Jan 15, 2025

Public workspaceisolation of Salmonella and pathogenic E. coli in broiler farms in Uganda

DOI
dx.doi.org/10.17504/protocols.io.kxygxw17ov8j/v1
  • 1Mbarara university of science and technology
Nalumaga Pauline Petra
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DOI: dx.doi.org/10.17504/protocols.io.kxygxw17ov8j/v1
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Protocol Citation: Thomas Ssemakadde, Nalumaga Pauline Petra, Jude Collins Busingye, Joel Bazira, Kabanda Taseera 2025. isolation of Salmonella and pathogenic E. coli in broiler farms in Uganda. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxw17ov8j/v1
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THE MANUSCRIPT IS UNDER REVIEW IN PLOS ONE (PONE-D-24-33064R2)
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 14, 2025
Last Modified: January 15, 2025
Protocol Integer ID: 118308
Keywords: Antimicrobial resistance, pathogenic E.coli, Salmonella species, One Health
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Abstract
This study investigated the prevalence, antimicrobial resistance, and extended-spectrum β-lactamase (ESBL) production in Escherichia coli and Salmonella isolated from broiler farms in Wakiso district, Uganda. A cross-sectional design was employed between August and September 2021. A total of 216 farms were selected using simple random sampling, and pooled samples of fresh fecal droppings and cloacal swabs were collected. Microbiological isolation and identification were conducted using on selective media, followed by phenotypic and genotypic assays to determine antimicrobial resistance profiles.

Antibiotic susceptibility testing was performed using the Kirby-Bauer disk diffusion method, and results were interpreted according to CLSI 2020 guidelines. Phenotypic ESBL production was screened using the double-disc synergy method. DNA extraction and conventional PCR were employed to confirm the presence of the blaTEM resistance gene. Sdata was analysed using Stata to identify significant associations between variables.
The findings highlight a significant prevalence of antimicrobial resistance and the emergence of ESBL-producing Escherichia coli and Salmonella in broiler farms. This calls for antimicrobial stewardship in poultry production.
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Materials
This study was conducted in Wakiso district a metropolitan district in the central region of Uganda that partly encircles Kampala district. The district coordinates are 00 24N, 32 29E. With a projected population of 2,915,200 million, Wakiso district is still Uganda's most populous Higher Local Government (HLG) (1). The population is projected to grow at 4.1% annually, which increases the demand for food, particularly poultry productsThe district was regarded as the top producer of poultry, producing 7.4% of all the chickens in the country (2).
Study design
This was a cross-sectional study focused on broiler farms from August to September 2021 based on the Food and Agriculture organization (FAO) categorization sector level 3 within selected counties in Wakiso district.
Sample Size Determination
The sample size was determined using Kishi-Leslie (1965) formula using 83% prevalence (3).
          n= Z2 P(1-P)
                     d2
n = study sample size required
Z= critical value associated with 95% confidence interval = 1.96
P = Estimated prevalence of 83%of E coli and salmonella d = margin of error = 0.05
 n= 1.962 (0.83x 0.17)     
                     0.052
n= 216 samples
Therefore, 216 farms within Wakiso District were considered. The farm managers of the farms were interviewed to understand the associated factors that predispose these pathogens with consent.
Sample Collection
Simple random sampling to select the sub-counties was employed while purposive sampling used the information provided by the district veterinary office to identify the villages.   For the selected and eligible farms three swabs from fresh fecal droppings and two swabs from the cloaca were collected from randomly selected old (5-7weeks) birds (4). Five swabs from one farm on different chickens were considered to represent one sample (Pooling of samples).   The swabs were placed into a plain labeled tube containing Amie’s transport media without charcoal. This was then put into a cool box and transported to the laboratory within 24 hours.
Isolation and identification of microorganisms
The pooled samples were aseptically mixed into 9 ml of autoclaved Buffered Peptone Water (BPW) (Hi Media M1494, Mubi, India) in a sterile 50ml falcon tube with a lid to generate a pre-enriched sample and incubated for 16-24 hours at 37oC aerobically.
The enriched sample was picked and inoculated onto a ChromagarTM of Salmonella designed for the presumptive identification, qualitative direct detection, and differentiation of Salmonella species (5). Plates were incubated at 370C for 24 hours. Presumptive colonies for Salmonella (mauve-pink, raised, and smooth colonies) were selected for identification.
