Abstract
SETTING:
Twenty poultry farms in five provinces of Nepal were selected for studying bacterial pathogens and their antimicrobial resistance (AMR) patterns.
OBJECTIVE:
To document the proportion of cloacal swabs collected from 3,230 broiler and 3,230 layer chickens from September to December 2021 that grew isolates of Escherichia coli, Enterococcus spp. and Salmonella spp. along with their AMR patterns.
DESIGN:
This was a cross-sectional descriptive study.
RESULTS:
In broiler birds, Enterococcus spp., Salmonella spp. and E. coli were identified in respectively 36%, 39% and 63% of swabs. In layer birds, Enterococcus spp., Salmonella spp. and E. coli were identified in respectively 31%, 48% and 60% of swabs. For both bird types, there was variation in bacterial prevalence between the regions. For all three bacterial isolates, the lowest antimicrobial resistance was found with amikacin. For the other nine antibiotics tested, >50% of bacterial isolates showed resistance; between 60% and 90% of isolates showed resistance to ciprofloxacin and trimethoprim-sulfamethoxazole. Multidrug resistance ranged from 45% to 46% for Salmonella spp., 37–44% for E. coli and 13–17% for Enterococcus spp.
CONCLUSION:
This study shows that a large proportion of poultry in Nepal are infected with potentially pathogenic bacteria, and these are frequently resistant to commonly used antibiotics. Nepal urgently needs to implement corrective measures.
Keywords: antimicrobial resistance, Nepal, broiler chickens, layer chickens, Escherichia coli, Salmonella spp., Enterococcus spp
Abstract
CONTEXTE :
Vingt fermes avicoles dans cinq provinces du Népal ont été sélectionnées pour étudier les pathogènes bactériens et leurs profils de résistance aux antimicrobiens (AMR).
OBJECTIF :
Documenter la proportion d’écouvillons cloacaux prélevés chez 3 230 poulets de chair et 3 230 poules pondeuses de septembre à décembre 2021 qui ont produit des isolats d’Escherichia coli, d’Enterococcus spp. et de Salmonella spp. ainsi que leurs profils d’AMR.
MÉTHODE :
Il s’agissait d’une étude descriptive transversale.
RÉSULTATS :
Chez les poulets de chair, Enterococcus spp., Salmonella spp. et E. coli ont été identifiés dans respectivement 36%, 39% et 63% des écouvillons. Chez les pondeuses, Enterococcus spp., Salmonella spp. et E. coli ont été identifiés dans respectivement 31%, 48% et 60% des écouvillons. Pour les deux types d’oiseaux, la prévalence bactérienne varie selon les régions. Pour les trois isolats bactériens, la résistance la plus faible a été observée avec l’amikacine. Pour les neuf autres antibiotiques testés, >50% des isolats bactériens présentaient une résistance ; entre 60% et 90% des isolats présentaient une résistance à la ciprofloxacine et au triméthoprime-sulfaméthoxazole. La multirésistance variait de 45 à 46% pour Salmonella spp, 37 à 44% pour E. coli et 13 à 17% pour Enterococcus spp.
CONCLUSION :
Cette étude montre qu’une grande proportion de volailles au Népal est infectée par des bactéries potentiellement pathogènes, et que celles-ci sont fréquemment résistantes aux antibiotiques couramment utilisés. Le Népal doit de toute urgence mettre en œuvre des mesures correctives.
