1.
INTRODUCTION
Diarrheal
diseases are on the rise worldwide, with an estimated 1.6 million deaths
annually affecting children under five years of age (Troeger et al.,
2018). Kotloff et al. (2013) reported that this increase is due to
several microbial pathogens, including Vibrio cholerae, Escherichia coli,
Salmonella, Shigella, and Clostridioides difficile (C. difficile),
which contribute to both healthcare-associated infections and
community-acquired (Kotloff et al., 2013). It has been reported the main
cause of antibiotic-associated diarrhea, especially nosocomial infections, is C.
difficile (Carroll &Bartlett, 2011). This bacterium is a motile,
rod-shaped, Gram-positive bacterium previously confirming that thirteen of the
100 identified species of Clostridium are considered to be extremely
harmful to humans or animals (Dupuy et al., 2006).
Several
studies have confirmed that the overuse of antibiotics, especially without a
prescription, can significantly alter the gut microbiota (Baines et al.,
2015; Sun &Hirota, 2015). This disruption in the gastrointestinal
tract increases the host’s susceptibility to infection, particularly C.
difficile infection (Sun &Hirota, 2015). The use of certain
antibiotics, including clindamycin, ampicillin, penicillin, tetracycline, and
cephalosporins, has been associated with a high risk of C. difficile infection
(Khashei et al., 2018).
The standard first-line treatment for C. difficile infections typically involves
metronidazole and vancomycin (Canas et al., 2023). However, antibiotic
resistance to these medications has been reported, complicating treatment and
making CDI management more challenging (Di et al., 2015). Moreover,
recent research has also shown that commonly used antibiotics used to treat diarrhea,
such as clindamycin and fluoroquinolones, and occasionally even the first-line
treatment vancomycin development of C. difficile (Canas et al.,
2023). The ability of C. difficile to antibiotic resistance, along with
its toxin production, expresses the severity of C. difficile infection
(CDI) and makes it a significant public health (Baines et al., 2015).
Recently,
antibiotic resistance mechanisms in C. difficile have become a
significant concern due to the emergence of various mechanisms (Van et al.,
2008). The presence of the erm (B) and erm (C) genes in this
bacterium encode rRNA methylases that modify the target site, conferring
resistance to macrolides such as erythromycin (Cassandra et al., 2020).
Additionally, the erm(B) gene also reduces the efficacy of clindamycin by encoding
an rRNA methylase (Miele et al.,1995).
Resistance to metronidazole has been associated with mutations in the rdxA and
frxA genes, which are involved in this bacterium activation. Moreover, the
acquisition of β-lactamase genes, such as blaOXA-11 and blaOXA-48, has led
to resistance to cephalosporins, including the Cefixime (Boekhoud et al.,
2021). Additionally, the presence of vanB and vanG operons in
some C. difficile isolates enables modification of the peptidoglycan
cell wall target, thereby reducing the efficacy of vancomycin (Lessa et al.,
2015). These antibiotic resistance genes poses are often located on plasmids
and transposons which are mobile genetic
elements, facilitating their spread between different C. difficile
strains and even other bacterial species. This poses a significant public
health threat, as it limits the available treatment options for serious C.
difficile infections (Canas et al., 2023).
Several
studies have highlighted a concerning rise in infections among humans and
animals leading to the overuse of antibiotics without prescription in Duhok
Governorate, located in the Kurdistan Region of Iraq (Saeed and Ibrahim, 2013;
Hasan and Ibrahim, 2022; Hami and Ibrahim, 2023; Ibrahim, 2023; Mohialdeen
& Ghaffar, 2023; Issa, 2024; Taher and Othman, 2024). This widespread and
often unnecessary use of antibiotics has raised significant public health
concerns, particularly regarding the development of antibiotic resistance which
has vice versa to affect the gut microbiome. To the best of our knowledge, this
study represents the first attempt to detect antibiotic-resistant genes in C.
difficile by analyzing DNA extracted from stool samples using the PCR
(polymerase chain reaction) technique. This research aims to shed light on the
prevalence of antibiotic resistance in C. difficile, contributing to a
better understanding of the challenges posed by antimicrobial resistance in the
region.
