Volume 9, Issue 2, February 2024 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

Isolation and Determination of the Spore -Forming

Gene in Pathogenic Bacteria by PCR Technique at the
Republican Hospital in Kirkuk
Shler Ali Khorsheed
College of Science Kirkuk, University

Abstract:- This study focuses on the isolation and I. INTRODUCTION

molecular quantification of bacteriostatic genes in
pathogenic bacteria prevalent in the Republican Bacterial survival mechanisms in harsh environmental
Hospital in Kirkuk. Spore formation plays a pivotal role conditions often involve the formation of spores, with
in the virulence and persistence of pathogenic bacteria, Gram-positive bacteria, particularly Bacillus and
making it essential to understand the presence and Clostridium species, being known for their proficiency in
diversity of spore-forming genes in the hospital producing endospores .The extremely specialized cellular
environment. Using polymerase chain reaction (PCR) forms known as bacterial endospores enable endospore-
technology, we aimed to develop a targeted and effective forming Formicates (EFF) to withstand extreme
method to detect bacteriostatic genes within bacterial environmental conditions. [1,2 ] .EFF are thought to be
isolates obtained from clinical samples from 200 common in natural settings, especially those that experience
patients of different ages and genders at the Republican stress. Apart from their presence in natural settings, EFF
Hospital. Bacterial isolates were collected from various frequently leads to contamination issues in man-made
clinical sources using standard microbiological locations like hospitals or industrial production units. [3,4].
protocols, and their identification was confirmed Spore-forming bacteria such as Clostridium perfringens
through traditional microbiological methods. Bacillus Bacillus cereus, and Bacillus subtilis have been implicated
subtilis, Clostridium perfringens, Pseudomonas in hospital-acquired infections and nosocomial outbreaks,
aeruginosa Escherichia coli, Streptomyces aureus. primarily through transmission via healthcare workers and
Enterobacter cloacae were isolated from 50% of contaminated surgical instruments [5,6,7]. These spore-
patients. Genomic DNA extraction was performed, and forming bacteria exhibit remarkable resistance to alcohol-
PCR primers were designed to specifically amplify the based disinfectants and various treatments that typically
spore-forming gene region. The resulting PCR products eliminate vegetative cells, including desiccation, heat, UV
were visualized using gel electrophoresis to confirm the and γ-radiation, mechanical stress, chemical exposure,
presence of target genes. The result was the isolation of hydrostatic pressure, and osmotic stress [8,9]. Hence, it is
the .spo0A gene. The study not only aimed to determine imperative to identify these spore-forming bacteria and
the prevalence of spore-forming genes in pathogenic elucidate the genes responsible for spore formation, with
bacteria, but also explored potential associations with spo0A being a pivotal gene involved in endospore
clinical outcomes and antibiotic resistance profiles. In development [10,11]. The ubiquitous presence of the
addition, bioinformatics tools were used to analyze regulatory protein Spo0A is a hallmark of stress-induced
genetic evolution, highlighting the genetic diversity and gene expression machinery and endospore formation
evolutionary aspects of bacteriostatic genes among [12,13]. Molecular tests targeting spore-forming genes can
isolates. This research, conducted at the Republican aid in classifying and characterizing these bacteria,
Hospital in Kirkuk, provides valuable insights into the distinguishing between species with and without
molecular characteristics of pathogenic bacteria in the sporulation genes [14,15]. Previous studies have
healthcare setting. The findings contribute to our determined gene homologs in various bacteria using
understanding of microbial dynamics within hospitals degenerate PCR [16,17]. PCR, a widely used laboratory
and may inform infection control strategies. technique, enables the rapid amplification of specific DNA
Furthermore, the PCR-based approach provides a and RNA sequences, making it invaluable for bacterial
rapid and sensitive diagnostic tool to detect spore- identification and detecting resistance genes [18]. Multiplex
forming genes, facilitating targeted interventions to PCR, an enhanced version, incorporates multiple primers in
mitigate the impact of spore-forming bacteria on patient a single reaction, facilitating the simultaneous analysis of
health. multiple genes and reducing costs and time [19,20,21].
Quantitative PCR (qPCR) is a precise method for
Keywords:- Polymerase Chain Reaction Technique (PCR)- quantifying gene frequencies in DNA extracts [22,23,24].
Spore-Forming Bacteria – Genome- Spo0a Gene. In this study, qPCR primers targeting the spo0A gene were
employed, tested, and validated in pure culture samples
[25,26,27]. In summary, understanding the mechanisms and
genes involved in bacterial spore formation is crucial for

