Monitoring the antimicrobial resistance of Salmonella and Escherichia coli in chickens and migratory birds

Monitoring the antimicrobial resistance of Salmonella and Escherichia coli in chickens and migratory birds

By

Nehal, M. Nabil^*, Reem, M. Reda^*, Maram, M. Tawakol^*

*Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Agricultural Research Center, Nadi El-Seid Street, Dokki, Giza 12618, Egypt.

ABSTRACT

Antimicrobial resistance is considered as one of the most important issue that has a negative impact on poultry industry worldwide as well as of public health concerns. In this study, antimicrobial resistance pattern of Salmonella species and Escherichia coli, isolated from broiler chickens and migratory birds in Egypt, was investigated. A total of 250 diseased broiler chickens and 100 migratory birds (five bird species) were collected. All collected samples were then subjected to Salmonella and E. coli isolation, identification and antimicrobial susceptibility pattern. Salmonella and E. coli were isolated from broiler chickens with an incidence of (14%) and (40%) respectively, and from migratory birds with an incidence of (10%) and (30%) respectively. Based on serotyping, three serotypes were detected from broiler chickens (S. typhimurium, S.kentucky and S. enteritidis) and four Salmonella serotypes from migratory birds (S. typhimurium, S. kentucky, S. infantis and S. enteritidis). Twelve E. coli serotypes found in samples collected from broiler chickens (O1, O6, O55, O86, O91, O124, O125, O128, O144, O158, O159 and O166) while E. coli serotypes obtained from migratory birds, were found as: O1, O2, O26, O55, O78, O91, O113, O121, O124, O 128, O146 and O158.Overall,the antimicrobial susceptibility testing of isolated Salmonella and E. coli from broiler chickens and migratory birds showed resistance to most of the used antimicrobial agents; Salmonella isolates obtained from broiler chicken farms were found totally resistant to amoxicillin, ampicillin-sulbactam, and tetracycline. E. coli isolates obtained from broiler chicken farms showed resistance ranged from 25% - 95%. Salmonella and E. coli isolates retrieved from migratory birds showed resistance which ranged from 60% - 100% and 66.7 % - 96.7% respectively. This study highlights the spread of antimicrobial resistance Salmonella and E. coli in both domestic chicken and migratory birds. Hence, strict biosecurity measurements and continuous monitoring of antimicrobial resistance among poultry population should be implemented in addition to avoid the misuse of antimicrobial agents.

INTRODUCTION

Avian salmonellosis and colibacillosis are on the top list of most commonly detected bacterial diseases in poultry all over the globe (Ibrahim et al. 2021 Lutful Kabir 2010 and Van Boeckel et al. 2019) and caused by pathogenic Salmonella species and E. coli (Ejeh et al. 2017). On the other hand, Salmonella is a another important bacterial pathogenposinga negative impaction poultry production due to high mortality rates in young birds and chronic carriers in adult birds (Soliman et al. 2018). E. coli is a Gram-negative bacterium and acts as normal inhabitant in the intestinal tract with the exception of few strains  which harbor virulence genes and have the ability to cause diarrhea (El-Demerdash et al. 2021) and colibacillosis (Jahantigh and Dizaji (2015) inbroiler, commercial layers and breeders (Koutsianos et al. 2021). Those strains usually associated with high morbidity and mortality in poultry species (Roseliza et al. 2016).

The resistance of poultry pathogens to antimicrobial agents has shown to result in treatment failure producing economic losses (Nhung et al. 2017). Several strains of Salmonella and E. coli were described previously with multiple resistances to antibiotics (Ejeh et al. 2017).

The emergence of antimicrobial resistance of E. coli has been described previously mainly due to the misuse of antimicrobial agents (Ibrahim et al. 2019). In addition to the negative impact on poultry, antimicrobial resistance threatens the public health via zoonotic strains.

Away from domestic bird species, migratory birds are considered as potential source of pathogenic microorganisms spread either biologically or mechanically (Benskin et al. 2009). Migratory birds have shown an important role in the ecology and circulation of different bacterial pathogens for example: Salmonella enterica, Campylobacter jejuni, Pasteurella multocida and Mycobacterium avium (Hubálek 2004). Migratory birds can transmit antimicrobial resistant bacteria to domestic birds and then to humans, as well as from one country to another (Elsohaby et al. 2021 Islam et al. 2021^a). (Bonnedahl and Järhult 2014). Therefore, this study aims to investigate the present status of antimicrobial resistance of Salmonella and E. coli among both domestic poultry (chickens) and migratory bird species in Egypt. Here we applied different methods commonly used in official antimicrobial resistance monitoring programs. This in turn will increase our knowledge in understanding the resistance profile in different migratory birds: Common teal (Anas crecca) Northern pintail (Anas Acuta), Northern Northern shoveler (Spatula clypeata), Common pochard (Aythyaferina) and Mallard ducks (Anas Platyrhynchos)) obtained from live bird markets (LBMs) in Egypt.

