Extended-Spectrum B-Lactamase (ESBL)-Producing Escherichia coli Isolated from Children under 5 years with and without Diarrhoea in Two Hospitals in Dschang, Cameroon

Research Article

J Bacteriol Mycol. 2023; 10(2): 1206.

Extended-Spectrum B-Lactamase (ESBL)-Producing Escherichia coli Isolated from Children under 5 years with and without Diarrhoea in Two Hospitals in Dschang, Cameroon

Lethicia Danaëlle Mafo1; Raspail Carrel Founou1,2,3*; Michel Noubom1,4; Brice Davy Dimani3; Jessica Ravalona Zemtsa5; Patrice Landry Koudoum3; Aurelia Djeumako Mbossi5; Diomede Noukeu Njinkui6,7; Flaurant Thibau Tchouangueu1; Luria Leslie Founou2,5,8; Donatien Gatsing9

1Faculty of Medicine and Pharmaceutical Sciences, Department of Microbiology-Haematology and Immunology, University of Dschang, Cameroon

2Antimicrobial Research Unit, College of Health Sciences, School of Health Sciences, University of KwaZulu-Natal, South Africa

3Antimicrobial Resistance and Infectious Disease(ARID), Research Institute of Centre of Expertise and Biological Diagnostic of Cameroon (CEDBCAM-RI)

4Annex Regional Hospital of Dschang, Dschang Cameroon

5Reproductive, Maternal, Newborn and Child Health (ReMARCH) Research Unit, Research Institute of the Centre of Expertise and Biological Diagnostic of Cameroon (CEDBCAM-RI)

6Faculty of Medicine and Pharmaceutical Sciences, Department of Pediatric, Maternal and Child Health, Dschang Cameroon

7Pediatric, Obstetrics and Gynecology Hospital of Douala, Douala Cameroon

8Bioinformatics & Applied Machine Learning Research Unit, EDEN Biosciences Research Institute (EBRI), EDEN Foundation, Yaounde Cameroon

9Faculty of Sciences, University of Dschang, Department of Biochemistry, Dschand, Cameroon

*Corresponding author: Raspail Carrel Founou Department of Microbiology-Haematology and Immunology, University of Dschang, Dschang, Cameroon. Email: czangue@yahoo.fr; czangue@gmail.com

Received: May 10, 2023 Accepted: June 02, 2023 Published: June 09, 2023

Abstract

Introduction: Extended-Spectrum β-Lactamase (ESBL) mediating resistance in Enterobacterales is a global public health issue, especially in Low-and Middle-Income Countries (LMICs) such as Cameroon. ESBL-producing Enterobacterales reduce therapeutic options and lead to the use of last resort drugs such as carbapenems even in vulnerable populations like children under five years. This study aims at determining the phenotypic and genotypic characteristics of ESBL-producing Escherichia coli (ESBL-Ec) isolated from children under five years with and without diarrhoea in two health care facilities in Dschang.

Materials and Methods: A cross-sectional study was conducted from 3 February to 19 May 2022 in two hospitals in the city of Dschang, Cameroon. Stool collected were cultured on Eosine Methylen Blue (EMB) medium. Enterosystem 18R kit was used for bacterial identification. Evaluation of the resistance patterns and detection of ESBL production were performed with, the Kirby Bauer disk diffusion method and CHROMagar® ESBL medium, respectively. The genomic DNA of ESBL-Ec was extracted using the boiling method and subjected to conventional and multiplex PCRs for detection of blaSHV, blaCTX-M and blaTEM genes. Data were entered into ExcelTM 2016. Epi info and R software were used for statistical analyses with a p-value <0.05 considered statistically significant.

Results: Out of the 125 children enrolled, 67.2% (84/125) were colonized by E. coli. Among these, 57.14% (48/84) were colonized by ESBL-Ec. The prevalence of ESBL-Ec per hospitals, was higher in H1 than that of H2 although without statistical significance (60.42% vs 39.58%, p=0.24). ESBL-Ec isolates showed high levels of resistance to amoxicillin-clavulanic acid (96.22%), cefotaxime (75.47%), ceftriaxone (73.58%), ofloxacin (67.92%), levofloxacin (56.6%) and ciprofloxacin (54.71%). The majority of ESBL-Ec isolates (52.83%; 28/53) were co-producers of blaCTX-M and blaTEM.

Conclusion: Infection prevention and control measures coupled with antimicrobial stewardship strategies need to be strengthened to reduce emergence and dissemination of ESBL-Ec among this vulnerable population.

Keywords: ESBLs; CTX-M; Escherichia. coli; Diarrhoea; Children;

Introduction

Diarrhoeal diseases are one of ten leading causes of death among children under five years, particularly in Low and Middle-Income Countries (LMICs) where inadequate Water, Sanitation and Hygiene (WASH) prevails [1-3]. Foodborne diseases caused 600 million illnesses and 420,000 deaths globally in 2010 [4]. Enterotoxigenic and enteropathogenic Escherichia coli were the leading bacteria involved in the largest number of cases and deaths, respectively [5,6].

