1. Veterinary Officer at Veterinary Laboratory, Surkhet, Nepal; prativavetdr@gmail.com
2. Veterinary Chief, Veterinary Laboratory, Surkhet, Nepal; drbikhashshrestha@gmail.com
3. Veterinary Officer at Veterinary Laboratory, Surkhet, Nepal; g10drashrestha@gmail.com
4. National Coordinator at FAO, Nepal; karki@fao.org
5. Himalayan College of Agriculture Science and Technology, Nepal; jasmineadhikary909@gmail.com

*             Correspondence: prativavetdr@gmail.com

Keywords: Escherichia coli, chicken meat, food borne infection, Surkhet

1. INTRODUCTION

Escherichia coli is a known most occurrence bacterial isolates food borne pathogens.It is found in contaminated chicken meat and importance indicator food borne infection with this bacterium ( Parvin, 2020 ). Antimicrobial resistance patterns of E. coli both clinical and food borne pathogens increased at a speedy rate. (NARMS). E.coli is most common species of facultative anaerobe found in the gastro- intestinal tract of both man and animals, most commonly encountered pathogen in the Enterobacteriaceae family (Lim et al., 2010). E.coli is one of the major food borne bacterial pathogen causing watery and bloody diarrhea e.g. E.coli 0157; H7, as life threatening disease such as hemorrhagic colitis, hemolytic uremic syndrome and thrombotic thrombocytopenic purpura in human (Ameer et al., 2021). In the poultry sector, the huge losses arises in the economic farmer and AGDP of country due to the effect of AMR( Mellata,2013). Unaware of antimicrobial use among poultry farmer worldwide have determined and addresses to the emergence and spread of AMR ( Alhaji NB,2018 ). Poultry farmers has also influenced for the use of the high range of antibiotics at one time or in a period of time. This high demands often drives the treatment and enhance production (Malana S.,2022). This issues can derived by the easily availability and sale of antibiotics in drug store and through street vendors (A1 Masud A.,2020 ). Globally presence of E.coli isolates and  antimicrobial resistance E.coli isolated from poultry at farm level (ByrneN,2022).The resistant patterns of E.coli spps is increasing in different antibiotics( Tetracycline 54.6% and ampicillin 54%) (Steward Mudenda,2023). At Veterinary Laboratory Surkhet 31% of postmortem samples were E.coli is positive.  Veterinary Disease Investigation Laboratory(VDIL), Surkhet of the Karnali Province is situated in Birendranagar Municipality-7, Surkhet and was established in FY 1988/1989 AD. It provides laboratory diagnostic services to 10 districts of Karnali Province and 6 districts of Lumbini Province  all together 16 districts. To provide the diagnostic livestock services smoothly, three districts (Banke, Dang and Jumla) have established basic laboratories. They perform parasitological examinations, microbiological culture and antibiotic sensitivity test.  The mission of the VDIL, Surkhet is to promote the health of livestock, poultry and ensure safe animal products for consumer by assisting VH and LSEC’s offices, veterinarians, clients, and others who are responsible for animal health in detection and prevention of animal diseases. Annual report AMR.  Central Veterinary Laboratory (CVL) is a national reference laboratory for antimicrobial surveillance so this active surveillance in Kathmandu district will be conducted by CVL and CVL will lead over all the activities related to AMR active surveillance in other veterinary laboratory as well.  There are many broilers and layers farms located in different VDCs at Surkhet area. Both commercial and semi commercial farms of different sizes are available in the Surkhet. So, the sampling sites were try to cover the poultry rearing area at Surkhet. According to the statistical analysis report of Ministry of agriculture ministry,Karnali Pradesh about 21,23003 poultry farm is over there.And in Surkhet Districts about 10,000 population of poultry of small and large farm is running. Mudenda 2023 studied that 92.9% (339/365) prevalence of E.coli isolates from Cloacal swab of live broiler chicken in Bangladesh .Saeed,2023 had found that out of the 320 samples, 164 (51.3%) were confirmed as E. coli isolates from cloacal swab of backward chicken farming in Pakistan .Mgaya 2021 had also found that 212 (55.2%) out of 384 samples were positive for E. coli from cloacal swab of poultry from different poultry swabs.Sapkota,2020 had studied and analysed 31 positive E.coli samples out of 100 samples of suspected E.coli from postmortem of poultry received at Veterinary Laboratory, Surkhet. Similarly another passive surveillance data of Veterinary Laboratory, Surkhet indicated that 49/50 postmortem samples were found E.coli positive.( F/Y 2080/81). J.Rahamani, 2020 research study had showed that out of 60 cloacal samples 58 is confirmed E.coli isolates. S.S. deven,2022 had studied that 91 E.coli isolates were confirmed from 320 cloacal swab of broiler chicken.Kitti,2021 had also studied that 402 cloacal sample from different poultry farms were found 100 % confirmed by Conventional and malditoff identification system. Amadi,2015 had found that out of 202 E.coli isolates resistance patterns were tetracycline (29.7%), followed by ampicillin (6.9%), gentamicin (5.9%), and trimethoprim-sulfamethoxazole (3%).Total 31 % were expressed resistance with 3 or more antibiotics. J. Rahamanni,2020 had interpreted 48% of samples were resistant to tetracycline, 38% resistance to ciprofloxacin, 16% resistance to chloramphenicol, 10% resistance to gentamicin accordingly. S.S. Deven,2022 had determined that most of the isolates were resistant to tetracycline (95.24%), chloramphenicol (85.71%), and sulphamethoxazole/ trimethoprim (85.71%). Kitti,2021 had found his research in E.coli isolates was the highest resistance was observed in ampicillin (100%), whereas the lowest resistance was recorded for gentamicin (10.3%). The (86.76%) of E.coli isolates were multidrug resistant.