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Volume 9, Issue 2, February 2024 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
addressing the challenges spore-forming bacteria poses, .Bacillus the lecithinase test produced negative results,
especially in healthcare settings. Molecular techniques such meaning that lecithinase activity was absent. The isolated
as PCR and qPCR play pivotal roles in this endeavour, colonies were notable for their notable features, which
enabling the identification and characterisation of these included a wrinkled surface on the agar medium, a notable
resilient microorganisms [28]. size, and yellow coloring. As a result of these
distinguishing features, the isolated bacteria were identified
II. METHODOLOGY as Bacillus subtilis , Clostridium perfringens, Pseudomonas
aeruginosa Escherichia coli, Streptomyces aureus.
A. Samples Enterobacter cloacae. Conversely, Mannitol-negative,
This research was conducted in one of Kirkuk's lecithinase-positive, large, flat, and grainy colonies on the
hospitals, which is Kirkuk General Hospital in Iraq, from agar surface were identified as belonging to the Bacillus
January 12, 2023 to 12 of May, 2023. A total of 200 species [32,33].
samples were collected from the wounds of patients who
underwent various surgical operations. D. DNA Extraction:
Characteristic bacterial colonies were selected and
B. Sample Collection: inoculated into a 5 mL Brain Heart Infusion (BHI) solution
Samples were collected by swabbing post-operative obtained from (Sigma-Aldrich, USA). The inoculated
wounds. These collected samples were immediately solution was incubated overnight at 35°C. Subsequently,
transported to the laboratory to ensure their microbial DNA extraction was performed from the cultured solution
integrity. Samples were inoculated onto blood agar, using the (QIA amp DNA mini kit) following the
chocolate agar, and MacConkey agar plates. After that, the manufacturer's instructions for the kit.
plates were incubated for 24 hours at 37°C. After
incubation, observe the agar plates for bacterial growth. E. DNA Quantification:
Isolate individual bacterial colonies by streaking them onto To evaluate the quality of the extracted DNA, agarose
fresh agar plates using the quadrant streak method or a gel electrophoresis was employed, and the concentration of
similar technique To obtain pure cultures, sub-culturing the isolated DNA was assessed at 260 nm using a Nano
streak isolated colonies onto new agar plates. Repeat this drop ND 2000 spectrophotometer from (Thermo Scientific,
process until a pure culture of the spore-forming bacteria is USA). [34,35,36].
obtained. Perform Gram staining and other preliminary
tests to identify the general characteristics of the isolated F. DNA Sequences for Spore Forming Genes:
bacteria. Subject the isolated bacteria to heat treatment To investigate the bacterial genome, particularly
(e.g., 80-85°C for 10 minutes) to induce sporulation. stain focusing on spore-forming genes, we employed primers
the bacterial smears with malachite green or another targeting the 16S rRNA gene, generating fragments of
suitable spore stain and observe under a microscope for the approximately 500 bp. These primers, Eub8f (5'-
presence of spores. Preserve pure cultures of spore-forming AGAGTTTGATCCTGGCTCAG-3') and Eub519r (5'-
bacteria by storing them at -80°C or in appropriate culture GTATTACCGCGGCTGCTGG-3'), were used for this
storage conditions, using a suitable cry protect ant like purpose [37,38 ,39].
glycerol. This protocol provides a basic guideline for
isolating spore-forming bacteria. [29].. In order to identify While the 16S rRNA gene provides phylogenetic
spore-forming bacteria among diverse bacterial species, information about the presence of various bacterial species,
suspected colonies were subjected to a series of we also needed to examine a functional indicator related to
biochemical tests, including catalase production, motility sporulation, specifically the spo0A gene. To achieve this,
assessment, root growth assessment, citrate utilization we utilized primers designed for the spo0A gene. These
reactions, and hemolysis assays. These species primers, spo0A166f (5′-GATATHATYATGCCDCATYT-
identification test procedures are well documented in the 3′) and spo0A748r (5′-GCNACCATHGCRATRAAYTC-
scientific literature [30, 31 ] 3′), were chosen based on their specificity, amplification
efficiency, fragment length, and sequence compatibility
C. Isolation of Spore-Forming Bacteria : with the spo0A gene, which plays a central role in the
The bacterial samples were examined under a spore-forming process [40,41].
microscope due to their Gram stain composition.
Simultaneously, the isolation procedure concentrated on G. PCR Technique:
detecting bacteria exhibiting ethanol resistance, a feature The reaction mixture consisted of 20 µL of Master
frequently linked to spore-forming bacteria. After isolation, Mix (2X) from Thermo Fisher, USA, along with 8.3 µL of
the size of the colony was measured, and a series of tests distilled water, 1 µL (10 µM) of each primer, and 2 µL
was used to distinguish between different kinds of bacteria. (approximately 50 ng) of template DNA. This reaction
The Mannitol test produced positive results, demonstrating mixture was then placed in a thermal cycler programmed
that the bacteria were using Mannitol. On the other hand. for one cycle at 92 °C for 18 minutes, followed by 30
the lecithinase activity of S. aureus is used in detection of cycles of denaturation at 92 °C for 30 seconds, annealing at
coagulase-positive strains, because of high link between 52°C for 30 seconds, and extension at 70°C for 1.5 minutes.
lecithinase activity and coagulase activity. Can be used to Finally, a single extension step at 70°C for 1.5 minutes was
differentiate between certain species within the genus performed. [42].

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Volume 9, Issue 2, February 2024 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
The PCR products were subsequently loaded onto a aureus (No 4 no band). , Pseudomonas aeruginosa (No 5
1.5% agarose gel and stained with ethidium bromide no band found), Clostridium perfringens (No 6 band
(ETBR) at a concentration of 0.5 μg/mL for the appeared 608 bp) respectively.
electrophoresis step. The PCR samples were visualized
using an ultraviolet trans illuminator, and the resulting IV. DISSCUTION
bands were analyzed using a gel documentation system for
documentation and analysis. [43,45]. The occurrence of post-operative infections caused by
microbial agents, particularly spore-forming bacteria, poses
 The Diagnosis Species were as Follows a significant medical challenge. Addressing this issue
The identified bacterial species in the study included requires effective interventions and increased attention to
Escherichia coli (found in 15 cases), Enterobacter cloacae public hospitals in Iraq. Consequently, the adoption of
(found in 30 cases), Bacillus subtilis (15 cases), accurate techniques, proper procedures, and swift detection
staphylococcus aureus . (14 cases), Pseudomonas methods for identifying infectious microbes from wounds is
aeruginosa (15 cases), and Clostridium perfringens (11 imperative According to studies conducted in 2008 by
cases). Isibor and in 2009 and 2010 by Pradhan, wound infections
happen in hospitals following surgical procedures, and the
Among these identified bacterial species, Bacillus degree of contamination in the wound, where it is located,
subtilis and Clostridium perfringens are well-known for and how long the patient stays there can all affect how well
their spore-forming capabilities. The primers used in this the patient does. [47,48, 46].
study targeted the spo0A gene, recognized as the key gene
responsible for initiating spore formation. The sequencing Polymerase chain reaction (PCR) stands out as a
results confirmed the presence of the spo0A gene in these sensitive, specific, and rapid molecular method extensively
spore- forming bacteria isolated from Kirkuk General employed for detecting microbial species in medical
Hospital [46]., as summarized in Table 1. specimens. In our current study, PCR is indispensable, as it
allows us to target a specific gene, namely the spore-
III. RESULT forming gene.spo0A in bacillus sabtilis and clostridium
perfringes as in the study of Hoon and Krieg in 2010 and
The results indicate that out of the total 200 samples 2009 that within the Firmicutes, endospore formers
analyzed, 100 of them tested positive for bacterial infection, constitute a paraphyletic group [50]. Only the first two
confirming an infection rate of 50% based on the sample classes of this phylum—Bacilli, Clostridia, and
set. The gel electrophoresis results, depicted in (Figure 1), Erysipelotrichi—contain species that generate endospores.
demonstrate the quality of the PCR products. Following the Bacilli are primarily aerobic bacteria, while Clostridia are
subsequent steps of cloning and sequencing, the bacterial primarily anaerobic kinds of bacteria [51].
species were successfully identified.
The majority of our understanding of the biology of
endospore-forming Firmicutes (EFF) has come from
investigations conducted in laboratories using cultivable
strains since the late 19th century [51]. PCR's capabilities
encompass gene extraction, gene sequence comparison, and
determination of a specific gene's presence within bacterial
genomes.

Our examination involved collecting samples from

100 patients with post- operative wounds using cotton
swabs. All collected samples underwent cultivation to
assess the presence of infection as in the study of Qadan in
2009 [52]. The results revealed that 50% of the wounded
individuals were infected, underscoring the urgency of
enhancing the surgical department's attention in public
hospitals each of Niska and Pittet have They have results
close to those of our study [53,54,55].. Various reactions,
such as gram staining (which identifies spore-forming
bacteria as gram-positive) and tests for catalase production,
Fig. 1: Agarose Gel Electrophoresis of PCR-Amplified motility, rhizoid growth, citrate reactions, and hemolysis,
Partial 16S rRNA Genes from Bacteria were conducted to achieve this goal. Subsequently, total
DNA was extracted and employed as a template for
As Lane M represents the DNA ladder , where lanes amplifying the 16S rRNA sequence as in studying both
from 1 to 6 represent the PCR amplified genes for the Madhavan and Jones have results an approach to the results
isolated bacteria , Escherichia coli ( No 1 no band found ) , of this study [56,57, 58]. Additionally, the spo0A gene was
Entrobacter cloacae (No 2 no band found ) , Bacillus targeted to identify spore-forming bacteria within mixed
subtilis ( No 3 band appeared 1000 bp ) Staphylococcus bacterial samples for the protection of regions within the

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Volume 9, Issue 2, February 2024 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
16S rRNA gene of bacteria, we utilized globally recognized for regulating spore formation, the spo0A gene. which were
primers this approach considered a group of bacteria Bacillus subtilis , Clostridium perfringens, as shown in the
responsible for infections. Furthermore, we employed study of Anand and Errington in (2001,2000). [59,60].
forward and reverse primers for the master gene responsible

Table 1: Test for Spo0A Amplification through the used Primers

Bacteria Series Temp of Endospore Amplificatio n of Sequences for Spo0a
Optimal Formation Spo0a Gene
Growth
Bacillus subtilis 30 0c Positive Positive sequences
spo0A166f (5′-
GATATHATYATGCC
DCATYT-3′) and spo0A748r (5′-
GCNACCATHGCRAT RAAYTC-3′)
Clostridium perfringens, 30 c0 Positive Positive sequences
spo0A166f (5′-
GATATHATYATGCC
DCATYT-3′) and spo0A748r (5′-
GCNACCATHGCRAT RAAYTC-3′)
Pseudomonas 30 c0 Negative Negative Absent
aeruginosa
Escherichia coli 30 c0 Negative Negative Absent
Staphylococcus areaus 30c0 Negative Negative Absent
Enterobacter cloacae 30 c0 Negative Negative Absent

V. CONCLUSION diseases. Additionally, the findings may have implications

for infection control practices within healthcare settings. In
The isolation and determination of the spore-forming conclusion, the isolation and determination of the spore-
gene in pathogenic bacteria using the Polymerase Chain forming gene using PCR at the Republican Hospital in
Reaction (PCR) technique at the Republican Hospital in Kirkuk represent a significant step forward in advancing our
Kirkuk hold significant implications for understanding and understanding of bacterial pathogenesis. This research has
managing bacterial infections. This sophisticated molecular the potential to inform clinical practices, guide treatment
biology approach allows for the identification and strategies, and contribute to the overall improvement of
characterization of spore-forming genes, providing crucial public health in the region.
insights into the virulence and persistence mechanisms of
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