Characteristics Of Blandm-1 Gene And How It Affects Human Health

Introduction

For the past decades, the presence of carbapenem- resistance Entrerobacteriaceae has affected different nations across the globe negatively. According by ( ) the carbapenem- resistance Entrerobacteriaceae (CRC) cause different type of infections that their treatment is not easy. The CRC produce an enzyme, carbapenamase, which hydrolyses the carbanems or the beta-drugs. There are different kinds if carbapenemase such as Ambler class A called Klebsiella pneumoniae carbapenemase (KPC) and Ambler class B called metallo-beta-lactases (MBLS), which was identified in the Pseudomonas Aeroginose. In 2009, there was a first case whereby New Delhi MBL (NDM) was identified in the K. pneumoniae and Escherichia coli isolated from a patient who was hospitalized, apparently drug resistant gram negative bacteria produced NDM was found in the community and also the health care with different types of gram negative genera having different blaNDM- harboring plasmids. BlaNDM genes are found in an array of plasmid replicon types and also in plasmids with a broad host range such as IncA/C.

Plasmids are small circular double stranded DNA molecules that are distinct from a chromosomal DNA. (Carattoli et al., 2005) says that they exist naturally in bacteria and they may also occur in some eukaryotes. Furthermore, they replicate autonomously as they are extrachromosomal (Carattoli, 2005). Plasmids are found in bacteria and as such may provide bacteria with genetic advantages e.g. antibiotics resistance. Carattoli (2005) also suggests that not only do plasmids aid in antibiotic resistance but they also contribute toward bacterial diversity. Plasmid incompatibility and mobility aids researchers in understanding mechanisms of transfer in bacteria. With this, bacterial drug resistance can be understood more and potential human health threats can be eliminated. Simille et al., (2010) suggests that in order to give a biology systems answer to the problems of plasmid dissemination e.g. dissemination of multidrug resistant bacteria then it is vital to understand plasmid mobility. This would help in reducing the recent increase in human mortality caused by bacterial diseases. Plasmid mobility has a biological function and provides agents of mobility as they allow for homologous recombination; it leads to integration of new genes that lead to adaptability into a new environment; is an essential lexicon of plasmid conjugation (Simille et al., 2010). Plasmids can be grouped into three types with regards to their mobility or lack thereof. A plasmid according to Simille et al., (2010) can either be mobilizable, non mobilizable or transmissible. Transmissible plasmids have their own mating pair formation complex, mobilizable use MPFs of other genetic elements and nonmobilizable are un-conjugative.Mobility can be assessed by understanding plasmid relaxases which are enzymes responsible for site- and strand-specific nicks of DNA strands Novick (1987) .

An emerging threat to human health is the incompatibility group IncA/C which are large, have low copy number and are theta-replicating plasmids (Johnson and Lang, 2012). This incompatibility group is associated with the emergence of multidrug resistance in enteric pathogens of humans. They have the plasmid New Delhi metallo-beta-lactase 1 (NDM-1) gene which is concerning to human health. This incompatibility group has an extremely broad range, coupled with an ability to spread via conjugative transfer within bacterial communities. They are associated with Klebsiella pneumoniae, Salmonella enterica, Escherichia coli etc. Johnson and Lang (2012) continue to state that IncA/C plasmids have remarkable genome structure that allows them to interact with bacterial host using multiple regulatory mechanisms and also mobilize other genetic elements. This genome contains an intron containing theta replicon, at least three integrative hotspots capable of acquiring a variety of mobile genetic elements. They have a putative transfer region that is similar to that of integrative conjugative element, transcriptional regulators belonging to many classes and a large number of hypothetical genes that appear to be derived from different sources (Johnson and Lang, 2012).

Although IncA/C is linked with a broad variety of hosts in the bacterial community, how the plasmid-bacteria relationship vary in different bacteria is unknown. This report examines the nature of IncA/C plasmid-bacteria relationship and blaNDM-1 gene and their implications on human health. For this purpose the blaNDM-1 gene mechanism and regulation of dissemination in conferring resistance is investigated. This analysis is important in determining ways of reducing the increasing human mortality rates caused by bacterial infections. (Peymani et al.2016) state that there is an alarming emergence of the IncA/C and this should be considered as a warning for the implementation of appropriate infection control and therapeutic policies. This incompatibility group is emerging in poultry and pigs [(Dame-Korevaar, (2017); Jahanbakhsh et al., (2005)]. This calls for intensified study on the plasmids mode of transfer and its family of genes.