2. MATERIALS AND METHODS

The sampling has been done to represent birds produced from small, medium, large farms and small subset of backyard birds. For this surveillance, following farms sizes would be considered as small, medium, and large farms which was finalized via expert opinion in  technical working group meeting  based on poultry farming scenario of  Nepal which  corresponds with commercial broiler and layers farm size in Nepal.

Table 01: Classification of farm sizes for broilers and layers

Birds	Farm sizes (number of birds)
	Small	Medium	Large
Broiler	<500	500- 2500	>2500
Layer	<1000	1000- 2500	>2500

The details of the number of samples for broilers and spent layers representing different farm sizes to be collected by each laboratory in their catchment area have been described in the surveillance protocol for active surveillance for poultry which has been finalized by the Technical Working Group – Animal Health based on the framework provided by the Massey University.  The prevalence of resistance to the various antibiotics in the different bacteria/bacterial groups is unknown (ref). The number of isolates required to estimate prevalence vary with the expected prevalence and the level of precision required for the prevalence estimate, as shown in Table 2. Highest numbers are required to estimate prevalence levels of 50% and numbers increase with higher levels of precision for the estimate.

Table 02: Number of bacterial isolates required to estimate prevalence of resistance to a specific antibiotic in an organism for two levels of precision (5% and 10%). (Extracted from OIE Terrestrial Animal Health Code)

Expected AMR prevalence	Number of samples
	Desired precision
	10%	5%
10%	35	138
20%	61	246
30%	81	323
40%	92	369
50%	96	384
60%	92	369
70%	81	323
80%	61	246
90%	35	138

Table 3 tells the number of bacterial isolates required for the estimation of prevalence of resistance in that bacterial genus or species, not the number of samples to be taken. The number of birds that need to be sampled to produce the target number of bacterial isolates will depend on the prevalence of each bacterial infection in the population being sampled. For example, if the expected prevalence of the organism of interest in the population is 50%, then the number of samples which are needed for the prevalence estimates in Table 3 is double the number shown in the Table. The prevalence of the different bacteria is highly variable, ranging from high prevalence levels for E coli and lower prevalence levels for Salmonella.

Table 03: Expected prevalence of different bacterial infections in poultry

Bacteria	Expected prevalence in poultry population
E. coli	80 – 100%
Salmonella	5 – 20%
Campylobacter	80%
Enterococcus	50%

The samples was collected from  slaughterhouses and farms. Sampling at collection points in the chicken marketing chain, such as slaughterhouse or near the live bird market is generally the most cost-effective location to collect samples. So, GPS coordinates of each samples was recorded. It will also help to find out the exact location of E,coli isolates. In both types, the birds ready for selling or slaughtering will be chosen. A total of 53 cloacal swab samples of poultry were taken along with questionnaire and GPS coordinates from different area of Surkhet i.e. Chinchhu,Gurvakot,Birendranagar,.

