Prevalence and Antimicrobial Susceptibility of Gram Negative Bacteria in Urine of Students of Federal Polytechnic Nekede, Owerri

Abba-Father C. A. M.*, Ibe I. J., Edward O. C., Etoamaihe U. E., Iroka P. C. and Ekere I. C

Department of Biology/Microbiology, Federal Polytechnic Nekede, Owerri, Imo State, Nigeria

INTRODUCTION

Most bacteria causes urinary tract infection (UTI), but the most common is Escherichia coli which is responsible for about 80-85% of UTI, Ortho women’s report, 2004 according to (Azubuike, C. N. et al., 2002), urinary tract infections are categorised as ascending or descending routes. Three years later, (Stamm, W. E & Newby, S. S. 2005) stated that infections occurs when microorganism usually bacteria from digestive tract cling to the opening of the urethra and begin to multiply. Four routes have been purposed the ascending routes, from the urethra to bladder, then by the urether to kidney, the haematogenous routes, with seeding of the urether to the kidney during the course of bacteraemia; intestine to kidney by way of lymphatic and direct infections. These bacteria may be present in the vaginal and rectal areas.

Gram negative bacteria are bacteria that do not retain their crystal violet dye in the gram staining protocol. They are differentiated by their cell wall structure. The following characteristics are displayed by gram negative bacteria as follows; Cytoplasmic membrane, thin peptidoglycan layer (much thinner than gram positive), outer membrane containing lipopolysaccharide outside the peptidoglycan layer, porin exists in the outer membrane, which acts like pores, there is a space between the layer of peptidoglycan and the secondary cell membrane called the periplasmic space, if present, flagella have four (4) supporting rings instead of two  No techoic acid or lipopolysaccharide.

Some examples of gram negative bacteria include; Escherichia coli, Salmonella species, Pseudomonas species, Klebsiella species, Proteus species, Helicobacter species, Mosoxella species, Cyanobacteria species, Spirochetes species.

They also constitute a serious problem in urinary tract infections in many parts of the world. Appropriate antimicrobial treatments are often critical to decreasing morbidity and mortality among hospitalized patients having the infections caused by the pathogens. Gram negative bacteria are non-spore forming bacilli that grow rapidly on ordinary laboratory media under both aerobic and anaerobic conditions. It has been estimated that symptomatic urinary tract infects (UTI) occurs in as many as 7million visits to emergency units and 100,000 hospitalised annually. UTI has been the most common hospital acquired infections, accounting for as many as 35% of nosocomial infection. It is the second most common cause of bacteraemia in hospitalised patients (Prah, J. K. et al., 2019). UTI is known to occur in all populations but has a particular impact on females of all ages and males at two extremes of life, immuno-compromised patients and anyone with function or structural abnormalities of the urinary and excretory system.

UTI is known to be the microbial invasion of any of the tissues of the urinary tract reaching from the renal cortex to the urethrameatus (Nicolle, L. E. 2000). It is also known to be the presence in two consecutive urine samples of greater than 100rods (10⁵ ) organisms per ml of a single bacterial strain in the urinary tract. UTI can be categorized in ascending or descending. Infections which are confined to the urethral or the bladder are ascending and referred to as uretitis or cystitis respectively. On the other hand, the pathogens spread from one or other infected body site to the kidney down along the ureter to the bladder. Such descending UTI cause severe kidney infection, a condition called pyelonephritis (Rahman, F. et al., 2009). This is potentially more serious; infections to the urethra are called urethritis and to the prostrate gland are called prostitis. This classification is the presence or absence of symptoms, reoccurrence or absence or presence of complicating factors which are host factors facilitating establishment and maintenance of bacteraemia or worsening the prognosis of UTI`s engaging the kidney.