MATERIALS AND METHODS

To access my gene of interest the National Center for Biotechnology Information webpage was used (https://www.ncbi.nlm.nih.gov ). Going on to the popular resources and clicking on genes and searching for NDM-1 beta-lactamase 31 items were found. To narrow the search the plasmid chosen was found in Klebsiella pneumoniae. One of the journals used to find information on the gene blaNDM-1 gene from PubMed under ID: 18983573. Information on the gene of interest was recorded on Table 1. Using the accession number NC_023908.1 a nucleotide-nucleotide BLAST search was run. A protein-protein BLAST search was also run to find sequence homologs. The webpage for BLAST search is (https://www.blast.ncbi.nlm.nih.gov/Blast.cgi ). For the protein-protein BLAST search protein ID used was WP_111672912.1. Datasets for both the nucleotide-nucleotide and protein-protein BLAST were downloaded in FASTA format. The following homologs were downloaded Escherichia coli, Escherichia coli, Klebsiella pneumoniae and Enterobacter eloacae. A gene prediction was done to identify regions of genomic DNA that encodes proteins. This was done by opening the Gene web page (http://exon.gatech.edu/GeneMark/ ). The genomic sequence was pasted into the GeneMark program and run. Regions of similarity were obtained using sequence alignments. These may show functional, structural and/or revolutionary relationships between homologs and the gene of interest. For pairwise sequence alignment WATER and MATCHER tools were used for local alignment. To access these programs the EBI site was used: https://www.ebi.ac.uk/Tools/psa/ . For pairwise alignment the homolog chosen for comparison against the gene of interest was Escherichia coli under accession number WP_06380858.1. All homologs were used for the multiple alignment sequence. For global alignment NEEDLE and STRETCHER programs were used. Multiple alignment sequence identifies highly conserved residues in homologous proteins. To show these highly conserved regions a boxshade was used as shown in. Fig 1. The homologs were downloaded in FASTA format and the file was uploaded for the MSA Clustal Omega program. For all these programs the protein sequence was used. The results were sent via email and the phylogenetic tree is shown on fig. 2. The multiple alignment sequences were aligned for publication (https://www.ebi.ac.uk/Tools/msa/tcoffee/). A boxshade was used to show highly conserved regions, (https://www.ch.embnet.org/software/BOX form.html). The alignment from T-Coffee was pasted into Boxshade input window. The option ‘other’ was chosen as the input sequence format and RTF new as the output format. ‘Run Boxshade’ option was chosen and the output number 1 to download the output file. The results were opened using Microsoft Word. These are done to generate high impact pictures from multiple sequence alignments. The sequence alignment logo was done using the website (https://www.weblogo.berekeley.edu/).

DISCCUSION

Table two shows the homologs that were selected, all of them were the from a gram negative genera. This shows that gram negative bacteria are the best for the transfer of blaNDM-1 through the plasmids. This proves that there was no common ancestor of this gene, the gene was transferred form one bacterium to another by the plasmids. Plasmid IncA/C are the best in the transference of the blaNDM-1 gene in the klebsiella pnuemoniae. Plasmid IncA/C are an incompatibility group, and they have routes for dissemination for the resistance gene. From the results obtained, it shows that the Escherichia coli, Enterobacter cloacae and Escherichia coli of DNA accession numbers of NC_019069.1, NC_023914.1 AND NC_015872.1 are similar, they have the same sequences. And this proves that horizontal transfer was used for the transfer of gene.

There is a relationship between blaNDM-1 gene and other genes of resistance that are found in the IncA/C plasmid. This relationship enables the gene to carry out its function under conditions that normally thrive the presence of carbapenem. IncA/C is also able to spread resistance genes across diverse bacterial communities and this poses as a threat to human and animal health (Dame-Korevaar et al., 2017).The boxshade in fig.1 shows that all homologs have an almost identical gene. There are highly conserved regions and this may be due to the fact that this is transferred therefore there are little or no alterations made to it. According by (………) blaNDM-1 gene offers resistance to Imipenem, Meronem, Ertapenem, Doripenem,Biapenem ,Tebopenem and this is becoming a global challenge in medicine.

It is noted by Khari et al. (2016) that detection of carbapenemase beta-lactamase is a huge problem in clinical microbiology since there are no procedures of Clinical and Laboratory Standards Institute for phenotypic detection of carbapenemase beta-lactamase producing bacterium. Though several tests are there, but none has been standardized and they are undependable and inappropriate and usually result in misinterpretation and treatment failures (Khari et al., 2016). Khari et al. (2016) continues to state that phenotypic tests do not distinguish between chromosomal and carbapenemase beta-lactamase genes however molecular characterization should be considered as the gold standard method. This suggests that for incoming experiments to characterize this gene at molecular level smaller amplicons should be used.

Conclusion

In conclusion, this study shows that multi drug resistant bacteria threaten human health. Their occurrence is a universal concern and as such improvement of new and effective antibiotics is needed. It has been suggested that there is a physical connection between blaNDM-1 resistance gene and other resistance genes that are carried on IncA/C and this linkage may be used to determine the relationship of the incompatibility group and bacteria. This will help in the right analysis and therapeutically tactics of treating diseases. To approach such it is suggested that researchers use Bioinformatics tools to design high power experiments that will be more productive than the conventional approaches.