Antimicrobial resistance (AMR) has become a major global health problem in both humans and animals, and is more frequent and severe in low- and middle-income countries than in high-income countries.1,2 The emergence of AMR is linked to irrational, indiscriminate and incorrect use of antibiotics in humans, as well as in the veterinary and agricultural sectors, and this is especially a problem in resource-poor countries.3 A recent study estimated that there were nearly 5 million human deaths associated with AMR globally, and 1.3 million deaths directly attributable to bacterial AMR.4
The WHO, the Food and Agriculture Organization (FAO) and the World Organization for Animal Health (OIE) are working together to develop strategic plans to better understand and curb the threat of AMR and enable better prevention and treatment of human and animal infections.5,6
In Nepal, more than half of the Escherichia coli, Enterococcus spp., Pseudomonas spp., Salmonella spp., Klebsiella pneumoniae, and Pasteurella spp. isolates in animals have been identified as being resistant to antibiotics.7 The volume of veterinary antibiotic sales in Nepal increased by >50% from 2008 to 2012, mainly as a result of sales through retailers without veterinarian prescription, as well as indiscriminate and extensive use of antibiotics due to the expansion of animal and poultry enterprises in the country.8
The availability of antibiotics over the counter, the use of antibiotics in feed supplements and antibiotic administration without prescription all point to the rampant and injudicious use of antibiotics, which almost certainly has contributed to increasing AMR in the poultry production system in Nepal.9 Moreover, the powder form of antimicrobials used as feed premixes and poultry feed supplements is a common practice in commercial poultry farms.10 It is estimated that about 50% of antibiotics are prescribed inappropriately,11 and about 70% of veterinary drugs are sold without a veterinarian’s prescription.12 Most of the time, poultry farmers themselves use antibiotics in subtherapeutic doses for the treatment of common infections or as growth promoters. These instances of antibiotic misuse affect the bacterial response to these antibiotics and lead to the development of multidrug-resistant (MDR) bacteria, which may be transmitted to humans indirectly by the consumption of poultry or poultry byproducts.13 These improper practices in poultry enterprises are contributing to the increasing risk of AMR in Nepal.
Several surveillance reports on AMR of zoonotic bacteria such as E. coli, Staphylococcus spp., and Enterococcus spp. in food animals, food and humans have been published across the globe.14 However, to date there have only been a few small-scale studies of AMR in Nepal in either human or animal medicine.
This study therefore aimed to document the prevalence of E. coli, Salmonella and Enterococcus spp. and their AMR patterns in the poultry production system in Nepal between September and December 2021. Specifically, key objectives were to identify the proportion of cloacal swabs from broiler and layer chickens that had isolates of E. coli, Enterococcus spp. and Salmonella spp. and to determine their AMR patterns.
METHODS
Study design
This was a cross-sectional study that involved the collection of cloacal swabs from poultry.
Study area and selection of farms
The poultry farms located in the headquarters and popular cities of five provinces: Province 1 (no official name), Province 3 (Bagmati), Province 4 (Gandaki), Province 5 (Lumbini) and Province 7 (Sudur Paschim) of Nepal were selected for the study (Figure). A total of 20 poultry farms were included in the study. There were two broiler and two layer farms, having an average flock size of 2,000 birds, which were selected from each of the five provinces for cloacal swab collection. A simple random technique formula was used to collect the cloacal swabs from each farm. According to this technique, 323 cloacal swabs were collected from each of the farms.
FIGURE.
Study area/farm locations for cloacal swabs in Nepal.
Sampling of cloacal swabs
Between September and December 2021, 6,460 cloacal swabs, 3,230 from broiler birds and 3,230 from layer poultry birds (from seven cities of five provinces of Nepal) were collected in 1% peptone water (5–7 mL) using aseptic techniques. The cloacal swabs were collected in the morning. Samples were transported immediately after collection to the Laboratory of Veterinary Microbiology and Pathology, Agriculture and Forestry University, Chitwan, Nepal, using a cooling box, and stored at refrigeration temperature for 48 h.
Bacterial culture and isolation
The samples were incubated at 37°C for 24 h before streaking onto the selective media to culture the target bacterial species. Enterococcosel and Brilliant Green Bile Agar were used to isolate Enterococcus spp., E. coli and Salmonella spp., respectively. After streaking the samples, they were further incubated in 37°C for 24 h to obtain the colonies. Based on the colony characteristics specific to each of the bacterial species, further biochemical testing was carried out to confirm the bacterial types.15 Biochemical tests were carried out (Table 1) to confirm and characterise the specific bacterial species.15
TABLE 1.
