1 | ||||||
| AUG | AMP | CEF | SPE | 1 | ||||||
| AUG | AMP | GEN | TET | 2 | ||||||
| AMP | GEN | SPE | TET | 2 | ||||||
| AUG | AMP | SPE | SSS | 1 | ||||||
| AUG | AMP | TET | SSS | 1 | ||||||
| AMP | SPE | TET | SSS | 1 | ||||||
| APR | GEN | NEO | SPE | 1 | ||||||
| GEN | SPE | TET | SSS | 5 | ||||||
| GEN | SPE | TET | SXT | 1 | ||||||
| AUG | AMP | CEF | 8 | |||||||
| AUG | AMP | TET | 5 | |||||||
| GEN | SPE | SSS | 3 | |||||||
| AMP | GEN | TET | 2 | |||||||
| NEO | TET | SSS | 2 | |||||||
| AMP | CEF | GEN | 1 | |||||||
| AMP | SPE | SSS | 1 | |||||||
| AMP | SXT | SSS | 1 | |||||||
| APR | NEO | TET | 1 | |||||||
| GEN | TET | SSS | 1 | |||||||
| AMP | TET | 5 | ||||||||
| AUG | AMP | 2 | ||||||||
| AMP | GEN | 2 | ||||||||
| AMP | APR | 1 | ||||||||
| APR | NEO | 1 | ||||||||
| TET | 23 | |||||||||
| AMP | 3 | |||||||||
| APR | 1 | |||||||||
| Pan-susceptible | 32 | |||||||||
| Other (Non-characterized) |
12 | |||||||||
3.2. Antimicrobial resistance of Enterococcus species
All Enterococcus isolates were resistant to at least one antimicrobial agent. Antimicrobial resistance phenotypes of Enterococcus isolates, in descending order, were TET (73.4%), CEF (51.9%), BAC (42.6%), ERY (31.2%), TYL (30.1), NEO (27.7%), GEN (8.98%), SPE (8.98%), PEN (7.8%), SXT (7.4%), ENR (5.1%), AMP (2.7%), CHL (2.7%), VAN (1.9%), CIP (1.6%), AUG (0.4%) and FLO (0.4%) (Figure 3). Only 3.9% (10/256) of Enterococcus isolates were resistant to high concentration of GEN. Multidrug resistance was seen in 44.9% Enterococcus isolates of which 25.8%, 14.4%, 2.3%, 0.8% and 1.6% of Enterococcus isolates were resistant to three, four, five, six, and seven classes of antimicrobials, respectively (Figure 4). No pan-resistant or pan-susceptible Enterococcus isolates were observed. The intrinsic resistance of Enterococcus isolates were noted for APR (98.83%) and LIN (96.88%).
Figure 3.
Antimicrobial resistance profile of Enterococcus species. The descending order of resistance was seen for tetracycline, ceftiofur, bacitracin, erythromycin and tylosin
Figure 4.
(Panel A) Resistant profile of Enterococcus species to different classes of antimicrobials and (Panel B) Multidrug resistance profile of Enterococcus species
AMR profiles of E. faecalis and E. faecium were summarized in Table 3. The descending order of AMR of E. faecalis were; TET (72.6%), CEF (46.2%), BAC (43.9%), ERY (31.4%), TYL (27.4%), NEO (26.9%), GEN (10.3%), SPE (6.3%), CHL (3.1%), SXT (1.3%), VAN (1.8%), PEN (1.8%), ENR (2.7%), CIP (0.9%), AMP (0.4%), AUG (0.4%) and FLO (0.4%) (Figure 5). Only 6.3% (14/223) of E. faecalis isolates were resistant to high concentration of GEN. Multidrug resistance was seen in 40.4% of E. faecalis isolates of which 26.5% of E. faecalis isolates were resistant to three classes of antimicrobials, 11.2% of E. faecalis isolates were resistant to four classes of antimicrobials, 1.8% of E. faecalis isolates were resistant to five classes of antimicrobials and 0.9% of E. faecalis isolates were resistant to six classes of antimicrobials (Figure 5). The resistance profiles of all E. faecalis isolates are demonstrated in Table 4. The most common resistance phenotype of E. fecalis was TET (R) + BAC (R) (37/223) followed by TET (R) + CEF (R) (23/223), TET (12/223) and TET (R) + ERY (R) + NEO (R) + TYL (R) (12/223).
