24 Feb 1

Journal of Science and Technology
ISSN: 2456-5660 Volume 9, Issue 02 (FEB -2024)

www.jst.org.in DOI:https://doi.org/10.46243/jst.2024.v9.i02.pp1-23
Plasmid Profiles of Resistant Strains of Escherichia coli in Men

Attending Prostate Clinics in a Tertiary Hospital in Enugu State,
Nigeria
Kelechi Nkechinyere mbah-Omeje1* Nebo Stephen Chinonso2 , Iloputaife Emmanuel

Jaluchimike3
1*
Senior lecturer, Department of Applied Microbiology and Brewing, Enugu State University
of Science and Technology, Enugu State, Nigeria, kelechimbahomeje@gmail.com
2
Department of Applied Microbiology and Brewing, Enugu State University of Science and
Technology, Enugu State, Nigeria,
3
Lecturer, Department of Applied Microbiology and Brewing, Enugu State University of
Science and Technology, Enugu State, Nigeria, emmanuel.iloputaife@esut.edu.ng
*Correspondence author;email: kelechimbahomeje@gmail.com
To Cite this Article
Kelechi Nkechinyere mbah-Omeje, Nebo Stephen Chinonso , Iloputaife Emmanuel

Jaluchimike, “Plasmid Profiles of Resistant Strains of Escherichia coli in Men Attending Prostate
Clinics in a Tertiary Hospital in Enugu State, Nigeria” Journal of Science and Technology, Vol. 09,
Issue 02,- FEB 2024, pp1-23
Article Info
Received: 10-01-2024 Revised: 20-01-2024 Accepted: 30-01-2024 Published: 08-02-2024
Abstract
Multi-drug resistant Escherichia coli has become a major threat and cause of many urinary tract
infections (UTI) in Enugu, Nigeria. The study was carried out to determine the resistant plasmids
of multidrug resistant Escherichia coli isolated from men attending prostate clinics at 82 division
Hospital, Enugu. A total of 50 urine samples were collected from patients attending prostate
clinic at 82 division Hospital, Enugu, presenting UTI: with their biodata. The samples were
inoculated on Eosin methylene blue agar and characterized by general microbiological standard.
Antimicrobial susceptibility testing was performed using Kirby-bauer disk diffusion method on
Mueller-hinton agar. Multi drug resistant isolates were selected for plasmid profiling. Plasmids
were extracted by the alkaline lysis method, electrophoresed on 0.8% agarose gel and profiled
using a gel-photo documentation system gel. Escherichia coli were isolated at 72%. E. coli
isolate showed high resistance to most of the test agents. E.coli strains were observed to resistant
to more than nine antibiotics. The resistant plasmid DNA were detectable in all of the 11 multi
drug resistant Escherichiacoli having single sized plasmids of the same weight 18kbp. 6 strains
(lanes 6,7,8,9,10 and 11) had multiple sized plasmids at 9,8,8,8 and 9kbp respectively. They
were all resistant to amoxycillin, ciproflox, nalidixic acid, reflaxine, pafloxacin and
chloramphenicol. The study has highlighted the emergence of multidrug resistant plasmid among
E.coli causing urinary tract infections in Enugu, Nigeria. There is a high level of resistance to
many antimicrobials that are frequently used in Enugu, Nigeria.
Key words: Plasmids, Antibacterial resistance, antimicrobials, Escherichia coli, Urinary tract
infections
1. Introduction
Urinary tract Infection (UTI)is a significant disease and a major cause of morbidity and mortality
32,33
and are commonly spread . Occurrence of urinary pathogens occurs among different age
groups, sex, catherization, hospitalization and previous exposure to antimicrobial39. Urinary tract
infections present with burning sensations during urination, dysuria, pain in the back or lower
abdominal pain, fever or chills and frequent or intense urine urge. In Nigeria, E. coli, Proteus spp
and klebsiella spp have been isolated in 90.0% of UTI reported cases15,19. E.coli is mostly
implicated in UTI and is associated with asymptomatic bacteriuria and symptomatic UTI caused
by uropathogenic E. coli 12,17.

E.coli is ubiquitous in humans and can act as an extraintestinal pathogen (EXPEC) causing both
community as well as hospital acquired urinary tract infections and sepsis often leading to
serious secondary health issues37. The widespread and injudicious use of antibiotics at the
community level cause a high level of resistance patterns of microorganisms to common
antibiotics. UTI caused by multidrug resistant (MDR)E.coli increases cost of treatment,
morbidity and mortality25,26. These MDR isolates has serious implications for the empiric
therapy against pathogenic isolates and for the possible co-selection of antimicrobial resistant,
mediated by multi drug resistant plasmid35,36.Antibiotic resistance in E. coli can arise by
mutations in diverse targets or by acquisition of preexisting genes whose products target
antibiotics for alteration or efflux10,30.Mobile resistance genes have the greatest potential for
spread of antimicrobial resistance in the microbiome. Bacterial plasmids are self-replicating,
extrachromosomal elements that are key agent of change in microbial population. They promote
the dissemination of a variety of traits, including virulence, enhanced fitness, resistance to
antimicrobial agents, and metabolism of rare substances. E. coli from clinical isolates are known
to harbor plasmids of different molecular sizes9. E.coli shows high resistance to ampicillin,
amoxicillin, tetracycline and trimethoprim – sulfamethoxazole30,32. Resistance patterns of
uropathogenic E. coli to various antibiotics has also been reported1. Multiple drug resistance
isolates causing UTI has serious implications for the empiric therapy against pathogenic isolates
and for the possible co-selection of antimicrobial resistant mediated by multi-drug resistant
plasmids. Antibiotic sensitivity patterns vary in different locations, the widespread occurrence of
drug resistant E.coli and other pathogens in developing countries and Nigeria in particular has
necessitated the need for regular monitoring of antibiotics susceptibility trends to provide basis
for developing rational prescription programs, making policy decisions and assessing the
effectiveness of both. In this study, we aimed to determine the prevalence and the resistant
plasmids of multi-drug resistant Escherichia coli isolated from men attending prostrate clinics in
parts of Enugu State, Nigeria.
II. Materials and Methods
Sampling
A total of fifty (50) clean-voided, mid-stream urine samples were collected from men attending
‘82 Division hospital, Enugu. Samples were collected from Jan to Feb 2023 and sent
immediately to microbiology lab, Enugu State University of Science and Technology.
Isolation of Organisms
All the media used were prepared according to manufacturer’s instructions and then autoclaved
at 1210C for 15 min. 0.1ml of urine sample was transferred on Eosin methylene blue agar and
spread with a sterile inoculating loop. The plates were incubated at 370C for 24hr. Resultant
colonies were purified by transferring to nutrient agar and incubated at 370C for 24hr. The
characteristic isolates were aseptically isolated and characterized using established
microbiological methods including colonial morphology, Gram’s staining reaction, haemolytic
reaction, catalase and coagulase tests. Identified E.coli colonies were stored on agar slants at 40C
for further use.
Inoculum Preparation
MacFaland’s turbidity standard was prepared by dissolving 1m1 of barium chloride (BaC12) into
9m1 of sulphuric acid (H2S04). One loopful of each of the pure isolates (E.coli ) were transferred
/into sterile 5m1 nutrient broth in a test tube and incubated at 28°C for 24hr. Each of the cultures
was then adjusted to 0.5 MacFaland’s turbidity standard.
Antibiotic Susceptibility Testing
The antibiotic susceptibility test was carried out as described by Kirby-Bauer disc diffusion
method and interpreted according to the National Committee for Clinical Laboratory Standards
(NCCLS). 0.1ml of each isolate already matched to 0.5 Macfarland’s standard turbidity were
transferred on Mueller hinton agar plates and incubated at 370C for 24 h. Ten common
antimicrobial drugs were use: ciprofloxacin (CPX) (10ug), Nalidixic acid (NA)(10ug),
Gentamycin(CN)(10ug),Amoxycillin(AMX)(30ug),septrin(S)(30ug),
reflacine(RD)(10ug),perfloxacin(PEF)(30ug),chloramphenicol
(CH)(30ug),ofloxacin(OFX)(10ug), levofloxacin (LEV)(30ug)(Maxicare medical
laboratory).The size of the area of suppressed growth(zone of inhibition) was determined by the
concentration of the antibiotics present in the area, therefore, the diameter of the inhibition zones
denotes, the relative susceptibility to a particular antibiotic. Inhibition zones of diameters were
measured after 24h of incubation at 370C.
Plasmid DNA Extraction
