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Gene & Protein in Disease K fragment for resistance gene hunting

to AMR each year . Without showing signs of decreasing, sequence that can enable cloning of antibiotic resistance
[1]
the mortality rate has been projected to reach 10 million in genes.
2050 if necessary and effective steps are not taken to curb
AMR [1,2] . The treatment options effective for controlling 2. Materials and methods
the infections caused by antimicrobial-resistant pathogens 2.1. Bacterial strains, culture conditions, and
are dwindling due to the emergence of new resistance antibiotics
genes and the spreading of resistant strains [3,4] . One of the
drivers promoting the dissemination of AMR is the usage Escherichia coli DH10B strain was used as a host organism
of antibiotics for the treatment of infections in animals and for cloning experiments. Staphylococcus aureus ADU1
humans, and as a food additive to promote the growth of and S. aureus ADU2 strains having chloramphenicol
livestock [5,6] . Due to prevalent usage, the antibiotics remain and erythromycin resistance, respectively, were obtained
active and accumulate in the environment . The antibiotic from our clinical isolates. They were cultured at 37°C in
[7]
resistance genes appear naturally or due to antibiotic aerobic conditions. Tryptic soy broth and agar were used
pressure. However, the dissemination of the resistance for cultivation.
genes can be accelerated by the antibiotic pressure in Ampicillin, chloramphenicol, and erythromycin
the environment, even if the genes occur naturally [8,9] . were used in this experiment, with final concentrations
Therefore, the alarming rise of the AMR scene is a wake-up of 100 µg/ml, 10 µg/ml, and 10 µg/ml, respectively. In
call for the improvement of new and efficient techniques addition to ampicillin, 50 µg/ml X-gal (5-bromo-4-
for the detection of known and unknown antibiotic chloro-3-indolyl β-D-galactopyranoside) and 1 mM IPTG
resistance genes derived from various environments, (isopropyl β-D-1-thiogalactopyranoside) were used for the
which is first crucial step for gauging the gravity of the preparation of AXI selective medium.
antibiotic resistance problem and formulating solutions to
overcome this issue. 2.2. Plasmid isolation
™
In fact, investigations on antibiotic resistance genes are Plasmids were isolated using Presto Mini Plasmid
an ongoing effort in the wake of the emergence of antibiotic- Kit (Geneaid, China) according to the manufacturer’s
resistant strains in clinical isolates and the insufficient instructions.
treatment of infectious diseases. Phenotypic methods such 2.3. DNA isolation
as antibiogram and microdilution, as well as genotypic
methods such as polymerase chain reaction (PCR), are DNA was extracted from S. aureus ADU1 using DNA4PCR
standard methods used in detecting antibiotic resistance in (RTech, Turkey). Briefly, colonies were suspended in
the microbes . However, phenotypic methods are unable distilled water. After centrifugation, the pellet was
[10]
to identify the gene or the dominant resistance gene/ suspended in DNA4PCR solution. The suspension was
mechanism contributing to antibiotic resistance, and a incubated at 56°C for 20 min. After vortexing, samples were
culture of microorganisms is needed if phenotypic methods incubated at 100°C for 8 min. Following centrifugation,
are employed. On the other hand, genotypic methods like the supernatant was used as a DNA source.
PCR cannot be used to detect unknown resistance genes Total DNA of S. aureus ADU2 was isolated with
and even the detection of a part of the gene cannot be a Bacteria Genomic DNA Purification Kit (Genemarkbio,
corroborative evidence of antibiotic resistance. Cloning of Taiwan Region). The lysozyme treatment was carried out
antibiotic resistance genes is a more reliable method, and with a slight modification. Lysostaphin solution with a
it has been used to clone the genes from different sources final concentration of 5 µg/ml was added. The remaining
using cloning vectors [11,12] . Functional metagenomics steps were processed according to the manufacturer’s
stand as another molecular option for cloning antibiotic recommendations.
resistance genes from different sources . However, the
[13]
main limitations of this method are as follows: (i) The 2.4. DNA manipulations
resistance gene intended to be cloned already exists in Restriction reactions were performed using FastDigest
the vector and (ii) the high costs and tremendous efforts enzymes (Thermo Scientific , USA). For digestion
™
involved in obtaining the vector needed. In this study, we with one enzyme, 10 µl of DNA, 2 µl of 10× buffer, 1 µl
constructed an extraordinary vector, PCR-based vector of restriction enzyme, and 6 µl of water were mixed. To
named K fragment and conceived a cloning method perform double digestion, 1 µl of each enzyme and 6 µl
involving the K fragment. Our results showed that PCR of water were pooled together before being added to the
amplicons generated from the amplification using the reaction mixture. The reaction mixtures were incubated
constructed vector contains plasmid origin of replication at 37°C for 15 min. For DNA ligation, T4 DNA Ligase

Volume 2 Issue 4 (2023) 2 https://doi.org/10.36922/gpd.1674