2.
MATERIALS AND METHODS
Stool Samples and DNA Extraction
Thirteen
bacterial DNA samples were extracted from stool specimens of diarrheic
pediatric patients diagnosed with C. difficile. This study was conducted
at Hivi Pediatric Teaching Hospital in Duhok, Iraq, between October 2021 and
May 2022. These identified samples were part of a larger cross-sectional study
that involved the collection of 200 stool samples from children suffering from
diarrhea aged from 6 months to 6 years.
All
samples were treated using a stool transport and recovery buffer (S.T.A.R,
Roche, Mannheim, Germany) prior to DNA extraction, following the manufacture
protocol of a High Pure PCR Template Preparation kit (Roche, Germany). DNA
concentration was achieved using a Nanodrop DeNOVIX (Wilmington, USA) with
values ranging from 120 to 256 µg/µL. The purity ratios of both 260/280 and
230/280 were falling between 1.95 and 2.2. Then, this DNA was immediately
stored to preserve its integrity and ensure a high-quality yield for further
DNS amplification.
The
purified DNA extracts of 13 C. difficile from hospitalized diarrheic
children (10 hospital-acquired and 3 community-acquired) previously identified by RT-PCR direct detection of C.
difficile 16S rDNA using the following primers: CIDIF-F CTT GAA TAT CAA AGG
TGA GCC A and CIDIF-R CTA CAA TCC GAA CTG AGA GTA (Eurofins, Ebersberg,
Germany) (Kikuchi et al., 2002). were analyzed for 5 different drug-resistance genes using the
conventional PCR technique (Table 1).
PCR technique
In this
study, these 13 samples diagnosed with C. difficile, were analyzed using
Polymerase Chain Reaction (PCR) to detect five antibiotic resistance genes.
Five specific different primers (Macrogen- South Korea) were employed to
amplify genes associated with resistance to clindamycin, erythromycin,
metronidazole and vancomycin. The targeted genes included Beta lactam
Cefotaximase-Munich gene (blaCTX-M), Erythromycin Ribosomal Methylase C
gene (ermC), Erythromycin Esterase A gene [ere(A)], Oxygen-insensitive
NADPH Nitroreductase A gene (rdxA) and Vancomycin resistance Regulatory Protein
R gene (vanR) as described in (Table 1).
The PCR
amplification was performed using an Applied Biosystems 9700 thermal cycler
(USA). Each reaction contented of 2X PCR master mix (Roche-Germany),
1μL of 20 pmol/μL each forward and reverse primers (Eurofins,
Ebersberg, Germany), 2μL template DNA, and 7μL nuclease-free water to
up to 20μL for reaction. The amplification was conducted considering the
following circumstances: initial denaturation at 94°C for 5 minutes, followed
by 35 denaturation cycles at 94°C for 30 seconds, annealing at different
temperatures for 30 seconds, elongation at 72°C for 1 minute, and final
extension at 72 °C for 10 minutes.
Following
amplification, the PCR products were separated by agarose gel electrophoresis
(Cleaver, UK). The amplicons, ranging between 299 and 850 base pairs, were
visualized under UV light, and images were captured to confirm the presence of
the target genes, as illustrated in Table 1.
Ethical
Approval
The
Research Ethics Committee of the Duhok Directorate of General Health provided
ethical permission. No.13072021-7-7.
Statistical
analysis
The
presence of antibiotic-resistance genes among isolates was examined using the
Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/).
4.
DISCUSSION
This cross-sectional study
aimed to detect drug-resistant genes among C. difficile from both
community and hospital-acquired diarrheic children against commonly prescribed
antibacterial genes. Cefotaxime resistance was highly prevalent, being present
in all 13 isolates (100%). This suggests that cefotaxime resistance is
widespread in the area due to the production of extended beta-lactamase enzymes
(ESBLs), such as bla-CTM-M genes carried on plasmids, which
can be easily spread via the conjugation process (Hassan and Ibrahim, 2022).