The DNA extraction was done using Zyppy™ Plasmid Miniprep. 1.5 ml of bacterial culture was
centrifuged for 5 min at maximum speed (1200 rpm), the supernatant was discarded and the
process repeated twice. A total of 600 μl of TE or water was added to the bacterial cell pellet and
resuspended completely. A total of 100 μl of 7X Lysis Buffer (Blue) was added and mixed by
inverting the tube 6 times. The sample was allowed to stand for 2 mins. A total of 7X Lysis
Buffer was added, color change was observed in the solution from opaque to clear blue,
indicating complete lysis. A total of 350μl of cold Neutralization Buffer (Yellow) was added and
mixed thoroughly. The sample turns to yellow when the neutralization is complete and a
yellowish precipitate was formed. The sample was inverted for 3 times to ensure complete
neutralization and centrifuged at maximum speed for 4 minutes. The supernatant (~900μl) was
transferred into the provided Zymo-Spin™ IIN column. Care is taken to avoid disturbing the cell
debris pellet. The column was placed into a collection tube and centrifuged for 30 secs. The
flow-through was discarded and the column placed back into the same Collection Tube. 200μl of
Endo-Wash Buffer was added to the column and centrifuged for 30 secs. A total of 400μl of
Zyppy™ Wash Buffer was added to the column and centrifuged for 1 min. The column was
transferred into a clean 1.5 ml microcentrifuge tube and 30 μl of Zyppy™ Elution Buffer was
added directly to the column matrix and allowed to stand for one min at room temperature
centrifuged for 30 seconds to elute the plasmid DNA.
Agarose Gel Electrophoresis of Extracted Plasmid DNA
An aliquot (3 µl) of the extracted plasmid DNA was mixed with 7 µl of DNA gel loading dye
(Thermo Scientific™). The mixture was analyzed on 1% Agarose gel stained with 1µg/mL of
ethidium bromide following protocol described by Lee et al. (2012). Electrophoresis was carried
out at 90 volts for 45 min and visualized/ illuminated under ultraviolet transilluminator. A 1 kbp
DNA ladder (New England Biolabs, USA) was used as DNA molecular weight marker.
Statistical Analysis
Frequencies were obtained and percentages were calculated for study variable. Chi-square was
used to calculate and determine significance. A p-value of less than or equal to 0.05 was
considered to be statistically significant(p<0.05).
III. Results
Growth and Morphology of Isolates
Out of 50 urine sample examined 36(72%) were blue black colonies with metallic sheen
characteristic of E. coli (Table1).
Prevalence of Isolates in The Study Area
E.coli was highly prevalent in the study area at 36(72%) (Fig 1) .
Microscopic appearance and Biochemical Characteristics of E. coli
E. coli appeared as Gram- negative rod shaped cells under the microscope and differed in their
biochemical characteristics (Table 2).
Prevalence of Symptomatic and Asymptomatic Individuals in the test Population
Pre-experimental activity involved sharing of structured questionnaire comprising of signs and
symptoms, information obtained from the respondents helped in grouping them into those that
observed symptoms and those who observed no signs and symptom. 36( 72%) respondents were
symptomatic while 14 (28%)were asymptomatic (Fig 2).
Macroscopic Analysis of Urine Samples from the Study Population
Physical analysis of urine from respondents recorded altered colour and smell(odour) at higher
occurrence of 44 (88%) followed by turbidity at 36 (72%) and bloody urine at 8 (16%) (Table 3).
Prevalence of Genital Symptoms and Clinical Signs in Men with Urinary Tract Infection
From the questionnaire, a total of 36 men indicated having symptoms of urinary tract infections.
Significant relationship at p<0.05 (0.0012) was observed between presence of symptoms and
infection with E. coli.(Table 5)
Pattern of Antimicrobial Susceptibility Result of Isolates
The results of antibiotic susceptibilities of E. coli from positive urine samples were shown in Fig
3. It was observed that E. coli isolates were resistant to most of the test agents at 100%.
Prevalence of Multi-drug Resistance Among the Isolates
Multi drug resistance was defined in the study as resistance to four or more of the antibiotics
tested. 100% of E. coli (Table 6).
Plasmid DNA profiling
Eleven (11) E. coli isolates were characterized by plasmid DNA profiling. Single plasmid bands
were observed in all the isolates with molecular weights of approximately 18kbp (lanes
1,2,3,4,5,6,7,8,9,10 and 11). Multiple plasmid bands were observed with molecular weight of
4kbp (lanes 7,8,9and 10). Lane 7 had an additional 7kbp (Table6).
Table 1: Growth and Morphology of Isolates
Media Growth morphology Organism isolated
Eosin methylene blue agar Blueblack colonies with Escherichia coli
greenish metallic sheen

