{"paper_id":"49e2e3e8-c735-4d3b-acb7-65e61174846b","body_text":"1 Prevalence, associated factors and antimicrobial susceptibility patterns of \n2 Salmonella species and pathogenic Escherichia coli isolated from broiler poultry \n3 farms in Wakiso district, Uganda. \n4 Thomas Ssemakadde1, Nalumaga Pauline Petra1, Jude Collins Busingye1, Joel Bazira1, \n5 Kabanda Taseera1\n6 1Department of Microbiology, Faculty of Medicine, Mbarara University of science and \n7 technology\n8 Corresponding author; ppaulinenalumaga@must.ac.ug\n9\n10 ABSTRACT\n11\n12 Background: \n13 The emergence and re- emergence of zoonotic bacterial infections and the upsurge \n14 reflected in current trends of antimicrobial-resistant bacteria is a major global concern. \n15 Salmonella spp and Escherichia coli (E. coli) are the two most important food-borne \n16 pathogens of public health interest incriminated in poultry products worldwide hence \n17 necessitating constant monitoring of microbial food safety measures. The purpose of this \n18 study was to determine the prevalence, associated factors and antimicrobial susceptibility \n19 patterns of Salmonella and E. coli in poultry farms in Wakiso District to provide detailed \n20 information of extent of spread to guide plans that influence safer poultry keeping practices \n21 in this era. \n22 Methods: This study was a cross sectional study that used a total of two hundred sixteen \n23 (216) poultry samples from cloacae swabs and fecal swabs collected from broiler poultry \n24 farms and cultured on Chromagar TM Salmonella and Sorbitol MacConkey agar for \n25 pathogenic E. coli. Biochemical tests, minimum inhibitory concentration, and polymerase \n26 chain reaction were utilized.  Assessment of the correlations between the resistance \n27 patterns of resistant and susceptible isolates was determined using mean, and multiple \n28 logistic regression.\n29 Results: A total of 40 (18.5%) Salmonella and 120 (55.6%) Pathogenic E. coli was \n30 isolated. In this investigation, extended beta lactamase (ESBL) production was detected \n31 in 18 isolates Salmonella and 57 pathogenic E. coli . Prevalence of blaTEM gene was \n32 expressed in 7/18 (39%)  Salmonella isolates and 42/57 (73.8%) Pathogenic E. coli isolates \n33 The associated factors that predispose these farms to Salmonella and Pathogenic E. coli \n34 identified in this study include: contact of poultry and wild birds (p -value =0.000), movement \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted August 16, 2024. ; https://doi.org/10.1101/2024.08.16.24312101doi: medRxiv preprint \nNOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.\n\n35 from one pen to the other by farm-handlers (P-Value = 0.030), use of untreated water ( P-\n36 Value =0.005) and food contamination of commercial poultry feeds (P-Value= 0.0021)\n37 Conclusion: Salmonella spp and Escherichia coli remain the two most important food-\n38 borne pathogens of public health interest incriminated in the poultry field, and it is evident \n39 from this study that these bacteria have resistant genes associated with them.\n40\n41 Key words; associated factors; antimicrobial susceptibility; Salmonella species; \n42 pathogenic Escherichia coli; broiler farms; Wakiso district; Uganda.\n43\n44\n45 INTRODUCTION\n46 The emergence and re- emergence of zoonotic bacterial infections and the upsurge \n47 reflected in current global trends of antimicrobial resistance is a major universal concern \n48 (1). Presence of bacteria that have resistant genes decreases the efficacy of the few \n49 available antibiotics culminating into infection and failure patterns in treatment which \n50 sometimes are implicated in morbidity and mortality trends across the globe (2). \n51 Globally poultry farming is one of the fast-growing low-cost investment ventures currently \n52 on the rise and this is partly explained by the significant growth in the population size that \n53 provides ready market for poultry products (3). As a result, poultry raised in rigorous, intense \n54 circumstances using antimicrobial drugs to hasten growth and prevent disease as a result \n55 of the method of raising the birds (4). Antibiotic-resistant bacteria in poultry could have \n56 resistant genes that could potentially be passed on to humans posing threat such as \n57 economic losses in animal production and treatment failure, leading to mortalities and \n58 morbidities (5).\n59 According to WHO nearly 33 million healthy years of life worldwide are lost each year as a \n60 result of unsafe practices related to food handling leading to consumption of unsafe food \n61 (6). In Africa today, over 91 million people fall ill due to food borne illnesses and \n62 approximately 137,000 deaths are registered representing a third (1/3) of the global death \n63 toll from food borne illnesses. Diarrheal diseases in the African continent are estimated to \n64 account for 70% of the burden of food borne diseases (6).\n65 Several studies done focusing on the disease trend patterns have highlighted the significant \n66 role of E.coli as a contributing member to the diarrheal disease burden in humans and this \n67 accounts for 4.0% of the global burden of disease daily thus contributing to an estimated \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted August 16, 2024. ; https://doi.org/10.1101/2024.08.16.24312101doi: medRxiv preprint \n\n68 1.8 million fatalities each year, of which children account for roughly 90% (7, 8). In the \n69 poultry industry, Avian pathogenic Escherichia coli (APEC) is a typical bacterium that is \n70 proportionally linked to tremendous economic losses that is due to a number of conditions \n71 which range from septicemia, salpingitis, chronic respiratory disease, and embrionary \n72 death. For the recent colibacillosis outbreaks across the globe that represent 80% of the \n73 disease cases worldwide, the strongly associated serotypes are O78 and O2 (9). \n74 According to disease surveillance and outbreak data published in the MOH weekly \n75 Epidemiological bulletin, there is an upsurge in the number of Typhoid cases seen in \n76 Kampala and Wakiso districts (10). Owing to the upward increase in the human population, \n77 the demand driven venture of poultry rearing and the increase in typhoid cases, there is \n78 need to investigate whether these poultry products play a part in perpetuating these \n79 infections. The existence of these food borne pathogens in poultry leads to negative effects \n80 on both humans, animal, and environmental health sectors and as such calls for \n81 investigations to draft better methods of eliminating potential AMR situations.\n82 This study will determine the antimicrobial susceptibility patterns, related variables, and \n83 prevalence of Salmonella and pathogenic E. coli in poultry farms in Wakiso District to \n84 provide detailed information of extent of spread, resistant genes and this will guide plans to \n85 influence safer poultry keeping practices in this era of increasing population pressure for \n86 poultry products. \n87 MATERIALS AND METHODS\n88  Study Area\n89\n90 This study was conducted in Wakiso district (as seen in figure 1) a metropolitan district in \n91 the central region of Uganda that partly encircles Kampala district. The district coordinates \n92 are: 00 24N, 32 29E. With a projected population of 2,915,200 million, Wakiso district is still \n93 Uganda's most populous Higher Local Government (HLG) (11).There is an annual \n94 population increase projected at 4.1% and this rapidly increasing population increases the \n95 demand of food including poultry products. The district was regarded as the top producer \n96 of poultry, producing 7.4% of all the chickens in the country (12).\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted August 16, 2024. ; https://doi.org/10.1101/2024.08.16.24312101doi: medRxiv preprint \n\n97 Study design\n98\n99 This was a cross-sectional study focused on broiler farms based on the FAO \n100 categorization sector level 3 farms whose chicken that was about to enter the food supply \n101 chain, within selected counties in Wakiso district. Fresh fecal droppings plus cloacal \n102 samples were collected during August to September period of the year 2021. \n103\n104 Sample Size Determination\n105 The sample size was determined using Kish -Leslie's (1965) standard formula. The sample \n106 size was derived using 83% prevalence (13).\n107 n= Z2 P(1-P)\n108 d2 \n109 n = study sample size required\n110 Z= critical value associated with 95% confidence interval = 1.96\n111 P = Estimated prevalence of 83%of E coli and salmonella d = margin of error = 0.05\n112  n= 1.962 (0.83x 0.17)\n113 0.052 \n114 n= 216 samples\n115 Therefore, a minimum of 216 samples were collected from the various farms in the different \n116 constituencies of Wakiso District. A subsequent number of farm managers with consent \n117 from these 216 farms were interviewed to understand the associated factors that \n118 predispose these pathogens to the farm.