Confirmation of bacterial species using biochemical tests
| Bacterial species | Laboratory test criteria for identification |
|---|---|
| E. Coli | Gram staining (Gram− rod), yellow green colony on BGA, metallic Sheen on EMBA, IMVic test− indole+, MR+, Voges-Proskauer broth−, Simon citrate−, TSI (yellow butt, yellow slant, H2S−, gas+) |
| Salmonella | Gram staining (pink colony on BGA, TSI agar (red-slant, yellow butt, H2S+, gas− and agglutination+ with O antiserum, KOH+, oxidase− |
| Enterococcus | Gram staining (Gram+), characteristic colony on Enterococossal agar, catalase−, KOH−, oxidase− |
− = negative; BGA = Brilliant Green Agar; EMBA = Eosin Methylene Blue Agar; IMVic = Indole, Methylene Red, Voges-Proskauer, Citrate + = positive; MR = Methylene Red; VP = Voges-Proskauer broth; TSI = triple sugar iron; H2S = hydrogen sulphide; KOH = potassium hydroxide.
Antibiotic sensitivity testing
The Kirby-Bauer test, known as the disk-diffusion method, was employed to perform antibiotic sensitivity testing (AST).16 The confirmed colonies were inoculated in nutrient broth, and turbidity was maintained in 0.5 Mc Farland using a turbidity meter/photometer. The colonies were then incubated for 1–2 h and then streaked onto Muller–Hinton Agar. A disk of commonly available and practiced antibiotics in the poultry production system such as amikacin (AMK) (10 μg), penicillin G (10 units), tetracycline (30 μg), doxycycline (30 μg), ciprofloxacin (CFX) (5 μg), enrofloxacin (30 μg), trimethoprim-sulphamethoxazole (5 μg), levofloxacin (5 μg) and chloramphenicol (30 μg) per disc were inoculated onto the medium and incubated for 24 h. The following day, zones of inhibition around each antibiotic disk were calculated manually using a metric ruler across the zone of inhibition. The measurement was compared to the criteria set by the Clinical and Laboratory Standards Institute (CLSI; Wayne, PA, USA).17 Based on these criteria, the organism was then classified as being resistant (R), intermediate (I) or susceptible (S) to the antibiotic in question. Internal quality control for the disk diffusion tests was done using E. coli American Type Culture Collection (ATCC) 25922 and S. aureus ATCC 25923 as the control organisms. The laboratory is accredited with the Nepal Veterinary Council.
At the time of the study, antibiotic disks containing vancomycin or teicoplanin for assessing Enterococcus spp., and antibiotic disks containing ceftriaxone and meropenem for assessing Gram-negative bacteria were not available either in the laboratory or in the market for purchase. Antibiotic susceptibility/resistance patterns against these antibiotics could not therefore be assessed. Broth microdilution testing for assessing colistin resistance was also unavailable.
Data analysis and definitions
Data were collected into paper-based proforma and entered into MS Excel v13 software (Microsoft, Redmond, WA, USA). A descriptive analysis was performed using frequencies and proportions. We used the term “possible multidrug resistance (MDR)” to indicate resistance of a bacterial isolate to three or more antibiotic classes out of the six classes of antibiotics that we assessed in the laboratory in Nepal. Assessing for full MDR would have required assessing the bacterial isolates against the glycopeptide class (vancomycin or teicoplanin) for Enterococcus spp. and the cephalosporin (ceftriaxone) and carbapenem (meropenem) classes for E. coli,18,19 which could not be done for reasons given earlier.
Ethics
The necessary ethical approval to conduct this study was obtained from the Nepal Veterinary Council, Kathmandu, Nepal (no 9/2078/79, dated 23 July 2021).
RESULTS
There were 3,230 cloacal swabs received from broiler flocks and 3,230 cloacal swabs were obtained from layer flocks in the five selected provinces of Nepal.
Prevalence and geographic distribution of target bacterial species in cloacal swabs
The prevalence of Enterococcus spp., Salmonella spp. and E. coli in broiler birds and layer birds is shown in Tables 2 and 3, respectively. In broiler birds, Enterococcus spp. and Salmonella spp. were identified in respectively 36% and 39% of all cloacal swabs, while E. coli prevalence was higher, at 63%. There were some variations between the provinces, but E. coli was the predominant pathogen isolated in all provinces.