28%
No Isolate
E.coli
72%
Fig 1: Distribution of E. coli isolates from the urine samples
Table 2: Identification and Characterization of Isolates
Biochemical characterization Inference

E .coli
Catalase Test +ve
Coagulase Test -ve
Indole Test +ve

Oxidae Test +ve
Citrate Test -ve
MethylRed Test +ve -
Sugar Fermentation Test
Lactose Acid/Gas
Fructose Acid
Maltose Acid
Glucose Acid
Microscopy Rod shape
Gram staining Gram –ve
Key
+ Positive
- Negative
A Acid
32%
Symtpomatic
Asymptomatic
68%
Fig 2: Prevalence of Symptomatic and Asymptomatic respondent in the Test population
Table 3: Macroscopic Analysis of Urine
Test (n=50) Normal % of Normal Abnormal %of Abnormal
Parameter Parameter Parameter Parameter
Colour Pale to dark 6 (12%) Altered colour 44 (88%)
yellow
Clarity Clear 3 (6%) Turbid 36 (72%)
Bloody 8 (16%)
Odour Slightly nutty 3 (6%) Altered smell 44 (88%)
Table 4: Distribution of E.coli Amongst Men with Abnormal Urine Parameter
Abnormal Parameter % of Abnormal Parameter % of those positive

with E.coli(n=36)
Altered colour 44 (88%) 36(81%)*
Turbid 36 (72%) 36 (100%)*

Bloody 8 (16%) 8(22%)*
Altered smell 44 (88%) 36 (81%)*
*significance p<0.05(0.0012)
Table 5: Prevalence of Genital Symptoms and Clinical Signs in Men with Urinary Tract
Infection
Criteria(Symptoms) % Positive % of men positive with

n=50 E.coli(n=36)
Pain in the scrotum 42 (84%) 36 (85%)*
Swelling and redness in the 40 (80%) 36 (90%)*

testicle
Blood in the semen 15(10%) 5 (33%)
Fever and chills 28 (53.6%) 28 (36%)*
Dysuria 44 (88%) 36 (85%)*
*There was significant relationship betweenE.coli and symptoms at p<0.05(0.0013).
120
100
100 97 97 97 97
95 95 95
86.3 86.3
80
60
Sensitivity%
Intermediate%
40
Resistivity %
20
13.6 13.6
4.5 4.5
2 2 2 2
0 0
0
Fig 3: Pattern of Antimicrobial Susceptibility Results of E. coli

Table 6: Prevalence of Multidrug Resistance Pattern among the Isolates
Types of isolates Resistance to Resistance to Resistance to Resistance to
4 agents 5 agents 6 agents 7agents
E. coli(n=34) 36 (100%) 34 (100%) 34 (100%) 29 (85%)
18kbp
DL 5 8 9 10 11 -ve
1 2 3 4 6 7
Fig 4. Plasmid DNA profiling. Lanes 1,2,3,4,5,6,7,8,9,10,11 have single bands.
Lanes 6,7,8,9,10&11 have multiple bands
Table 7: Plasmid Characterization isolate from E.coli strains, showing numbers and
sizes and pattern of resistant antibiotics
Isolate Number Size of Resistant

