Enterococci are members of Gram-positive
In fact, enterococci species have low virulence factors. However, under conditions in which the host encountered immune suppression or the integrity of the gastrointestinal or genitourinary tract has been disrupted, they spread to normally sterile sites and cause various infections. These infections include urinary tract infections, wound infection, sepsis, endocarditis, intra-abdominal abscesses, and biliary tract infections [
According to Centers for Disease Control and Prevention (CDC) nationwide report, enterococci are one of the leading causes of nosocomial infections worldwide, with healthcare-associated infections occurring more frequently in resource-limited settings than in developed countries [
Different risk factors have been reported to be associated with the spread of enterococci infections including concurrent infections, history antibiotic use, surgery, catheterization, longer duration of hospitalization, and underlying immunosuppressing diseases such as HIV, diabetics, and cancer [
The emergence and spread of antimicrobial resistance among enterococcus species pose enormous challenges for clinicians, especially in the management of severe infections. The increased prevalence and dissemination of multidrug-resistant enterococcus has narrowed the therapeutic options globally, as the majority of enterococcus isolates exhibit a high level of resistance to ampicillin, penicillin, and vancomycin, which are indeed the most historically useful anti-enterococci antibiotics [
In 2018, the national survey for antimicrobial resistance in Europe report indicated that vancomycin-resistant enterococcus species increased significantly from 10.4% in 2014 to 14.9% in 2017 in different countries [
The prevalence and drug resistance figures are significantly high in developing countries. In Africa and Ethiopia, some reports indicated the overall prevalence of enterococci and antibiotic resistance ranged from 2.2% to 76% and 6.3% to 95.5%, respectively [
A hospital-based cross-sectional study was conducted from February 2019 to May 2019 at Dessie Referral Hospital, South Wollo Zone of Amhara regional state, Northeast Ethiopia. Dessie Referral Hospital is located in Dessie city, 400 km from the capital, Addis Ababa, and 471 km far from Bahir Dar, the capital of the Amhara regional state. The city has one referral hospital, one general hospital, three private general hospitals, five health centers, eight private higher clinics, and one Public Health Research Institute. Dessie Referral Hospital provides emergency, antiretroviral therapy (ART) services, chronic care, surgical, dental, medical, pediatric, gynecologic, obstetric, and other services. The hospital serves patients from all parts of the region which comprises a population of more than 4 million people. Dessie Referral Hospital pediatric services consist of inpatient and outpatient departments. The outpatient pediatric department consists of 15 beds and five rooms, while the inpatient pediatric ward consists of 13 rooms and 49 beds, with an average of about 7 children admitted per day.
All children younger than 15 years old, attending DRH and who were requested for laboratory investigation during the study period, were included in this study. On the other hand, pediatric patients who received antibiotics within the past 2 weeks were excluded. Moreover, as enterococci are normal flora of some specific sites including respiratory, genital, and gastrointestinal tracts, samples such as sputum, throat swab, stool, and vaginal swabs were excluded from the study.
The sample size was determined using a single population proportion formula considering 50% prevalence, marginal error of 5%, and
Data were collected using a short interview guided by pretested structured questionnaire consisting of the client’s sociodemographic, clinical, and risk factor data. Clinical samples were collected from each study participant aseptically. About 5 ml of the blood sample was collected from children and dispensed into blood culture bottle prepared with 25 ml of Tryptic Soya Broth (FL Medical, Italy) aseptically. Ten ml of freshly voided midstream urine specimen was collected using wide mouth, leak-proof, sterile, plastic container under the supervision of the principal investigator and processed within 2 hours of collection. Approximately 5 ml of cerebrospinal fluid (CSF) sample was collected aseptically into sterile tube by lumbar or ventricular puncture performed by a physician and processed within one hour of collection. Wound swab, pus, eye, and ear discharges were obtained using sterile cotton tip applicator stick aseptically. The blood sample was transported with the blood culture broth while all swabs were transported within BHI broth (HiMedia™). The collected specimens were then stored in a cold box and transported to the Department of Microbiology Laboratory, Amhara Public Health Institute (APHI), Dessie Branch. Immediate inoculation had been performed for all specimens on arrival to the laboratory.