MATERIAL and METHODS

2.1. Sampling

A total of 250 diseased broiler chickens (30 to 35 days of age), showing diarrhea, inability to move, ruffled feather with higher mortality rates, were collected from Dakahlia Governorate, Egypt. Five individual chickens were collected from each farm and subjected to postmortem examinations. Liver and spleen were collected for isolation of Salmonella and E. coli isolation meanwhile cecum tissues were collected for Salmonella isolation only. All samples were obtained aseptically and shipped directly to the Reference Laboratory for Veterinary Quality Control on Poultry Production (RLQP), Animal Health Research Institute (AHRI) for further laboratory examinations. In addition, a total of 100 cloacal swabs were collected from five species of 100 migratory birds from live bird markets (LBMs) in Dakahlia and Damietta Governorates (60 birds from Dakahlia and 40 birds from Damietta) (Table 1). Swab samples were collected on non-selective media (Buffered Peptone Water) for Salmonella and E. coli isolation and identification.

. Bacteriological examinations

The obtained tissue samples and cloacal swabs were subjected to Salmonella isolation according to ISO 6579 (2017) and serotyped according to Kauffman- white scheme (Kauffman, 1974) to detect somatic (O) and flagellar (H) antigens (Cruickshank et al. 1975) and (WHO 2007). Also the collected tissue samples and cloacal swabs were subjected to E. coli isolation and identification as described previously by Edward and Ewing (1986), and further serotyped according to Kok et al. (1996).

2.3. Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed using agar disc diffusion method on Muller Hinton agar plates as described elsewhere Finegold and Martin (1982). Ciprofloxacin (5µg), Ampicillin - sulbactam (20µg), tetracycline (30µg), amoxicillin (25 µg), erythromycin (15 µg) and doxycycline (30µg) were used. The inhibition zones diameters were measured and assessed according to the manufactures´ instructions (Oxoid), and then were categorized into susceptible or resistant according to the CLSI guidelines (CLSI 2016).

3. RESULTS

3.1. Incidence of Salmonella and E. coli isolation, identification and serotyping in broiler chickens 

Salmonella and E. coli were isolated from the randomly collected chickens with an incidence of (14%) and (40%) respectively (table 2).

The serotyping of the isolated Salmonella and E. coli revealed the detection of three Salmonella serovars (S. typhimurium, S.kentucky and S. enteritidis) and 12 E. coli serogroup (O1, O6, O55, O86, O91, O124, O125, O128, 

DISCUSSION

Avian salmonellosis and colibacillosis are widely spread food-borne disease affecting birds and threaten poultry industry worldwide via their negative impact on meat and egg production as well as associated mortality(Ibrahim et al. 2021) and (Lutful Kabir 2010). Over the last decades, from 2000 to 2018, the proportion of antimicrobial resistance in chickens has been increased from 0.15 to 0.41 (Van Boeckel et al. 2019). Migratory birds have shown a clear important role in the interspecies transmission and intercontinental dissemination of antimicrobial resistant Salmonella (Wei et al. 2020) and act as reservoirs of antimicrobial resistant E. coli (Islam et al. 2021^b).

In the present study, Salmonella was isolated from 250 broiler chickens with (14%). The findings in the current study agreed to some extent with Diarrassouba et al. (2007) Adam et al. (2018) and Limawongpranee et al.(1999) who isolated Salmonella from chickens with (11.2%), (11.6%) and (14.3%) respectively. Previous studies conducted by Lebert et al. (2018) and Abd El-Ghany et al. (2012) isolated Salmonella from chickens in Ontario with (0.3%)and from Egypt with (7.03%) respectively and these percentages were lower than this study. Three Salmonella serotypes were reported from chicken farms in this study; (S. typhimurium, S.kentucky and S. enteritidis). These results matched with a study conducted by Abd El-Ghany et al. (2012) who isolated S. typhimurium, S. enteritidis and S. kentucky from chickens in Egypt, and Limawongpranee et al. (1999) who isolated S. enteritidis from chickens in Japan. Salmonella, isolated from broiler chickens, showed antimicrobial resistance to amoxicillin, ampicillin-sulbactam, tetracycline, ciprofloxacin, doxycycline and erythromycin (table 4). These findings agreed to some extent with previous studies performed by Adam et al. (2018) who recorded resistance of all Salmonella isolates against amoxicillin, ampicillin and erythromycin, and Das et al. (2021) who reported resistance to ampicillin (98.8%), tetracycline (94.2%) and ciprofloxacin (50%) respectively.