TABLE 2.
Prevalence of Enterococcus spp., Salmonella spp. and E. coli in broiler birds in the different provinces of Nepal
| Provinces | Cloacal swab n | Enterococcus spp. % | Salmonella spp. % | E. coli % |
|---|---|---|---|---|
| 1 | 646 | 30 | 47 | 62 |
| 3 | 646 | 27 | 41 | 69 |
| 4 | 646 | 47 | 36 | 62 |
| 5 | 646 | 38 | 35 | 80 |
| 7 | 646 | 28 | 38 | 48 |
| Total | 3,230 | 36 | 39 | 63 |
TABLE 3.
Prevalence of Enterococcus spp., Salmonella spp. and E. coli in layer birds in the different provinces of Nepal
| Provinces | Cloacal swab n | Enterococcus % | Salmonella % | E. coli % |
|---|---|---|---|---|
| 1 | 646 | 20 | 63 | 70 |
| 3 | 646 | 27 | 54 | 81 |
| 4 | 646 | 36 | 36 | 32 |
| 5 | 646 | 39 | 34 | 61 |
| 7 | 646 | 28 | 38 | 54 |
| Total | 3,230 | 31 | 48 | 60 |
In layer birds, the same overall pattern was observed. Enterococcus spp. and Salmonella spp. were identified in respectively 31% and 48% of all cloacal swabs, while E. coli prevalence was higher, at 60%. There were again some variations between the provinces, but in all provinces, except Province 4, E. coli was the predominant pathogen isolated.
Antimicrobial resistance patterns in broiler birds
AMR patterns for E. coli, Salmonella spp. and Enterococcus spp. in broiler birds are shown in Table 4. For all bacterial isolates, the lowest levels of resistance were observed with AMK. For E. coli, >50% isolates showed resistance to all the other nine antibiotics, except for enrofloxacin, at 48%. Resistance was highest with doxycycline, at 83%. For Salmonella spp., >50% of isolates showed resistance to all the other nine antibiotics, except for penicillin (23%) and enrofloxacin (40%). Resistance was highest with CFX, at 81%. For Enterococcus spp., >50% of isolates showed resistance to all the other nine antibiotics except for penicillin, at 41%. Resistance was highest with trimethoprim-sulfamethoxazole, at 84%.
TABLE 4.
Antibiotic resistance patterns to bacterial isolates in broiler chickens
| Antibiotics tested | Bacterial isolates | ||
|---|---|---|---|
|
| |||
| Resistance to E.coli (n = 2,047)* n (%) | Resistance to Salmonella spp. (n = 1,264)* n (%) | Resistance to Enterococcus spp. (n = 1,156)* n (%) | |
| Amikacin | 153 (7) | 90 (7) | N/A |
| Penicillin | N/A | N/A | 474 (41) |
| Tetracycline | 1,419 (69) | 822 (65) | 867 (75) |
| Doxycycline | 1,696 (83) | 834 (66) | 613 (53) |
| Ciprofloxacin | 1,229 (60) | 948 (75) | 855 (74) |
| Enrofloxacin | 1,085 (48) | 506 (40) | 890 (77) |
| Trimethoprim-sulfamethoxazole | 1,468 (72) | 1,023 (81) | 971 (84) |
| Levofloxacin | 1,456 (71) | 809 (64) | 659 (57) |
| Chloramphenicol | 1,264 (62) | 544 (43) | 821 (71) |
* Positive isolates.
N/A = not available.
Antimicrobial resistance patterns in layer birds
AMR patterns for E. coli, Salmonella spp. and Enterococcus spp. in layer birds are shown in Table 5. For all bacterial isolates, as with the broiler birds, the lowest resistance was observed with AMK. For E. coli, >50% of isolates showed resistance to all the other nine antibiotics. Resistance was highest with CFX, at 88%, but resistance >80% was also found with trimethoprim-sulfamethoxazole, levofloxacin and enrofloxacin. For Salmonella spp., >50% of isolates showed resistance to all the other nine antibiotics, except for penicillin (24%). Resistance was highest with tetracycline, at 80%. For Enterococcus spp., >50% of isolates showed resistance to all the other nine antibiotics except for penicillin, at 45%. Resistance was highest with CFX and trimethoprim-sulfamethoxazole, at 80%.