Table 3.
Antimicrobial resistance profile of E. faecalis and E. faecium.
| Drug class | Drug | Disk potency | Resistance percentage |
|
|---|---|---|---|---|
|
E. faecalis |
E. faecium |
|||
| (n = 223) | (n = 21) | |||
| β-lactam | AUG | 30μg | 0.4 | 0 |
| AMP | 10 μg | 0.4 | 28.6 | |
| PEN | 10G | 1.8 | 85.7 | |
| CEF | 30 μg | 46.2 | 95.2 | |
| Phenicols | CHL | 30 μg | 3.1 | 0 |
| FLO | 30 μg | 0.4 | 0 | |
| Fluoroquinolones | ENR | 5 μg | 2.7 | 42.9 |
| CIP | 5 μg | 0.9 | 14.3 | |
| Macrolides | ERY | 15 μg | 31.4 | 38.1 |
| TYL | 60 μg | 27.4 | 38.1 | |
| Aminoglycosides | GEN | 10 μg | 10.3 | 4.8 |
| NEO | 30 μg | 26.9 | 47.6 | |
| SPE | 100 μg | 6.3 | 33.3 | |
| Tetracyclines | TET | 30 μg | 72.6 | 61.9 |
| Folate pathways inhibitors | SXT | 1.25–23.75 μg | 1.3 | 66.7 |
| Cyclic polypeptides | BAC | 10 IU | 43.9 | 42.9% |
| Glycopeptides | VAN | 30 μg | 1.8 | 0 |
Figure 5.
(Panel A) Antimicrobial resistance profile of E. faecalis and (Panel B) indicates Multidrug resistance profile of E. faecalis.
Table 4.
Summary of resistance profiles of E. faecalis (n = 223)
| Resistance profile | Number of isolates | ||||||
|---|---|---|---|---|---|---|---|
| TET | BAC | CEF | ERY | TYL | NEO | 1 | |
| TET | BAC | CEF | ERY | TYL | 2 | ||
| TET | BAC | CEF | NEO | GEN | 1 | ||
| TET | BAC | CEF | GEN | 2 | |||
| TET | BAC | CEF | 6 | ||||
| TET | BAC | GEN | 1 | ||||
| TET | BAC | ERY | TYL | GEN | 1 | ||
| TET | BAC | ERY | TYL | NEO | 1 | ||
| TET | BAC | ERY | TYL | 6 | |||
| TET | BAC | 37 | |||||
| TET | CEF | ERY | NEO | TYL | 8 | ||
| TET | CEF | ERY | NEO | 1 | |||
| TET | CEF | GEN | 5 | ||||
| TET | CEF | NEO | 6 | ||||
| TET | CEF | 23 | |||||
| TET | 12 | ||||||
| TET | ERY | TYL | 7 | ||||
| TET | ERY | 1 | |||||
| TET | ERY | GEN | NEO | TYL | 1 | ||
| TET | ERY | NEO | TYL | 12 | |||
| TET | GEN | 8 | |||||
| TET | NEO | 6 | |||||
| BAC | CEF | ERY | NEO | 2 | |||
| BAC | CEF | ERY | GEN | 1 | |||
| BAC | CEF | ERY | 4 | ||||
| BAC | CEF | NEO | 2 | ||||
| BAC | CEF | 5 | |||||
| BAC | ERY | NEO | TYL | 1 | |||
| BAC | 2 | ||||||
| CEF | ERY | TYL | NEO | 2 | |||
| CEF | ERY | TYL | 6 | ||||
| CEF | NEO | GEN | 1 | ||||
| CEF | NEO | 3 | |||||
| CEF | 5 | ||||||
| ERY | TYL | 2 | |||||
| ERY | TYL | NEO | 1 | ||||
| AUG | GEN | TET | 1 | ||||
| AMP | CEF | ENR | PEN | 1 | |||
| CHL | BAC | ERY | TET | TYL | 4 | ||
| CHL | BAC | ERY | ENR | TET | TYL | NEO | 1 |
| CHL | BAC | ERY | ENR | TET | TYL | 2 | |
| CIP | CEF | ENR | PEN | 1 | |||
| FLO | CEF | GEN | TET | SXT | VAN | 1 | |
| PEN | CEF | BAC | TET | 1 | |||
| PEN | CEF | CIP | ENR | 1 | |||
| SPE | BAC | NEO | TYL | ERY | 1 | ||
| SPE | BAC | CEF | NEO | 5 | |||
| SPE | BAC | CEF | 4 | ||||
| SPE | BAC | NEO | 1 | ||||
| SPE | BAC | 3 | |||||
| SXT | TET | NEO | 1 | ||||
| SXT | TET | NEO | CEF | 1 | |||