of plasmid(kb) antibiotics
plasmid
isolated CPX
NA MX S RD PEF CH FX LEV
E1 1 18kb CPX NA MX S RD PEF CH FX LEV -
E2 1 18kb CPX NA MX S RD PEF CH FX LEV -
E3 1 18kb CPX NA MX S RD PEF CH FX LEV CN
E6 2 18kb, 9kb CPX - MX - RD PEF CH FX LEV CN
E7 3 18kb,8kb, CPX - MX - RD PEF CH FX LEV CN
7kb
E8 2 18kb,8kb - NA - S RD PEF CH FX LEV CN
E9 2 18kb,8kb CPX - - S RD PEF CH FX LEV CN
E10 2 18kb, 8kb CPX NA MX S RD PEF CH FX LEV CN
E11 2 18kb, 9kb CPX NA MX S RD PEF CH FX LEV CN
IV. DISCUSSION
UTI caused by the multi drug E. coli has increased in the current years probably
due to the increasing and irrational use of antibiotics3. In this study, E. coli occurred at a
high prevalence of 34(75%) (Fig 1). This is in line with the works of 3 who found E.coli
the most predominant species in their study population. E coli has been reported as the
leading causes of acute and uncomplicated UTI in ambulatory patients 4,5. Other reporters
populated that in Nigeria, E. coli, Proteus spp and Klebsiella spp have been isolated in
22,27,28, 29,26
90.0% of UTI reported cases . This present study revealed E. coli isolates as
Gram –ve rod shaped strains. (Table 2) The isolates differed in their biochemical
reactions. In this study, information obtained was based on respondents. Those classified
as asymptomatic men were those who did not report observing any signs and symptoms
while symptomatic men observed signs and symptoms (Fig 2).
In this study only 2 (4%) recorded as asymptomatic respondents while 42 (84%) were
33
symptomatic. According to unpathogenic E. coli (UPEC) causes symptomatic urinary
tract infection (UTI) and little is known about the mechanisms by which the strains
colonize the human urinary tract. E. coli has also been implicated as the most common
organism associated with asymptomatic bacteriuria (ABU). E.coli is responsible for more
33,2
than 80.0% of all UTI causing both ABU and symptomatic UTI’s . This present study
tries to make association of E. coli with signs and symptoms from respondents. The
pattern of physical urine analysis showed altered colour at 44 (88%); turbid at 36 (72%);
bloody at 8 (16%) and altered at smell 44 (88%). There was association between these
criteria with isolated (Table 3). This work observes a strong association between genital
symptoms and clinical signs in men positive with E. coli. Clinical signs for the isolates
were 42 (84%) for pain in the scrotum at 8% for swelling and redness in the testicle
(10%) for blood in the semen at 5 (10%), 56% for fever and chills and 88% for dysuria.
In the study, there was association between clinical signs in men with urinary tract
infection and presence of E.coli as causative agents (Table 4). In this study, a high
prevalence of resistance to antimicrobial agents was recorded, E. coli was resistant at
100% to amoxicillin resistance to fluoroquinolones drugs were recorded thus:
Pafloxacin(97%); Reflacine (97%); Ofloxacin (97%); and Ciprofloxacin(95%) followed
by Gentamycin and septrin respectively(Fig 4.3). Adnan 2015 and Akingbade et al.,2014
showed resistance of E. coli from urinary tract infection to cotrimazole at 66.7% and 70%
respectively. Varied results have been recorded in other studies. Susceptibility to
gentamycin (85.8%), levofloxacin (80%), ciprofloxacin (72.5%) and Ofloxacin (60.8%)
was recorded in the works of 2, this is in contrast with the present study. Susceptibility or
40,7,14,3,2
resistance pattern have been demonstrated in different geographic location , all
recorded varied reaction in their susceptibility patterns. E.coli from clinical isolates are
known to harbor plasmids of different molecular sizes9. According to 31

bacteria harbor
antibiotic resistant genes which can be horizontally transferred to other bacteria. Multiple
drug resistance in isolates capable of causing urinary tract infection is a serious public
health problem as empirical therapy becomes compromised and triggers possible co-
35,36
selection of antimicrobial resistant pathogens . In the present study, 100% of E. coli
isolates showed multi drug resistance to test agents. 12% were completely resistant to 10
agents (Fig 3). The increasing resistance of E. coli strains to antibiotics makes guidance
of therapy by antibiotic susceptibility test of paramount importance.
Resistance to numerous antimicrobial agents in this study may be due to indiscriminate
and widespread use of these antibiotics in Enugu, Nigeria which may lead to the spread
of mutant strains, insusceptible to medical treatment. All multi-drug Escherichia coli
isolates in the study possessed plasmids with similar molecular weights of 18kbp. Sample
8 and 10 had additional bands of 9kbp while sample had an additional band of 10 kbp
(Fig4). These observations suggest that a number of different plasmids and plasmid
combination occur in E. coli strains. This agrees with previous studies of Smith et al.,
2003 who reported that 47 of the E.coli isolated form animals in Lagos harbor detectable
plasmids which ranged in sizes from 0.564kb to > 23kb. Dambara et al.,1987 reported
that E.coli isolates possessed small plasmids of molecular sizes which ranged between
3.9kb 50kb. Umolu et al.,2008 also reported that E. coli isolates with high multi-drug
resistance profiles were found to possess plasmids, though with large sizes in the range of
6.557- 23. 130kb.
V. Conclusion
This study showed that there was a high prevalence of E. coli. There was also emergence of
multidrug resistant plasmids among Escherichia coli causing urinary tract infections in men
attending prostate clinic in Enugu, Nigeria. It is of public health concern as most of the
susceptible drug of choice in other studies were found completely resistant in the study.
Conflict of interest statement
Authors declare that they have no conflict of interest.
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Journal of Science and Technology