Specimens were inoculated on appropriate culture media to isolate the enterococci bacteria. The blood culture bottles were incubated at 37°C and observed after 24 hrs daily for consecutive 5 days for the presence of turbidity, hemolysis, gas formation, or color changes which are evidence of microbial growth. If the culture bottle does not show any growth within 7 days, it was reported as negative. Whenever visible growth appears, the bottle was opened aseptically, a small amount of broth was taken with a sterile loop and subcultured on Bile Esculin Azide agar (BEAA) (Oxoid Ltd., UK). Urine samples were inoculated on BEAA media with a 1
The antimicrobial susceptibility testing of enterococci isolates was performed using the Kirby-Bauer disk diffusion technique as modified by the Clinical and Laboratory Standard Institute (CLSI) in 2019 [
Data quality was ensured by using standardized data collection materials. The questionnaire was pretested on 5% of the sample size in the nearby Boru Meda Hospital before the actual study commenced to make sure whether the questionnaire is appropriate and understandable. All the questions in structured questionnaire were prepared in a clear and precise way and translated into local language (Amharic). The collected data were checked daily for completeness. Moreover, all laboratory analyses were performed by maintaining quality control procedures. Standard operating procedures (SOPs) were strictly followed verifying that media meet expiration date and quality control parameters per CLSI guideline. All culture media were prepared following the manufacturers’ instructions. Batch of prepared media was checked for pH, performance, and sterility test by incubating samples of the plate at 37°C for 24 hrs, and reagents for Gram stain and biochemical tests were checked using known standard strains of
Data was entered and analyzed using Statistical Package for Social Sciences (SPSS) version 25.0. (IBM, USA), and descriptive statistics, binary, and multivariate logistic regression were computed. The bivariate analysis using maximum likelihood estimates of the categorical variables was used to determine the association of each variable with the dependent variable. Furthermore, variables with
A total of 403 study participants were included during the study period. Of these, 188 (46.7%) were males, while 215 (53.3%) were females. The age of the study participants ranged from 15 days to 14 years. The age distribution of participants indicated that majority 159 (39.5%) of the study participants were in the age group of 5 to 9 years followed by 124 (30.8%) belonging to the age group ≤4 years. Slightly majority of the study participants 214 (53.1%) were rural dwellers. Moreover, the clinical data showed that outpatient was the predominant group among the study participants, with a proportion of 67.2%, while the remaining 32.3% were from inpatient (Table
Sociodemographic characteristics of pediatric patient study participants at DRH from February to May 2019.
