, Idoko Alexander2
, Iroha Ifeanyichukwu Romanus3
PREVALENCE OF MULTIDRUG RESISTANT ESCHERICHIA COLI AND KLEBSIELLA PNEUMONIAE SPECIES ISOLATED FROM URINE SAMPLES OF PATIENTS AT ALEX EKWUEME FEDERAL UNIVERSITY TEACHING HOSPITAL ABAKALIKI, EBONYI STATE, NIGERIA
Onu Henrietta Chinaemerem1, Idoko Alexander2, Iroha Ifeanyichukwu Romanus3
1Alex Ekwueme Federal University, Ndufu-Alike, P.M.B. 1010. Ebonyi State, Nigeria.
2Department of Biochemistry, Faculty of Natural Sciences, Caritas University, Amorji – Nike, P.M.B. 01784, Enugu, Nigeria.
3Department of Applied Microbiology, Faculty of Science, Ebonyi State University, Abakaliki, P.M.B. 053, Ebonyi State, Nigeria.
Aim and Objective: In hospitals and community, morbidity and mortality are attributed to urinary tract infections (UTIs). This study investigated the prevalence of multi-drug resistant isolates of Escherichia coli and Klebsiella species from urine samples of patients visiting Alex Ekwueme Federal University Teaching Hospital Abakaliki (AE-FUTHA), Ebonyi State.
Method: With the use of standard microbiological and biochemical techniques for analysis, a total of 300 mid-stream samples of urine were collected in sterile bottles. Phenotype screening for extended spectrum beta lactamase (ESBL) production was achieved by double disc synergy test (DDST). Disc diffusion method was used to check for ESBL producing bacteria susceptible to antibiotics. With specific primers, the presence of temoniera (TEM) and sulfhydryl variable (SHV) beta lactamases genes was determined using polymerase chain reaction (PCR).
Results: 88 isolates of E coli and Klebsiella species were isolated from the urine samples; 29 were E coli and 59 were K. pneumonia species. 47 ESBL positive isolates were identified with E. coli, 19 (40%) and Klebsiella, 28 (60%). Escherichia coli isolated from GOPD patients, gynecology, and men's surgery ward were 100% resistant to ofloxacin, ceftazidime, amoxicillin, nitrofurantoin, chloramphenicol, and aztreonam, while Klebsiella species isolated from gynecology department, maternity ward, and psychiatric ward were 100% resistant to ofloxacin, Nitrofurantoin and chloramphenicol. Multiple antibiotics resistance index (MARI) of E. coli and Klebsiella species isolated recorded an average of 0.64 for E. coli and 0.41 for Klebsiella species.The molecular analysis revealed that 44.4% of SHV beta lactamase and 65.6% TEM-type β-lactamase genes were present in ESBL producing E. coli while 54.5% of SHV beta lactamase gene and 45.5% TEM-type β-lactamase genes were present in ESBL producing Klebsiella species.
Conlusion: In conclusion, TEM and SHV-type β-lactamase genes are the primary cause of β-lactam antibiotic resistance in E. Coli and Klebsiella species resulting in increased infections caused by organisms harboring the ESBL gene. Nitrofurantoin, ceftazidime, cefalexin, and cefotaxime may be the antibiotic of choice in the treatment of UTIs.
Keywords: Antibiotics, β-lactamase, Escherichia coli, Klebsiella species, multiple antibiotics resistance, sulf-hydryl variable, temoniera.
INTRODUCTION
Attention is gradually shifted to antimicrobial resistance as a global public health concern and emergency1. Exhaustive use of antimicrobials for food animals may pose challenge in the treatment of infections because resistance among bacterial pathogens from animals and humans could be selected2. Resistant bacteria from animals to humans could be transferred through environment, direct interaction with animals or food chain, and resistant infections may result3. In Africa, such as in Nigeria, resistance to gentamicin was seen in isolates of E. coli in connection with urinary tract infections4. The arrival of the new generation of quinolones antimicrobial agents, fluoroquinolones provided a choice option for the treatment of various life threatening infections resulting from multi-drug resistant bacteria5. Meanwhile, the widespread use of these drugs in clinical settings gave rise to bacteria resistance4. Treatment of UTIs in hospitals and communities was often handled with fluoroquinolones and their application has resulted in increased resistance in E. coli responsible for UTIs6. In E. coli strains, DNA replication is inhibited by quino-lones. These agents mark the E. coli DNA enzymes such as topoisomerase IV (parC) and topoisomerase II (gyrase). Meanwhile, E. coli resistance to quinolones occurs due to alteration in single amino acid in topoisomerase IV or gyrase as a result of gyrA, gyrB, parC, and parE mutated genes1. Human gastro-intestinal tracts (HGITs) are frequently colonized by E. coli strains, and are responsible for several infections including diarrhea, UTIs, meningitis and septicemia5. However, E. coli’s harmless strains persist as commensals in HGITs but become virulent and thereby cause infection and diseases when they become genetically modified1. In both hospital and community, these E. coli strains are increasingly reported to have acquired antibiotic resistance genes mostly for UTIs, yet with scarce therapeutic intervention5.
