Detection of 11 Multidrug Resistance Genes among the Strains of A. Baumannii by Computational Approach

Background: Acinetobacter baumannii is typically short, rod shaped gram negative bacterium. The World Health Organisation has declared it as an opportunistic pathogen in humans. Multi drug resistance involves different genetic determinants making the pathogen difficult to treat. So this study is undertaken to characterize the 11 different drug resistant genes from 19 virulent strains of A. baumannii using in-silico PCR. Aim: Detect the 11 multidrug resistance genes among strains of A. baumannii by computational approach. Materials & Methods: 11 multidrug resistance genes of A. baumannii were selected. Forward and reverse primers of the 11 genes as reported from earlier studies were used for in-silico PCR amplification. 19 strains of A .baumannii set as default strains on the server were chosen and the amplicon bands were observed. Result: Among the 11 multidrug resistance genes only blaOXA-51like and blaADC were detected among the 19 virulent strains of A. bauamannii. Original Research Article Noufal et al.; JPRI, 33(60B): 3776-3785, 2021; Article no.JPRI.74422 3777 Conclusion: The findings of the study documents the frequency of blaADC and blaOXA-51 like from the selected strains of A. baumannii. However further experimental validation must be done towards the periodical surveillance on the drug resistant strains of A. baumannii in hospital settings.


INTRODUCTION
Acinetobacter baumannii is typically short, almost round, rod shaped gram negative bacterium. WHO (World Health Organization) has declared it as an opportunistic pathogen in humans, affecting people with compromised immune systems and is becoming increasingly important as a hospital derived infection [1]. A. baumannii are multidrug resistant bacteria, which have characteristics such as aerobic, pleomorphic, non-motile and coccobacilli bacteria. A. baumannii is classified under the moraxellaceae family, which remains difficult to treat. The prevalence of drug resistant strains are continuously increasing and therefore the treatment options are considerably limited [2]. Propensity of multi-drug resistance in A. baumannii is a significant reason for its major transformation as a nosocomial pathogen. There are three different types of multidrug resistance properties, each has their own systemic quality. MDR-A. baumannii, alludes to strains which display protection from more than at least three antimicrobial drug classes. XDR-A. baumannii, alludes to all strains impervious to everything except two medication classes. Pan-drug resistance, alludes to opposition shown by the strains to all medication classes, and a development of culture safe A. baumannii was accounted for including the segregates that were impervious to carbapenems, colistin and polymyxins [3]. This is due to multiple mechanisms of drug resistance shown by A. baumannii via impermeable outer membrane, production of enzymes such as different beta lactamases classes which allows resistance towards carbapenems, porin channel alteration, efflux pumps and genetic materials that leads resistance towards fluoroquinolones [4].
Carbapenems have an ability of hydrolysing the beta lactamases (carbapenamases) which belong to molecular class D (OXA enzyme) that have constantly emerged around the globe with high prevalence in East Asia. The class D (OXA carbapenems) of Acinetobacter species are divided into several phylogenetic subgroups: blaOXA-23like, blaOXA-51like, blaOXA-141like. In recent findings it has been reported that enzymes belonging to the subgroups blaOXA-51 like are intrinsic to A. baumannii, that occurs in most (or) all strains, even though very variably expressed [5]. There were identification of two clones showing carbapenems resistance, these are produced by blaOXA-23like enzymes, they are named as OXA23 clone-1 and OXA23 clone-2.The third group called as SE clone, shows variable carbapenem resistance acquired by insertion sequence [6].
According to Ambler classification, the taken enzyme belongs to class-B metallo-beta lactamase (MBLs) and the multidrug resistance class D OXA type carbapenemases and most of them are mediated by plasmids. MBL's are further divided into several groups such as blaVIM, blaIMP, blaGIM and a recent finding has documented blaNDM in almost many clinical strains. In the MBL's activity the presence of divalent cations are needed as co-factors with the action of one or two zinc ions, for the catalytic ability of chelating as an inhibitory agent. All these mentioned 4 MBL's are having high potential for hydrolyzing all beta-lactam antibiotics except for the specific monobactams such as aztreonam [7], [8].
Most studies have documented A. baumannii as MDR strains, and show high variations in both their phenotypes and genotypes as nosocomial pathogens. blaTEM, blaSHV-type and blaCTXtype of genes, are responsible for extended spectrum beta-lactamase production that could be mediated by both plasmids and chromosomes. This plays an important role in exhibiting the resistance against later generations cephalosporins such as cefepime, cefotaxime and ceftazidime [9].
In the same line, sulbactam is a beta lactamase inhibitor, when combined with penicillin it lacks the ability of antimicrobial activity in most bacterial species. It possesses both bacteriostatic and bactericidal effects against A. baumannii through several mechanisms. With many studies documenting the drug resistance properties among A. baumannii, the present study was undertaken to evaluate the frequency of 11 genetic determinants of resistance genes among the 19 different strains of A. baumannii by computational approach.

