Comprehensive analysis of imipenemase (IMP)-type metallo-Beta-lactamase showing global distribution threating Asia

: Antibiotic resistance, particularly beta-lactam resistance, is a major problem worldwide. Imipenemase or IMP-type metallo-beta-lactamase (MBL) has become a more prominent enzyme, especially in Asia, since it was discovered in the 1990s in Japan. There are currently more than 91 variants of IMP-type enzymes. The most commonly identified variant of IMP-type enzymes is IMP-1 variant. IMP-type MBLs have been identified in more than 10 species in Enterobacterales . Pseudomonas aeruginosa is the most frequent carrier of IMP-type enzymes worldwide. In Asia, IMP-type MBLs have been distributed in many countries in the region. This work investigated a variety of currently available IMP-type MBLs in both global level and regional level. Out of 88 variants of IMP-type MBLs reported worldwide, only 32 variants were found to have susceptibility profiles. Most of the IMP-type MBLs were resistant to Carbapenems, especially Imipenem and Meropenem, followed by the 3 rd generation cephalosporins, and interestingly, monobactams. Our results comprehensively indicated the distribution of IMP-type MBLs in Asia and raised the awareness of the sit-uation of antimicrobial


Introduction
Multidrug resistance organisms, especially β-lactamase-habouring pathogens, is a major global public health problem worldwide resulting in high mortality, high morbidity and rising economic costs [1]. The β-lactamase enzyme, that can be produced by both gram-positive bacteria and gram-negative bacteria, inactivates β-lactam antibiotics, i.e. penicillin, cephalosporin, carbapenem and monobactam, by hydrolysing the amide bond of β-lactam ring [2]. Currently, there are more than 7,270 enzymes available in the β-lactamase database (www.bldb.eu). β-lactamase can be classified into four classes, based on Ambler classification; class A, C, D are serine protease-derived β-lactamases while class B is the metallo-or zinc dependent β-lactamase (MBL) [3].
Even though IMP-type MBLs are important and widely distributed around the world, a comprehensive review of this enzyme has not been conducted. Moreover, a previous phylogenetic construction was restricted due to the number of available sequences. To understand the comprehensive picture of blaIMP gene, a review of relevant literature and a phylogenetic tree reconstruction was performed to investigate the distribution of IMP-type MBLs, phylogenetic relationship of the genes, and the association between phylogenetic cluster and antibiotic susceptibility.

Review of literatures
A comprehensive literature search was performed by PM and PP on Pubmed/Medline and EMBASE until 30th November 2021 to obtain relevant articles. The search terms used were "IMP and beta-Lactamases". A list of references was stored and the duplicates were removed using Endnote. PM and PP separately screened and selected the titles and the abstracts mentioning IMP metallo-beta-lactamase. Articles were included when the prevalence of blaIMP gene was reported. Articles were excluded when the English version was not available.

blaIMP gene sequence retrieval and analysis
A total number of 88 sequences of IMP-type metallo-beta-lactamase genes (blaIMP) were found and downloaded from both beta-lactamase databases [7] (last accessed November 2021) and GenBank database in November 2021. IMP-36, -50 and -57 could not be found and retrieved from both databases. Multiple sequence alignment of both nucleotide sequences and amino acid sequences was processed using an iterative refinement algorithm in MUSCLE with default parameters [8] and manually edited in MEGA software version 11 [9].

Phylogenetic tree estimation
Prior to the construction of phylogenetic tree, the model test was conducted to estimate the most appropriate model using built-in functions in MEGA (Kumar, 2018

Distribution of IMP-type MBLs
A search of NCBI database and EMBASE using "IMP and beta-Lactamases" for gene encoding blaIMP demonstrated a variety of variants of IMP-type MBL genes as well as species of IMP-carrying organisms. There were 88 variants of IMP-type MBL genes currently deposited on NCBI's GenBank. These 88 variants were identified in 29 species across 32 countries (Table 1). According to the genes submitted to GenBank and literature search, the detection of blaIMP was frequently reported from Japan (25%), followed by China (17%) and France (7%) ( Figure 1A).