The E. coli strains also encodes resistance to oxyimino-cephalosporins in the expression of Extended Spectrum Beta Lactamases (ESBLs), thus treatment to its infections may be difficult7. In another case, E. coli strains was recently reported to pose resistance to carbapenems, which are known to be effective in treating infections from ESBL-producing E. coli, by generating a hydrolyzing enzyme, called carbapenem-hydrolases4. In the antibiotic munitions, amino-glycosides essentially useful for treating infections and UTIs caused by E. coli, that are serious threat to life but the devastating widespread antibiotic resistance has made some them ineffective in treating infections caused by E. coli7. The combined rates of biofilm formation in Uropathogenic E. coli (UPEC) isolates were achieved as 84.6% (95% CI: 72.7-91.9)8. Clinicians well recognized Klebsiella as a cause of community-acquired bacterial pneumonia, seen in chronic alcoholics and revealing peculiar radiographic abnormalities as a result of relentless pyogenic infection with a high fatality rate if not treated9. Most of the infections of Klebsiella are reported to be hospitalization associated. Majorly, the pathogenic Klebsiella spp. attacks the hospitalized individual whose immunity has been compromised due to existing chronic pulmonary obstruction or diabetes mellitus9. The immunity of patients in intensive care unit (ICU) tend to be compromised most time and are at risk of infections due to nosocomial as a result of existing untreated disease or may be due to the exposure to invasive procedures. Similarly, such patients who are also administered broad spectrum antibiotics, could be vulnerable to pseudomembranous colitis and candidiasis infections since their protective microbiota has been damaged1. Penicillin, broad and narrow spectrum cephalosporins and monobactams (Aztreo-nam), are reported to be hydrolyzed effectively by plasmid-mediated β-lactamase, which are also known as extended spectrum Beta lactamase (ESBLs), while they cannot hydrolyze cephamycin or carbapenems (imipenem, meropenem)1. ESBL generating strains frequently found in K. pneumoniae and Escherichia coli are hindered by inhibitors of β-Lactamase, which include tazobactam, clavulanic acid and sulbactam1. A resistant strain to inhibitors of β-Lactamase is the plasmid-mediated AmpC β-lactamase, found in the chromosome, inhibits clavulanic acid and hydrolyzes cephamycin1. Carbapenemase is reported to to exhibit multipurpose hydrolytic abilities on carbapenems, cephalosporins, monobactams and penicillins9. The therapeutic intervention of E coli and Klebsiella species acquire antibiotic resistance genes for UTIs and other bacterial infections seem to be limited. Therefore this study was aimed at investigating the prevalence of multidrug resistant isolates of Escherichia coli and Klebsiella species from urine samples of patients visiting Alex Ekwueme Federal University Teaching Hospital Abakaliki, Ebonyi State.
MATERIALS AND METHODS
Media for Bacteriological Screening
Nutrient broth, Nutrient agar, Mueller-Hinton agar and MacConkey agar (Oxoid, UK), were the media used and their preparation was based on instruction of the manufacturer.
Equipment
The following are the equipment used; microscope (Olympus optical Co. Ltd. UK), autoclave (Searchtech, England), thermocycler, refrigerator (Gallenkamp Germany), weighing balance (Ohaus, USA) and incubator (Newlife England).
Reagents and Chemicals
Chemicals and reagents used include; crystal violet, normal saline, lugols iodine, oxidase reagent, methyl-red reagent, immersion oil, Kovacs reagent, hydrogen peroxide, acetone alcohol and decolourizer.