| VAN | TYL | TET | NEO | ERY | CEF | 1 | |
| VAN | BAC | ERY | TET | TYL | 1 | ||
| VAN | CEF | 1 | |||||
| Other(Non-characterized) | 6 | ||||||
The descending order of resistance of E. faecium was CEF (95.2%), PEN (85.7%), SXT (66.7%), TET (61.9%), NEO (47.6%), BAC (42.9%), ENR (42.9%), ERY (38.1%), TYL (38.1), SPE (33.3%), AMP (28.6%), CIP (14.3%) and GEN (4.8%). No E. faecium was found resistant to AUG, CHL, FLO and VAN (Figure 6). Multidrug resistance was seen in 85.7% of E. faecium isolates of which 19.0% of E. faecium were resistant to three classes of antimicrobials, 38.1% of E. faecium were resistant to four classes of antimicrobials, 9.5% of E. faecium were resistant to five classes of antimicrobials and 19.0% of E. faecium were resistant to seven classes of antimicrobials (Figure 6). The resistance profiles of all E. faecium isolates were shown in Table 5. The most common resistance phenotype was CEF (R) + NEO (R) + TET (R) + SXT (R) + PEN (R) (4/21).
Figure 6.
(Panel A) Antimicrobial resistance profile of E. faecium and (Panel B) indicates multidrug resistance profile of E. faecium.
Table 5.
Summary of resistance profiles of E. faecium (n = 21)
| Resistance profile | Number of isolates | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CEF | NEO | TET | SXT | PEN | 4 | |||||||
| AMP | CEF | ENR | PEN | SXT | BAC | ERY | NEO | TET | SPE | TYL | 2 | |
| AMP | CEF | ENR | PEN | SXT | 2 | |||||||
| CIP | AMP | BAC | CEF | ENR | ERY | NEO | PEN | SPE | TET | SXT | TYL | 1 |
| CIP | ENR | GEN | SXT | 1 | ||||||||
| CIP | AMP | CEF | ENR | ERY | PEN | SXT | 1 | |||||
| BAC | CEF | TET | PEN | ENR | ERY | NEO | SPE | SXT | TYL | 1 | ||
| BAC | CEF | TET | PEN | SPE | 1 | |||||||
| BAC | CEF | TET | PEN | ERY | NEO | TYL | 1 | |||||
| BAC | CEF | TET | PEN | SPE | 1 | |||||||
| BAC | CEF | TET | PEN | ENR | ERY | TYL | 1 | |||||
| BAC | CEF | 1 | ||||||||||
| CEF | PEN | SXT | 1 | |||||||||
| CEF | SPE | TYL | 1 | |||||||||
| CEF | ERY | PEN | TYL | 1 | ||||||||
| CEF | NEO | TET | SXT | PEN | 1 | |||||||
4. Discussion
The emergence of AMR is a serious threat to global health, and thus the WHO has recently declared a priority list of pathogens which need novel antibiotic development [28]. Multidrug resistance is a worldwide concern due to failures in treating infectious diseases. The resistance genes are often on mobile genetic elements, including plasmids, integrons, and transposons [29]. The resistance genes are transferred among bacteria via horizontal gene transfer, conjugation, transformation and transduction, which ultimately encodes for multidrug resistance [30]. The present study was designed to investigate the antimicrobial resistance profiles of E. coli and Enterococcus species isolated from non-viable chicken embryos, an overlooked niche concerning the emergence of multidrug-resistant bacteria.
