Biofilm Inhibitory Effects of Lactobacillus Spp Against Streptomycin-
resistant Uropathogenic Escherichia Coli
NWANEKWU KENNETH EMEKA
Department of Microbiology, Faculty of Biological Science, Imo State University, Owerri, NIGERIA
Abstract: The biofilm inhibitory effects of Lactobacillus spp against Streptomycin-resistant uropathogenic
Escherichia coli (UPEC) were evaluated using the crystal violet test method. Lactobacillus spp were isolated
from milk samples while fifty strains of Uropathogenic Escherichia coli were isolated from urine samples from
Urinary Tract Infection patients attending Federal Medical Centre (FMC) Owerri, Nigeria. Ten of the E. coli
strains resistant to streptomycin antibiotics were screened for their susceptibility to antibiofilm effect of
Lactobacillus secondary metabolites extracts. From the result obtained, only one of the E. coli strains was
susceptible while nine strains were resistant. This result shows clearly that the metabolite extracts from
Lactobacillus sp were not effective in the antibiofilm activity of the E. coli strains and thus not a good candidate
for the management of UTI caused by E. coli.
Keywords: Uropathogenic, Antibiofilm, UTI, Lactobacillus, E. coli
Received: July 2, 2022. Revised: September 14, 2023. Accepted: October 19, 2023. Published: November 27, 2023.
1. Introduction
Uropathogenic Escherichia coli (UPEC) is a
nonsporulating, flagellated, facultative anaerobic
Gram-negative rod belonging to the family
Enterobacteriaceae (Yi-Te et al., 2020). It is the
most significant causative agent of UTIs in humans
accounting for about 75% of cases (Flores-Mireles et
al., 2015). It is also a major food contaminant
causing serious food spoilage and food borne
infection (CDC 2012). E. coli has the capability to
cause cause UTI and other diseases because of the
various virulence factors it possesses. These factors
include, acid tolerance, toxin production and biofilm
formation (Wiles et al., 2008). Biofilm is a mass of
microbial cells attached to a surface and enclosed in
a matrix of polysaccharide (Donlan, 2002). Biofilms
formation serves as a survival mechanism for
bacteria during extreme environmental conditions
such as nutrient deficiency and antimicrobial actions
(Nandakumar et al., 2013). These organisms have
also been found to harbor a large number of
antibiotic inactivating enzymes such as beta-
lactamases leading to antimicrobial resistance
(Davies & Davies 2010). Several studies have
reported cases of antimicrobial resistance among
UPEC especially among commonly used antibiotics
such as ciprofloxacin, trimethoprim-
sulphamethoxazole, streptomycin among others (Ali
et al., 2016; Neupane et al., 2016).
Streptomycin is the first discovered aminoglycoside
antibiotic, originally isolated from the bacteria
Streptomyces griseus. It has activity against several
aerobic gram-negative bacteria including E. coli
(Zhu et al., 2001). Its broad-spectrum activity
against gram-negative and gram-positive bacteria
has been greatly diminished, largely due to
developing antibiotic resistance (Daniel, 2005). The
mechanism of resistance appears to be associated
with the inhibition of its active transport into the
bacterial cell. Commonly resistant bacteria include
Enterobacteriaceae and most Streptococci species
(Akhtar et al., 2016; Azam et al., 2019).
Streptomycin is a drug of choice in the treatment of
E. coli infections. They have been used in several
local communities in the management of UTI caused
by E. coli. However, there have been several cases
of treatment failures and incidence of streptomycin
resistance by E. coli and other gram-negative
organisms lately, necessitating the need for other
natural products alternatives.
Lactobacilli are widespread in nature and reside in a
variety of natural habitats, ranging from plants to the
mammalian oral, gastrointestinal or vaginal cavities
(Kenreigh &Wagner 2006). Lactobacilli are known
for their ability to inhibit the growth of bacteria due
to the production of antimicrobial materials such as
bacteriocins, biosurfactants and lactic acid
(Soleimani et al., 2010). These secondary
metabolites produced by Lactobacillus have been
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Nwanekwu Kenneth Emeka
E-ISSN: 2945-0454
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shown to possess antibacterial activity against most
pathogenic organisms (Mejlholm and Dalgaard
2015). However, to combat the problem of
antimicrobial resistance, these metabolites are used
against the virulence factor (biofilm formation)
which is responsible for the pathogenicity of the
disease rather than the inhibitory activity against the
organism. Thus, the present study is therefore aimed
at evaluating the antibiofilm activity of metabolites
secreted by Lactobacilli against Streptomycin-
resistant uropathogenic E. coli (UPEC).