Antibiotics disk
Antibiotics used are all of Oxoid Ltd, Basingstoke, United Kingdom and they include: ampicillin (10 μg), amoxicillin (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), cefoxitin (30 μg), nitrofurantoin (10 μg), chloramphenicol (10 μg), azetreonam (5 μg), cefepime (25 μg), ertrapenem (10 μg) and ofloxacin (30 μg).
Determination of sample size
In this study, Cochran’s sample size formula was employed for the determination of sample size. Using 95 % confidence interval (CI) at a 0.05 precision, the sample size was calculated and the Cochran formula;
n=Z2pq/e2
Where: n=sample size; Z=standard normal deviation at 95% confidence interval (which was 1.96); p=proportion of target population (prevalence estimated at 50%; this implies 75/100=0.75); q=alternate proportion (1-p), which was calculated as: 1–0.5=0.5; e=desired level of precision (degree of precision/significance).
This was taken as 0.05; Thus, using the sample size determination formula:
n=Z2pq/e2; (1.96)2 x 0.75(1-0.75)=291.8 0.052
Thus, in this study, the sample size of 300 was approximately and randomly collected.
Ethical Clearance
The ethical committee of Alex Ekwueme Federal University Teaching Hospital Abakaliki, Ebonyi State approved the design of this study.
Sample collection
The microbiology laboratory unit of the hospital was the sample collection centre and 300 mid-stream urine samples were collected. In order to carry out bacterio-logical analysis, these samples were transported to Microbiology laboratory unit of Applied microbiology department of Ebonyi State University Abakaliki.
Phenotypic Determination of ESBL Production by Double disk synergy test (DDST)
The phenotypic confirmation of ESBL producing E. coli from the isolates was done by the method of double disc synergy test (DDST) as described in a previous study6. Briefly, adjustment to 0.5 McFarland turbidity standards of the overnight suspensions of the 2nd and 3rd generation Cephalosporin resistant bacteria was achieved and on a solidified MH agar (Oxoid, UK), they were aseptically swabbed. At the center of Mueller-Hinton agar plates, antibiotic disks containing amoxicillin acid (10µg) was placed and adjacent to the central disk (amoxicillin acid) at a distance of 15 mm, was the antibiotic disk containing ceftazidime (30 µg) and cefotaxime (30 µg) positioned. For 18- 24 hours, the incubation of the plates at 37oC was done. And increase (a ≥ 5 mm) in the inhibition zone diameter for either of the cephalosporins tested in combination with the central disk versus its zone when tested alone confirms ESBL production phenotypically by the DDST method6.
Antibiotic susceptibility testing
Employing the Kirby-Bauer disc diffusion Method described by the Clinical and Laboratory Standards Institute11, ESBL producing isolates of E. coli and Klebsiella species’s antibiotic susceptibility test of was carried out. On the entire Mueller-Hinton agar plate was the bacteria inoculum equivalent to 0.5 McFarland standard of the isolate smeared and pre-diffusion was allowed for 5 minutes. At 37oC and for 24 hours, the antibiotics aseptically saturated on the agar plates and incubated include; Amoxicillin (10 µg), Ceftazidime (30 µg), Ampicillin (30 µg), Ertapenem (10 µg), Nitrofurantoin (10 µg), Chloramphenicol (10 µg), Cefepime (25 µg), Aztreonem (5 µg), Cefotaxime (10 µg), Ofloxacin (30 µg) and Cefalexin (30 µg), (Oxoid Ltd, Basingstoke, United Kingdom). And the level of susceptibility to each antibiotic was measured and interpreted using the diameter of zone of inhibition11.
Determination of Multiple Antibiotic Resistance (MAR) index
The formula MAR=a/b, was used to measure the multiple antibiotic resistance (MAR) index. Where a is the number of antibiotics to which test isolate displayed resistance and b is the total number of antibiotics it was subjected to12.
Molecular studies
Preparation of bacterial stock prior to PCR analysis
Screening for the presence of SHV and TEM beta lactamases was achieved by selecting 6 ESBL producing E. coli which were representative of each sample source and 7 ESBL producing K. Pneumoniae which represented each sample source. Using standard microbiological culturing technique10, preparation of stock cultures for ESBL producing E. coli and K. pneumoniae isolates for gene amplification was done. E. coli and K. pneumoniae cultures were reassigned from stock cultures in the refrigerator and inoculated on double strength nutrient broth (Oxoid, UK). In order to resuscitate the isolates, inoculation of the culture tubes at 37oC were done overnight. Then on nutrient agar plates, a looping through the turbid suspension, inoculation was done and purification achieved after overnight incubation, and the plates were subsequently incubated for 18-24 hours at 37oC. A stock culture for DNA template and bacteria cells for molecular studies was prepared from 8 colonies of the overnight cultures which were inoculated in 5 ml peptone water.
