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ORIGINAL ARTICLE
Year : 2019  |  Volume : 7  |  Issue : 2  |  Page : 71-75

Vancomycin-resistant enterococcus faecalis among patients with urinary tract infection at aminu kano teaching hospital


1 Department of Medical Laboratory Science, Faculty of Allied Health Sciences, College of Health Sciences, Bayero University Kano, Kano, Nigeria
2 Department of Medical Microbiology and Parasitology, Faculty of Clinical Sciences, Bayero University, Kano, Nigeria

Date of Submission31-May-2019
Date of Decision29-Sep-2019
Date of Acceptance07-Oct-2019
Date of Web Publication02-Apr-2020

Correspondence Address:
Dr. Abdulhadi Sale Kumurya
Department of Medical Laboratory Science, Faculty of Allied Health Sciences, College of Health Sciences, Bayero University Kano, Kano
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njecp.njecp_16_19

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  Abstract 


Background: Emergence of vancomycin-resistant enterococci (VRE) has become a serious issue for nosocomial infection control worldwide. The increase in antibiotic resistance among enterococci, specifically to vancomycin, has become a major clinical and epidemiological problem. Aim: The study aimed at the identification, phenotypic characterization of Enterococcus faecalis, and susceptibility pattern of vancomycin on E. faecalis associated with urinary tract infection (UTI) on patients admitted to the female medical ward and postnatal ward of Aminu Kano Teaching Hospital, Kano, Nigeria. Materials and Methods: A total of 114 urine samples were aseptically collected from patients suspected with UTI and cultured on blood agar and cystine–lactose–electrolyte-deficient agar and incubated under anaerobic and aerobic environment, respectively, at 37°C for 24 h. Bacterial growth was subjected to standard methods for the identification of E. faecalis and tested for their antibiotic susceptibility patterns on vancomycin (30 μg), ciprofloxacin (10 μg), streptomycin (10 μg), chloramphenicol (30 μg), tetracycline (10 μg), and co-trimoxazole (25 μg) (Oxoid, UK) using disk diffusion method. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of vancomycin were determined. ATCC E. faecalis 29,212 was used as control organism for every test run. Data generated were analyzed using descriptive statistics. Results: A total of 8 (7.0%) E. faecalis were isolated from 114 samples studied. The prevalence of 50.0% and 25.0% VRE was obtained by disk diffusion and broth dilution methods, respectively. The result of antibiotics susceptibility pattern revealed that 6 (75.0%) of the E. faecalis isolates show multiple resistance to tetracycline (30 μg), co-trimoxazole (1.25/23.75 μg), and streptomycin (10 μg), but more sensitive to ciprofloxacin (5 μg) and chloramphenicol (30 μg). The MIC result revealed that vancomycin has high effect at lower concentration of 4 and 2 μg/ml and loses its effect at increase concentration of 8, 16, 32, and 64 μg/ml, and the result obtained from the MBC of vancomycin revealed that it has only a bacteriostatic effect against E. faecalis. Conclusion: Therefore, ciprofloxacin and chloramphenicol are the best therapeutic options to treat infection with VRE in Kano, and treatment with the resistant drugs obtained may affect feature treatment and management of infection with these drugs.

Keywords: Antibiotics, infection, resistance, urinary tract infection, vancomycin


How to cite this article:
Kumurya AS, Idris AM, Ali MM. Vancomycin-resistant enterococcus faecalis among patients with urinary tract infection at aminu kano teaching hospital. Niger J Exp Clin Biosci 2019;7:71-5

How to cite this URL:
Kumurya AS, Idris AM, Ali MM. Vancomycin-resistant enterococcus faecalis among patients with urinary tract infection at aminu kano teaching hospital. Niger J Exp Clin Biosci [serial online] 2019 [cited 2020 Aug 5];7:71-5. Available from: http://www.njecbonline.org/text.asp?2019/7/2/71/281618