The result was lower than those found by Boekhoud et al. (2021) in the
Netherlands, who reported a resistance rate of 35.5% in C. difficile. This
disparity in outcomes can be ascribed to differences in antibiotic usage
policies. In our region, antibiotics are easily accessible without a
physician's prescription and are widely prescribed without proper control. Among
the studied C. difficile, 76.9% and 69.2% were positive for resistant
genes against clindamycin and erythromycin, respectively. Clindamycin
is effective against anaerobic Gram-negative bacilli, which are the predominant
normal flora of the colon and create a symbiotic environment that allows C.
difficile (Duffy et al., 2020). The results of the current study
were similar to those of Tamma et al (2022), who found that 60% of C.
difficile isolates were resistant to clindamycin. Erythromycin resistance
ranges from 40 to 80% globally, which aligns with the results of this study.
Metronidazole resistance was
observed in only one of the 13 C. difficile isolates (7.69%). This
suggests that metronidazole continues to be an effective treatment option for C.
difficile infections, likely due to the relatively low occurrence of
resistance mechanisms. These findings are consistent with a study conducted by
Lessa et al (2015), who reported that metronidazole resistance in C.
difficile isolate is generally low, with resistance rates typically below
5% in most studies.
Our finding aligns with the results of Al-Rawe et al. (2023),
conducted in Iraq; they found that eight genes are present in all isolates and
contribute significantly to drug resistance through ribosome defence,
antibiotic efflux, and antibiotic deactivation. The authors concluded that mutations
in the functional domains of the tetA (P), tetM, and ermB genes were the most
promising new therapeutic targets.
Similarly, vancomycin resistance was infrequent, with only two out of the 13 C.
difficile isolates showing resistance (15.38%). This aligns with the
results of Baines et al (2011), who found that vancomycin resistance in C.
difficile remains relatively uncommon, with most studies indicating
resistance rates under 5%. Vancomycin is a narrow-spectrum antibiotic that is
particularly effective against Gram-positive cocci and is recommended for
severe cases of pseudomembranous colitis when metronidazole resistance is
present.
However, it is worth noting that severe cases of C. difficile
infection are rare in our area. As a result, these antimicrobial drugs are not
commonly prescribed by physicians, but they remain effective treatment options.
In regards to the origin of the isolates, three out of thirteen isolates were
obtained from children with community-acquired diarrhea. All of these isolates
tested positive for resistant genes against cefotaxime, clindamycin, and
erythromycin but negative for metronidazole and vancomycin. It is noteworthy
that the resistance gene with the lowest occurrence was metronidazole at 10%,
followed by vancomycin at 20%. Conversely, the highest percentage was observed
for cefotaxime at 100%, followed by clindamycin at 70% and erythromycin at 60%.
These findings clearly indicate the circulation of drug-resistant genes among
community-acquired infections, likely due to the extensive usage of
antibiotics. Moreover, it is important to acknowledge that these genes have the
potential to transfer among unrelated bacteria easily.
CONCLUSIONS
Based on this study's
findings, it can be concluded that C. difficile isolates that circulated
in the region commonly show antibiotic resistance genes, especially for
cefotaxime, clindamycin, and erythromycin, which are commonly used antibiotics
for various bacterial infections. However, lower levels of resistance were
observed for metronidazole and vancomycin, providing reassurance regarding
their effectiveness as first-line treatment options.
Acknowledgements
The author thanks the Department of Biology, College of Science, University of Duhok, Duhok, Iraq.
Statements and
Declarations
Conflict of
interest: The author declared no potential conflict
of interest.
Consent to
Participate: The author has consented to
submit the article to this journal.
Consent to
Publish: The author has agreed to publish the
article in this journal.
Funding: This study did not receive specific funding from public,
commercial, or non-profit organizations.
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