For isolation of pathogenic E.coli, the pre-enriched sample was inoculated on Sorbitol MacConkey agar plates and incubated at 37 °C for 24 hours. The colonies of pathogenic E. coli were smooth, raised, had entire margins, and were colorless on SMAC.
The presumptive organisms were subcultured on nutrient agar and identified through gram staining, Triple Ion Sugar (TSI) test, the IMViC test, Methyl Red, Voges- Proskauer, Sulphur indole motility, and Simmons citrate utilization (figure 2). All reagents used were from (Oxoid, England) (6).
Bacterial antibiotic susceptibility testing
Bacterial suspension was adjusted to 0.5McFarand standard and inoculated on Muller Hinton agar medium (Oxoid CM0337 Basingstoke, England) using surface spreading method. The antibiotic discs listed in Table 1 (all from Oxoid, England) were used. The plates were incubated for 24 hours at 37°C. The results were read and interpreted according to Clinical Laboratory Standards Institute, 2020 (7). E. coli ATCC 25922 (American Type Culture Collection, Rockville, MD, USA) was used as a reference control strain.
Table 1: Commonly used antibiotics at NaLIRRI, 2022 obtained from Oxoid Ltd suppliers, and the discs were selected based on CLSI (28th Edition, 2020) and their availability at the NALIRRI (National Livestock Resources Research Institute) Microbiology laboratory.
Antibiotic Class Antibiotics used for both E. coli and Salmonella isolates
Aminoglycosides Gentamicin (GM) (10 μg/ml)
Carbapenem Meropenem (MEM) (10 μg/ml)
Cephalosporins class III Ceftriaxone (CRO) (30 μg/ml)
Amphenicol Chloramphenicol (CHL) (30 μg/ml)
Cephalosporins class IV Cefepime (CPM) (30 μg/ml), Cefotaxime (CTX)(30µg/ml), Ceftazidime (CTZ) (30µg/ml)
Quinolones Ciprofloxacin (CIP) (5 μg/ml)
Macrolides Erythromycin (EM) (30 μg/ml)
Penicillin Ampicillin (AMP) (10 μg/ml)
Tetracyclines Tetracycline (OXT) (30 μg/ml)
Sulphonamides/ Trimethoprim Co-trimoxazole (CXT) (25 μg/ml)
Phenotypic Screening of Enterobacteriaceae for Extended-spectrum β-lactamases (ESBLs) production
Cefotaxime (30μg) and Ceftazidime (30μg) antibiotic discs were used to phenotypically test Enterobacteriaceae for the development of ESBLs and incubated overnight at 37 0C on Muller Hinton agar (7).
Double Disc Synergy method
A sterile cotton swab was used to surface spread the bacterial suspension onto a Mueller Hinton agar plate. The antibiotic discs used were Ceftazidime (CAZ) (30μg) alone and Ceftazidime in combination with Clavulanic acid (CAL) (30/10μg) (8). The discs were spaced approximately 30 mm apart. The plates were incubated at 37° C overnight. Both the single disc and the combined disc's zones of inhibition were measured. The results were interpreted according to CLSI guidelines (8).
Genomic DNA extraction
Bacterial genomic DNA extraction was conducted on all 18 Salmonella and 57 Pathogenic E.coli following manufacturer instructions of the Bio line ISOLATE II genomic DNA kit (Cat No. Bio-52065 Lot No. IS502-B054750). The presence of genes encoding for ESBL (blaTEM gene) was detected using conventional PCR amplification using primers listed in (table 2).
Table 2: Primers used in the PCR reaction.
blaTEM (F) TGG GTG CAC GAG TGG GTT AC 526 58 Tenover et al., 1994
(R) TTA TCC GCC TCC ATC CAG TC
The PCR master Mix reagents were prepared by mixing 12.5 µL master mix consisting of One Taq quick load two times master mix/w standard buffer, dNTPs and Taq polymerase (M0486S), 1.5 µL forward (100 µM), 1.5 µL primary reverse (one hundred µM), and5 µL DNA template and RNAse-free dH2O up to 25 µL.
The PCR process was carried out in a thermocycler (Perkin Elmer, Wellesley, MA, USA) with a pre-denaturation cycle of 95°C for 15 min, followed by a DNA amplification stage with 30 cycles (94°C for 1 min, 58°C for 1 min, and 72°C for 1 min) and final extension cycle of 72°C for 5 min.  