  Introduction Top


Enterococci are Gram-positive cocci which occur in pairs or short chains, catalase negative, nonspore forming, facultative anaerobic organisms that belong to the family Enterococcaceae.[1] The organism mostly lives in the intestinal tract and the female genital tract as colonizer. However, Enterococcus can cause an infection if it gets into the urinary tract, bloodstream, or skin wounds.[2]Enterococcus reported as the most commonly isolated bacteria after  Escherichia More Details coli and Staphylococcus aureus from nosocomial infection in Europe.[3]Enterococcus faecalis accounts for about 80%–90% of nosocomial infections caused by Enterococcus species, whereas Enterococcus faecium accounts for only 5%–10%.[4] Vancomycin is an antibiotic used to treat infections caused by enterococci. However, now some Enterococcus organism become resistance to this drug and is known as vancomycin-resistant Enterococcus or vancomycin-resistant enterococci (VRE).[5] Recently, the emergence of VRE has become a serious challenge for clinicians in patient management with enterococcal infections worldwide. In Africa, including Nigeria, there have been very few reports of VRE, which are still not enough to state the true picture of the VRE. VRE cause a wide variety of infections involving the urinary tract, wound, and bloodstream, among other sites most commonly in hematological malignancy patients and transplant recipients.[6] These cases are increasing in Nigeria.[7],[8],[9] This study aimed to detect vancomycin-resistant E. faecalis from urine samples of urinary tract infection (UTI) patients' admitted to female medical ward (FMW) and postnatal ward (PNW) at Aminu Kano Teaching Hospital (AKTH).


  Materials and Methods Top


Study area

This study was conducted at AKTH, a tertiary health-care institution, and as well a referral center for all secondary and primary health-care providers within the geopolitical zone, located along Zaria road by the east and along Maiduguri road by the north within Kano metropolis, Kano state. The state is located at the North-Western region of Nigeria layed between latitude 11°30'N and longitude 8°30'E with a total land area of 20,760 km2.[10]

Study population

The study populations were patients who have been admitted to FMW and PNW of selected study site that consented willingly to participate in the study.

Sample collection and processing

A total of 104 clean-catch midstream urine samples were collected and cultured onto blood agar and cystine–lactose–electrolyte-deficient agar and incubated at 37°C for 18 h. The suspected enterococcal isolates were confirmed using standard bacteriological procedure, which includes Gram reaction, catalase reaction, and growth on bile esculin agar, 6.5% sodium chloride media, and sorbitol fermentation test.[11]

Ethical consideration

The ethical permission was obtained from the Ethical Committee of AKTH prior to the commencement of the study (AKTH/MAC/SUB/12A/P-3/VI/2490).

Antibiotic sensitivity testing

Antimicrobial susceptibility testing was done using modified Kirby–Bauer disk diffusion method on Mueller-Hinton agar based on the Clinical and Laboratory Standards Institute.[12] Isolated enterococci were tested against vancomycin (30 μg), ciprofloxacin (10 μg), streptomycin (10 μg) chloramphenicol (30 μg), tetracycline (10 μg), and co-trimoxazole (25 μg) single disks (Oxoid, UK).

Minimum inhibitory concentration

Inoculums suspension

About 4–5 colonies of isolated E. faecalis were emulsified in a sterile plain test tube containing 10 ml normal saline which compared to match with 0.5 MacFarland turbidity standards.[11]

Antibiotics suspension

A vancomycin powder (Mylan, Italy) was used to prepare a stock solution. Using analytical weighing balance 0.64 mg which is equivalent to 64 μg of vancomycin powder was weighed and dissolved in 10 ml sterile distilled water to yield 64 μg/ml as a stock solution.[13]