Demographic characteristics | Frequency in number | Frequency in percent |
---|---|---|
Age | ||
≤4 | 124 | 30.8% |
5-9 | 159 | 39.5% |
10-14 | 120 | 29.8% |
Sex | ||
Male | 188 | 46.7 |
Female | 215 | 53.3% |
Residence | ||
Urban | 189 | 46.9% |
Rural | 214 | 53.1% |
Patient setting | ||
Outpatient | 271 | 67.2% |
Inpatient | 132 | 32.8% |
Family educational status | ||
Illiterate | 178 | 44.2% |
Read and write | 111 | 27.5% |
High school | 47 | 11.7% |
Diploma | 40 | 9.9% |
First degree and higher | 27 | 6.7% |
A total of 11 (2.7%) enterococci isolates were isolated from all clinical samples. Seven out of 11 isolates were identified from outpatients. Relatively higher frequency of enterococci infection (5/11) was observed in the age group of ≤4 years compared to the other age groups (Table
Prevalence of enterococci infection among pediatric patients (
Demographic characteristics | Enterococci infection | |
---|---|---|
Positive (%) | Negative (%) | |
Age | ||
≤4 | 5 (45%) | 119 (96.0%) |
5-9 | 2 (18.2%) | 157 (98.7%) |
10-14 | 4 (36.8%) | 116 (96.7%) |
Sex | ||
Male | 5 (45.4%) | 183 (97.3%) |
Female | 6 (54.5%) | 209 (97.2%) |
Residence | ||
Urban | 4 (36.3%) | 185 (97.9%) |
Rural | 7 (63.7%) | 207 (96.7%) |
Patient setting | ||
Outpatient | 7 (63.7%) | 125 (94.7%) |
Inpatient | 4 (36.3%) | 267 (98.5%) |
Family educational status | ||
Illiterate | 5 (45.4%) | 173 (97.2%) |
Only read and write | 2 (18.2%) | 109 (98.2) |
Completed high school | 3 (27.3%) | 44 (93.6%) |
College diploma | 1 (9.1%) | 39 (97.5) |
First degree and higher | 0 (0.0%) | 27 (100.0%) |
Total | 11 (2.7%) | 392 (97.3%) |
Distribution of enterococci infection in different clinical specimens of pediatric patients at DRH from February to May 2019.
In this study, a total of 14 independent variables were considered during the bivariate analysis of risk factors for enterococci infection. In the multivariate analysis, the presence of enterococci infection was significantly associated with having a history of invasive procedure (
Bivariate and multivariate analysis of associated factors for acquiring enterococci infection among pediatric patients attending DRH from February to May 2019.
Variables (total no.) | Enterococci infection | COR (CI 95%) | AOR | |||
---|---|---|---|---|---|---|
Positive (%) | Negative (%) | |||||
Age | ||||||
≤4 (124) | 5 (4%) | 119 (96.0%) | 1.21 (0.319-4.65) | 0.77 | NA | |
5-9 (159) | 2 (1.3%) | 157 (98.7%) | 0.360 (0.06-2.05) | 0.25 | ||
10-14 (120) | 4 (3.3%) | 116 (96.7%) | 1 | |||
Sex | ||||||
Male (188) | 5 (2.7%) | 183 (97.3%) | 1.05 (0.31-3.50) | 0.93 | NA | |
Female (215) | 6 (2.8%) | 209 (97.2%) | 1 | |||
Residence | ||||||
Urban (189) | 4 (2.1%) | 185 (97.9%) | 1.56 (0.45-5.42) | 0.48 | NA | |
Rural (214) | 7 (3.3%) | 207 (96.7%) | 1 | |||
Patient setting | ||||||
Inpatient (271) | 7 (5.3%) | 125 (94.7%) | 3.73 (1.07-13.00) | 0.038 | 0.33 (0.10-2.13) | 0.48 |
Outpatient (132) | 4 (1.5%) | 267 (98.5%) | 1 | |||
Family educational status | ||||||
Illiterate (178) | 5 (2.8%) | 173 (97.2%) | 0.63 (0.12-3.33) | 0.59 | NA | |