Our data showed a high degree of resistance of E. coli to β-lactam antimicrobials; AMP (50.6%) and AUG (40.0%). Our data in regards to AMP resistance is comparable with AMP resistance of E. coli isolated (43%) from poultry products in Canada by the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) in 2016 [31]. A recent study has described the emergence of extended-spectrum β-lactamases (ESBLs)-encoding plasmids from E. coli isolates in poultry with a similar rate of prevalence as observed in humans which warrants regular monitoring of AMR in the broiler industry [32]. We observed a relatively high prevalence of CEF resistance in E. coli (29.4%) which justifies the voluntary withdrawal of this antimicrobial from poultry production in 2014 [31]. It would be interesting to study CEF resistance in E. coli from chicken embryo mortality a few years; hence, since CEF resistance of E. coli in poultry hatcheries may impose a risk of dissemination to humans. It has been reported that E. coli of poultry origin are closely related to E. coli-associated extra-intestinal infections in humans [33]. When compared to GEN resistance reported by CIPARS in poultry products (9%), it’s a higher prevalence in E. coli isolated in dead embryos [31]. CIPARS represents data of the overall Canadian poultry industry, which may under-represent this emerging ecological milieu in western Canada. However, both Canadian Antimicrobial Resistance Surveillance System (CARSS) and CIPARS have well-documented an increased trend in GEN resistance in E. coli isolates of poultry origin during 2004–2014 [34]. GEN is used in the poultry industry to reduce neonatal poultry mortality and for growth promotion [35]. Hence, we can speculate the association of GEN use and increased resistance in the poultry industry in western Canada. In our study, 52.9% of E.coli was TET resistant, which is comparable with CIPARS data as they have observed 50% of E. coli resistant to TET in 2016 [31]. This trend may be explained by the heavy use of TET in the poultry industry in Canada [36]. There are currently 38 different TET resistance genes described [37], and further investigation is needed to characterize these genes in isolates recovered in our study to determine the resistance mechanisms.
We have seen 1.9% VRE in dead chicken embryos although VAN has not been used in the broiler chicken industry in Canada. The mean VRE increased from 6.2% in 2011 to 7.9% in 2014 in Europe. The frequency of VRE ranged from 0% (Estonia, Finland, Iceland, and Malta) to 45.1% (Ireland). In 2014, increasing trends of VRE were seen in Bulgaria, Croatia, Denmark, Hungary, Ireland, Italy, Slovakia, and United Kingdom from 2011 to 2014 [38]. A study conducted in British Columbia, Canada in 2010 investigating Enterococcus isolates obtained from faecal and caecal contents of commercial poultry, demonstrated that none of the enterococci were resistant to VAN [39]. Enterococci of foodborne origin were not identified as a direct cause of resistant enterococci in humans, but they could pose a risk in transfer of resistance determinants to human-adapted strains of the same genus or other genera, as shown in VAN resistance in S. aureus and TET and ERY resistance in Listeria monocytogenes [40–42].