TABLE 5.
Antibiotic resistance patterns to bacterial isolates in layer flocks
| Antibiotics tested | Bacterial isolates | ||
|---|---|---|---|
|
| |||
| Resistance to E.coli (n = 1,945)* n (%) | Resistance to Salmonella spp. (n = 1,553)* n (%) | Resistance to Enterococcus spp. (n = 989)* n (%) | |
| Amikacin | 139 (7) | 78 (5) | N/A |
| Penicillin | N/A | N/A | 445 (45) |
| Tetracycline | 1,310 (67) | 1,242 (80) | 762 (77) |
| Doxycycline | 1,383 (71) | 1,118 (72) | 742 (75) |
| Ciprofloxacin | 1,705 (88) | 1,118 (72) | 791 (80) |
| Enrofloxacin | 1,619 (83) | 761 (49) | 722 (73) |
| Trimethoprim-sulfamethoxazole | 1,698 (87) | 978 (63) | 791 (80) |
| Levofloxacin | 1,622 (83) | 761 (49) | 673 (68) |
| Chloramphenicol | 1,524 (78) | 745 (48) | 534 (54) |
* Positive isolates.
N/A = not available.
Possible multidrug resistance in broiler and layer birds
The prevalence of possible MDR was high (Table 6). For broiler and layer birds, possible MDR ranged from 37% to 44% for E. coli, 45–46% for Salmonella spp. and from 13% to 17% for Enterococcus spp.
TABLE 6.
Possible MDR to E. coli, Salmonella spp. and Enterococcus spp. in broiler and layer birds
| Types of bird | Bacterial isolates | Total n | MDR n (%) |
|---|---|---|---|
| Broiler birds | |||
| E.coli | 2,047 | 757 (37) | |
| Salmonella spp. | 1,264 | 581 (46) | |
| Enterococcus spp. | 1,156 | 150 (13) | |
| Layer birds | |||
| E.coli | 1,945 | 856 (44) | |
| Salmonella spp. | 1,553 | 699 (45) | |
| Enterococcus spp. | 989 | 169 (17) |
MDR = multidrug resistance (defined as resistance to three or more classes of antibiotics out of the six classes which were assessed).
DISCUSSION
This study in five provinces of Nepal assessing the prevalence of selected bacteria and their AMR patterns in poultry production had two key findings. First, between one third and one half of the birds that were studied using cloacal swabs were infected with Enterococcus or Salmonella spp. In contrast, nearly two-thirds of the birds were infected with E. coli. In broiler birds, there was a higher prevalence of Enterococcus spp. and E. coli compared with layer birds, while in layer birds there was a higher prevalence of Salmonella spp.
While Salmonella spp. and E. coli have been reported in the poultry system previously in Nepal, we have found no previous reports on Enterococcus spp. However, in other countries Enterococcus spp. have been found in commercial poultry flocks, and the bacteria was associated with an increased death rate.20–22
While there have been a few studies in Nepal assessing the prevalence of non-typhoidal Salmonella spp. in environmental samples from poultry farms, chicken meat, necropsy samples and slaughter house samples,23–28 we believe that this is the first study in Nepal to assess the prevalence of this bacteria in cloacal swabs taken from live birds. Our study found higher prevalence rates of Salmonella spp. than in these previous studies,23–28 and this variation may be due to the different sampling methods used and the season during which the studies took place. Cloacal swabs are likely to provide a more accurate picture of bacterial burden due to minimal cross-contamination; two studies conducted in India and Uganda showed prevalence rates similar to what was found in our study.29,30
The high prevalence of E. coli was not surprising. In Nepal, colibacillosis caused by E. coli is a leading cause of disease in the poultry enterprise, where it contributes to 26% of the entire burden of illness in poultry flocks.25 The high prevalence of E. coli in our study is similar to that found elsewhere,31,32 although higher prevalence rates of ⩾75% have been reported in Egypt.33
Second, although there was a low prevalence of resistance to AMK in our study, levels of resistance to the other nine tested antibiotics in layer birds were high, indicating a higher prevalence of resistance than in broiler birds.
For E. coli, which may cause urine tract infections in humans, the levels of resistance observed among the commonly used antibiotics to treat these infections, such as CFX and trimetho-prim-sulfamethoxazole, ranged from almost 60–95%. These findings concur with other studies,34 although lower levels of resistance have been found elsewhere.29,33 The varying levels of resistance relate to differences in farming and veterinary practices, as well as the injudicious use and misuse of antibiotics in different settings. For Salmonella spp., levels of resistance were similarly high, which ranged from 60% to 80% for the same two antibiotics, CFX and trimethoprim-sulfamethoxazole. These findings concurred with what was reported previously from Egypt.35 Our study found high levels of antibiotic resistance to Enterococcus spp. Possible MDR prevalence of the three bacteria was high. Resistance was highest for Salmonella spp., approaching nearly 50%, followed by E. coli and then Enterococcus spp., in which the prevalence was <20%. These results agree with findings from elsewhere.30,31,35
The strengths of the study were the large numbers of broiler and layer birds included in the study and sampling from different regions of the country. The collection of cloacal samples and the microbiological work were also done to high standards.
However, there were some limitations. The most important of these was our inability to procure antibiotic disks for assessing susceptibility/resistance to vancomycin, ceftriaxone and meropenem. While these antibiotics are not commonly used in poultry practice in Nepal, they are important for managing infections in humans caused by these zoonotic pathogens. Their availability would have expanded the number of antibiotic classes, and thus the denominator, with which to formally determine MDR prevalence.18,19 As a result of this limitation, the MDR rates determined in the study are probably an underestimation of the true rate; hence, our use of the term “possible MDR”. It is intended to revisit susceptibility testing of the isolates against vancomycin, ceftriaxone and meropenem as part of a separately reported study. Other limitations included difficulties in moving samples around the country while COVID-19 restrictions were in place, and our lack of qualitative research to understand more about the use of antibiotics in the poultry system.
Despite these limitations, the study highlights the large number of boiler and layer birds in Nepal whose cloacal swabs contain important potentially pathogenic bacteria (E. coli, Salmonella and Enterococci spp.), a large proportion of which are resistant to commonly used antibiotics. This poses an enormous threat to animal, as well as human health, with transferable antibiotic resistance passing from animals to humans, either directly through the food chain or indirectly, through the use of animal manure for vegetables and through contaminated soil and water. Resources for a better understanding of the most common inappropriate uses of antibiotics and the enactment of legislation to ensure their cessation or severe restriction are urgently required.
CONCLUSION
In five provinces of Nepal, 6,460 cloacal swabs (3,230 from broiler and 3,230 from layer poultry birds) were collected and examined for the presence of three bacteria (E. coli, Salmonella spp. and Enterococcus spp.) and their AMR patterns. Between one third and one half of the birds were infected with Enterococcus or Salmonella spp., while nearly two-thirds were infected with E. coli. Apart from AMK, there were high levels of resistance to the nine other antibiotics assessed, including CFX and trimethoprim-sulfamethoxazole, as well as high levels of possible MDR. There is an urgent need to better understand the most common inappropriate usages of antibiotics in Nepal and institute legislation to ensure that these are minimised as far as possible.
ACKNOWLEDGEMENTS
The authors thank everyone at the poultry farms who assisted with the collection of samples and the microbiology laboratory personnel for their work in identifying bacterial isolates and analysing AMR patterns.
Funding Statement
This study was funded through the small grants scheme of the Special Programme for Research and Training in Tropical diseases (TDR), WHO, Geneva, Switzerland.
Footnotes
Conflicts of interest: none declared.
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