1. Introduction
Enterococci are facultative anaerobic Gram-positive bacteria, immobile and non-encapsulated, present in the digestive tract of animals and humans [1] . Some species are useful in the manufacture of dairy products, playing a role in the conservation and bacteriological quality of food while maintaining their nutritional and organoleptic properties. But others are often opportunistic and cause infections in adults and newborns especially Enterococcus faecalis and Enterococcus faecium which rank among the leading causes of nosocomial bacteremia and urinary tract infections [2] [3] . They often persist on hospital surfaces due to their ability to withstand adverse environmental conditions (low or high temperatures, high pH, and high salinity). The global Enterococcus faecalis—Enterococcus faecium ratio is currently shifting towards Enterococcus faecium. The acquired antibiotic resistance traits of this latter species can explain this evolution [4] .
The advent of antibiotic therapy in the 1940s allowed a significant evolution in the management of infectious diseases. Unfortunately, antimicrobial resistance has quickly become a global health problem [5] . Enzymatic inhibition, reducing membrane permeability, modification of antibiotic targeted binding sites and the production of efflux pumps are some of the mechanisms used by organisms to fight against antibiotics. Some of the main reasons for antimicrobial resistance are self-medication, incorrect use of antibiotics (wrong dosage and prolonged use), lack of standards for health workers and misuse in animal husbandry [5] .
Enterococci present variable resistance levels to some antibiotics. This is the case for aminoglycosides, beta-lactams and cephalosporins [6] . In 2017, WHO ranked Enterococci among priority pathogens for research and development of new antibiotics. In America, a study carried out by Faron et al., in 2016 indicated that the prevalence of vancomycin-resistant Enterococci (VRE) over the past decade was increasing [7] . In 2015 Abamecha et al.; in Ethiopia evaluated the resistance profile of Enterococci and demonstrated that 36% of the isolated strains were resistant to ampicillin, 54.4% to streptomycin and 34.2% to gentamycin [8] . While in Algeria, Benzaid et al., in 2022 reported a prevalence of 7.81% of nosocomial infections due to Enterococcus faecalis [9] . In Cameroon, a study conducted in 2015 by Gonsu et al.; on the antibiotic sensitivity profile of Enterococci showed that 94% of Enterococcus faecalis strains were resistant to erythromycin, 59% to trimethoprim + sulfonamides and 53% to tetracycline. They stated that the majority of strains were isolated from 72% of hospitalized patients, and 27.58% of non-hospitalized patients [10] .
However, few studies have been carried out in Cameroon to fully understand the burden of antibiotic resistance as a result of Enterococci in hospital acquired infections. Therefore, the objective of our study was to determine the antibiotic resistance profile of Enterococcus faecalis and Enterococcus faecium isolates from urine and pleural fluid in two hospitals of Cameroon.
2. Materials and Methods
2.1. Study Duration and Location
A cross-sectional descriptive, laboratory and analytical study was carried out between June to August 2023 in two public hospitals; One in the Centre region (Saint Porres Dominican Hospital Center) and the other in Littoral region (Laquintinie hospital of Douala). Sampling and analysis was done in the laboratories of both hospitals.
2.2. Sampling Method and Study Population
Our study population included all hospitalized and non-hospitalized patients which a cytobacteriological test of urine and pleural fluid were done. The minimum sample size was obtained using the Lorentz formula. Data related to potential risk factors were collected using a questionnaire.
2.3. Sample Analysis
The samples were inoculated on CLED Agar for urine and on Chocolate + polyvitex and blood agar (prepared from Columbia agar) for pleural fluid samples and incubated at 37˚C for 18 to 24 hours. Identification of isolates was carried out using the API 20 STREP micro gallery (Biomerieux, France) and tested for antimicrobial susceptibility.