\n119 Sampling method\n120\n121 The study employed a variety of methods including simple random sampling to select the \n122 sub counties, purposive sampling using information provided by the district veterinary office \n123 to identify the villages and the respective farms from which samples were picked. \n124 For the selected and eligible farms, three swabs from fresh fecal droppings and two swabs \n125 from the cloaca were collected while adhering to the appropriate bio safety measures at the \n126 farm as stated in the WHO bio safety manual fourth Edition. Five swabs from one farm on \n127 different chicken were considered to represent one sample (Pooling of samples). \n128\n129 Sample Collection\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted August 16, 2024. ; https://doi.org/10.1101/2024.08.16.24312101doi: medRxiv preprint \n\n130 Three fresh fecal droppings of chicken from the target shed with oldest flock were randomly \n131 selected. Using a sterile swab, adequate fecal material was obtained by swabbing the top \n132 of the freshly deposited fecal matter. \n133 Two random chickens from the target shed with oldest flock were randomly selected. Using \n134 a sterile swab, adequate fecal material was obtained from the cloaca. The swabs were \n135 placed into a plain tube containing Amies transport media without charcoal. A Stopper was \n136 used to seal the tube, the sample containing tube was labeled with the sample identifier/ \n137 Farm research generated IDs, location and date and stored in a rack or zip lock bag. This \n138 was then put into a cool box and transported to the laboratory within 24 hours.\n139\n140 Isolation and identification of microorganisms\n141\n142 For Pathogenic E. coli and Salmonella spp\n143\n144 The pooled samples were aseptically mixed into 9 ml of autoclaved Buffered Peptone Water \n145 (BPW) (Hi Media M1494, Mubi, India) in a sterile 50ml falcon tube with a lid to generate a \n146 pre- enriched sample, vortex, and incubated for 16-24 hours at 37oC aerobically.\n147\n148 The enriched sample was picked and inoculated onto a chromogenic agar (Chromagar TM \n149 Salmonella which contains Agar, Peptone and yeast extract +Chromagenic& selective mix) \n150 which is a particular chromogenic culture medium designed for the presumptive \n151 identification, qualitative direct detection, and differentiation of Salmonella (14). Incubation \n152 of the plates was done for 24hrs at 37 0C. Presumptive colonies for Salmonella (mauve- \n153 pink, raised and smooth colonies) were selected for identification \n154 For isolation of pathogenic E.coli, the pre-enriched sample was inoculated on Sorbitol \n155 MacConkey agar plates and incubated at 37 °C ± 1°C for 24 hours. The colonies of \n156 pathogenic E. coli were smooth, raised, had entire margins and were colorless on SMAC.\n157 The presumptive organisms were subcultured on nutrient agar and identified through gram \n158 staining, Triple Ion Sugar (TSI) test, the IMViC test, Methyl Red, Voges- Proskauer, Sulphur \n159 indole motility, and Simmons citrate utilization. All reagents used were from (Oxoid, \n160 England) (15).\n161\n162 Bacterial antibiotic susceptibility testing\n163 Bacterial suspension that was adjusted to 0.5McFarand standard and inoculated on Muller \n164 Hinton agar medium (Oxoid CM0337 Basingstoke, England) using surface spreading \n165 method. The antibiotic discs listed in table 1 (all from Oxoid, England) were used. The plates \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted August 16, 2024. ; https://doi.org/10.1101/2024.08.16.24312101doi: medRxiv preprint \n\n166 were incubated for 24 hours at 37°C. The results were read and interpreted according to \n167 Clinical Laboratory Standards Institute standards Version 14th edition, 2020 (16).\n168 E. coli ATCC 25922(American Type Culture collection, Rockville, MD, USA) was used as \n169 reference control strain.\n170 Table 1: Commonly used antibiotics at NaLIRRI, 2022 obtained from Oxoid ltd \n171 suppliers and the discs were selected based on CLSI (28 th Edition, 2020) and their \n172 availability at NALIRRI Microbiology laboratory.\nAntibiotic Class\nAntibiotic used for both E. coli and \nSalmonella isolates \nAminoglycosides Gentamicin (GM) (10 μg/ml)\nCarbapenem Meropenem (MEM) (10 μg/ml)\nCephalosporins class III Ceftriaxone (CRO) (30 μg/ml)\nAmphenicol Chloramphenicol (CHL) (30 μg/ml) \nCephalosporins class IV\nCefepime (CPM) (30 μg/ml), Cefotaxime \n(CTX)(30µg/ml), Ceftazidime (CTZ) (30µg/ml)\nQuinolones Ciprofloxacin (CIP) (5 μg/ml)\nMacrolides Erythromycin (EM) (30 μg/ml)\nPenicillin Ampicillin (AMP) (10 μg/ml)\nTetracyclines Tetracycline (OXT) (30 μg/ml)\nSulphonamides/ Trimethoprim Co-trimoxazole (CTX) (25 μg/ml)\n173\n174 Phenotypic Screening of Enterobacteriaceae for Extended-spectrum β-lactamases \n175 (ESBLs) production\n176\n177 Cefotaxime (30μg) and Ceftazidime (30μg) antibiotic discs were used to phenotypically test \n178 Enterobacteriaceae for the development of ESBLs and incubated overnight at 37 0C on \n179 Muller Hinton agar (16). \n180\n181 Confirmation of ESBL producing Enterobacteriaceae\n182\n183  Double Disc Synergy method\n184\n185 A sterile cotton swab was used to surface