2. Materials and Methods
Escherichia coli
The Uropathogenic E. coli strains were isolated from
patients with urinary tract infection in the clinical
diagnostic Laboratory of Federal Medical Centre
Owerri, Nigeria using standard bacteriological
methods. Urine samples from 50 women were
collected in sterile specimen screw-capped bottles
and transported to the Lab immediately for analysis.
One milliliter of the urine specimen was inoculated
into 19mL molten agar and poured into a sterile petri
dish. The agar was allowed to solidify and then
incubated at 37oC for 24hours. After incubation, the
colonies formed were subjected to conventional
biochemical tests to confirm the presence of E. coli
strains.
Lactobacillus spp
Lactobacillus spp were isolated from different milk
samples according to the method described by
Mahsa et al (2017). One hundred (100) ml of the
liquid milk samples were collected in sterile conical
flasks and allowed to ferment at room temperature
for 3 days. Ten-fold serial dilutions of the samples
were made and 0.1ml of suitable dilution inoculated
unto MRS agar. The pH of the medium was adjusted
to 5.5 by adding HCl. The set plates were incubated
anaerobically at 35oC for 48hours. Colonies were
tested for catalase activity. Catalase negative
organisms were sub-cultured onto fresh sterile MRS
Agar to obtain pure culture. The isolated
microorganisms were sub-cultured unto a
maintenance culture medium of MRS broth
containing 12% v/v glycerol. This was incubated at
30oC until growth was detected and then stored at
4oC in refrigerators.
2.1 Preparation of Lactobacillus metabolite
extract
Metabolites extracts from the Lactobacillus spp was
prepared by the method described by Rao et al.,
(2015). Broth cultures of all the LAB isolates were
first prepared by simply inoculating a loopful of
culture into fresh 20ml MRS broth in 25ml sterile
bottles and incubated at 35oC in an anaerobic jar for
72 hours. The 72hour broth culture of Lactobacillus
spp was used in the preparation of crude extract. Ten
mil (10ml) of the LAB broth culture was transferred
into tubes and centrifuged at 5000 revolution per
minute (r.p.m) for 15 minutes to obtain clear
sedimentation of the pellets. The supernatant was
decanted into separate containers. The supernatant
fluid was adjusted to pH 6.5 by adding NaOH and
then treated with 5mg/ml catalase. The supernatant
fluid was then filter sterilized through a 0.45µm pore
size cellulose acetate filter. The product was
designated as LAB metabolite extract.
2.2 Antibiotics susceptibility testing
The antibiotics susceptibility test was carried out
using the Agar disk diffusion method (Syukur, et al.,
2014). A volume of 100 μl of an overnight culture of
each UPEC isolate on Mueller-Hinton broth with the
turbidity of 0.5 McFarland was streaked on Mueller-
Hinton agar plates. The routinely used 10 antibiotic
discs, including Reflacin, Nalidixic acid,
Augmentin, Gentamycin, Ampicillin, Ofloxacin,
Streptomycin, Septrin, Ampicillin and Ciprofloxacin
were placed on the surface of the inoculated plates.
The plates were incubated at 37° C for 24 hr.
2.3 Investigation of the anti-adhesive effect of
lactobacilli supernatant
To evaluate the anti-adhesive effect of the
Lactobacilli, polystyrene microtiter plate 100 was
used. First, 75 µl of the lactobacilli supernatant and
then 75 µl culture suspension of UPEC were added
to the wells. The microtiter plates were incubated at
37°C for 24 hours. Each of UPEC (without
lactobacilli) was poured into the control wells. Then,
the contents of the wells were removed and each
well was washed three times by PBS. Ethanol 96%
w/w-1 (for 15 min) and 2% w/w-1 crystal violet (for
10 min) were used for stabilizing the cells and
staining, respectively. Then, the polystyrene
microtiter plate was rinsed with a gentle stream of
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water. When the wells were dried by exposing to the
air, 33% w/w-1 acetic acid was added to the wells as
a solvent, and optical absorbance was measured at
492 nm for each well using spectrophotometer. The
test was carried out in duplicate (Mahsa et al., 2017).
3. Results and Discussion
3.1 Isolation and Identification of
Lactobacillus spp
Morphological and physiological characteristics of
the Lactobacillus isolates were carried out and
presented in Table 1. The isolates were motile,
Gram-positive, non-spore forming and catalase-
negative rods. These are typical characteristics of
Lactobacilli as commonly isolated from milk and
other fermented products (Crowley et al., 2013).