Further, E. coli was isolated from 250 examined broiler chickens with an incidence of (40%). These findings agreed to some extent in the isolation percentages of E.coli as described previously in Jordon by Ibrahim et al. (2019) and Indonesia by Hardiati et al. (2021) who mentioned that E. coli was isolated with (53.4%) from chickens in Jordan andwith (55.6%) from broiler farms in Indonesia. Previous studies recorded higher percentages of E. coli isolation than this study such as Lebert et al. (2018) and Al Azad et al. (2019) who isolated E.coli with (99%) from chicken flocks in Ontario, and with (100%) from broiler in Bangladesh respectively. Meanwhile a study conducted by Adam et al. (2018), recorded a lower E. coli isolation (21.6%) than the current study. The serotyping in this study revealed the detection of 12 E. coli serogroups (O1, O6, O55, O86, O91, O124, O125, O128, O144, O158, O159 and O166) (Table 2). These results agreed with previous studies performed by Ibrahim et al. (2019) and Amer et al.(2015) who recoded O1 from chickens in Jordan, and O86, O91, O125 and O158 from broiler chickens in Egypt respectively. E. coli that isolated from broiler chickens showed antimicrobial resistance which ranged from 25 % - 95% respectively. The isolated E. coli showed resistance to amoxicillin, ampicillin-sulbactam, tetracycline, ciprofloxacin, doxycycline and erythromycin (table 4) and these findings were in near similarity with Adam et al. (2018) who recorded resistance of E.coli isolates against amoxicillin, ampicillin, erythromycin, tetracycline, streptomycin and doxycycline. However previous researcher such as Hardiati et al. (2021) showed resistance of E. coli isolates to ampicillin (100%), erythromycin (92%), tetracycline (88%) and ciprofloxacin (88%), and Talebiyan et al. (2014) recorded resistance to erythromycin (71.70%), doxycycline (16.98%), ciprofloxacin (7.55%).

In addition, the findings of this study revealed the presence of several strains of Salmonella in several species of migratory birds in two different geographical locations in Egypt. Previous studies conducted by Shalaby et al. (2012) and Islam^a  et al. (2021) indicates the presence of Salmonella in migratory birds in Egypt and Bangladesh with an incidence of (28.26%) and (21.21%) respectively. Meanwhile Elsohaby et al. (2021) and Grigar et al. (2017) isolated Salmonella from migratory birds in Saudi Arabia and Texas with percentages of (2.4%) and (0.5%) respectively and their findings were lower than the current study.

 In the current study S. typhimurium and S. kentucky were isolated from Northern pintail and Mallard ducks, S. infantis and S. enteritidis were isolated from Northern pintail and Common pochard. Meanwhile a study conducted by Wei et al. (2020) isolated S. enterica strains (S. typhimurium, S. berta, and S. virchow) from Mallard duck, Northern pintail, Spot-billed duck, Intermediate egret, Eastern great egret and Eurasian wigeon migratory birds in from South Korea. Our results differ from Grigar et al. (2017) who isolated S. Thompson and S. braenderup from water fowl in Texas, and Antilles et al. (2015) who recorded that all of examined free living birds in north eastern Spain were negative for Salmonella.

A higher incidence of E. coli isolation was found compared to Salmonella in migratory birds mainly in Common teal in both locations of this study.  A study performed in Pakistan by Mohsin et al. (2017) isolated ESBL- E. coli from mallard duck, red headed pochard, common pochard, shoveler duck, Eurasian wigeon, Eurasian coot and rosy starling with an incidence of (17.3%) which was lower than recorded in this study. However another reports performed by Islam^b et al. (2021) Shobrak and Abo-Amer (2014) isolated E. coli from migratory birds with percentages of (93.94%) and (94%) respectively and these incidences were higher than this study. The isolation of E.coli from mallard ducks in this study (18.4%) was lower from a study conducted in Germany by Ewers et al. (2009) who isolated E. coli from wild mallard ducks with (82.4%), however it was higher than Seleem et al. (2021) who reported E.coli (O86) from migratory ducks and (O125) quails in Egypt with (0.57%).

Overall, with regard to the results of antimicrobial susceptibility in this study; Salmonella and E. coli isolated from migratory birds showed higher antimicrobial resistance which ranged from 60% - 100% and 66.7 % - 96.7% respectively. Majority of the isolated Salmonella and E.coli showed multidrug resistance. These findings were in agreement with Mohsin et al. (2017) Seleem et al. (2021) Islam^bet al. (2021) and Yuan et al. (2021) who reported multidrug resistance of E. coli isolated from migratory birds in Pakistan, Egypt, Bangladesh and China respectively.

CONCLUSION

Our study shows that Salmonella and E. coli were isolated from broiler chickens with an incidence of (14%) and (40%) respectively, and in100 migratory birds (obtained from live bird market) with an incidence of (10%) and (30%) respectively. This calls for continuous monitoring of antimicrobial resistance in Egypt and highlights the needs for future genetic studies including whole genome sequencing to understand the genetic relatedness between isolates retrieved from domestic and migratory birds.