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Gene & Protein in Disease K fragment for resistance gene hunting

A bacteria harboring resistance gene need to be cultivable
under laboratory conditions, but the challenges lie in the
non-cultivability of numerous micro-organisms or the
exorbitant costs and immense labor that need to be invested
for culturing. Molecular methods are ideal for the detection
of known genes but they are incapable of characterizing
unknown genes. Thus, cloning and determining resistance
B genes remain the most feasible approach to characterizing
unknown resistance genes. In this study, K fragment was
developed to clone antibiotic resistance genes, and its
functionality was tested by cloning resistance genes from
PCR amplicon and genomic DNA. The present study also
demonstrated the capabilities of the newly developed
method involving K fragment in phenotypic and molecular
characterization of resistance genes.

One of the earliest and most common molecular
methods for resistance gene cloning is shotgun cloning [17,18] .
Figure 7. Experiment results of cloning genes from genomic DNA into K Functional metagenomics represent a modified shotgun
fragment. (A) Erythromycin-resistant colonies. Erythromycin resistance
gene was cloned into K fragment. The photograph on the left shows the method designed for cloning antibiotic resistance genes
colonies grown on the plate on which the transformants containing the from different environmental sources [19,20] . Despite its
erythromycin-resistant gene cloned into K fragment (with the aid of usefulness, the shotgun method requires a cloning vector
HindIII enzyme) were plated. The photograph on the right shows the carrying at least one antibiotic resistance gene as a marker
colonies grown on the plate on which teh transformants containing and thus cannot be used for cloning all antibiotic resistance
the erythromycin-resistant gene cloned into K fragment (with the aid
of Sau3aI [BamHI] enzyme) were plated. (B) The colony PCR results genes. K fragment does not carry any antibiotic resistance
of Erythromycin-resistant colonies. H1 and H2 represent different gene, and it allows cloning and expression of all resistance
colonies were obtained by cloning the erythromycin resistance gene into genes. A conspicuous limitation of using K fragment
K fragment (with the aid of HindIII enzyme). S1-6 represent different as a vector is some of the antibiotic resistance cannot be
colonies were obtained by cloning the erythromycin resistance gene into observed in transformed bacteria, rendering the colony
K fragment (with the aid of Sau3aI [BamHI] enzyme). P: Positive control,
N: Negative control, M: Lambda-PstI marker. identification process challenging after the bacteria are
plated on the agar plates. For instance, as the competent
were different, measuring at 2000 bp, 2300 bp, and 2700 bp bacteria, E. coli, are intrinsically resistant to vancomycin,
(Figure 7B). The sequencing results showed that ermC, all bacteria will grow on selective media whether or not
[21]
erythromycin resistance gene, was cloned in K fragments in it contains vancomycin resistance genes in K fragment .
different orientations. The repL gene encoding a replication The shotgun method also shares the same limitation. We
protein in Staphylococcus plasmid was cloned to K fragment believe that K fragment has a great potential in functional
together with ermC. These results revealed that ermC metagenomics for cloning antibiotic resistance gene
gene was carried by a plasmid in S. aureus ADU2 strain. obtained from various environments (Figure 8). Another
The constructed plasmid that can replicate in E. coli was popular method for the detection of resistance genes is the
transferred to S. aureus RN4220 strain by electroporation total DNA sequencing of a given sample. The metagenomic
for confirmation of its ability to replicate in Gram-positive method is used for the detection of resistance genes in
[22,23]
bacteria. After transformation, colonies were selected on total DNA obtained from different sources . However,
the resistance genes detected in metagenomic analysis are
erythromycin-containing agar plates and confirmed ability not inconsistent with the phenotypic and microbiological
of the construct containing K fragment and ermC fragment results . Metagenomic methods are still able to detect
[24]
to replicate in both E. coli and S. aureus. DNA sequence of a resistance gene even if it is not complete
4. Discussion or functional, but the detectable entities for this method
category are only limited to the known resistance genes.
Characterization of antibiotic resistance genes from Unfortunately, the continuously emerging resistance genes
different sources is the foremost yet important endeavor for in future will only add to the inexplicable burden that we
deterring the spreading of AMR. Typically, molecular and are facing right now with characterizing the enormous
phenotypic methods are employed in the characterization number of unknown resistance genes. Thus, various
of resistance genes. In phenotypic methods, however, the approaches have been proposed to efficiently identify

Volume 2 Issue 4 (2023) 7 https://doi.org/10.36922/gpd.1674