The resistance of enterococci to TET (73.4%), BAC (42.6%) and TYL (30.1%) was remarkable in our study. It has also been suggested that commensal microbiota of poultry can be a reservoir of BAC resistance, and this BAC resistance can be readily transferable to E. faecalis in human [43]. Genes encoding resistance to TET, tetL and tetM, are frequently associated with ermB which encodes resistance to macrolides, lincosamides, streptograminB and quinupristin-dalfopristin. Since BAC is commonly used as a growth-promoting antibiotic in the Canadian poultry industry, resistance to BAC and other antibiotics mentioned above can be co-selected [43]. A recent study conducted in Asia looked at determining AMR of uropathogenic E. coli and APEC and found multidrug resistance in 98% of isolates where most of them were resistant to at least five antimicrobials tested [44]. Moreover, emerging extended-spectrum β-lactamases (ESBL) producing E. coli were resistant to aminoglycosides and fluoroquinolones [45]. Among them, a classic example of globally disseminated, multidrug-resistant E. coli strain sequence type (ST) 131 (ST131) which causes significant amounts of the urinary tract and bloodstream infections in humans [46].
5. Conclusion
In the present study, we have observed that chicken embryos harbour a significant number of multidrug-resistant E. coli and enterococci, revealing that this niche can be a substantial source of superbugs in the environment. The current antimicrobial resistance surveillance systems predominantly focus on monitoring resistance in poultry farms and processing plants. Embryonated eggs represent a critical niche that can reveal the nature of AMR that would be passed on to the production farms and ultimately to humans via the poultry products. Our data suggest that the screening of antimicrobial resistance, particularly at the level of embryonated eggs, is quintessential in AMR surveillance to understand AMR dissemination for developing appropriate control measures.
Disclosure statement
No potential conflict of interest was reported by the authors.
References
- [1].Ciorba V, Odone A, Veronesi L, et al. Antibiotic resistance as a major public health concern: epidemiology and economic impact. Ann Ig. 2015;27:562–579. [DOI] [PubMed] [Google Scholar]
- [2].Osman KM, Kappell AD, Elhadidy M, et al. Poultry hatcheries as potential reservoirs for antimicrobial-resistant Escherichia coli: a risk to public health and food safety. Sci Rep. 2018;8:5859. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [3].Heuer H, Schmitt H, Smalla K.. Antibiotic resistance gene spread due to manure application on agricultural fields. Curr Opin Microbiol. 2011;14:236–243. [DOI] [PubMed] [Google Scholar]
- [4].Van Loo E, Caputo V, Nayga RM Jr., et al. Effect of organic poultry purchase frequency on consumer attitudes toward organic poultry meat. J Food Sci. 2010;75:S384–97. [DOI] [PubMed] [Google Scholar]
- [5].Casewell M, Friis C, Marco E, et al. The European ban on growth-promoting antibiotics and emerging consequences for human and animal health. J Antimicrob Chemother. 2003;52:159–161. [DOI] [PubMed] [Google Scholar]
- [6].Nannapaneni R, Hanning I, Wiggins KC, et al. Ciprofloxacin-resistant Campylobacter persists in raw retail chicken after the fluoroquinolone ban. Food Addit Contam. 2009;26:1348–1353. [DOI] [PubMed] [Google Scholar]
- [7].Topp E. Agriculture and agri-food Canada’s research program on antimicrobial resistance. Can Commun Dis Rep. 2017;43:224–227. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [8].Hussain A, Shaik S, Ranjan A, et al. Risk of transmission of antimicrobial resistant escherichia coli from commercial broiler and free-range retail chicken in India. Front Microbiol. 2017;8:2120. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [9].Velasquez CG, Macklin KS, Kumar S, et al. Prevalence and antimicrobial resistance patterns of Salmonella isolated from poultry farms in southeastern United States. Poult Sci. 2018;97:2144–2152. [DOI] [PubMed] [Google Scholar]