Minimum inhibitory concentration assay

A nutrient broth was prepared according to the manufacturer's instruction. A prepared nutrient broth was placed in a water bath to prevent it from solidifying before used. Eight sterile plane test tubes were arranged and dispensed with 1 ml nutrient broth each. The tubes were labeled 64, 32, 16, 8, 4, and 2 μg, whereas tubes 7 and 8 are labeled positive and negative control, respectively. One ml of antibiotics stock solution was dispensed in the first test tube labeled 64 μg from the first tube 1 ml of the mixture was aspirated and dispensed to the second tube, the procedure was repeated from the second tube to the third tube. The serial dilution was repeated up to six tubes to yield 32, 16, 8, 4, and 2 μg/ml. One ml of the E. faecalis suspension was added to the tubes, respectively, including seven tubes were labeled as positive control and leaving the last one (tube 8) as negative control. All the tubes were incubated at 37°C for 18 h, the procedure was repeated for all the isolated E. faecalis.[14] The tubes were checked for the presence or absence of turbidity after the incubation, the absence of turbidity shows evidence of antibiotics activity.

Minimum bactericidal concentration

All the tubes from minimum inhibitory concentration (MIC) that showed the absence of turbidity was sorted out for minimum bactericidal concentration screening. The test was done to find whether the antibiotics activity is bacteriostatic or bactericidal. The mixture contained in the tubes was poured onto a solid nutrient agar free from any antibiotic and incubated at 37°C for 18 h.[12] Plate that showed growth after incubation indicates bacteriostatic effect, whereas other plates that show no growth indicated the antibiotic activities are bactericidal.

Control

ATCC E. faecalis 29,212 was used as control organism for every test run.

Statistical analysis and interpretation

Data obtained were analyzed using the statistical package for the social sciences version 20. 0 (IBM Inc, Armonk, New York, USA). It was cleaned by checking for errors, missing values, and extreme values or outliers, and it was summarized using percentage and frequency. Finally, the result was presented in the forms of tables.


  Results Top


More than half of the samples were obtained from FMW 80 (70.2%) and 34 (29.8%) from PNW. A total of 111 (97.4%) participants were hospitalized for 1–5 days and 105 (92.1%) participants were on antibiotics [Table 1].
Table 1: Clinical history of the study participants

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From all the 114 samples collected, 8 (7.0%) E. faecalis were recovered. All the isolated E. faecalis showed to be Gram-positive, gamma hemolysis, and ferment lactose [Table 2].
Table 2: Distribution of the Isolated Enterococcus faecalis based on their morphological features

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The result of the biochemical test of all isolated E. faecalis showed 8 (100.0%) catalase negative, bile esculin and 6.5% sodium chloride test positive, and 7 (87.5%) of the isolated E. faecalis ferment sorbitol [Table 3].
Table 3: Biochemical test for the screening of Enterococcus faecalis

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The antibiotics susceptibility test revealed that six out of the eight E. faecalis (75.0%) isolates have multiple resistance to tetracycline (30 μg), co-trimoxazole (1.25/23.75 μg), and streptomycin (10 μg) but sensitive to ciprofloxacin (5 μg) and chloramphenicol (30 μg) [Table 4].
Table 4: Antibiotics susceptibility pattern of  Enterococcus faecalis Scientific Name Search tes (n=8)

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The MIC of vancomycin was found to have high effect at lower concentrations of 4–2 μmg/ml, which showed no evidence of growth to 2 (25.0%) E. faecalis each and 1 (12.5%) E. faecalis at 32 and 8 μmg/ml each [Table 5].
Table 5: Minimum inhibitory concentration of vancomycin on isolated Enterococcus faecalis (n=8)

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The minimum bactericidal concentration of vancomycin was found to have no effect at all concentrations, which showed evidence of growth to all the E. faecalis isolates tested [Table 6].
Table 6: Minimum bactericidal concentration of vancomycin on isolated Enterococcus faecalis (n=8)