In silico analysis of IMP-type MBLs
In silico analysis of IMP-type MBL genes was conducted to investigate the diversity of enzymes. By using multiple sequence alignment of 88 variants of IMP-type MBLs, the conserved sequences of active sites were identified as follows: His95, Phe96, His97, Asp99, Ser100, His157, Cys176, and His215 (numbered according to IMP-1; Figure S1). These   By combining antibiotic susceptibility profile with phylogenetic tree to investigate the relationship between clustering and susceptibility, it was found that susceptibility pattern was not associated with phylogenetic tree (Figure 3).

Discussion
The importance of clinically important bacteria has been increasing due to the multidrug resistance caused by the production of drug-inactivating enzymes, especially beta-lactamases [18]. More critically, carbapenemase enzyme has been increasingly identified in pathogens that are associated with nosocomial infections [19,20]. This study is the first to comprehensively investigate the epidemiology and diversity of IMP-type MBLs, a class B beta-lactamase with carbapenemase ability.
An IMP-type MBL is encoded by blaIMP-N gene (N = no. of variant) which can be located on the chromosome or the plasmid, which facilitates the transfer of blaIMP genes via horizontal gene transfer [21,22]. Our study showed that the blaIMP gene was detected in clinically relevant species, including P. aeruginosa and A. baumannii, which are associated with hospital-associated infection and listed in "Priority 1: CRITICAL" list of antibiotic resistant pathogens by WHO (https://www.who.int/news/item/27-02-2017-whopublishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed). Interestingly, our analysis revealed that the top 3 countries that blaIMP genes were detected were all Asian countries: Japan, China and Thailand. Japan is the first place where IMP-type MBLs (IMP-1) were reported [6]. There were 28, 15, 7, and 5 variants of blaIMP gene identified in Japan, China, Thailand, and Singapore, respectively. A recent study revealed that carbapenemases -derived P. aeruginosa -are distributed thoroughly in every part of Thailand [23]. However, the epidemiological study of IMP variants in Japan and China has not been conducted. It is, therefore, important to note that blaIMP gene is one of the causes of antibiotic resistance in Asia.
Phylogenetic tree is commonly used to investigate the evolutionary relationship of genes or organisms. Our findings revealed that a reconstructed phylogenetic tree using 88 blaIMP variants clustered the genes into three main groups (Figure 2). In a broad picture, this tree was similar to a previous version [23]. Nevertheless, group Ia, which was previously clustered in group II, was currently identified in group I with high bootstraps.
It is important to note that the structure of phylogeny of blaIMP is nearly well-defined except that some branches remain dynamic depending on the number of genes added to the tree.
The change of position on the phylogenetic tree could be caused by the increased number of tested genes in our study.
A search for antibiotic susceptibility profiles revealed that only 32 variants (out of 88) were tested for their susceptibility. The profile showed that 3 rd generation cephalosporins and carbapenem were less effective against most strains with blaIMP. Interestingly, Aztreonam remained active to the strains with some types of blaIMP. However, the association between susceptibility and phylogenetic tree was absent. This is supported by the finding showing the sequence of the active site (catalytic site) was highly conserved within the members of MBLs [24]. It is of note that nucleotide or amino acid substitutions outside the active site might not affect the beta-lactam-hydrolysing activity of the enzyme.
In addition, the susceptibility profile of strains containing each blaIMP variant must be performed to ensure the association between substitution/phylogenetic tree and antibiotic resistance pattern. All in all, the finding of this work demonstrated that antibiotic resistance-associated genes distributed to several regions around the world. This emphasised that the need of discovering or inventing novel antibiotic agents and enforcing antibiotic stewardship is urgent.

Conclusions
Carbapenemase, especially IMP-type MBLs, has caused public health problems worldwide. This study is the first to comprehensively analyse all currently available variants of IMP-type MBLs and associated susceptibility. Asian countries, especially Japan and China, are presently under a wide spread of blaIMP-carrying bacteria, listed in the WHO's antibiotic-resistant bacteria. An unrooted phylogenetic backbone of blaIMP gene variants demonstrated two separate groups without susceptibility or geographical association. This strengthens antibiotic stewardship policy on a global level to control antibiotic resistance problems.

Supplementary Materials:
The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Figure S1: Multiple sequence alignment of amino acid sequence of 88 blaIMP variants.
Author Contributions: Conceptualization, P.P.; methodology, P.P; formal analysis, P.P and P.M.; writing P.P. and P.M.; visualization, P.P. and P.M. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding