Journal of Science and Technology

ISSN: 2456-5660 Volume 6, Issue 3, May-June 2021

www.jst.org.in DOI: https://doi.org/10.46243/jst.2021.v6.i3.pp01-06

Isolation and Characterisation of UTI Pathogens and Their

Antibiotic Susceptibility
Bhushan P. Bhusare1, Mukund S. Ambawade2, Sachin B. Deokar3, Mansi V.
Damale4, Shaheen B. Inamdar5
1, 4
(Department of Microbiology, Sandip University Nashik, Maharashtra, India)
(Department of Microbiology, P.G.K.M’s Haribhai V. Desai College, Pune , Maharashtra, India)
2, 3
5
(Department of Biotechnology, Modern College of Agricultural Biotechnology, Paud, Pune, Maharashtra, India)
Corresponding Author: [email protected]

To Cite this Article

Bhushan P. Bhusare, Mukund S. Ambawade, Sachin B. Deokar, Mansi V. Damale, Shaheen B. Inamdar, “Isolation
and Characterisation of UTI Pathogens and Their Antibiotic Susceptibility”, Journal of Science and Technology,
Vol. 06, Issue 03, May-June 202021, pp01-06

Article Info
Received: 25-01-2021 Revised: 10-04-2021 Accepted: 12-04-2021 Published: 15-04-2021
Abstract: The present study aimed to identify the causative organisms present in urine specimen and their
antibiotic sensitivity profile among patients suspected with urinary tract infection (UTI). Ten mid-stream urine
samples were collected from patients who suspected for UTI. Total 50 isolates were isolated by culturing on
MacConkeys, Cetrimide, and Mannitol salt agar plates. Gram staining resulted that, 74% isolates were Gram
negative and 34% were Gram positive. The isolates were processed further for identification, and the result reveals
that, Escherichia coli aspredominant isolate which constituting 70%, followed by Enterobacter spp. (20%) and
Staphylococcus aureus(10%) of total isolates. Antibiotic susceptibility and resistance result had shown that, E. coli
is susceptible to Ampicillin as well as Amikacin and resistance to Ceftriaxone as well as Amoxicillin. S. aureus is
susceptible Ampicillin, Amikacin as well as Ceftriaxone, and resistance toAmoxicillin. While, Enterobacter
spp.showed resistance to all used antibiotics (Amikacin, Ampicillin, Ceftriaxone and Amoxicillin). Present work will
be helpful for clinicians in order to improve the UTI treatment and prevent antibiotic resistance.
Keywords:Antibiotics, E. coli, Enterobacter, Isolates, UTI
_____________________________________________________________________________________
I. Introduction
Urinary tract infections are the second most serious health infections worldwide1. The most common cause
of UTI is gram-negative bacteria from Enterobacteriaceae family. However, gram positive bacteria of same family
were also reported to cause UTI infection in rare cases. Many researcher groups reported that the members of
Enterobacteriaceae family are the most frequently detected in UTIs, causing 84.3% of the UTIs in both community,
and healthcare settings2,3,4,5. E. coli, E. faecalis, K. pneumoniae, P. aeruginosa, P. mirabilis, Proteus vulgaris, S.
aureus, S. marcescens, and S. saprophyticus are the most common bacteria causing UTI in humans3,6. Among these,
E. coli accounts more than 80% of community acquired and ~50% of hospital acquired UTIs 7,8,9. Different factors;
age, gender, immune-suppression and urological instruments may affect prevalence of UTIs. Incidence of UTI is
reported to be higher in women than men of whom 40% to 50% will suffer at least one clinical episode during their
lifetime10. Women suffered more and risk factor may increase because of short urethra, absence of prostatic
secretions, pregnancy and easy contamination of urinary tract with faecal flora11. ~ 90% of pregnant women develop
ureteral dilation, which will persist until delivery12. It may contribute to increase urinary stasis and ureterovesical
reflux. With this, during pregnancy period, increase in plasma volume decreases urine concentration and up to 70%
of pregnant women developing glycosuria, which is responsible to boost the growth of the bacteria in urine 2.
Catheter-associated UTIs are one of the most dangerous health risks contributing 34% of all health care associated
infections13.
The emergence of extended-spectrum beta-lactamases has threatened the empirical use of cephalosporins and
ciprofloxacin 14,15. Hence, microorganism mediated drug resistance strategy is very helpful. Microorganisms use
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 Isolation and Characterisation of UTI Pathogens and Their Antibiotic Susceptibility

various mechanisms to develop drug resistance, such as recombination of foreign DNA in bacterial chromosome,
horizontal gene transfer and alteration in genetic material16. Resistance pattern of microorganisms may vary from
country to country, state to state, large hospital to small hospital and hospital to community. There is no systematic
national surveillance of antibiotic resistance and less data is available to quantify the problem17. Also, it is essential
to detect UTI causing pathogens and resistance of these pathogens to commonly prescribe antibiotics in clinical set
up for improvement and effectiveness in treatment18. To consider all above major issues, the objective of the
present study was to highlight the bacterial etiology of UTIs and to determine the antibiotic sensitivity which will
helpful to clinicians for improving the UTI treatment and prevent antibiotic resistance.

II. Material And Methods

Study Design and Collection of Urine Sample
This study was carried out in Department of Microbiology, H. V. Desai College, Savitribai Phule Pune
University, Pune (MS), India. A total of 10 outpatients (5 males and 5 females in mean age of 42.1 years) were
included in this study that was infected with UTI. Ten ml of clean and mid-stream of urine samples were collected
in sterile containers (Himedia, India) from outpatients who visited private pathology laboratory from Pune city
(Godbole Pathology Laboratory, Pune). All containers were labelled according to gender of each patient. The urine
samples were processed further for bacterial cultivation and identification19.
Isolation and identification of bacterial isolates
Considering the common causative pathogens of UTI, three types of agar media (Mannitol salt agar,
Cetrimide agar, and Mac-Conkey’s agar) were selected for isolation. Urine samples were inoculated by sterile loop
(Himedia-India) on plates prepared with different agar media. The plates were incubated aerobically at 37 °C for 24-
48 h. All single and pure bacterial colonies were identified on the basis of morphological and biochemical
characteristics. Colony morphology, motility, gram stain, sugar fermentation and utilisation, oxidase, catalyse,
nitrate reduction and IMViC tests were used for characterisation 19,20,21.
Antibiotics sensitivity testing and MDR
Antibiotic susceptibility testing was done by Kirby Bauer (1996) disc diffusion method following the
guidelines of the National Committee of Clinical Laboratory Standards (NCCLS 1999). Bacterial inoculums were
prepared by suspending the freshly-grown bacteria in 25 mL sterile nutrient broth.A sterile cotton swab was used to
streak the surface of Mueller Hinton agar plates. By sterile forceps, all antibiotics disc were placed onto the surface
of Mueller Hinton agar (Himedia, India) and incubated aerobically at 37 C for 24 h. These antibiotics disks were
used for antibiotic sensitivity as well as resistance pattern against isolated UTI causing pathogens.Four antibiotics
used in the current study were obtained from Himedia Labs, Mumbai, India;Amikacin (AK 30μg), Ampicillin (AMP
10µg), Amoxicillin (AM 10µg), Ceftriaxone (CRO 30µg). E. coli ATCC-25922 was used as a control
isolate.Inhibition zone diameter (mm) of each antimicrobial disc was measured, and the isolates were classified as
resistant, and susceptible.
Data analysis
The experiment of antimicrobial analysis were replicated three times and the results are presented as
mean±SD. Data were calculated by using ‘t-test’ and analysed using in SPSS software.

III. Results
Isolation and Identification of isolates
A total 50 isolates of bacteria were collected from the 10 urine samples. In this study, 54% of isolates were
recovered from females, and the remaining 46% were from males. The morphology of UTI causing isolates showed
different characteristics including; size (1-4 mm), Shape (either circular or irregular), Colors (colorless, off-white,
light, and dark pink, yellowish, greenish, and violet), margin (entire, curved or undulate), Opacity (opaque or
transluscent), elevation (flat, raised, convex or undulate) and consistency (moist, rough, smooth, and dry). Few
isolates showing maximum number of similar colonies MSA and MacConkey agar plates that are selected for
identification. Whereas no growth was obtained on cetrimide agar (Table 1).

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 Isolation and Characterisation of UTI Pathogens and Their Antibiotic Susceptibility

Table 1: Isolation of UTI pathogens on selective agar medium

Urine Samples MacConkey agar Mannitol salt agar Cetrimide agar

I + - -
II + - -
III + - -
IV + - -
V + - -
VI + + -
VII + - -
VIII + - -
IX + - -
X + - -
(+ sign indicates the presence of growth; - sign indicates No growth)
Gram staining resulted that, 90% isolates were Gram negative and 10% were Gram positive. Collected isolates were
identified. Here, the isolates were checked with different biochemical test such as Indole, MR, VP, Citrate, Catalase
and Urease. Result reveals that, 70% of the isolates from MacConkey agar plate showed pink colonies with dark
centers and positive test for indole which indicates the presence of E. coli. Colorless colonies, negative test for indol
(-), and, positive test for urease (+) confirmed the presence of Enterobacter spp. Whereas 10% of the total isolates
from MSA plate showed positive test for catalase indicating the presence of S. aureus (Table 2; Graph 1).

Table 2: Biochemical test and Identification of bacteria

Isolate Biochemical test Identified Organism

Indole MR VP Citrate Catalase Urease
1 + + - - NA NA E. coli
2 + + - - NA NA E. coli
3 - - + + NA + Enterobacter spp.
4 + + - - NA NA E. coli
5 + + - - NA NA E. coli
6 - + + + + NA S. aureus
7 + + - - NA NA E. coli
8 + + - - NA NA E. coli
9 - - + + NA + Enterobacter spp.
10 + + - - NA NA E. coli
(Voges Proskauer (VP); Methyl Red (MR); +: sign indicates the positive test; - : sign indicates negative test; NA:
Not Applicable)

Graph 1: Percentage of Causative organism found in samples of UTIs

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 Isolation and Characterisation of UTI Pathogens and Their Antibiotic Susceptibility

Antimicrobial susceptibility testing and MDR

In-vitro sensitivity test for antimicrobial agents on Muller Hinton agar using the Kirby-Bauer disk diffusion
method was carried out for the representative isolates, which include gram-positive organism, gram-negative
organisms. The susceptibility was measured as a zone of inhibition (mm). Absence of zone of inhibition indicates
the resistance. We have observed that, E. coli is susceptible to Ampicillin (12.3±0.7) as well as Amikacin (22.5±0.8)
and resistance to Ceftriaxone as well as Amoxicillin. S. aureus is susceptible Ampicillin (10.3±0.9), Amikacin
(29.2±1.0) as well as Ceftriaxone (9.4±0.4), and resistance toAmoxicillin (Table 1; Fig 1a and b). It was also
recorded that, two species of Enterobacter genusi.e. E. cloacae and E. hormaechei, both showed the resistance to
all used antibiotics (Amikacin, Ampicillin, Ceftriaxone and Amoxicillin)(Table 3; Fig 1b).

Table 3:Antibiotic susceptibility and resistant pattern of UTI causing bacteria

Sr. No. Name of the Used Antibiotics and zone of Inhibition (mm)
Bacteria
Ampicillin Amikacin Ceftriaxone Amoxycillin

01 Enterobacter 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0

spp.
02 S. aureus 10.3±0.9 29.2±1.0 9.4±0.4 0.0±0.0
03 E. coli 12.3±0.7 22.5±0.8 0.0±0.0 0.0±0.0

(The experiment was replicated three times and the results are presented as mean±SD)

Figure 1 (a): Antibiotic sensitivity testing; isolates showing antibiotic susceptibility

Figure 1 (b):Antibiotic sensitivity testing; isolates showing antibiotic resistance

IV. Discussion
In developing countries and among all age group, UTI infection considered most common infection of
bacterial etiology22,23. In elderly people, UTI infection found to be the most common cause of hospitalisation for
infection. Also in primary treatment, it is the most common cause of antibiotics prescriptions. It also constitutes over
30% of all infectious complications in patients after kidney transplantation 24,25. Still, it is challenging to clinicians to

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 Isolation and Characterisation of UTI Pathogens and Their Antibiotic Susceptibility

treat and diagnose the upper and lower urinary tract infections because of their frequent appearance, recurrence, and
a worldwide increase of antibiotic resistance. This indicates that urine culturing and antimicrobial susceptibility
testing is necessary for a definitive diagnosis and treatment of UTI 22. Hence, the present work aimed todetermine
the antibiotic susceptibility isolates of bacteria from the urine of patients suffering with UTI. In this study, total fifty
isolates were isolated from ten different samples (five male and five female urine samples) using selective media. In
present work, we have observed the prevalence of E. coli in UTI, as well as their antibiotic sensitivity pattern.
Similar to our study, Raeispour and Ranjbar in 2018 reported that E. coli is considered as the cause of 80–90% of
UTIs and it is one of the most common bacterial infections in today’s life26. Gupta and co-researcher reported,
among 65 patients (including children, adults, and women) of UTI, E. coli found to be causative agents for UTI in
53 patients27. In the present study, identified the presence of S. aureus in samples collected from UTI infected
patients. Akortha and Ibadin reported the gram-positive bacteria, particularly S. aureus as the most commonly
implicated pathogen in patients with UTIs because of virulent nature of the organism, which gives it the ability to
overcome body defence mechanisms and resistance to antibiotics28. After isolation of different bacteria and their
confirmation through different biochemical tests, we performed antibiotic sensitivity test. The knowledge of
antibiotic sensitivity of pathogen is very necessary and helpful for guidance and treatment of pathogens29. We have
observed that, E. coli are susceptible to Ampicillin as well as Amikacin and resistance to Ceftriaxone and
Amoxicillin. Similar types of results were reported by Bano et al. 2012, E. coli showed the highest sensitivity (56%)
to Amikacin and low susceptibility (5%) to Ciprofloxacin 1. Some of the researcher also reported that highest
effectiveness of Amikacin against E. coli while Ciprofloxacin showed the lowest sensitivity against this pathogen30.
Sabir et al. (2014), reported that the E. coli was highly resistant to Amoxicillin31. S. aureus is resistant to only
Amoxicillin andsusceptible to other used antibiotics (Ampicillin, Ceftriaxone and Amikacin). Shittu and Mandere in
1999, reported the Sensitivity patterns of S. aureus to antibiotics, results reveals the 100% sensitivity to gentamicin
and cephalosporin, but resistant to Amoxicillin/Clavulanate and Nitrofurantoin 32. Abuse or misuse of antibiotics
among general population favored the emergence of resistance strains which may show the differences in sensitivity
pattern of S. aureus33. We have observed that, two species of Enterobacter spp. are resistance to all used antibiotics
(Amikacin, Ampicillin, Ceftriaxone and Amoxicillin). Davin-Regli and Pagès reported that the E. aerogenes and E.
cloacae is naturally resistant to Ampicillin, Amoxicillin–Clavulanic acid, Cephalothin, and Cefoxitin by low
production of the natural inducible cephalosporinase of Bush group 134.

V. Conclusions
Maximum Gram negative and few gram positive isolates were recorded in present studywhich indicates the
gram-negative bacteria were responsible for urinary tract infections. Present study concluded that, E. coli. recorded
aspredominant isolate followed by Enterobacter sp. and then S. aureusout of total isolates collected from the
samples of patient suffering from UTI’s. Antibiotic sensitivity results showed that, E. coli are (susceptible;
Ampicillin, Amikacin and resistance; to Ceftriaxone and Amoxicillin) and S. aureus is resistant to only Amoxicillin
andsusceptible to other used antibiotics (Ampicillin, Ceftriaxone and Amikacin). While, Enterobacter spp. showed
resistance to all used antibiotics (Amikacin, Ampicillin, Ceftriaxone and Amoxicillin). This study will be helpful to
guide physicians in making right choice of drugs while treating patients thus ensuring effective and treatment of the
infection and preventing antibiotic resistance.

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