Read and write (111) | 2 (1.8%) | 109 (98.2) | 2.35 (0.54-10.25) | 0.25 | ||
High school (47) | 3 (6.4%) | 44 (93.6%) | 0.88 (0.10-7.80) | 0.91 | ||
Diploma (40) | 1 (2.5%) | 39 (97.5) | 1.02 (0.21-6.37) | 0.99 | ||
First degree and higher (27) | 0 (0.0%) | 27 (100.0%) | 1 | |||
History of | ||||||
Antibiotic administration | 10 (4.3%) | 221 (95.7%) | 7.81 (0.99-61.66) | 0.052 | 10.17 (0.50-206.3) | 0.131 |
Invasive procedure | 5 (17.2%) | 24 (82.8%) | 28.2 (7.77-102.23) | <0.001 | 26.91 (4.96-148.99) | <0.001 |
Malnutrition | 2 (8.3%) | 88 (91.7%) | 3.73 (0.76-18.35) | 0.101 | 6.31 (0.5-28.09) | 0.076 |
Burn | 1 (14.3%) | 6 (85.7%) | 6.43 (0.70-58.53) | 0.260 | NA | |
Animal contact | 3 (6.4%) | 44 (93.6%) | 2.96 (0.75-11.59) | 0.110 | 0.11 (0.15-0.93) | 0.043 |
Chronic illness | 6 (9.2%) | 59 (90.8%) | 6.77 (2.00-22.91) | 0.021 | 16.21 (2.98-88.08) | <0.001 |
Contact with health profess | 5 (6.1%) | 77 (93.9%) | 3.40 (1.01-11.46) | 0.042 | 1.78 (0.32-9.91) | 0.508 |
Admission | 8 (11.8%) | 60 (88.2%) | 14.75 (1.36-15.59) | 0.020 | 13.73 (3.01-62.59) | <0.001 |
Hospital stay >48 hr | 7 (15.7%) | 39 (84.7%) | 3.41 (0.39-29.74) | 0.261 | NA |
The enterococci isolates showed a higher level of drug resistance to tetracycline (10/11), chloramphenicol (9/11), augmentin (8/11), and erythromycin (7/11), while 5/11 resistance rates were reported for the rest of the antibacterial agents. Moreover, nearly half (5/11) of the isolated enterococci were vancomycin-resistant (Table
Antimicrobial susceptibility patterns of enterococci isolates from pediatric patients attending DRH from February to May 2019.
S. no. | Antibiotics | Susceptible (%) | Resistance (%) |
---|---|---|---|
1 | Penicillin | 6 (54.5) | 5 (45.5) |
2 | Ampicillin | 6 (54.5) | 5 (45.5) |
3 | Vancomycin | 6 (54.5) | 5 (45.5) |
4 | Erythromycin | 2 (33.7) | 4 (66.7) |
5 | Ciprofloxacin | 6 (54.5) | 5 (45.5) |
6 | Nitrofurantoin | 2 (33.4) | 4 (66.7) |
7 | Tetracycline | 1 (9.1) | 10 (90.9) |
8 | Chloramphenicol | 1 (16.3) | 5 (83.7) |
9 | Amoxicillin/clavulanic acid | 3 (27.3) | 8 (72.7) |
All detected enterococcus isolates (11) were resistant to at least one antimicrobial agent, whereas 9 isolates were resistant to ≥2 antimicrobials. Multidrug resistance (defined as nonsusceptibility to at least one agent in three or more antimicrobial categories) was seen in 6/11 of the enterococci isolates (Table
Drug resistance pattern of enterococci isolates from pediatric patients attending DRH from February to May 2019.
Resistance rate | Combination of antibiotics | Number of isolates |
---|---|---|
R1 | TET | 2 |
R2 | TET, AUG | 2 |
R2 | TET, VAN | 1 |
R4 | TET, P, AMP, AUG | 1 |
R4 | TET, VAN, AUG, CIP | 1 |
R5 | TET, P, AMP, E, CPR | 1 |
R5 | TET, P, AMP, VAN, CPR, | 1 |
R8 | TET, P, AMP, VAN, CPR, NIT, E, C | 2 |
Total | 11 |
E: erythromycin; TET: tetracycline; AMP: ampicillin; P: penicillin; AUG: amoxicillin/clavulanic acid; C: chloramphenicol; CIP: ciprofloxacin; VAN: vancomycin; NIT: nitrofurantoin; R1: resistance to one; R2: resistance to two; R4: resistance to four; R5: resistance to five; R8: resistance to eight drugs.