2.4. Antibiotic Susceptibility Testing
All isolates of Enteroccocus spp confirmed were inoculated using a bacterial inoculum (following the 0.5McFarland standard) on Müeller-Hinton medium and incubated for 24 hours at 37˚C in order to test their sensitivities to different families of antibiotics using the agar diffusion method. The interpretation was made according to the Antibiogram Committee of the French Society of Microbiology (CA-SFM-2023 v.1.0). We used 11 different antibiotic discs per strain: Ampicillin (2 µg), Gentamicin (30 µg), Vancomycin (5 µg), Teicoplanin (30 µg), Doxycycline, cotrimoxazole (1.25/23.75 µg), Erythromycin (15 µg), Streptomycin (300 µg), Norfloxacin (10 µg), Rifampicin (5 µg), and Imipenem (10 µg). The detection of Enteroccocus isolates resistant to glycopeptides was done by observing the reduced inhibition diameter around the Vancomycin and Teicoplanin disks.
2.5. Data Evaluation and Analysis
The data on socio-demographical and potential risk factors were recorded on the Microsoft Excel 2016 software and analysed using Epi info version 7.0. The results were expressed using descriptive statistics and associated to their confidence interval at 95%. The research of potential risk factors associated to the presence of Enterococci, was done using the logistic regression in univariate and multivariate analysis. P value < 0.05 was considered as significant.
2.6. Ethical Considerations
An ethical clearance was obtained on the basis of the evaluation and validation of the research protocol by the Institutional Ethics Committee of Université des Montagnes (Autorisation N˚ 2023/176/UdM/PR/CEAQ). Free and informed consent of patients were obtained and sample collection authorizations were equally obtained from the two hospitals.
3. Results
3.1. Sociodemographic Characteristics of the Study Population
A total of 511 patients made up of (63.99%). females were recruited for the study. The majority 90.22% of the patients were not hospitalised and 80.63% had not consumed antibiotics for at least two weeks. Our specimen were mostly made up of wound (67.71%) (Table 1).
3.2. Distribution of Enterococcus spp Infection among Patients
Enterococcus spp were isolated in 27.79% of our samples with Enterococcus faecalis mostly encountered (Figure 1).
3.3. Antibiotic Resistant Pattern of Enterococcus spp Isolates
3.3.1. Antibiotic Susceptibility Testing
Enterococcus spp showed high level of resistances to penicilline (99.3% to Ampicilline), macrolides (66.2% to Erythromycin) and cyclines (85.2% Doxycycline) (Figure 2). Eleven antibiotics were tested following EUCAST 2023.
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Figure 1. Distribution of Enterococcus spp infection among patients.
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Table 1. Distribution of the study population according to their socio-demographic characteristics.
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Figure 2. Antibiotic susceptibility test of Enterococcus spp.
3.3.2. Prevalence of Glycopeptide Resistant Enterococcus
A few 6.65% of Enterococcus spp isolates were resistant to Glycopeptide (Figure 3).
3.4. Analysis of Risk Factors Related to Infection with Enterococcus spp
Risk factors related to infection with Enterococcus spp were sought with a 95% confidence interval. P-values were significant for values less than 0.05 (Table 2).
Hospitalization, Health facility, type of urine specimen and hand hygiene were risk factors related to infection with Enterococcus spp.
3.5. Univariate Analysis of Risk Factors to Glycopeptide Resistant Enterococcus spp Infection
Risk factors to glycopeptide resistant Enterococcus spp were sought with a 95% confidence interval. P-values were significant for values less than 0.05 Hospitalization, health facility and hand hygiene were risk factors related to glycopeptide resistant Enterococcus spp (Table 3).
4. Discussion
The current study highlights the significance of the fight against antibiotic resistance, contribute to the care of patients suffering from multi-resistant bacteria and reduce nosocomial infections. The study revealed a high frequency of Enterococcus faecium mostly encountered as compared to Enterococcus infection with significant resistance to 11 antibiotics.
The average age of the participants in the current study was 35 years given that the African population is mainly made up of young people. Furthermore, our participants were predominantly women (66.99%) than men (36.01%). This is explained by the fact that women are more exposed to urinary infections due to the anatomical particularities of the urinary system; their urethra is shorter, making it easier for bacteria to enter the bladder [11] .