3.2 Isolation and Identification of E. coli
strains
Echerichia coli strains were isolated and identified
by their morphological and physiological
characteristics and presented in Table 2. All the
isolates were Indole and MR positive, VP, Citrate
and Urea negative respectively. These characteristics
represent typical physiological properties of E. coli
as reported in previous research (Bukh et al., 2009;
Tenaillon et al., 2010).
Table 1: Morphological and Biochemical Characteristics of Lactobacillus Isolates
Isolate Gram Shape Spore Motility Catalase Nitrate Growth Suspected Organism
formation at 5% NaCl
I + Rod - + - - + Lactobacillus acidophilus
II + Rod - + - - + Lactobacillus acidophilus
III + Rod - + - - + Lactobacillus acidophilus
_____________________________________________________________________________________
Table 2: Morphological and Biochemical Characterization of E. coli strains
Isolate Gram Shape Motility MR VP Indole Citrate Urea Suspected Organism
I - Rod + + - + - - Escherichia coli
II - Rod + + - + - - Escherichia coli
III - Rod + + - + - - Escherichia coli
IV - Rod + + - + - - Escherichia coli
V - Rod + + - + - - Escherichia coli
VI - Rod + + - + - - Escherichia coli
VII - Rod + + - + - - Escherichia coli
VIII - Rod + + - + - - Escherichia coli
IX - Rod + + - + - - Escherichia coli
X - Rod + + - + - - Escherichia coli
3.3 Antibiotics susceptibility test
Nine antibiotics were tested against the E. coli
strains isolated and the results presented in Table 3.
From the results obtained, the organisms showed
varying reactions to the tested antibiotics. The
antibiotics Reflacin, Ciprofloxacin, Augmentin,
Gentamycin and Ampicillin inhibited the growth of
the organisms, while the E. coli strains were resistant
to Oflocxacin, Septrin, Nalidixic acid and
Streptomycin. This result is in line with previous
reports of the established resistance of E. coli to a
number of antibiotics including those reported in this
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study (Osungunna & Onawunmi 2018). Ten of the
E. coli strains tested were all remarkably resistant to
Streptomycin while been susceptible to
Ciprofloxacin. This is in clear disagreement with the
report of Mahsa et al., (2017) which reported that
the biofilm forming strains of E. coli were resistant
to Ciprofloxacin. This could be as a result of the
degree of exposure and use of the various antibiotics
in the treatment of UTI in different countries and
geographical locations. It however agrees with Dabo
et al., (2019) and Rafique et al., (2020) who reported
E. coli resistance to Streptomycin.
3.4 Antibiofilm-adhesive effect of lactobacilli
supernatant against E. coli isolates
Results of the antibiofilm adhesive effect of
Lactobacillus supernatant against E. coli strains are
presented in Figure 1. The figure showed that the
Lactobacilli metabolites did not inhibit or affect the
biofilm adhesive capabilities of nine of the E. coli
strains, while only one E. coli strain EC1among the
ten strains were susceptible to the effects of the
Lactobacilli. The EC5 strain was the most resistant
isolate to Lactobacilli metabolite. This shows that
the Lactobacilli metabolites are not effective in
inhibiting the effect of Uropathogenic E. coli
biofilm. The results obtained in this study did not
agree with the report of Mahsa et al., (2017) and
Abedi et al., (2013) which reported that probiotic
Lactibcilli had anti-adhesive effect.
Table 3: Antibiotic susceptibility test
Antibiotic EC1 EC2 EC3 EC4 EC5 EC6 EC7 EC8 EC9 EC10
Reflacin + + + + + + + + + +
Ciprofloxacin + + + + + + + + + +
Augmentin - + + + + + + + + +
Gentamycin + + + + + + + + + +
Ampicillin - + + + + + + + + +
Ofloxacin + - - - - - - - - +
Streptomycin - - - - - - - - - -
Septrin - - - + - - - - + -
Nalidixic acid - + - - - - + - - -
+ Susceptible; - Resistant
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Fig 1: Antibiofilm adhesive effect of Lactobacilli supernatant against E. coli isolates
4. Conclusion
Based on the findings of this study, it is therefore
imperative to say that secondary metabolite extracts
from Lactobacillus spp doesn’t have antibiofilm
effects against Streptomycin-resistant Uropathogenic
E. coli and as such cannot be used in the
management of UTI as alternative natural product to
antibiotics.
There is therefore the need to continue the search
and screening of other natural product candidates
such as medicinal plants and other probiotic
organisms as potential and alternative sources of
antibiotics in the management and treatment of
microbial infections and combat the incidence of
antibiotics resistance. Further work will be done in
looking at medicinal plant extracts against biofilm
formation and other virulence factors produced by
antibiotic resistant E. coli.
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