- [10].Davis GS, Waits K, Nordstrom L, et al. Antibiotic-resistant Escherichia coli from retail poultry meat with different antibiotic use claims. BMC Microbiol. 2018;18:174. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [11].Moreno MA, Garcia-Soto S, Hernandez M, et al. Day-old chicks are a source of antimicrobial resistant bacteria for laying hen farms. Vet Microbiol. 2019;230:221–227. [DOI] [PubMed] [Google Scholar]
- [12].Jimenez-Belenguer A, Domenech E, Villagra A, et al. Antimicrobial resistance of Escherichia coli isolated in newly-hatched chickens and effect of amoxicillin treatment during their growth. Avian Pathol. 2016;45:501–507. [DOI] [PubMed] [Google Scholar]
- [13].Davies R, Breslin M. Observations on Salmonella contamination of eggs from infected commercial laying flocks where vaccination for Salmonella enterica serovar Enteritidis had been used. Avian Pathol. 2004;33:133–144. [DOI] [PubMed] [Google Scholar]
- [14].Chousalkar K, Flynn P, Sutherland M, et al. Recovery of Salmonella and Escherichia coli from commercial egg shells and effect of translucency on bacterial penetration in eggs. Int J Food Microbiol. 2010;142:207–213. [DOI] [PubMed] [Google Scholar]
- [15].Liljebjelke KA, Hofacre CL, Liu T, et al. Vertical and horizontal transmission of Salmonella within integrated broiler production system. Foodbourne Pathog Dis. 2005;2:90–102. [DOI] [PubMed] [Google Scholar]
- [16].Rossi DA, Fonseca BB, Melo R, et al. Transmission of Campylobacter coli in chicken embryos. Braz J Microbiol. 2012;43:535–543. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [17].Zhao S, Wang CL, Chang SK, et al. Characterization of Escherichia coli isolated from day-old chicken fluff in taiwanese hatcheries. Avian Dis. 2019;63:9–16. [DOI] [PubMed] [Google Scholar]
- [18].Humbert F, Salvat G. The risk of transmission of salmonellae in poultry farming: detection and prevention in Europe. Rev Sci Tec. 1997;16:83–90. [PubMed] [Google Scholar]
- [19].Karunarathna R, Popowich S, Wawryk M, et al. Increased Incidence of enterococcal infection in nonviable broiler chicken embryos in Western Canadian hatcheries as detected by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Avian Dis. 2017;61:472–480. [DOI] [PubMed] [Google Scholar]
- [20].van den Bogaard AE, Stobberingh EE. Epidemiology of resistance to antibiotics: links between animals and humans. Int J Antimicrob Agents. 2000;14:327–335. [DOI] [PubMed] [Google Scholar]
- [21].Agunos A, Léger D, Carson C. Review of antimicrobial therapy of selected bacterial diseases in broiler chickens in Canada. Can Vet J. 2012;53:1289. [PMC free article] [PubMed] [Google Scholar]
- [22].Yassin AK, Gong J, Kelly P, et al. Antimicrobial resistance in clinical Escherichia coli isolates from poultry and livestock, China. PloS One. 2017;12:e0185326. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [23].van Veen SQ, Claas E, Kuijper EJ. High-throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories. J Clin Microbiol. 2010;48:900–907. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [24].CLSI VET01-S2 Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; 2nd informational supplement. Wayne (PA): Clinical and Laboratory Standards Institute; 2014. [Google Scholar]
- [25].CLSI M100-S24 Performance standards for antimicrobial susceptibility testing; 24th informational supplement. Clinical and Laboratory Standards Institute. Wayne, Pennsylvania, USA; 2015. [Google Scholar]
- [26].EUCAST European Committee on Antimicrobial Susceptibility Testing breakpoints tables for interpretation of MICs and zone diameters. Basel, Switzerland: European Society of Clinical Microbiology and Infectious diseases; 2014. [Google Scholar]
- [27].Magiorakos AP, Srinivasan A, Carey R, et al. Multidrug‐resistant, extensively drug‐resistant and pandrug‐resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012;18:268–281. [DOI] [PubMed] [Google Scholar]
- [28].Organization WH Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. Geneva: World Health Organization; 2017. [Google Scholar]
- [29].Frost LS, Leplae R, Summers AO, et al. Mobile genetic elements: the agents of open source evolution. Nat Rev Microbiol. 2005;3:722. [DOI] [PubMed] [Google Scholar]