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Figure 3. Prevalence of glycopeptide resistant Enterococcus spp.
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Table 2. Univariate analysis of risk factors related to Enterococcus spp infection.
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Table 3. Univariate analysis of risk factors to glycopeptide resistant Enterococcus spp infection.
The Enterococcus spp were isolated in 27.79% of our samples. This result is higher than the 8.44% found by Akhter et al. in Tunisia in 2014 in which they isolated Enterococci from urine only [12] . Enterococci ranks among the leading bacteria responsible for urinary tract infection [13] [14] .
In the current study, E. faecium predominates over E. faecalis (66.2%); these results are similar to those of Gonsu et al.; who also demonstrated the predominance of 52% of E. faecium [10] . Recent studies have demonstrated that the worldwide ratio of Enterococcus faecalis—Enterococcus faecium is changing in favor of E. faecium due to the Intrinsic and acquired antimicrobial resistance traits of this species [15] [16] .
The antibiotic susceptibility testing of the current study revealed of a strong resistance to penicillins, tetracyclines, and macrolides. This is similar to that of Muylaert and Mainil who reported this resistance to macrolides, tetracycline betalactams. Intrinsic resistance to beta lactams is maintained in Enterococci through the overproduction of penicillin binding proteins. Concerning aminoglycoside resistance, the low permeability of the well to the large aminoglycoside molecules could explain the moderate resistance [17] . Furthermore, 6.65% of Enterococcus spp isolates were resistant to Glycopeptide. Resistance to glycopeptides is mainly the result of the presence of resistance genes carried by plasmids. The most common being vanA, vanB and vanC [18] .
The univariate analysis of risk factors in our study allowed us to highlight the main factors linked to infection due to Enterococcus spp such as hospitalization (OR = 5.37; P-value = 0.00), hand hygiene (OR = 2.57; P-value = 0.01) and the specimen type (OR = 2.37; P-value = 0.01). Our results corroborate with a similar study carried out by Stucki et al. in Switzerland in 2014 [19] . These values could be explained on the one hand by the fact that when hygiene and environmental conditions are not favorable, the promiscuity of patients in hospital rooms favors transmissible infections; during their stay at the hospital, patients are exposed to bacteria through several sources including infected patients especially when hygiene rules are not respected [20] . On the other hand, the length of stay hospital is also a factor because the more a hospitalized person is bedridden, the more they are exposed to the risk of infections due to Enterococcus spp [21] . These factors were equally linked to glycopeptide resistant Enterococcus.
5. Conclusions
Our study revealed that the frequency of Enterococcus infection was 27.79% with Enterococcus faecium mostly encountered (66.2%). Significant resistance to penicillin (99.3% Ampicillin), macrolides (66.2% Erythromycin) and cyclines (85.2% Doxycycline) were observed. Equally, 6.65% of Enterococcus spp isolates were resistant to Glycopeptide.
Risk factors related to infection with Enterococcus spp were related to the type of health facility, urine specimen and hand hygiene practices while risk factors related to infection with Enterococcus spp were hospitalisation, health facility and hand hygiene being related to glycopeptide resistant Enterococcus.
Acknowledgements
Our thanks go to all those who contributed to this research study.
Authors’ Contributions
Conceptualization: CID; Project administration: CID, and AMA; Resources: CID, LPM, EMNM, NMN; Methodology: CID, FW, GYK, CSN, CPK, LPM, EMNM, NMN and AMA; Investigation: FW, GYK, CSN, CPK, LPM, EMNM, NMN, HKT, MJTT, and YVYM; software: CID, and CSN; Formal analysis: CSN and FW; Visualization: CID, and AMA; Writing-original draft: CID, FW, GYK, and CPK; Writing-review and editing: CID, FW, and CSN; Validation: CID, and AMA; Supervision: AMA; Data curation: CID. All authors have read and agreed to the published version of the manuscript.