Figure 01: Sample Location

According to active surveillance protocol of Fleming Fund as shown,from each farm one samples were collected.

Table 04:  Sampling area of Surkhet valley

VDCs of Surkhet	Total Farm	No. of Samples
Bheriganga	11	11
Melkuna	11	11
Gurvakot	13	13
Birendranagar	18	18
Total	53	53

Collected Cloacal swab samples were brought to the laboratory with cold chain maintain and sterilization procedure..As much as soon, these samples were kept in incubator for 37 ⁰C for 24 hours. Conventional method of Active surveillance protocol methodology for E.coli isolates were applied. All confirmed positive E.coli isolates were further applied Kirby-Beaur’s method i.e. Disc diffusion Test for the determination of antibiotic sensitive and resistance patterns. The standard CLSI Guidelines were followed for interpretation of the results.

Table 05: Antibiotic susceptibility profile for selected antibiotics for E.coli isolate

S. No.	Antibiotic name	Concentration	Sensitive	Intermediate	Resistance	Remarks
1.	Tetracycline	30 μg	≥ 15	12-14	≤ 11
2.	Gentamicin	10 μg	≥ 15	13-14	≤ 12
3.	Ampicillin	10 μg	≥ 17	14-16	≤ 13
4.	Cotrimoxazole	25 μg	≥ 16	11-15	≤ 10
5.	Ciprofloxacillin	5 μg	≥ 21	16-20	≤ 15
6	Cefoxitime	30 μg	≥22	18-20	≤ 21

Source: CLSI Standard (2019)

After findings the confirmed no. of E.coli isolates of different area of Surkhet Valley.The no. of E.coli isolates percentage is calculated by the formula (no. of positive E.coli cases/total no. of samplesX100).Similarly the sensitive and resistance percentage of E.coli isolates had been performed accordingly.Significant difference on different commonly used antibiotics association with different area of Surkhet area has been analyzed by P-value  using Chi square Test by using Excel programme. GPS coordiantes were also recorded and analyzed by putting the value of longitudinal and lattitude value of each sample area to indicates the present of E.coli isolates over there.

3. RESULTS AND DISCUSSION

The AMR was detected in 96.5% (n = 50/53) of the isolates, of which 100% (n = 50) were multidrug-resistant (MDR). E. coli was highly resistant to cotrimox (100%) and ampicillin (100%), tetracycline (96%), resistance to Ciprofloxacillin (50%), and were represents of each area of surkhet valley is presented in the Table no.6.

Table 06: No. of E.coli isolates found in different area of Surkhet Valley

VDCs of Surkhet	Total Farm	No. of Samples	Positive E.coli samples
Bheriganga	11	11	11
Melkuna	11	11	11
Gurvakot	13	13	13
Birendranagar	18	18	15
Total	53	53	50

Table 07: E.coli Sensitive and Resistance Patterns from 50 Cloacal swab of Poultry

S.no.	Place	G @		Cip @		Tet @		Cot @		Cx @		Ap @		Total
		S	R	S	R	S	R	S	R	S	R	S	R
1.	Bheriganga	1	10	7	4	0	11	0	11	1	10	0	11	66
2.	Gurvakot	1	12	7	6	2	11	0	13	0	13	0	13	78
3.	Melkuna	0	11	5	6	0	11	0	11	1	10	0	11	66
4.	Birendranagar	0	15	6	9	0	15	0	15	8	7	0	15	90
Total		2	48	25	25	2	48	0	50	10	40	0	50	50

Of the 53 samples, E.coli was isolated from 94% (n =50 ) from this active surveillance study. Percentage of E.coli isolates from Gurvakot 100%(13/13),Bheriganga100%(11/11),Melkuna100%(11/11),Birendranagar83.3%(15/18) is observed is given in the table

Table 08: Cefoxitime Antibiotics sensitivity significance difference analysis of E.coli

Area	IS	R	Row Totals
Bheriganga	1  (3.08)  [1.40]	10  (7.92)  [0.55]	11
Birendranagar	9  (4.20)  [5.49]	6  (10.80)  [2.13]	15
Gurvakot	1  (3.64)  [1.91]	12  (9.36)  [0.74]	13
Melkuna	3  (3.08)  [0.00]	8  (7.92)  [0.00]	11
Column Totals	14	36	50  (Grand Total)

The chi-square statistic is 12.2322. The p-value is .006629. The result is significant at p < .05. Since the  p value>0.05,we fail to reject null hypotheis.So, there is There is no difference in the resistance of E coli against Cefoxitime across the local level of Surkeht (P>0.05).