DNA Extraction using Quick-DNA™ Miniprep Plus Kit (Catalog Nos. D4068 & D4069)
With modification, the Quick-DNA™ Miniprep Plus Kit, genomic DNA was extracted. Before us, isotonic buffer was used to re-suspend the cultured cells. Into a micro-centrifuge tube, 200 µl of the sample was pipette. This was then followed by addition of 200 µl of biofluid lysis buffer and 20 µl of Proteinase K, meticulously mixed for 10-15 seconds using a vortex mixer. Incubation was done for 10 minutes at 55°C in to this mixture was 420 µl of genomic binding buffer (GBB) added and meticulously mixed for 10-15 seconds using a vortex mixer. In a Zymo-SpinTMIIC-XLR columnin, the mixture was transferred to a collection tube and centrifuged at 12,000 x g for 1 minute. The collection tubes were discarded with the flow through after centrifuging. Into a new collection tube of 400 µl of DNA pre-wash buffer was added to the spin column and for 1 minute, centrifuged at 12,000 x g, and then the tubes were emptied. Into the spin column, 700µl of g-DNA wash buffer was added and centrifuged at 12,000xg for 1 minute and the collection tubes were emptied. To the spin column again, 200µl of g-DNA wash buffer was added and centrifuged again for 1 minute at 12,000 x g. The collection tubes were discarded with the flow through after centrifuging. Into a clean micro-centrifuge tube, the spin column was transferred and then direct addition of 65 µl of DNA elution buffer on the matrix and at room temperature, this was incubated for 5 minutes. In order to elude the DNA, for 1 minute, the mixture was centrifuged at maximum speed, and at -20°C the eluted DNA was stored until further amplified by PCR.
PCR screening for ESBL genes
By the aid of PCR technique with specific primers as described13, different ESBL genes that encode ESBL production in the resistant ESBL producing Escherichia coli and K. pneumonia isolates was were detected.
Agarose Gel Electrophoresis of PCR products
Of the agarose powder, 2g was weighed and mixed with 100 ml of 1xTAE in a microwaveable flask. In a microwaveable flask, 2g of agarose powder was weighed and mixed with 100 ml of 1xTAE for the preparation of electrophoresis. After 3 minutes microwaving, the agarose gel mixture in a flask dissolved completely and was cooled to 50oC. Under the ultraviolet (UV) light, a 10 µl EZ vision DNA stain was added which bound to the PCR product to allow its visualization. Into a gel tray with the well comb in place, was the agarose transferred and at room temperature and for 30 minutes, it was allowed to solidify. By adding gel, the well comb was removed with care. To each of the PCR fragment, the loading buffer was added and inserted in the electrophoresis unit. The gel box covering the gel surface was sealed using 1xTAE buffer. Into the first lane of the gel was the molecular weight ladder packed and into the additional wells of the gel was the PCR product meticulously packed. For about 1hour 30 minutes, the gel was run at 80-150 V. Under UV trans illumination, the DNA fragment was visualized after electrophoresis was completed14.
Statistical Analysis
In the course of this study, the raw data collected were presented in tables and bar charts as mean ± standard deviation while simple descriptive statistics such as minimum, maximum, and one-way analysis of variance (ANOVA) was used to interpret relevant data with the help of Microsoft Excel 2013 software and IBM Statistical Package for Social Sciences (SPSS) version 22. And statistically significant was set at p<0.05.
RESULTS
Amplification conditions for the PCR technique and Oligonucleotide primers Selection
Table 1 reveals the specific primers that were used to determine the presence of ESBL resistance genes.
Morphology, microscopy and biochemical characteristics of bacterial isolates from urine samples of patients in different wards of AE-FUTHA
Out of the 300 urine samples collected from patients in Alex Ekwueme Federal University Teaching Hospital (AE-FUTHA), 88 isolates of bacteria were identified. Table 2 shows the microscopic, morphological and biochemical characteristics of the bacteria isolated. It showed the biochemical test, the colour, shape and Gram staining reaction.
Distribution of Bacteria from Urine samples of Patients in Different Wards in AE-FUTHA
Table 3 shows the occurrence of isolated bacteria from different wards in AE-FUTHA is shown. The highest E. coli frequency of 7 (24 %) is from female medical ward, while Labour ward, Male medical, Orthopedic and Male surgical wards all with a frequency of 1 (3 %) had the least isolated. Isolation of K. pneumonia was mostly from Female medical ward with a frequency of 30 (59 %), while Psychiatric ward had the least isolates with a frequency of 1 (2 %).
Table 3 shows result of detected ESBL. This result showed that from 300 urine samples screened for presence of ESBL, 47 ESBL positive bacteria was gotten in which 19 (40 %) were E. coli, and K. pneumonia had the highest number of ESBL positives with a frequency of 28 (60 %), out of the 88 isolated bacteria.
Antibiotic Susceptibility Pattern of ESBL Producing E. coli and K. pneumonia Isolated from Urine Samples of Patients in Different Wards in AE-FUTHA
Table 4 and Table 5 shows the antibiotic susceptibility pattern of E. coli and K. pneumonia isolated from urine samples of patient in different wards in AE-FUTHA.
There was 100% resistant to the different antibiotics used in this study which includes; ofloxacin, ceftazidime, amoxicillin, nitrofurantoin, chloram-phenicol and aztreonam (Table 4), by E. coli isolated from patients in GOPD, Gynaecology and Men Surgical ward. While in Table 5, there was 100 % resistant to ofloxacin, nitrofurantoin and chloram-phenicol by K. pneumonia isolated from Gynecology ward, Labour ward and Psychiatric ward.
Multiple Antibiotic Resistance Index (MARI) of ESBL producing E. coli and K. pneumonia Isolated from Urine Samples of Patients in AE-FUTHA
The results of multiple antibiotics resistance index (MARI) of E. coli and K. pneumonia isolated from different hospital wards in AE-FUTHA is shown in Table 6. For E. coli, the average MARI was read at 0.64 E. coli and Klebsiella specie was read at 0.41.
Molecular Analysis
Figure 1 displayed the gene image of SHV amplifications of SHV beta lactamases. Isolates 1-6 represent E. coli from GOPD, A&E, Female Medical, Gynecology, Female Surgical and Labor wards. Isolates 1, 2, 3 and 6 (44.4 %) were positive for SHV among ESBL producing E. coli while isolate 4 is negative. Isolates 7-13 from GOPD, A&E, Female Medical, Gynecology, Female Surgical, Labor and Psychiatric wards are ESBL producing K. pneumoniae and all were positive for SHV except isolate 8. Figure 2 shows the gene amplifications of TEM beta lactamases. Isolates 1-6 represent E. coli from GOPD, A&E, Female Medical, Gynecology, Female Surgical and Labor wards. Isolates 1,2,3,5 and 6 (65.6 %) were positive for TEM among ESBL producing E. coliwhile isolate 4 was negative. Isolates 7-13 are ESBL producing K. pneumoniae and all were positive for TEM except isolates12 and 13 from GOPD, A&E, Female Medical, Gynecology, Female Surgical, Labor and Psychiatric wards.
DISCUSSION
ESBLs producing MDR pathogen E. coli and Klebsiella species were respectively 40 % and 60 % as revealed in this study. Several reports showed varied prevalence rates of ESBLs producing Entrobacteri-aceae from countries and regions, capable of causing UTIs. The popularly reported ESBL-positive species are K. pneumoniae and E. coli, while plasmid-mediated ESBL genes are hosted by Enterobacteriaceae14,16. ESBL were produced by 40.9 and 40.3% E. coli and Klebsiella species respectively as reported by by16 from Egypt and Blair15 in Saudi Arabia, whose findings were lower than this finding.
It was reported that E. coli was 10% prevalent among 100 patients studied, of the age group of ≤17 years, females (11.5%) had higher prevalence than males (8.3%)17. In this study, the isolates of E. coli were found to be highly susceptible to nitrofurantoin (70%) and cefotaxime/clavulanic acid (100%) but were resistance to nalidixic acid (90%), cefuroxime (100%), ciprofloxacin (90%) and ceftazidime (100%). It was reported that ESBL production was 71.5% and 82.3% of the Gram-negative bacilli (E. coli and Klebsiella species), which was higher than results of this study19. This study showed that form the 88 isolates of E. coli and Klebsiella species, 47 (53%) were pheno-typically confirmed as ESBL producers. ESBL producing Enterobacteriaceae prevalence highly differs among country and among the hospitals within the country, as below 1% to more than 70% ESBLs producers is reported across the globe20.
In 400 urine samples (68.6%) out of which 134 E. coli isolates were identified, Uropathogens were reported to be present and 80 (59.7%) of the uropathogenic E. coli (UPEC) were recognized as ESBL-producers and while they were all susceptible to meropenem, multi-drug resistance (62%) was observed in more than half of the ESBL-producers20. The prevalence of ESBL production as observed in E. coli was higher than K. pneumonia in this study. Similar report was given by Anago18, but contradicted in a previous study12, where 19 (66 %) E. coli isolates were ESBL producers from a total of 29. This also agrees with the findings of a previous study18,19. This study also showed that 28 (47 %) of Klebsiella spp. isolates were ESBL producers out of 59 samples. This is in agreement with the report of Rossolini19, but disagrees12,18. From the analyzed samples, it was reported that 283 (29.5%) E coli and Klebsiella pneumonia were the most frequently isolated organisms, with positive cultures21.
All isolates were susceptible to carbapenems in this study and for treatment of ESBL producing bacteria, carbapenems are the most common alternative drugs employed. This was in agreement with another study greater than 89% of the ESBL producers were susceptible to imipenem and meropenem18. Carba-penem resistance in another organism could result in the use of other drugs as alternative in the broad spectrum as first line for treatment of ESBL-positive bacteria which treatment cost might be very high12,19. It was also reported that in ESBLs producing E. coli and Klebsiella species, presenting therapeutic problem, resistance to non- beta lactams classes of antibiotics occured18,22.
For consistent epidemiological analysis of anti-microbial resistance drug, molecular characterization of β-lactamase gene is imperative18. Several studies showed that the predominant of ESBL gene was dissimilar18. It was reported that with PCR, 35 isolates of E. coli were confirmed positive from the 58 ESBL screened positive E. coli23. The ESBL gene reported predominant was blaTEM while among MBL genes, blaSPM was the most prevalent. CTX-M, SHV and TEM genes were said to be the most prevalent type of ESBL genes. Meanwhile, the most common types of β-lactamase genes reported in the past were SHV and TEM genes types but lately, CTX-M type has reportedly said to have a wide range of global spread than SHV and TEM19.
Several studies reported the predominance of CTX-M in many countries such as 74% in Iran, 70% in Morocco and North Africa, and 93.7% in India18,19. This study is in agreement with other studies since the predominant ESBL gene in uropathogenic E. coli was TEM-type β-lactamase gene (65.6 %)12,19. Similarly, in Portugal (40.9%), Italy (45.4%) and Turkey (72.7%), the predominance of TEM-type β-lactamase gene was reported12,24. In E. coli and Klebsiella pneumonia from UTIs TEM was predominance was reported in india followed by that of SHV18, which is consistent with this study. In 14.5% (29/200) participants, UTIs were confirmed and E. coli was 30.6% (11/36) predomimant25.
In conclusion, the two ESBL-producing E. coli and Klebsiella species are a rising problem and spread is occurring in the study area. The dominant variant in our study was the TEM gene, but the SHV gene was also on the increase. The TEM and SHV beta-lactamase genes are the main cause of beta-lactam resistance in E. coli and Klebsiella, which may lead to an increase in urinary tract infections and thus pose a major challenge to the management of ESBL infection. Ertapenem, nitrofurantoin, ceftazidime, cefalexin, and cefotaxime constitute a reasonable treatment option for UTI based on the status of the findings. As the prevalence of MDR- and ESBL-producing E. coli and Klebsiella isolates reduces treatment options and increases hospital cost, it is imperative to update the prevalent pattern of resistance in any setting that will aid in appropriate antimicrobial therapy.
AUTHOR’S CONTRIBUTION
This work was completed through the combine efforts of the authors. Authors OHC and IIR designed the study, managed the literature searches and performed the statistical analyses. Authors IA, OHC and IIR wrote the first draft of the manuscript and the final manuscript was read and approved by all authors.
ACKNOWLEDGEMENTS
Appreciation goes to I.R. Iroha and A. C Nwuzo for their academic guardians and support, in making this works a reality.
CONFLICT OF INTEREST
Authors declare that no conflict of interest exist.
REFERENCES