Characterization of Extended-Spectrum Beta-Lactamase-Producing Escherichia Coli From Domestic Free-Range Poultry In Agogo, Ghana

Background: Extended-spectrum beta-lactamase (ESBLs) producing bacteria in poultry meat has been suggested as one of the sources of resistant genes, that can lead to dicult-to treat infections in humans. This study aims at determining the frequency of ESBL-producing E. coli, the genetic characterization and antibiotic prole among domestic free-range poultry in Agogo, Ghana. Faecal samples were collected from domestic free-roaming chicken and cultured on ESBL screening agar. Strain identication and antibiotic susceptibility were performed using the VITEK 2 compact system. ESBL-producing E. coli were conrmed using the Double Disk Synergy (DDS) test. Molecular detection and further sequencing of bla TEM, bla SHV and bla CTX-M genes was performed using standardized methods. Results: 56.2% (n/N=81/144) of collected faecal samples were positive for ESBL-producing E. coli. All (n/N=81/81) strains were resistant to ampicillin, ceftazidime, cefpodoxime, cefotaxime and cefuroxime. High rates of resistance were also observed for tetracycline (93.8%, n/N=76/81) and trimethoprim-sulfamethoxazole (66.67; n/N=54/81). Resistance to Carbapenems was not found. The majority of ESBL producing E. coli carried bla CTX-M genes with bla CTX-M-15, 95.1% (n/N=77/81) being the dominant genotype.


Conclusions:
We report high ESBL rates, which is a potential infection source of animals as well as humans, and that a control of antibiotic overuse and animal hygiene/sanitation measures are important from a one health perspective.

Background
The rapid increase of resistance to commonly used antibiotics in human medicine and animal husbandry has become one of the leading global health concerns (1). Besides, overuse of antibiotics in both humans and animal farming has been considered as the driving force for the increase in antimicrobial resistance (AMR) worldwide. Currently, the World health organization (WHO) considers AMR as a 'One Health issue' as well as a "One World issue" (2). Without preventive measures in place, by 2050 the annual deaths due to infections with AMR bacteria are expected to have reached 10 million (3).
AMR infections, including infections caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in sub-Saharan Africa (SSA), are a major concern. E. coli are gram-negative bacteria found as commensals in the gut of humans and animals but are also implicated in a variety of infections, including life-threatening sepsis (4).
A recent study from Ghana indicated poultry as a likely source for ESBL-producing bacteria leading to di cult-to-treat infections in humans (5). This has also been shown in a global systemic review (6). ESBL-producing pathogens can be acquired by direct contact with animals or by consumption of meat products. In addition, ESBL-producing bacteria are often linked to concomitant resistance to other classes of antibiotic agents leading to multidrug resistance (MDR) (7). Therefore, there are global ongoing efforts to address this issue and the issue of AMR as a whole (8,9).
In Ghana, poultry farming is one of the predominant animal businesses with an estimated number of 74.5 million birds per year (10). Of these, approximately 25 million are kept on household level, freely roaming within communities (11). Amongst all meat products, poultry is the most popular, largely consumed in all regions of the country. In the quest to meet this demand, poultry keepers employ antimicrobials as growth promoter, and to prevent and treat infections (12).
Several studies on ESBL-producing bacteria, particularly E. coli, in commercial poultry farming have been published (5,13,14). However, to our knowledge, such studies have not been conducted on free-range poultry (household) farming-level in Ghana. Nevertheless, due to the animals' proximity to humans, such studies are essential to identify possible ESBL-transmission reservoirs within communities. This study aims at determining the frequency of ESBL-producing E. coli, their genetic characterization and antibiotic pro le among domestic free-range poultry in Agogo located in the Ashanti Region of Ghana.

Discussion
The study examined ESBL-producing E. coli among domestic free-range poultry in a rural community of Ghana. The considerably high rate of ESBL-producing E. coli exceeds what was found in commercial poultry farming in Ghana (5). This is not surprising, as Paintstil and colleagues demonstrated that 43% of domestic farmers from the same study area use antibiotics in poultry farming (15), which in turn increases the risk of higher ESBL acquisition in the normal gut ora of the animal (16). In general, not exclusively on household farm-level, frequencies of ESBL-producing bacteria found in chicken seem high, on the African continent. For example, a study showed 20% ESBL-producing E. coli in poultry from Zambia (17). A review by Alonso and colleagues has reported 42,0% and 55.5% ESBL-producing E. coli among chicken faeces from Tunisia and Algeria respectively, as well as 61.6% from chicken meat in Egypt (18).
Generally, in resource limited countries, inadequate sanitation aside the overuse of antibiotics are likely to play signi cant roles in the selection and transmission of antibiotic resistance (19). This is probably particularly true for areas where free-range (domestic) chicken is raised in communities with close contact to their owners. In sub-Saharan African countries, the potential transmission risk of antimicrobial resistant bacteria from animals or animal products to humans has been demonstrated before (20).
Besides, high level of resistance to beta-lactam antibiotics, the degree of resistance seen for other classes of antibiotics such as tetracycline and trimethoprim-sulfamethoxazole but also the much lower levels as seen for gentamycin and piperacillin is in line with what was found in other studies around the globe (21)(22)(23). Resistance in this study was re ected by the overall usage and availability of drugs in Ghana (24,25). Also uoroquinolones, such as cipro oxacin are important drugs in Ghana for the treatment of infections (26, 27). The level of cipro oxacin resistance (35.8%, n/N=29/81) seen was in line with 35.7% that was reported from a study in poultry from Nigeria (14) but lower than 59% that was reported from a previous study in Ghana on poultry meat (13). The difference seen might be attributed to the fact that cipro oxacin usage in farming on household level is not as common in this study area (15).
Globally also in most SSA countries, bla CTX-M positive E. coli have been shown to be the most common genotype found in clinical isolates from humans and animals (28, 29) with bla CTX-M-15 being the most prevalent gene associated with human infections (30). This was also true for the present study, where bla CTX-M-15 was found to be the dominant genotype amongst the ESBL-positive isolates. This is in line with data from a previous study analysing chicken meat in Ghana (5). bla CTX-M-15 as predominant genotype among the ESBL-producing strains is of particular interest, as it has been associated with clinical E. coli strains carrying several virulence factors (31,32). bla CTX-M-15 is often found on incompatibility group FII plasmids (33), known as "epidemic resistance plasmids" (34) due to their tendency to acquire other resistance genes, and their high potential to be transmitted through horizontal gene transfer (35).
Notably, the coexistence of bla CTX-M-15 and bla TEM-1b observed in this study was considerably lower, with only 1.2% of isolates harbouring both resistance genes, than what was reported from Nigeria (8.1%) (14). As in the present study ESBL genes itself were analysed only, no assumption can be made on whether these were located on the chromosome or the plasmid. Discrimination between chromosomal or plasmid location of ESBL genes in E. coli was previously analysed by Falgenhauer and colleagues (5) including isolates collected in the same area in which this study was conducted. The study revealed chromosomal and plasmid-mediated ESBL genes in the study area. Thus, we can assume that for some of our isolates carrying ESBL, the resistance can be located on the plasmids.
Our study has limitations, which have to be considered when interpreting the results. Faecal samples were collected from only three communities in the study area and from seven households. This may not be representative for other parts of Ghana. Also, the sample size was rather small. Therefore, to generalize the interpretation of this study, faecal samples from additional communities should be evaluated to cover a representative study area. Seasonality was not captured within this study as households were visited once within the 6 months study period only.

Conclusion
This study demonstrates a high frequencies of ESBL-producing E. coli in free-range chicken in rural Ghana. It also shows a high carriage level of CTX-M-15 producing E. coli isolates in household chicken. Hence, poultry, even when bred on household level, might be an important source for ESBL-producing bacteria in this part of the world.
Therefore, monitoring of antibiotic use and restrictions in poultry farming must be encouraged as well as the implementation of surveillance systems that monitor antimicrobial use and inform on emerging antibiotic resistant bacterial strains.

Study site and sample collection
Fresh faecal samples from poultry were randomly collected from six households in three different communities in Agogo in the Asante Akyem North Municipality within the Ashanti Region of Ghana (Fig. 1). Sample collection took place between June 2019 to December 2019. Faecal samples were collected from households where chickens were kept at the backyard. Each household possessed approximately 25 chicken and was visited once during the study period. Samples were transported at 2°C -8°C in a cool box to the microbiology laboratory at the Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR) within 2-4 hours after sampling for further analyses.

Identi cation And Antibiotic Susceptibility Testing
On arrival to the laboratory, samples were cultured on two MacConkey agars containing 1mg/L Ceftazidime and 1mg/L Cefotaxime, respectively. Plates were incubated at 35°-37°C for 18-24h at normal atmosphere. All morphological different lactose fermenters colonies (not exceeding 3 colonies) were subsequently sub-cultured on Columbia blood agar. Isolate identi cation and antibiotic susceptibility were tested using the VITEK 2 compact system (bioMérieux, Marcy L'Etoile, France). Con rmed E. coli were classi ed as S (Susceptible, standard dosing regimen) I (Susceptible, increased exposure) or R (Resistant) to commonly used antibiotics according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines (version 10.0, 2020; http://www.eucast.org/breakpoints/). Antibiotics tested included: ampicillin, ceftazidime, cefpodoxime, cefotaxime cefuroxime tetracycline, imipenem, meropenem, gentamicin, cipro oxacin, ampicillinsulbactam trimethoprim/sulfamethoxazole, ertapenem piperacillin-tazobactam and tigecycline and ESBL screening. MDR was de ned as resistance to more than 2 classes of antibiotics.
Production of ESBLs was con rmed by the combined double-disk test with ceftazidime and cefotaxime alone and in combination with clavulanic acid (Becton, Dickinson Company, Sparks, MD, USA) as de ned by EUCAST (EUCAST guidelines, version 10.0, 2020; http://www.eucast.org/breakpoints/).
Con rmed strains were saved in microbanks (Pro-lab Diagnostics, Richmond Hill, ON, Canada) and stored at -80˚C for further analysis. Quality control of cephalosporin-containing agar was performed using the ATCC 25922 and a bla CTX-M-15 positive E. coli.

Genotyping Of Esbl Genes By Polymerase Chain Reaction
DNA extraction for E. coli was performed using the boiling method as described by Tellevik and colleagues (36). DNA was subject to molecular characterization by polymerase chain reaction (PCR) for the detection of the resistance genes bla TEM (temoneira), SHV (sulphydryl variable enzyme) and bla CTX-M, (cefotaximase-Munich) respectively as described elsewhere (37). To further differentiate the bla CTX-M groups, speci c target primers (Table 1) were used for ampli cation and sequencing of the PCR amplicons (37). The PCR amplicon was sent to Microsynth Seqlab (Göttingen, Germany) for Sanger sequencing. The resulting sequences were aligned and identi ed by comparison with known sequences using CLC Sequence Viewer 8.0 (http://www.clcbio.com) and Res nder 4.1 (https://cge.cbs.dtu.dk/services/ResFindeer) Descriptive statistics were applied to analyse study data. All analysis were done using R studio software (/https://www.rstudio.com).  Antibiotic resistance among ESBL E. coli isolated from domestic poultry.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. CHARITYTRIMMEDFASTA.txt