ISSN: 2456-5660 Volume 9, Issue 02 (FEB -2024)

Plasmid Profiles of Resistant Strains of Escherichia coli in Men

Kelechi Nkechinyere mbah-Omeje1* Nebo Stephen Chinonso2 , Iloputaife Emmanuel

To Cite this Article

Kelechi Nkechinyere mbah-Omeje, Nebo Stephen Chinonso , Iloputaife Emmanuel

Received: 10-01-2024 Revised: 20-01-2024 Accepted: 30-01-2024 Published: 08-02-2024

many antimicrobials that are frequently used in Enugu, Nigeria.

by uropathogenic E. coli 12,17.

community level cause a high level of resistance patterns of microorganisms to common

antibiotics. UTI caused by multidrug resistant (MDR)E.coli increases cost of treatment,

mediated by multi drug resistant plasmid35,36.Antibiotic resistance in E. coli can arise by

mutations in diverse targets or by acquisition of preexisting genes whose products target

spread of antimicrobial resistance in the microbiome. Bacterial plasmids are self-replicating,

the dissemination of a variety of traits, including virulence, enhanced fitness, resistance to

amoxicillin, tetracycline and trimethoprim – sulfamethoxazole30,32. Resistance patterns of

parts of Enugu State, Nigeria.

II. Materials and Methods

immediately to microbiology lab, Enugu State University of Science and Technology.

characteristic isolates were aseptically isolated and characterized using established

microbiological methods including colonial morphology, Gram’s staining reaction, haemolytic

for further use.

was then adjusted to 0.5 MacFaland’s turbidity standard.

Antibiotic Susceptibility Testing

(CH)(30ug),ofloxacin(OFX)(10ug), levofloxacin (LEV)(30ug)(Maxicare medical

measured after 24h of incubation at 370C.

Plasmid DNA Extraction

centrifuged for 30 seconds to elute the plasmid DNA.

Agarose Gel Electrophoresis of Extracted Plasmid DNA

considered to be statistically significant(p<0.05).

Growth and Morphology of Isolates

characteristic of E. coli (Table1).

Prevalence of Isolates in The Study Area

E.coli was highly prevalent in the study area at 36(72%) (Fig 1) .

Microscopic appearance and Biochemical Characteristics of E. coli

biochemical characteristics (Table 2).

Prevalence of Symptomatic and Asymptomatic Individuals in the test Population

Pre-experimental activity involved sharing of structured questionnaire comprising of signs and

symptomatic while 14 (28%)were asymptomatic (Fig 2).

Macroscopic Analysis of Urine Samples from the Study Population

infection with E. coli.(Table 5)

Pattern of Antimicrobial Susceptibility Result of Isolates

Prevalence of Multi-drug Resistance Among the Isolates

tested. 100% of E. coli (Table 6).

Plasmid DNA profiling

4kbp (lanes 7,8,9and 10). Lane 7 had an additional 7kbp (Table6).

Table 1: Growth and Morphology of Isolates

Media Growth morphology Organism isolated

Eosin methylene blue agar Blueblack colonies with Escherichia coli

greenish metallic sheen

Fig 1: Distribution of E. coli isolates from the urine samples

Table 2: Identification and Characterization of Isolates

Biochemical characterization Inference

Catalase Test +ve

Coagulase Test -ve

Indole Test +ve

Oxidae Test +ve

Citrate Test -ve

MethylRed Test +ve -