An In-Silico Analysis of Acquired Antimicrobial Resistance Genes in Pseudomonas Aeruginosa Plasmids

Introduction: The aim of this study to reveal the prevalence of acquired antimicrobial genes in sequences of P. aeruginosa plasmids by using in silico methods. Methods: This study included 828 items with using 'Pseudomonas aeruginosa and plasmid’ keywords for searching in NCBI database. The sequences of 94 plasmids were retrieved from GenBank and analysed for detection of acquired antimicrobial resistance (AMR) genes with using ResFinder 2.1 database/webserver, KmerResistance 2.2 database. and ResFinderFG 1.0. Plasmids Sequence were aligned with using MEGA X Molecular Evolutionary Genetics Analysis across Computing Platforms. Results: 67 out of 94 plasmids sequences were qualied as AMR containing plasmids. For detected 9 classes of AMR genes, aminoglycosides 39.6% were highest rate. The next frequencies were beta-lactams (19%), sulphonamides (14.5%), and uoroquinolones (10.1%). For nine classes of antibiotics, 74 AMR gene were identied. Prevalent of sulphonamide resistance gene Sul1 was 32 out of 277 gene. In AMR plasmids, 6 ARD family were detected. 15 representative genomic sequences selected from each clade and three clades were revealed from them. The relationship of clades with drug resistance was not signicant (p-value = 0.682). Conclusions: Analyzing the Information in annotated sequence is reveal the mechanism of spreading of resistance gene in plasmids. Detection the trace of AMR genes in world papulation can help to nd more response to question in regards to spread of infection and analysis of AMR gene sequences give more insight to scientist to control of Pseudomonas aeruginosa infections.


Introduction
Pseudomonas aeruginosa habitats are very wide including soil and aquatic environments. This species can cause diseases in humans, animals and plants. P. aeruginosa is highly prone to drug resistance mostly by means of plasmids. plasmids are tools for horizontal gene transfer (HGT), in bacterial genetics evolution and adaptation. These mobile genetic elements are very diverse and have a high range hosts and ecological niches. Based on genetic organization, size and host range, 14 incompatibility groups of plasmids have been detected in Pseudomonas [1,2]. Incompatible groups are using same replication control. This groups are very mobile and capable to gene transfer called promiscuous. Promiscuity gave great role in antibiotic resistance spreading among bacterial populations [3].
Antimicrobial resistance (AMR) is a more growing issue in P. aeruginosa infections that cause health system crisis. The empirical use of antibiotics in veterinary and medicine promote and creating resistance plasmids that circulate in universe populations. Spread of plasmid harbor AMR genes as threat to public health and environment leads to mortality and morbidity. Antimicrobial resistance (AMR) in P. aeruginosa is a worldwide health problem and need more attention in diagnosis and nding the map of spreading by plasmids. We aimed in this study to reveal the prevalence of acquired antimicrobial genes in sequences of P. aeruginosa plasmids by using in silico methods.

Plasmid selection
This study included all 'Pseudomonas aeruginosa plasmid. Overlay 828 item were found with using 'Pseudomonas aeruginosa and plasmid' keywords for searching in NCBI database (Table 1). Based on web data this sequence has been published from 1991 until 2020. Size of plasmid ranged between 2140 bp _ 555265 bp and GC content were 30.1-65.8%.