Majority of pathogens are gram negative species with predominance of members of Enterobacteriace (Salles, M. J. C. et al., 2013). Escherichia coli accounts for majority of urinary tract infections in young women but other gram negative

Majority of pathogens are gram negative species with predominance of members of Enterobacteriace (Salles, M. J. C. et al., 2013). Escherichia coli accounts for majority of urinary tract infections in young women but other gram negative rods of different genera such as proteus species and pseudomonas aeruginosa an aerobic gram negative rod is also troublesome. As a urinary tract pathogens because of its resistance to antimicrobial medicine make it difficult to treat successfully.

Antibiotics are used for the control of bacterial infections in human. Generally, gram negative bacteria are sensitive to many antimicrobial agents but strains from different patients and carriers differ in the pattern and degrees of sensitivity to different drugs. Increasing antimicrobials resistance in bacterial pathogen is a worldwide concern. The prevalence of antimicrobial resistance among urinary tract infectious agents is also increasing (Mathai, D. et al., 2001; Karlowsky, J. A. et al., 2001) and its treatment has become more complicated due to increasing resistance and empirical therapy leading to treatment failures of most associated with gram negative bacteria. The present study investigated the pattern of gram negative uropathogens and their antimicrobial resistance pattern among the clinical isolates to the commercially available antibiotics that are often prescribed in urinary tract infectious cases. Therefore the aim of this study is to find out the prevalence of gram negative organisms in the urinary tract among some students of Federal Polytechnic Nekede, to investigate their antibiotic sensitivity pattern to enable formulation of drugs for urinary tract infection in the school community using age and sex prevalence, to determine the prevalence of bacterial strains and their antimicrobial  susceptibility in urine, to find the pathogenic bacteria commonly responsible with UTI and susceptibility patterns this will help the clinicians to choose the right empirical treatment.

MATERIALS AND METHODS:

Collection of Samples:

A total of 50 mid-stream urine (15-20mL) samples were collected from both male and female students referred to the medical center laboratory of the Federal Polytechnic Nekede Owerri for suspected cases of urinary tract infection (UTI) in sterile, dry, wide-necked, leak-proof containers. The samples were collected between May 2021 and July 2021, and taken to microbiology department laboratory of the Federal Polytechnic Nekede, Owerri for microbial analysis according to (Cheesbrough, M. 2006; Dielubanza, E. J., & Schaeffer, A. J. et al., 2011). All the samples were kept in a fridge, so that the bacteria in the urine sample will not multiply. On the urine sample were indicated the name, age, sex, including the date and time of collection. Each of the samples were observed with the naked eyes to ascertain if the urine was clear or cloudy (turbid) and the colour of the urine was also observed. Only students who had not been on antibiotic preceding one week of sample collection were enrolled.

Isolation and Characterization:

The urine was mixed thoroughly by rotating the container clockwise and anticlockwise directions. A loopful of the urine samples were inoculated on Cystine-Lactose-electroyte-deficient agar (CLED) and nutrient agar by streaking and incubated at 37⁰c aerobically for 24hrs. The plates were examined macroscopically. The bacterial colonies were counted and multiplied by 100 to give an estimate of the number of bacteria present per millimetre of urine. A significant bacterial count was taken as any count equal or in excess of 10,000 cfu ml. Representative of growing colonies were picked with repeated streaking. Resulting pure cultures obtained were used for biochemical test aimed at identifying the bacteria isolates and its microscopy was also carried out. The pure cultures were further characterized microscopically by Gram staining and biochemically using the following biochemical test,; Indole test, Methyl Red – Voges-Proskauer test, Citrate utilization test, Sugar fermentation test, Urease test and Motility test as described by (Cowan, S. T. & Steel, K. J. 2013).

Antimicrobial Susceptibility Test:

Antimicrobial sensitivity was tested for each isolated organism using the disk diffusion method of each isolated organism using the disk diffusion method of Kirby-Baurer as described by the (Clinical Laboratory Standard Institute. 2015).the multi disc contains the following antibiotic for gram negative organisms and they are (Septrin) (SXT) Chloranphenical (CH) sparfloxacin (sp) Ciprogloxaan (CPX) Amoxacillin (AM) Augmentin (AU) Gentamycin (CN) Pefloxacin (PEF) Tarivid (OFX), Streptomycin (S) Ofloxacin and Cephalexin (CEPH). Two fold dilution was carried out by extraxcting 1ml of the test organisms from the test tube, into another test bube containing Iml of peptone water. Labelled as TUBE 1 with a dilution factor of 10^-1. From TUBE 1, 1ml of the mixture was extracted into TUBE 3, with the dilution factor of 10^-3 to dilution factor of 10^-5 and the last was discarded. 10-2 dilution factor was used, because it matches Macfarland standard of 0.5ml. 0.5ml of the test organism was extracted from the dilution factor of 10-2 into each petridish containing muller-Hinton Agar and it was spread uniformly on the surface of the medium using glass bent rod.

A cmmercially prepared gram-ve sensitivity disc was placed and impregnated on the medium containing the 0.5ml of test organism. The anti-microbial diffused from the disc to the medium and the growth of the organism was inhibited at a distance from the disc, where as resistant strains had smaller zones of inhibition. 5-8mm zones of inhibition signifies sensitivity/susceptibility while below 5mm were regarded as resistance.

A disc of blotting paper was impregnated with a known volume and appropriate concentration of an antimicrobial and it was placed in a plate of sensitvity testing agar that was uniformly inoculated with test organism. The antimicrobial diffused from the disc to the medium and the growth of the organism was inhibited at a distance from the disc that was associated to the sensitivity of the organism. Strains that were sensitive to the antimicrobial were inhibited at a distance from the disc where as resistant strains had smaller zones of inhibition. Zones of inhibitions from 8-12mm signifies sensitivity while and below 8mm were regarded as resistance.

RESULT:

A total of 50 samples were collected from some student of Federal Polytechnic Nekede Owerri students were investigated. Out of the 50 samples, 24 samples were females while 26 samples were males with age range of 22-28yrs. Out of the 50 samples, 28 samples were positive (gram-ve), 13 were female while 15 were males. The remaining samples that showed no significant bacterial growth were discarded. Out of 27 samples from males 13 were positive with prevalence rate of 46.4% while 23 samples from females gave 15 were positive with prevalence rate of 56.7% as shown in table 1.Therefore, the prevalence rate of positive cases of (gram-ve bacteria) for male and females were 40.9% and 50.1% respectively. Out of the 50 student examined pseudomonas spp were isolcated and identified from 12 student, proteus spp from 7 student while E.coli from 3 student.

Pseudomonas had the highest prevalence rate of 50% followed by E.coli spp 25%, Proteus spp 10.70% and the other gram positive organisms put together had 21.4% as shown in table 2.

It was also observed that Gentamycin, Chloraphenical and tarivid were the most sensitive antibiotics in the study while tetracycline Cephalexin, septrin, sparfloxacin perfloxacin and Amoxacillin gave poor sensitivity or resistance. As shown in table 3 from this measures above. Gentamycin , Chloraphenical and Tarivid proved the best antibiotics against gram negative bacteria.

Table 1: Shows Sex Distribution of cases and their Prevalence rate (22-28yrs)

Table 2: Shows the bacteria distribution of positive cases with prevalence rate.

Calculation of prevalence as shown in the Table 2 above.

E. coli- spp 7 x 100

28 1 = 25%

Pseudomonas spp 12 x 100

28 1 = 42.9%

Proteus spp 3 x 100

28 1 = 10.7%

Others (Gram + ve) 6 x 100

28 1 = 21.4%

Overall prevalence for positive cases is

28 x 100

50 1 = 56%

Table 3: Shows the sensitivity pattern of bacteria isolated and their antimicrobial resistance

Key:

0-5mm shows antibiotics resistivity.

5-8mm shows antibiotic sensitivity.

SXT => Septrin

CH => Chloranphenicol

SP => Sparfloxacin

CPX => Ciprofloxacin

AM => Amoxacillin

AU => Augmentin

CN => Gentamycin

PEF => Pefloxacin

OFX => Tarivid

S => Streptonycin

Table 4: Urinalysis result from the student midstream urine samples