The emergence and escalation of Extended-Spectrum Β-Lactamase producing Enterobacterales (ESBL-E) is a major public health concern worldwide. ESBL-E including ESBL-producing E. coli (ESBL-Ec) exhibit resistance to third generation cephalosporins and also usually to several other antibiotic families such as aminoglycosides and fluoroquinolones leading to the use of last resort drugs [2,7,8]. ESBL-Ec has been incriminated in both nosocomial and community-acquired infections [5]. Numerous studies focusing on faecal carriage revealed a high prevalence of ESBL-Ec in Nepal (74%) [8], Egypt (71%) [9] and Morocco (58%) [10] in community settings; while, lower rates were observed in hospitals in Gabon (12%) and Ethiopia (17%) [11,12]. A study performed in Cameroon reported a 54% prevalence of ESBL-E faecal carriage in participants including outpatients, inpatients and hospital workers [13].

However, there is limited comprehensive data on ESBL-Ec colonizing or causing diarrhoeal diseases in children under five in Cameroon. This study aimed at determining the prevalence, risk factors, phenotypic and genotypic characteristics of ESBL-Ec isolated from faecal samples of children with or without diarrhoea in two hospitals of Dschang, Cameroon.

Material and Methods

Study population

Children under five years old with and without diarrhoea, attending the pediatric departments of two major hospitals, encoded for ethical consideration as H1 and H2, in the Western region of Cameroon were considered; during a four-month period (the 03rd February 2022 to 19th May 2022). H1 is a district hospital which receives an average of 300 children per year with about 20 cases of gastroenteritis per month. It is equipped with a biomedical analysis laboratory divided into three buildings and the hospital has 200 beds and provides care for more than 1,000 patients per year. The H2 which is a confessional hospital, receives an average of 30 children per month with about 5 children suffering from gastroenteritis. The hospital has 100 beds and provides care for more than 700 patients per year.

Recruitment and sample collection

The parent or legal guardian of any child that met the inclusion provided written informed consent and answered a questionnaire about the socio-demographics and clinical information of participants. Stool samples were collected in a sterile container after informed consent. Stool samples were cultured onto EMB agar (produced by Rapid Lab) and, incubated for 18-24h at 37°C in presence of oxygen. The growing colonies on EMB agar were analysed using biochemical tests through Enterosystem 18R as per the manufacturer instructions.

Antimicrobial susceptibility testing and Screening Confirmation for ESBL

Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method [14]. A panel of nine antibiotics were tested including amoxicillin/clavulanic acid (30μg), ceftriaxone (30μg), cefotaxime (30μg), chloramphenicol (30μg), ciprofloxacin (30mcg), levofloxacin (5μg), ofloxacin (5μg), tobramycin (10μg) and gentamicin (10μg). The different diameters of the inhibition zones were measured and interpreted as susceptible (S), intermediate (I) or resistant (R) according to the criteria defined by CA-SFM 2021 [14]. In addition, two screening methods were used for ESBL detection namely the champagne cork or funnel shaped synergies using double discs (clavulanic acid and ceftazidime) and chromogenic media CHROMagar™ ESBL.

E. coli ATCC 35218, K. pneumoniae ATCC 700603 and P. aeruginosa ATCC 27853 were used as quality control strains for the antimicrobial susceptibility testing and ESBL screening.

Genomic DNA Extraction

The genomic DNA of ESBL-Ec was extracted using a modified boiling method as described previously [15]. Briefly, one pure ESBL-Ec colony was suspended into 400μL of Tris-EDTA (10mMTris, 0.1mMEDTA) and then vortexed for five seconds. The suspension was then incubated for 25 min at 95°C in a dry bath digital (MIULab DKT200-1, Lasec International Ltd., Johannesburg, South Africa). After incubation, the suspension was centrifuged for 5 min at 9500rpm. The supernatant containing DNA (300μl) was subsequently transferred to a new eppendorf tube and stored at -40°C for future analysis.

Conventional Singleplex-Polymerase Chain Reaction (PCR)

Amplification of blaSHV gene was performed by conventional PCR using a thermal cycler BIO-RAD T100 (Bio-Rad Laboratories, Marnes-la-Coquette, France). The reaction took place in a 10μL reaction mixture consisting of 5μL of Dream Taq Green Polymerase Master Mix 2x (ThermoFisher Scientific™, Vilnius, Lithuana), 2.8μL of nuclease-free water, 0.1μL of each forward and reverse primer [10μM] and 2μL of DNA. The amplification steps were as follows: initial denaturation (95°C for 3 min), 30 cycles of denaturation at 95°C for 4s, annealing at 46.9°C for 45s, elongation at 72°C for 60 s, and final elongation at 72°C for 5 min as previously described [16,17].

Conventional Multiplex-Polymerase Chain Reaction (PCR)

The detection of blaCTX-M and blaTEM genes among ESBL-Ec isolates was performed by multiplex PCR method using a thermal cycler BIO-RAD T100 (Laboratoires Bio-Rad, Marnes-la-Coquette, France). The reaction was done in a 10μL reaction mixture consisting of 5 μL of Dream Taq Green Polymerase Master Mix 2x (ThermoFisher Scientific™, Vilnius, Lithuana); 2.6μL of nuclease-free water, 0.1 μL of each forward (CTX-Mu-F and TEM-F) and reverse (CTX-Mu-R and TEM-R) primers [50μM] and 2μL of DNA. Amplification steps were as follows: initial denaturation (95°C for 3 min), 30 cycles of denaturation at 95°C for 4 s annealing at 46.9°C for 45s, elongation at 72°C for 60 s and final elongation at 72°C for 5 min. The annealing temperatures and primer sequences of primers are shown in Table 1.