Study Setting
This is an observational in-silico study done in the Department of Microbiology, Saveetha Dental College and Hospital. This is an original research study where we have selected 19 strains of A. baumannii set as default in the in-silico PCR server [10]. These strains are the default organisms given in the server and the their resistance is actually not determined. The genes of target were blaOXA-23like, blaOXA-51like, blaOXA-141like, blaVIM, blaIMP, blaGIM, blaNDM-1, blaTEM, blaSHV, blaCTX and blaADC. Upon the amplification command, the server produced the amplicon bands for evaluation of the band size. From the amplicon bands, the frequency of the distribution of the drug resistant genes among the vital virulent strains of A. baumannii were evaluated Further evolutionary relationships were compared with the phylogenetic analysis as done in earlier reports [11].

RESULTS
The investigation on the prevalence of the drug resistant genes from 19 different strains of A. baumannii (Table 1) using an in-silico amplification server was promising. The results showed the starting position of the amplification in the chromosome or plasmid and the length of each amplicon. Amplicons obtained in each chromosome or plasmid have been tabulated (Table 2) with target genes, primers used, sequenced of primer (5' to 3'), annealing temperature, estimated size of base pair and the frequency of the target genes among the study strains. Among the 11 multidrug resistant genes, we observed 78.94% positivity of blaOXA-51like and 68.42% for blaADC. All the other 9 genes were not present in the selected 19 different strains of A. baumannii. We further assessed the evolutionary pattern of the distributed genes among the strains.

DISCUSSION
Higher frequency of blaOXA51 and blaADC, were observed in the present study. In the present study blaOXA-51like and blaADC was observed in 78.94% [ Fig. 1] and 68.42% [Fig. 2] respectively. In a previous study 93% of the strains showed the presence of blaOXA-51like [12]. Another study has documented the presence of blaADC in a higher percentage when compared with our study [13]. In-silico analysis holds to be a promising computational approach to detect the presence of the target genes [14,15]. Selection of A.bauamannii was done based on the earlier studies related to the frequency of the resistance among the global strains [16][17][18][19]. Computational based approach is also a reliable platform to design novel target against potent pathogens and also to detect natural biocompounds to inhibit the same [20-234]. The immune-informatic approach is also been applied for the detection of virulence factors from A.baumannii [24-26]. [   Thus the present findings of the study were in both correlating and in contrast with many other earlier studies [30][31][32]. This suggests, the insilico PCR amplification is best suited for the preliminary identification of the vital genes. However, further studies with the clinical strains in-vitro can render the actual results on the prevalence and distribution of the virulent and resistant genes among A. baumannii. The limitation of this study is that it involved only the set default strains in the tool, thus requiring the same evaluation with the clinical strains. The limitation of the study was that the distribution of the resistant determinants was observed as a computational approach. Thus the future prospects are set to evaluate the same using specific in-vitro and in-vivo study models.

CONCLUSION
The present study had detected the frequency of two vital genetic determinants of resistance among the 11 genes targeted for the study. blaOXA-51 like and blaADC were highly distributed among the selected 19 strains A. baumannii. The in-silico PCR tool was efficient in the identification of the genetic determinants at a preliminary level. However, further experimental evaluations must be done using the clinical isolates and to periodically monitor the resistance pattern for epidemiological surveillance of A.baumannii associated infections.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.