Discussion

The study was focused mainly on presence of E. coli isolates with its sensitive resistance pattern with different but common antibiotics in districts of Surkhet valley. Overall percentage of E. coli isolate in the present study was found to be 94%. The E. coli isolates found in the present study simulate with that found by Kitti et.al. (2021) who had found E.coli in 100% of cloacal samples of poultry. J. Rahamaanni et al. (2020)   had found E. coli isolate 98% in retail shop meat producing broiler, which was also similar to the present findings. Little higher percentage (92.9% (339/365)  of prevalence of E.coli isolate was recorded in poultry cloacal swab collected by Mudenda (2023) in Bangladesh. Lamichhaane Udhav,2020 had studied that the 36% of E.coli were positive from suspected case of poultry liver at Veterinary Laboratory,Surkhet. This active surveillance study indicated that the highest resistance patterns of different antibiotics from E.coli isolates( cotrimox(100%) and ampicillin (100%), tetracycline (96%), resistance to Ciprofloxacillin (50%) and 100% of E.coli isolates showed MDR resistance. The similar findings  by Kitti,2020 had found Ampicillin 100% resistant.s.Deven,2022 had determined the 95.24% and 85.7% in Tetracycline and Trimethosulprim.Kitti,2020 also found out the 86.76% E.coli isolates showed 86.76%MDR patterns.amadi,2015 had find out the minimum resistance percentage in Tetracycline 29.7%, Ampicillin 6.9%, Gentamycin5.9%,trimethosulprim 3% and 31% of E.coli showed 31% only. On the other hand, Cefoxitime which are not commonly used were also showed 90% resistance.The commonly used antibiotics are easily affordable and available over the counter (Lupindo A.M.,2014).Farmers were also unaware about the use of antibiotics and may not follow withdrawal period(Kimra ZI,2020). Thus this study indicates that the number of E.coli isolates increasing day by day. Different active surveillance study had also determined that the E.coli isolates present in the environment as a commensal organisms. Similarly, the antibiotic sensitive and resistance patterns of E.coli isolates were present in maximum number.This study also highlighted the MDR E.coli is also present in environment. These findings also indicates and suggestions supporting the more research need for their continuous surveillance. The finding of common antibiotics resistance may directly effect along the food chains in other ecosystems(Salah FD,2019).Therefore strict hygienic regulations of antibiotic used should be prepared and implemented. This type of antibiotics should be banned their use in animal food production.

4. CONCLUSION

This surveillance indicates that high number of E.coli isolates are present in the environment as a commensal microorganism in poultry species. This study reveals that the higher percentage of E.coli isolates were present in the poultry samples. With relation to the antimicrobial resistance, this study found that the higher percentage of antibiotic resistant E.coli is present even with the limited samples which could be an adverse health consequence for animal and human. Therefore, with the huge support from Fleming Fund country Grant FHI 360 Nepal had also facilitated the biorepository management of different bacteria isolated in the Laboratory. Veterinary Laboratory, Surkhet had also developed Biorepository management with protocol as given in Annexes. 5 of this E.coli isolates by sending each copy of E.coli to Central Veterinary Laboratory, Tripureswor for further findings and research. Following are the key Findings of this study:

• E.coli isolates are major findings pathogens in poultry industries.
• Resistance patterns of E.coli isolates with common used antibiotics is higher.
• Besides that resistant patterns of uncommon used antibiotics like Cefoxitime is also showed higher.
• Multi drug resistant E.coli were observed 100% from selected caecal swab of poultry at different area of Surkhet Valley.

5. RECOMMENDATION

From this active surveillance cross sectional study, we strongly recommended the revised and updates the act and policies related to antimicrobial use and withdrawn period of different commonly used antibiotics in different sectors.  This AMR problem causing adverse effect on animal health, human health and environment. So to minimize the antimicrobial resistance effects, we have to go through One Health approach program to tackle the AMR problems in the country. Therefore, Fleming Fund Mott Macdonald provided the huge contributions and capacity building and infrastructure development through One Health program. Fleming fund country Grant FHI 360 Nepal initiated this step from 2018 to till now.

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