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  Discussion Top


VRE have become a serious issue for nosocomial infection control worldwide. In Africa including Nigeria, there have been very few reports of VRE, most being from South Africa. VRE cause a wide variety of infections involving the urinary tract, wound, and bloodstream among other sites, most commonly in hematological malignancy patients and transplant recipients.[6] These cases are increasing in Nigeria.[7],[8],[9]

The result of this study showed that 8 (7.0%) of 114 samples collected and screened for UTI yielded positive E. faecalis isolates. This has again proven E. faecalis as one of the causes of UTI infection in women. The result of biochemical testes obtained in this study confirms the recovery of E. faecalis phenotypically. The prevalence of 7.0% E. faecalis in this study is slightly higher than what was obtained in another study done by Iregbu and Nwajiobi-Princewill, at central part of Nigeria, who report the prevalence of 6.2%, E. faecalis in their study.[15] In another study done by Akinjogunlaand Enabulele,[16] low prevalence of 3.8% E. faecalis was reported.

The isolated E. faecalis obtained from UTIs in this study has been found to be resistant to most chemotherapeutic agents, though the antimicrobial susceptibilities of these pathogens are highly predictable. Development of resistance to these antimicrobial agents in UTI cases will therefore affect future treatment and management of infections with these drugs. Majority of the treatments begin or are done completely empirically, the knowledge of the organisms, their epidemiological characteristics and their antibacterial susceptibility are therefore important. The isolated E. faecalis was resistant to tetracycline (30 μg), co-trimoxazole (1.25/23.75 μg), and streptomycin (10 μg). Resistance to these drugs may probably be an indication of earlier exposure of the isolates to these drugs and misuse of these drugs by the patients in the study area.

Vancomycin is one of the most important alternative treatments against multidrug-resistant enterococcal infections.[17],[18]

The prevalence of VRE in this study is 50.0% by disk diffusion techniques and 75.0% by dilution techniques at different MIC is consistent with a report from Benin (67.5%) by Ahoyo et al.[19] and from pediatric hematology/oncology patients in Egypt 75.0%.[20],[21] A lower isolation rate of VRE was reported from Iran (23.7%). These differences might be due to the study design. A study from a University Hospital in Belgium revealed the incidence of VRE to reached 46.5%.[22] Minimum bactericidal concentration of VRE found to have no effect at all concentrations, which shows to have bacteriostatic effect at all concentrations which showed the evidence of growth when plating the culture broth obtained from different MIC tube that shows no turbidity. This showed that vancomycin has only bacteriostatic effect against the isolated E. faecalis obtained in this study.


  Conclusion Top


The present study indicated that Gram-negative bacteria is one of the major causes of UTI in patients admitted to FMW and PNW of AKTH. The finding of this study suggests that multiple bacterial resistance in E. feacalis is becoming a more serious problem in the study area as observed in antibiotics susceptibility test by disk diffusion method. From this study, ciprofloxacin and chloramphenicol are the best therapeutic options to treat infection with VRE, and E. faecalis was confirmed to be more resistant to vancomycin at higher concentration and more effective at lower concentration.

The study revealed the increased prevalence (7.0%) of vancomycin-resistant E. faecalis in this study area.

Recommendations

  1. Considering the high prevalence of VRE (7.0%) proper patient management and adopting antibiotic stewardship in the study area which can help to reduce the incidence
  2. Ciprofloxacin and chloramphenicol could be the therapeutic options in the treatment of infection VRE faecalis in Kano, Nigeria.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Andrews JM. Determination of minimum inhibitory concentrations. J Antimicrob Chemother 2001;48 Suppl 1:5-16.  Back to cited text no. 13
    
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Nicolle LE. Uncomplicated urinary tract infection in adults including uncomplicated pyelonephritis. Urol Clin North Am 2008;35:1-12, 5.  Back to cited text no. 14
    
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Iregbu KC, Nwajiobi-Princewill PI. Urinary tract infections in a tertiary hospital in Abuja, Nigeria. Afr J Clin Exp Microbiol 2013;14:169-73.  Back to cited text no. 15
    
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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