- [30].Juhas M, Van Der Meer JR, Gaillard M, et al. Genomic islands: tools of bacterial horizontal gene transfer and evolution. FEMS Microbiol Rev. 2009;33:376–393. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [31].CIPARS The Canadian integrated program for antimicrobial resistance surveillance. 2017. https://wwwcahssca/media/uploads/cipars-national-meeting/documents/17-11-14_22-02/CIPARS_2016_Integrated_Results__aeox1pvpdf
- [32].Wu C, Wang Y, Shi X, et al. Rapid rise of the ESBL and mcr-1 genes in Escherichia coli of chicken origin in China, 2008–2014. Emerg Microbes Infect. 2018;7:30. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [33].Bergeron CR, Prussing C, Boerlin P, et al. Chicken as reservoir for extraintestinal pathogenic Escherichia coli in humans, Canada. Emerg Infect Dis. 2012;18:415. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [34].CARSS Canadian antimicrobial resistance surveillance system report. 2016. https://wwwcanadaca/en/public-health/services/publications/drugs-health-products/canadian-antimicrobial-resistance-surveillance-system-report-2016html
- [35].Marshall BM, Levy SB. Food animals and antimicrobials: impacts on human health. Clin Microbiol Rev. 2011;24:718–733. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [36].Diarra MS, Silversides FG, Diarrassouba F, et al. Impact of feed supplementation with antimicrobial agents on growth performance of broiler chickens, Clostridium perfringens and Enterococcus counts, and antibiotic resistance phenotypes and distribution of antimicrobial resistance determinants in Escherichia coli isolates. Appl Environ Microbiol. 2007;73:6566–6576. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [37].Roberts MC. Update on acquired tetracycline resistance genes. FEMS Microbiol Lett. 2005;245:195–203. [DOI] [PubMed] [Google Scholar]
- [38].da Costa ME, Machado HS. Evolution of antimicrobial resistance in Europe: a factual review. J Allergy Ther. 2017;8:1000250. [Google Scholar]
- [39].Diarra MS, Rempel H, Champagne J, et al. Distribution of antimicrobial resistance and virulence genes in Enterococcus spp. and characterization of isolates from broiler chickens. Appl Environ Microbiol. 2010;76:8033–8043. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [40].Hummel A, Holzapfel WH, Franz CM. Characterisation and transfer of antibiotic resistance genes from enterococci isolated from food. Syst Appl Microbiol. 2007;30:1–7. [DOI] [PubMed] [Google Scholar]
- [41].Aslam M, Diarra MS, Checkley S, et al. Characterization of antimicrobial resistance and virulence genes in Enterococcus spp. isolated from retail meats in Alberta, Canada. Int J Food Microbiol. 2012;156:222–230. [DOI] [PubMed] [Google Scholar]
- [42].Hayes JR, English LL, Carter PJ, et al. Prevalence and antimicrobial resistance of Enterococcus species isolated from retail meats. Appl Environ Microbiol. 2003;69:7153–7160. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [43].Chen M-Y, Lira F, Liang H-Q, et al. Multilevel selection of bcrABDR-mediated bacitracin resistance in Enterococcus faecalis from chicken farms. Sci Rep. 2016;6:34895. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [44].Kazemnia A, Ahmadi M, Dilmaghani M. Antibiotic resistance pattern of different Escherichia coli phylogenetic groups isolated from human urinary tract infection and avian colibacillosis. Iran Biomed J. 2014;18:219. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [45].Rogers BA, Sidjabat HE, Paterson DL. Escherichia coli O25b-ST131: a pandemic, multiresistant, community-associated strain. J Antimicrob Chemother. 2010;66:1–14. [DOI] [PubMed] [Google Scholar]
- [46].Petty NK, Zakour NLB, Stanton-Cook M, et al. Global dissemination of a multidrug resistant Escherichia coli clone. Proc Natl Acad Sci U S A. 2014;111:5694–5699. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Citations
- CIPARS The Canadian integrated program for antimicrobial resistance surveillance. 2017. https://wwwcahssca/media/uploads/cipars-national-meeting/documents/17-11-14_22-02/CIPARS_2016_Integrated_Results__aeox1pvpdf