In the past 1970s and 1980s, enterococci have been changed from being intestinal normal flora of little clinical significance to becoming one of the most common nosocomial pathogens associated with significant morbidity and mortality worldwide [
In this study, enterococci species were most predominantly isolated from urine specimens followed by blood and wound swabs. This was similar to previous reports conducted in Addis Ababa Ethiopia [
The present study showed that enterococci isolates had an overall multidrug resistance rate of 54.5%. This finding was in line with a study report from Ethiopia 60% [
Bacterial drug resistance for the commonly used antibiotics is currently spreading globally. In our study, 5/11 (45%) of the enterococci isolates were resistant to ampicillin, penicillin, vancomycin, ciprofloxacin, and nitrofurantoin. This was in line with the report from India, 52.6% [
The rate of 5/11 (45.5%) vancomycin-resistant enterococci (VRE) in the current study has made the scenario worse as it is the preferred choice of drug in the case where this bacterium became resistant to other antibiotics in the area. Even though the result was comparable with the study report undertaken in all age groups in Gondar, 41.5% [
The present study revealed that nearly half (45%) of the isolated enterococci showed resistance to ciprofloxacin, which was comparable with reports from Addis Ababa, 53.3% [
In this study, higher antimicrobial resistance was seen against commonly used antibiotics including tetracycline (91%), amoxicillin/clavulanic acid (72.7%), chloramphenicol (83%), and erythromycin (66.7%). This finding was higher than reports from southern Ethiopia [
The multivariate analysis of the present study showed that having a history of invasive treatment procedure was significantly associated with pediatrics enterococci infection (
The present study also showed that having a history of chronic illness (
Species investigation was not performed due to financial issue and shortage of reagents and materials.
The prevalence of enterococci from pediatric patients in this study was relatively low compared to other studies. The risk of infection became high when children have a history of different chronic illness and history of admission and undergoes invasive treatment procedures. A significant rate of multidrug-resistant enterococci including VRE was identified from clinical samples in the study area. Such occurrence of MDR and VRE showed limited antibiotic treatment options for enterococci infections. Therefore, efforts should be made to prevent nosocomial enterococci infections and spread of multidrug-resistant enterococci. Periodic evaluation of drug susceptibility pattern is also essential for rational and appropriate use of antibiotics. Moreover, appropriate prescription and use of antibiotics and large-scale species identification and genotypic studies at national level are necessary.
Amhara Public Health Institute
American Type Cell Culture
Bile Esculin Azide agar
Brain heart infusion
Centers for Disease Control and Prevention
Clinical and Laboratory Standard Institute
Cerebrospinal fluid
Dessie Referral Hospital
Healthcare-associated infections
Human immunodeficiency virus
Infection prevention and control
Multidrug resistance
Muller Hilton agar
Standard operating procedures
Statistical Package for Social Science
Urinary tract infection
Vancomycin-resistant enterococci.
Data supporting the conclusions of this article are within the manuscript.
Ethical clearance was obtained from the School of Biomedical and Laboratory Sciences, University of Gondar Ethical Review Committee, and permission was obtained from the Dessie Referral Hospital and Amhara Public Health Institute, Dessie Branch. Confidentiality was kept throughout the study, and positive findings for enterococci infection were communicated to the respective physicians with their drug susceptibility patterns for treatments.
Prior to commencing the study, written parental/guardian consent and/or patient accent was obtained for each study participant before data collection.
The authors declare that they have no conflicts of interest in this research work.
AA, MT, and MAB were involved in proposal writing and designing the study and participated in analysis and interpretation of data. AA, MT, SG, and MAB were involved in data collection and drafting of the manuscript. AA, MT, SG, and MAB finalized the write up of the manuscript. All authors critically revised the manuscript and read and approved the final manuscript for publication.
The authors would like to acknowledge the Department of Medical Microbiology, School of Biomedical and Laboratory Sciences, College of Medicine and Health Sciences, University of Gondar Hospital, for giving the opportunity to conduct this thesis work and all the study participants, APHI staffs especially Microbiology Department, and the staffs of DRH for their participation and support during the sample collection. This work only received institutional support from the Amhara Public Health Institute, Dessie Branch.