DNA Amplicons were electrophoresed using 1.5% agarose gel, in Tris-Borate EDTA buffer (TBE) 1×concentration, Safe View ClassicTM DNA stain, 6x loading dye (Thermo Scientific), and DNA ladder/marker 100 bp (Sigma-Aldrich, Inc., Saint Louis, MI, USA)DNA Bands were visualized on a Dark reader Transilluminator.
Data Management and Analysis
All records of the analysis were recorded in the lab register as a hard copy backup. Samples were assigned codes and Excel spreadsheets were used to enter the raw data, which were then exported to Stata (Version 12, Special Edition, College Station, Texas USA) for analysis. Double entry of data was done to rule out any errors. Frequency tables and graphs were used to present descriptive statistics.
 The 95% level of significance where the Pearson value (p < 0.05) determined the variables of statistical significance of Quantitative data.
Ethical Consideration
Approval was obtained from Mbarara University of Science and Technology; the Institutional Ethical Review Committee (MUST-2021-141), and at the ministry level, the permanent secretary Ministry of Agriculture, Animal Industries, and Fisheries, the district's chief administrative officer, and the district veterinarian.
Before engaging the farms, we sought clearance from the farm owners to access and collect samples from their farms. Data and bacterial isolates obtained for this investigation was handled with confidentiality.
Safety warnings
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Ethics statement
"Approval was obtained from Mbarara University of Science and Technology; the Institutional Ethical Review Committee (MUST-2021-141), and at the ministry level, the permanent secretary Ministry of Agriculture, Animal Industries, and Fisheries, the district's chief administrative officer, and the district veterinarian."
Before start
The emergence and re-emergence of zoonotic bacterial infections and the upsurge reflected in current trends of antimicrobial-resistant bacteria is a major global concern. Salmonella spp and Escherichia coli (E. coli) are the two most important food-borne pathogens of public health interest incriminated in poultry products worldwide hence necessitating constant monitoring of microbial food safety measures.
Prevalence and antimicrobial resistance of Salmonella and pathogenic E. coli in broiler farms, Wakiso district, Uganda
Prevalence and antimicrobial resistance of Salmonella and pathogenic E. coli in broiler farms, Wakiso district, Uganda
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Protocol references
1.           Statistics UE. Uganda Bureau of Statistics. Energy. 2022;95(13.6):14.5.
2.           Sebuliba-Mutumba R, Kibwika P, Kyazze F. Drivers of collective action and innovation adoption in successful poultry farmer groups in Wakiso District in Uganda. African Journal of Rural Development. 2017;2(3):353-65.
3.           Varga C, Guerin MT, Brash ML, Slavic D, Boerlin P, Susta L. Antimicrobial resistance in fecal Escherichia coli and Salmonella enterica isolates: a two-year prospective study of small poultry flocks in Ontario, Canada. BMC Veterinary Research. 2019;15(1):464.
4.           Mensah GI, Adjei VY, Vicar EK, Atsu PS, Blavo DL, Johnson SAM, et al. Safety of retailed poultry: analysis of antibiotic resistance in Escherichia coli from raw chicken and poultry fecal matter from selected farms and retail outlets in Accra, Ghana. Microbiology Insights. 2022;15:11786361221093278.
5.           Hoorfar J, Baggesen DL. Importance of pre-enrichment media for isolation of Salmonella spp. from swine and poultry. FEMS microbiology letters. 1998;169(1):125-30.
6.           Sarba EJ, Kelbesa KA, Bayu MD, Gebremedhin EZ, Borena BM, Teshale A. Identification and antimicrobial susceptibility profile of Escherichia coli isolated from backyard chicken in and around ambo, Central Ethiopia. BMC Veterinary Research. 2019;15:1-8.
7.           CLSI. Performance standards for antimicrobial susceptibility testing, document M100. Clinical and Laboratory Standards Institute Wayne, PA; 2020.
8.           Yılmaz M, Ak Ö, Hacıseyitoğlu D, Alkan S. A comparative study of VITEK-2, Double Disc Synergy and Combined Disc Methods for detection of ESBL (Extended Spectrum Beta-Lactamase) production in Escherichia coli and Klebsiella pneumoniae strains. Journal of Contemporary Medicine. 2022;12(1):116-20.