Comparison of IncL/M Plasmids Using the Neighbor-Joining Method on Basis repA and excA Genes

Corresponding Author: Touati Abdelaziz Laboratoire d’Ecologie Microbienne, FSNV, Université de Bejaia, 06000 Bejaia, Algérie Email: ziz1999@yahoo.fr Abstract: The aim of this study is to make comparison of IncL/M groups on basis of two genes candidates’ repA and excA genes. The sequences of 27 plasmids were compared using the neighbor-joining method. This method was used to construct a phylogenetic tree for the nucleotide sequences of two genes (repA and excA), using the program MEGA X software. The evolutionary distances were computed using the maximum composite likelihood method. The neighbor-joining method analysis showed different results based on the gene used for comparison. The repA gene was more accurate than excA gene to distinguish between different incL/M plasmids. This study suggested that IncL/M plasmids harboring different antibiotic resistance genes have evolved differently.


Introduction
The dissemination of antimicrobial resistance in Gram-negative bacteria has been largely attributed to the horizontal transfer of plasmid-located resistance genes (Carattoli, 2013). A plasmid is defined as a doublestranded, circular DNA molecule able of autonomous replication. By definition, plasmids do not carry genes essential for the growth of host cells under no stressed conditions (Carattoli, 2009).
Carbapenem antibiotics are generally considered the most effective antibacterial agents for the treatment of multidrug-resistant bacterial infections. However, with the widespread use of carbapenem, the prevalence of Carbapenem-Resistant Enterobacterales (CRE) has increased rapidly and has become a serious threat to public health. The production of carbapenemases is the major mechanism underlying carbapenem resistance in CRE throughout the world (Cui et al., 2019). Carbapenemases belong to Ambler class A (i.e., KPC types), class B (i.e., MBLs: VIM, IMP and NDM types) and class D (i.e., OXA-48). The KPC, NDM, IMP, VIM and OXA-type enzymes are the most common global carbapenemases among Gramnegative bacteria (Pitout et al., 2019). OXA-48-type carbapenem-hydrolyzing class D β-lactamases are widely distributed among Enterobacteriaceae, with significant geographical differences (Mairi et al., 2018).
Genes encoding carbapenemases are mostly located on conjugative plasmids that allow their efficient dissemination among Enterobacterales species. Although the prevalence of particular plasmids may vary depending on the source and geographical site, they have been increasingly isolated from bacteria of human, animal and environmental origin (Rozwandowicz et al., 2018;Touati and Mairi, 2019). In a recent review, Touati and Mairi concluded that the diffusion of OXA-48 in Algeria is probably linked to plasmid diffusion. This plasmid is largely conjugative between Enterobacterales members and assigned to IncL/M group .
The aim of this study was to compare plasmids of IncL/M group encoding different β-lactamases on the basis of two genes repA and excA.

Plasmid Characterization
The sequences of 14 plasmids carrying the blaOXA-48 gene were already published  and served as a matrix for bioinformatics comparison. Briefly, these plasmids were originated from different species of Enterobacterales strains obtained from different ecological niches in Algeria. They were sequenced on the Illumina genome analyser IIX system by GenoScreen SA (Lille, France). The sequences of these plasmids were deposited in the GenBank (MK121443.1 to MK121456.1).

Bioinformatic Analysis
To generate comparisons, 13 sequenced plasmids carrying different β-lactamases genes were obtained from the NCBI nucleotide database (www.ncbi.nlm.nih.gov/nucleotide). Results were filtered to exclude plasmid sequences that did not belong to the IncL/M group. Antimicrobial resistance genes were identified using Resfinder tool 3.2 (https://cge.cbs.dtu.dk/services/ResFinder/). The sequence of two genes was extracted from these plasmid sequences including repA and excA genes.
Complete sequence alignments of these two genes were then undertaken using CLUSTALW on the CLUSTAL Omega website of the EMBL-EBI (www.ebi.ac.uk/Tools/service/web). The neighborjoining method was used to construct a phylogenetic tree for the nucleotide sequences of these two genes, using the program MEGA X software. The evolutionary distances were computed using the Maximum Composite Likelihood method.

Results and Discussion
The data concerning the 27 analyzed plasmids are presented in Table 1. The size of the plasmids ranged from 49257 pb (pRAY) to 133208 pb (pIMP-HB623).
The repA gene has 1056 pb in size while the excA gene has 654 pb in size. All the 14 plasmids reported in our study had the same size (61881).
When compared using the neighbor-joining algorithm, the plasmids compared using repA gene were divided into three clusters (Fig. 1): Cluster 1 contained all plasmids harboring the blaOXA-48 gene (n = 23), the cluster 2 contained the two plasmids encoding IMP-like carbapenemase and the cluster 3 contained the remaining two plasmids carrying respectively the blaKPC-4 and blaFOX genes. However, when we use the excA gene for comparison, the neighbor-joining algorithm showed three clusters in which the plasmid pOXA48-Pm encoding blaOXA-48 is assigned in the same cluster of the plasmid encoding blaKPC-4 (Fig. 2). Thus, these two plasmid analyses showed different results depending on the gene used for comparison. It's interesting to highlight these all of these plasmids are assigned to the same incompatibility group IncL/M. Our aim to compare plasmids on the basis of conserved genes, her the repA and excA genes, is to give a useful simplest tool to track the evolution of plasmids.
One limitation of our study is the number of plasmids tested and the number of gene candidates to analyze. It's recommended to validate our results using other types of plasmids and other genes to get a correct view of the plasmids' evolution.
In conclusion, this study suggested that IncL/M plasmids harboring different antibiotic resistance genes have evolved differently.