Aim
The aim of this study was to contribute towards ameliorating the management and treatment of diseases linked to Enterobacteriaceae in Cameroon; providing epidemiological data on prevailing Enterobacteriaceae and their resistance patterns and risk factors.
Design
The design used was a cross-section design.
Study site
The West Region with an area of 13,892 km² is a territory located in the central-western portion of the Republic of Cameroon located at 5°30′N 10°30′E. It borders the North West Region to the northwest, the Adamawa Region to the northeast, the Centre Region to the southeast, the Littoral Region to the southwest, and the Southwest Region to the west. The West Region is the smallest of Cameroon's ten regions in area, yet it has the highest population density; a population of 1,865,394 (2013) with density of 134/km2. Its Capital is Bafoussam in the Mifi Division. It has 8 Divisions: Bamboutos, Upper-Nkam, Upper-Plateau, Koung-Khi, Ménoua, Mifi, Ndé and Noun36. The Region is found in the Grass field plateaus of the Western highlands with a cold climate and the main ethnic groups are the Bameliké and Bamoum22.
Laboratory work was carried out in the Research Unit of Microbiology and Antimicrobial Substances (RUMAS) in the University of Dschang.
Sample collection
In Cameroon, research on livestock is under the supervision of the Ministry of Livestock, Fisheries and Animal Industries (MINEPIA). Poultry and other livestock farms are registered under this Ministry which follows the health of the animals while still in the farm and before sales. A research authorisation (reference number 68/18/L/DREPIA-O/SRAG of 04/06/2018) addressed to the Divisional Delegates was obtained from the Regional Delegation of this Ministry. With the authorisation, the Divisional Delegates connected us to registered farms in the various Divisions. Consent from the farmers was verbal upon presentation of research authorisation and researchers’ credentials and affiliations. The study was performed on cloacal swabs from healthy broiler chicken of any age from 28 farms in 11 locations in 5 out of the 8 Divisions of the West Region of Cameroon. We used stratified sampling with Divisions as the strata.
Cloacal swabs were collected by rubbing a 50 cm2 area of the cloaca with a sterile swab pre-wetted with physiological water. Each swab was immersed in 5 ml of a sterile physiological solution in swab sampling tubes. The samples were transported in cool thermos flasks to the laboratory for analysis37.
Sample enrichment and culture
The swabs were dissolved in buffered peptone water and incubated for 24hrs at 38°C38. The enriched samples were cultured on EMB agar by plate streaking1 and incubated for 48 hours.
Isolation of isolates and preservation
Isolated colonies on the EMB agar were identified based on colony characteristics, picked and conserved in a conservation medium; a mixture of glycerol and Muller Hilton broth at 1 part : 3 parts and stored in a refrigerator39,40.
Phenotypic characterisation of isolates
A total of nine (9) rapid screening biochemical tests1,18 were performed on the isolates including: glucose and lactose fermentation, and hydrogen sulphide (H2S) and gas production using Kligler Iron Agar (KIA), urease activity and indole production using urea broth and Kovacs’ reagent, mannitol fermentation and motility using mannitol agar, and citrate fermentation using citrate agar.
Antibiotic susceptibility testing
Fresh colonies were used to prepare 0.5 McFarland solution inoculums using sterile physiological water. Antibiotic susceptibility testing was carried out on Mueller Hilton Agar by the Kirby-Bauer disc diffusion method2,18. Screening for antibiotic resistance in the isolates was done using following antibiotics41:
Class
|
Antibiotics
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Beta lactamins
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Amoxicillin, amoxicillin/clavulanic acid, ceftriaxone, cefotaxime and imipenem
|
Aminoglycosides
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Gentamicin and amikacin
|
Quinolones
|
Ciprofloxacin and levofloxacin
|
Tetracycline
|
Doxycycline
|
Phenotypic determination of resistance by penicillinase production was tested by combination disc test with amoxicillin/clavulanic acid with comparison to amoxicillin. Extended spectrum beta-lactamase production was tested by observing resistance to amoxicillin followed by resistance to ceftriaxone or cefotaxime42, and then the double disc synergy test was conducted by placing the ceftriaxone or cefotaxime 3 cm from amoxicillin/clavulanic acid giving a clear zone of intersection between the two discs43.
DATA ANALYSIS
Interpretation of phenotypic reactions
This research work generated information on Enterobacteriaceae carriage in broilers, identity of Enterobacteriaceae organisms, their prevalence, their antibiotic susceptibility profiles (susceptible, resistant, multidrug resistant and ESBL producing) and, odds ratios and correlations between outcomes (Enterobacteriaceae carriage, prevalence, resistance, MDR and ESBL production) and risks.
Enterobacteriaceae carriage was determined by the count of the different colonies on the Enterobacteriaceae-specific growth medium (EMB agar) based on colony aspect and morphology. The identity of the organism was gotten by interpreting the colony aspect and the results of the phenotypic reactions using a KIA user guide alongside the identification software: Feuille de calcule pour l’identification microbienne developed by J-Noël JOFFIN (8 Dec 2007 version). Susceptibility to antibiotics was evaluated using standard values as given by Sigma Aldrich®44. MDR was assessed as resistance of an isolate to two or more antibiotics belonging antibiotic classes of choice used against Enterobacteriaceae (cephalosporins, carbapenems, quinolones or aminoglycosides). ESBL production was interpreted from double disc synergy test between amoxicillin/clavulanic acid disc and ceftriaxone or cefotaxime disc.
Simple arithmetic operations and conversions were done using Microsoft Excel sheets while frequencies, prevalence, odds ratios, correlations and diagrams were done using IBM SPSS Statistics 20.