spread an Enterobacteriaceae suspension on to \n186 a Mueller Hinton agar plate after turbidity was set to the 0.5 McFarland standards. The \n187 antibiotic discs used were Ceftazidime (CAZ) (30μg) alone and Ceftazidime in combination \n188 with Clavulanic acid (CAL) (30/10μg) (17). The discs were spaced approximately 30 mm \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted August 16, 2024. ; https://doi.org/10.1101/2024.08.16.24312101doi: medRxiv preprint \n\n189 apart, and the discs and plates containing the test organism were incubated at 37° C \n190 overnight. Both the single disc and the combined disc's zones of inhibition were measured. \n191 Clavulanic acid-containing discs with an increase in zone diameter of ≥5mm in comparison \n192 to those without Clavulanic acid were deemed to have an ESBL (17).\n193\n194 Genomic DNA extraction\n195  \n196 Bacterial genomic DNA extraction was conducted on all the 18 Salmonella and 57 \n197 Pathogenic E coli from the pure plate colonies that were extracted. The Bio line ISOLATE \n198 II genomic DNA kit (Cat No. Bio-52065 Lot No. IS502-B054750) was used for the bacterial \n199 extraction following manufacturer’s instruction.  The presence of genes encoding for ESBL \n200 (bla-TEM gene) was detected using conventional PCR amplification using primers listed in \n201 (table 2).\n202\n203 Table 2: Primers used in the PCR reaction.\nResistanc\ne Gene Primer Sequence (5′→3′ Direction) Amplicon \nSize (bp)\nAnnealing \nTemperature \n(°C)\nReference\n(F) TGG GTG CAC GAG TGG GTT \nACblaTEM\n(R) TTA TCC GCC TCC ATC CAG TC\n526 58 Tenover et \nal., 1994\n204\n205 The PCR master Mix reagents was prepared as follows:\n206  12.5 µL master mix consisting of One Taq quick load two times master mix/w standard \n207 buffer, \n208  dNTPs&Taq polymerase (M0486S), \n209  1.5 µL forward (100 µM), \n210  1.5 µL primary reverse (one hundred µM), \n211  5 µL DNA template and RNAse-free dH2O up to 25 µL. \n212 PCR Cycling: The PCR process was carried out in a thermo cycler (Perkin Elmer, \n213 Wellesley, MA, USA) with a pre-denaturation cycle of 95°C for 15 min, followed by DNA \n214 amplification stage with 30 cycles (94°C for 1 min, 58°C for 1 min, and 72°C for 1 min) \n215 and final extension cycle of 72°C for 5 min.  \n216 DNA Amplicons were electrophoresed using 1.5% agarose gel, in Tris-Borate EDTA \n217 buffer (TBE) 1×concentration, Safe View ClassicTM DNA stain, 6x loading dye (Thermo \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted August 16, 2024. ; https://doi.org/10.1101/2024.08.16.24312101doi: medRxiv preprint \n\n218 Scientific), and DNA ladder/marker 100 bp (Sigma-Aldrich, Inc., Saint Louis, MI, \n219 USA)DNA Bands were visualized on a Dark reader Transilluminator.\n220\n221 Data Management and Analysis\n222\n223 All records of the analysis were recorded in the lab register as a hard copy back up. \n224 Samples were assigned codes and excel spreadsheets were used to enter the raw data, \n225 which were then exported to Stata (Version 12, Special Edition, College Station, Texas \n226 USA) software for statistical analysis. Frequency tables, graphs were used to present \n227 descriptive statistics. This was done at a univariate analysis level.\n228 Bivariate and multivariate analyses was carried out for the study objectives, quantitative \n229 data evaluations was made in relation to the 95% level of significance/confidence, the \n230 Pearson value (p-value ) to determine the objective variables' statistical significance, \n231 statistics were judged to be significant for any p < 0.05.\n232\n233 Ethical Consideration\n234 Approval was obtained from Mbarara University of Science and Technology ;Institutional \n235 Ethical Review Committee (MUST-2021-141), and at the ministry level, permanent \n236 secretary Ministry of Agriculture, Animal Industries, and Fisheries, district's chief \n237 administrative officer, and district veterinarian. \n238 Prior engaging the farms, the researcher sought for clearance from the farm owners to \n239 access and collect samples from their farms. This consent was requested voluntarily to \n240 participate in the study. The researcher treated all Farm's data and bacterial isolates \n241 obtained for this investigation in strict confidence. \n242  \n243 COVID19 Prevention and Management Plan\n244\n245 The researcher received the recommended two doses of COVID-19 vaccination. In addition \n246 to this, the researcher adhered to the government recommended standard operating \n247 procedures such as avoiding touching surfaces at the poultry farms on any place while \n248 carrying out this study, frequent washing/cleaning of the hands with soap or sanitizers, strict \n249 face masking while carrying out this study and the respondents will also be encouraged to \n250 put on their face masks. Social distancing was also adhered while interacting with persons \n251 at the time of data collection.  \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted August 16, 2024. ; https://doi.org/10.1101/2024.08.16.24312101doi: medRxiv preprint \n\n252\n253  Quality assurance and Quality control procedure:\n254\n255  Involved the use of reference controls (both positive and negative). During the data entry \n256 and analysis, the researcher ensured double entry of data to rule out clerical errors.\n257 RESULTS\n258 Prevalence of Salmonella and E. coli from the cultured samples:\n259\n260 Of the total of 216 samples collected, a total of 40 (18.5%) Salmonella and 120 \n261 (55.6%) Pathogenic E. coli was isolated as seen in the flow chart (figure 2). \n262\n263 Demographic and socio-economic characteristics of the respondents\n264\n265 Out of 216 farm managers selected from ten (10) different sub counties in Wakiso district \n266 that took part in the research and were interviewed, 87 (40.28%) were Males and 129 \n267 (59.72%) were females. The age group of31-45years had the most participants at \n268 113 (52.31%) while the age group of 46 and older had the fewest 50 (23.15%). In regards \n269 Education level training; at least all the farm managers had some basic training. \n270 Participants who completed primary level were 58 (26.85%), Secondary level were 80 \n271 (37.04%), vocational training were 33 (15.28%) and University level were 45 (20.83%). \n272 When examining the various farm managers' sources of income, the survey discovered \n273 that 146 participants (67.59%) relied primarily on poultry, others had mixed agricultural \n274 practices like livestock farming (including either cattle, Sheep, Goats or Piggery) alongside \n275 poultry 22 ( 10.18%), crop farming with poultry 17 (5.56%) and others had \n276 additional funds arising from self-employment in other sectors alongside the \n277 poultry 31 (14.35%) as seen in table 3.\n278\n279 Table 3: Farm characteristics and demographics:\nCharacteristics Percentage\n(Variable)\nCategory Frequency \n(N=216) (%)\nMale 87 40.28Gender\nFemale 129 59.72\n15-30 53 24.54\n31-45 113 52.31\nAge(years)\n>=46 50 23.15\nPrimary 58 26.85Education level\nSecondary 80 37.04\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted August 16, 2024. ; https://doi.org/10.1101/2024.08.16.24312101doi: medRxiv preprint \n\nVocational Training 33 15.28\nUniversity 45 20.83\nKitabi 25 11.57\nNamugongo 28 12.96\nKiira 32 14.81\nMakindye 28 12.96\nNabweru 19 8.79\nKakiri 21 9.72\nBusukuma 11 5.09\nWakiso TC 16 7.40\nMasulita 12 5.55\nSub counties\nNsangi 24 11.11\nPoultry alone 146 67.59\nLivestock farming (Cattle, goats, \nsheep, or Pigs) with poultry\n22 10.18\nCrop farming with Poultry 17 5.56\nSource of \nincome\nSelf-employed off-farm. (Others) 31 14.35\nOnce per Month 13 6.02\nOnce per week 117 54.17\nhygiene/rate of \ncleaning of the \nfarm\nDaily 86 39.81\nOther Birds (Ducks, Turkey, and \nGeese)\n68 31.48Presence of \nother animals \n(Pigs, Cows, \nDucks and \nTurkey)\nLivestock 148 68.52\nDeep Liter 103 47.69\nBattery Cage system 82 37.96\nmixed farming with turkey and ducks 19 8.76\nType of \nproduction \nsystem used\nSemi intensive (Free range system) 12 5.56\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted August 16, 2024. ; https://doi.org/10.1101/2024.08.16.24312101doi: medRxiv preprint \n\n11\n281 Factors associated with Salmonella and Pathogenic E. coli in broiler poultry farms \n282 in Wakiso District.\n283\n284 Salmonella and pathogenic E. coli were most frequently found in broiler poultry farms where \n285 there was contact between poultry and other bird species like turkeys and geese (P-value= \n286 0.019), contamination of commercial poultry feeds (P-value=0.012), movement of farm \n287 workers between pens (P-value=0.167)and use of untreated water (P-value=0.117). \n288 Therefore, there is a significant association between the above variables and the presence \n289 of the organisms of interest in the research (table 4).\n290\n291 Table 4: Showing factors associated with Salmonella and E. coli in poultry \n292 farms in Wakiso District\n293\nSalmonella E. coli\nVariable Category Frequency \n(n)=40\nP-value Frequency \n(n)=120\nP-value\nLivestock 83 (69.2) 65 \n(81.25%)\nContact of \npoultry and \nother bird \nspecies\nOther birds  25 (20.8)\n0.384\n15 \n(18.75%)\n0.020\nDaily 96 (80) 49 (61)\nOnce a week 21 (17) 26 (33)\nFrequency of \ncleaning and \ndisinfection of \nthe Bird's \nHousing.\nOnce a Month 03 (3)\n0.498\n05 (6)\n0.113\nMixed with \nWater\n95 (79.2 56 (70)contamination \nof commercial \npoultry\nMixed with other \nmaterials\n25 (20.8)\n0.242\n24 (30)\n0.002\nFrom one pen 58 (48.3) 55 \n(68.75%)\nmovement \nfrom one pen \nto the other \nby farm-\nhandlers \nFrom more than \none pen\n62 (51.7) 0.244 25 \n(31.25%)\n0.017\nUse of well \nwater\n38 (31.7) 32 (40)use of \nuntreated \nwater\nUse of tap water 82 (68.3) 0.392 48 (60)\n0.018\nHome made 46 (38.3) 25 (31)Source of \npoultry feeds Commercial 74 (61.7) 0.066 55 (69) 0.237\n294\n295\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted August 16, 2024. ; https://doi.org/10.1101/2024.08.16.24312101doi: medRxiv preprint \n\n12\n296\n297 Antibiotic susceptibility patterns of Salmonella and Pathogenic E. coli towards \n298 commonly used antimicrobials in poultry: \n299\n300 The phenotypic resistance profile for Salmonella organisms from broiler poultry samples \n301 when subjected to the selected antibiotics demonstrates that ampicillin 32 (80%) is the drug \n302 with the highest level of resistance, followed by erythromycin 28 (70%), tetracycline 27 \n303 (67.5%), and ciprofloxacin 25 (62.5%). On the contrary, low resistance was observed to \n304 ceftazidime 10 (15%), Gentamicin 3 (8%), Chloramphenicol 3 (8%), cefepime 2 (5%) and \n305 meropenem2 (5.0%) as shown in table 5.\n306\n307 Table 5: showing antibiotic susceptibility patterns of Salmonella towards commonly \n308 used antimicrobials in Poultry:\nAntibiotics\nResistant\nn (%)\nIntermediate \nResistance n \n(%)\nSusceptible\nn (%)\nGentamicin (GM) (10 μg/ml) 3 (7.5) 7 (17.5) 30 (75.0)\nMeropenem (MEM) (10 μg/ml) 1 (2.5) 2 (5.0) 37 (92.5)\nCeftriaxone (CRO) (30 μg/ml) 18(45) 4 (10) 18 (45)\nChloramphenicol (CHL) \n(30 μg/ml) 3 (7.5) 3 (7.5) 34 (85)\nCefepime (CPM) (30 μg/ml) 2 (5.0) 3 (7.5) 35 (87.5)\nCiprofloxacin (CIP) (5 μg/ml) 25 (62.5) 10 (25) 5 (12.0%)\nErythromycin (EM) (30 μg/ml) 28 (70) 7 (17.5) 5 (12.5)\nAmpicillin (AMP) (10 μg/ml) 32 (80) 2 (5) 6 (15)\nTetracycline (OXT) (30 μg/ml) 27 (67.5) 6 (15) 7 (17.5)\nSulfamethoxazole-trimethoprim \n(SXT) (25 μg/ml) 20 (50) 5 (12.5) 15 (37.5)\nCefotaxime (CTX)(30µg/ml) 10 (25) 2 (5) 28(70)\nCeftazidime (CTZ) (30µg/ml) 15 (37.5) 5 (12.5) 20 (50)\n309\n310 E. coli exhibited the highest resistance to Erythromycin at 88 (73%), followed by Ampicillin \n311 86 (72%), Chloramphenicol 85 (71%), Tetracycline at 82 (68%) and sulfamethoxazole-\n312 trimethoprim at 78 (65%). However, it’s worth noting that there was observed very low \n313 resistance to Ciprofloxacin 9 (8%), meropenem 4 (3%), and cefipime 3 (2.5%) as seen in \n314 table 6.\n315\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted August 16, 2024. ; https://doi.org/10.1101/2024.08.16.24312101doi: medRxiv preprint \n\n13\n316 Table 6: showing antibiotic susceptibility patterns of pathogenic E. coli Spp \n317 towards commonly used antimicrobials in Poultry.\nAntibiotics\nResistant           \nn (%)\nIntermediate \nResistance n \n(%)\nSusceptible      \nn (%)\nGentamicin (GM) (10 μg/ml) 10 (8.0) 7 (6.0) 103 (86.0)\nMeropenem (MEM) (10 μg/ml) 4 (3) 6 (2.5) 110 (92.0)\nCeftriaxone (CRO) (30 μg/ml) 57 (47.5) 5 (4.2%) 58 (48.3)\nChloramphenicol (CHL) (30 μg/ml) 85 (71.0) 25 (21.0) 10 (8.0)\nCefepime (CPM) (30 μg/ml) 3 (2.5) 8 (6.5) 109 (91)\nCiprofloxacin (CIP) (5 μg/ml) 9 (7.5) 66 (55) 45(37.5)\nErythromycin (EM) (30 μg/ml) 88 (73) 15(12.5) 17 (14.5%)\nAmpicillin (AMP) (10 μg/ml) 86(72) 12 (10) 18 (15)\nTetracycline (OXT) (30 μg/ml) 82 (68.0) 18 (15) 20 (17.0)\nsulfamethoxazole-trimethoprim (SXT) \n(25 μg/ml)\n78(65) 32 (27.0) 10 (8.0)\nCefotaxime (CTX)(30µg/ml) 51(42.5) 10(8.3) 59(49.2)\nCeftazidime (CTZ)(30µg/ml) 48(40) 8(6.7) 64(53.3)\n318\n319 Detection of ESBL (blaTEM), gene encoding resistance to commonly used antibiotics \n320 used in poultry in Salmonella and Pathogenic E. coli\n321\n322 Out of the 18 Salmonella samples analyzed for genotypic expression, 7/18 (39%) samples \n323 expressed presence of the bla TEM genes while 11 (61.1%) samples did not have this \n324 gene.\n325 Out of the 57 Pathogenic E. coli samples analyzed for genotypic expression, 42/57 \n326 (73.8%) samples expressed presence of the bla TEM genes while fifteen (26.3%) samples \n327 did not have this gene.\n328 DISCUSSION\n329 The prevalence of selected Salmonella and pathogenic E. coli in selected poultry \n330 farms\n331\n332 According to this study, a prevalence of 18% and 56%, respectively, was identified for \n333 Salmonella and pathogenic E coli in broiler poultry farms in the Wakiso district. In line with \n334 other studies carried out, this result was comparable to a study conducted by (18). This \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted August 16, 2024. ; https://doi.org/10.1101/2024.08.16.24312101doi: medRxiv preprint \n\n14\n335 revealed the prevalence of Salmonella and pathogenic E. coli  as 21.1% and 56.3% in \n336 Uganda.\n337 However, it can be argued that this prevalence rate is low when compared to a study done \n338 by (19) found an overall prevalence of 83%, of which 90.8% and 73% were from chicken \n339 in Lira and Kampala districts from the antibiotic susceptibility profiles of fecal Escherichia \n340 coli isolates from Dip-Litter broiler chicken in Northern and Central Uganda.\n341 This discrepancy can be due to the different study sites, sample methods, poultry sector, \n342 and sampling times used during the research. \n343 It is worth noting that this study had a lower prevalence of Salmonella spp compared to \n344 other studies such as one done by (20) in Ruiru Sub-County, Kenya which was at 28% with \n345 almost similar prevalence of pathogenic E. coli of 58%. Another study on antimicrobial \n346 resistance in Salmonella and Escherichia coli isolates from chicken droppings in Nairobi \n347 (21) found lower levels of Salmonella (12% vs. 57% in our study) and nearly similar \n348 prevalence of E. coli (57%) in the analyzed samples.\n349 The differences in environmental contamination levels, poultry management practices, \n350 breed, sample size, sampling, testing methodologies, and challenges in Salmonella \n351 detection methods may account for this similar trend of reduced Salmonella isolation and \n352 prevalence (22) or further still the practice of better bio safety and bio security practices at \n353 farms overseen by the established of Kenya Accreditation society (KENAS),competitive \n354 exclusion of sick birds, breeding for genetic resistance and vaccination.\n355 In Uganda as evidenced from the essential veterinary medicines list, vaccines exist only in \n356 private practitioners’ clinics and cannot be accessed freely by the Bio security level three \n357 farmers that were of interest in this study.\n358\n359  The factors associated with Salmonella and Pathogenic E. coli in poultry farms in \n360 Wakiso District.\n361 Presence of Salmonella and pathogenic E. coli in the poultry farms was significantly \n362 correlated with contact of poultry with other avian bird species such as ducks, geese, and \n363 guinea fowls in the same farms and pens, as well as not frequently cleaning the poultry \n364 farms by removing the manure or beddings. \n365 The study further investigated other factors associated with proper poultry practices such \n366 as the implementation of strict bio security interventions such as having restricted access \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted August 16, 2024. ; https://doi.org/10.1101/2024.08.16.24312101doi: medRxiv preprint \n\n15\n367 to the farmers by visitors and handling of these birds, implementation of a solid well \n368 established sewer system. These, however, did not significantly increase the risk of \n369 Salmonella and E. coli. This is comparable to a study conducted in Nigeria that \n370 focused on the risk factors related to Salmonella spp. In broiler and layer flocks, it \n371 was discovered that the presence of rodents, farm workers moving between pens, running \n372 and parking trucks close to poultry farms (p<0.05) and drinking untreated water (p<0.05) \n373 were all independently associated with a higher risk of Salmonella infection (23).\n374 Broilers are known for consuming large amounts of feed, and this habit encourages constant \n375 feces loss, raising the possibility of their environment becoming contaminated with various \n376 bacterial strains (18).\n377 Furthermore, many broiler farms had high stock densities, which could make environmental \n378 management efforts to reduce bacteria in the houses more difficult. As a result, workers \n379 (especially those handling large flocks) must be strictly supervised because it is claimed \n380 that they may neglect their responsibilities for maintaining hygiene . Therefore, poor \n381 management of poultry could lead to increased transmission of Salmonella and E. \n382 coli in poultry.\n383\n384 The antimicrobial Susceptibility patterns of Salmonella and E. coli in poultry farms\n385\n386 The highest resistance to ampicillin was found in both Salmonella and E. coli isolates, 32 \n387 (80%) and 86(72%), followed by erythromycin (28%) and 88(73%) and tetracycline (27) \n388 and 82(68.0%). \n389 Our research was comparable to a study published by (24) and (18). Therefore, there is \n390 great increase in antimicrobial resistance to the different drugs most especially ciprofloxacin \n391 in Uganda.\n392 According to reports from Uganda and other nations (25), tetracyclines are frequently used \n393 to treat bacterial illnesses and promote animal growth (26). Therefore, it is not surprising \n394 that pathogens have developed broad resistance to them. Bacteria like commensal E. coli \n395 experience selection pressure as a result of ongoing exposure to antimicrobials (27).\n396 In most nations, the rise in human cases of antimicrobial resistance is attributed to the \n397 increase and spread of infections from poultry to humans. In this work, we discovered that \n398 pathogenic E. coli and Salmonella spp. isolated from chicken in the Wakiso district had \n399 high resistance to widely used antibiotics used in both people and animals. As a result, \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted August 16, 2024. ; https://doi.org/10.1101/2024.08.16.24312101doi: medRxiv preprint \n\n16\n400 greater research on AMR in Salmonella spp. and E. coli clinical isolates from poultry is \n401 needed. \n402\n403 ESBL producing genes present among Salmonella and pathogenic E. coli isolated \n404 from poultry farms.\n405\n406 The majority of studies on poultry have noted the presence of genes such as; blaTEM, \n407 AmpC like lactamase gene, for antibiotic resistance (28), (29) and (30). A study conducted \n408 in Malaysia reported a lower prevalence of blaTEM (31) while a study in British Columbia, \n409 2007 reported slightly higher levels of the blaTEM gene, being 81.5 and 80% of \n410 amoxicillin and ampicillin resistant E.coli and Salmonella spp. isolates (32). \n411 Therefore, the community’s health is at risk because of the possibility that antimicrobial \n412 resistance genes in poultry waste will spread to humans and other fowl.\n413\n414 CONCLUSION\n415\n416 The two most significant food-borne pathogens of public health concern linked to poultry \n417 are still Salmonella spp. and Escherichia coli, and it is evident from this study with a \n418 prevalence of 18% and 56% respectively. These bacteria have resistant genes associated \n419 with them seen in 38.9% and 73.8% Salmonella and pathogenic E. coli samples. \n420\n421\n422 Limitations\n423\n424 i. Our focus in this study was strictly broiler poultry that were about to enter the food chain, \n425 this is does not paint a full picture in terms of the overall burden of the disease in poultry \n426 including all other avian birds such as layers, geese, guinea fowls and ducks to \n427 effectively understand the disease transmission dynamics and effect policies that will \n428 effectively halt further spread.\n429 ii. Only one gene blaTEM was focused on during this study and it would be vital to conduct \n430 different genetic manipulations of the same bacterial DNA to targeting different ESBL \n431 genes such as bla CTM, bla SHV etc.\n432\n433 Recommendations\n434\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted August 16, 2024. ; https://doi.org/10.1101/2024.08.16.24312101doi: medRxiv preprint \n\n17\n435 Based on the above the study findings, its pertinent that government of Uganda should \n436 strengthen the antimicrobial stewardship program and ensure that it is supported to carry \n437 out its mandate of coordination that supports the proper use of antimicrobials (including \n438 antibiotics), improves patient outcomes, lowers microbial resistance, and limits the spread \n439 of diseases brought on by multidrug-resistant organisms, the organization needs help.\n440 This should be achieved through the one health platform, a forum that brings together all \n441 key stakeholders from the four different line ministries of Health, Ministry of Agriculture \n442 Animal Industries and Fisheries, Ministry of Water and sanitation and Ministry of tourism, \n443 trade, and antiquities.\n444\n445 DECLARATION\n446\n447 Conflicts of Interest\n448 The authors confirm no conflicts of interest pertaining the publication of this article.\n449\n450 Author contributions\n451 TS and KT developed the study concept, TS, JCB and NPP provided input in data collection. \n452 TS and JCB analysed the data. KT and JB over saw the entire study. NPP, KT and JB wrote \n453 the final manuscript. All authors read and commented on the paper and agreed on the final \n454 version.\n455 Funding\n456 The authors received no financial support for the research, authorship, and publication of \n457 this article.\n458 Availability of data and materials\n459 The analyzed datasets are available from the corresponding author upon request.\n460 Consent for publication\n461 Not applicable\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted August 16, 2024. ; https://doi.org/10.1101/2024.08.16.24312101doi: medRxiv preprint \n\n18\n462 REFERENCES\n463\n464 1. Mthembu TP, Zishiri OT, El Zowalaty ME. 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CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted August 16, 2024. ; https://doi.org/10.1101/2024.08.16.24312101doi: medRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}