Screening for acquired AMR genes in plasmids
The sequences of 94 plasmids were retrieved from GenBank. This sequence were conducted in silico analysis for detection of acquired antimicrobial resistance genes with using ResFinder 2.1 database/webserver which 67 plasmids has AMR genes ( Table 1) [4]. Fifteen classes of antibiotic presented in database considered for screening that included, aminoglycoside (AG), beta-lactam, colistin, a uoroquinolone (FQ), fosfomycin, fusidic acid, glycopeptide, macrolide-lincosamide-streptogramin B (MLS), nitroimidazole, oxazolidinone, phenicol, rifampicin (RP), sulphonamide (SM), tetracycline (TC), and trimethoprim (TP). Search setting parameters for all 15-drug classes were adjusted to 90% as a minimum for percent identity and 100% for perfect alignment, also 60% for minimum length of sequence nucleotides to overlap with resistant genes. DNA of plasmid were submitted into database online software. [5] Analysis results including, predicted phenotype of resistance gene, database accession number, starting contig position of the gene, and alignment highscoring segment pair (HSP) query length, were gathered from program. All date were recorded in excel software. For all plasmids the index of potential multiple antibiotic resistance (p-MAR) were calculated based on screened 15 classes of antibiotics [6,7]. The analysis of p-MAR data's, can classify plasmids into three groups as: multidrug-resistant (MDR), extensively drug-resistant (XDR) or pan drug-resistant (PDR) by using the previously reported standards [5,8] This index is a good marker for epidemiology detection of isolates origin antibiotic use and the rate of 0.2 indicates a 'high-risk' of social health contamination [6,9]. Veri cation of acquired AMR genes in Aeromonas plasmids Further assessments of plasmid containing AMR genes were done with KmerResistance 2.2 database [10,11].
Analysis was conducted with default setting including, 70% for identity threshold and 10% for depth correction. Results were saved on excel sheet and compared with the results of ResFinder program.

Probing of plasmids for antibiotic resistance determinants (ARD)
The resistance phenotype of gene were explored for plasmids with AMR genes with ResFinderFG 1.0 [12] server, that screened resistance with functional metagenomic antibiotic resistance determinants. This database server setting was considered 98% for per cent identity and 60% for minimum query length. Results which screened for more than 13, ARD families were saved in excel sheet as 'assembled contigs/genomes and sequences.

Phylogenetic analysis of the retrieved Plasmids genomes
Plasmids Sequence were aligned with using MEGA X Molecular Evolutionary Genetics Analysis across Computing Platforms (MEGA X; https://www.megasoftware.net/). software by ClustalW approach. phylogenetic tree was done by a maximum parsimony (MP) approach in MEGA X software. Support value were considered 1,000 bootstrap replicates.

Statistical Analysis
Correlation between GC% and plasmid size were quali ed with using XLSTAT with principal component analysis (PCA) analysis with setting included mean, standard deviation, and correlation (Pearson).
Connection of plasmid with containing AMR gene with sequences available in NCBI databases and similarity between them were determined using BLAST online tool. Sequences were aligned with MEGA X using likelihood model in considering bootstrap procedure (1000 replicates).

Results
Size and GC content of Pseudomonas plasmids PCA were used to determine the relation between size and GC content, of 67 plasmids carrying AMR genes. Size of plasmid ranged between 2140 bp _ 555265 bp and GC content were 30.1-65.8%.  Total 67 For detected 9 classes of AMR genes as shown in Table 3, aminoglycosides 39.6% were highest rate. The next frequencies were beta-lactams (19%), sulphonamides (14.5%), and uoroquinolones (10.1%). For nine classes of antibiotics, 74 AMR gene were identi ed. Prevalent of sulphonamide resistance gene Sul1 was 32 out of 277 gene as shown in Fig. 1.

Antibiotic resistance determinants in Aeromonas plasmids
In AMR plasmids, 6 ARD family were detected. High frequency families were aminoglycosides acetyltransferases and beta-lactamase with 38.30% and 31.91%, respectively as shown in Fig. 2. phylogenetic analysis of plasmids Complete genome sequences of 67 plasmid containing AMR genes were analyzed using software with consideration exclusion and inclusion criteria. Representative sequence from each group were selected with maximum parsimony tree approach. Finally, 15 representative genomic sequences selected from each clade were more analyzed (Fig. 3). As shown in Fig. 3 the evolutionary lineages and common ancestry of all plasmid sequences were revealed by the phylogenetic tree. Three clades were revealed from them. The relationship of clades with drug resistance was not signi cant (p-value = 0.682) as shown in Table 4.

Discussion
Antibiotic resistance is a result of selection pressure due to the overuse and frequent misuse of antibiotics in health systems. The pro le of AMR gene in bacterial population is depend on horizontal transfer of mobile genetic elements. Plasmids are one of the major roles as mobile genetic elements. Speci cation of plasmid in bacterial give more insight in determination of ability of plasmid to transfer resistance gene and evaluation of genome. Pseudomonas aeruginosa plasmids has been sequenced and evaluated statistically, based on PCA analysis there was the negative Pearson's correlation (r = -0.191, α = 0.95) between GC content and plasmid size. The negative correlation between plasmid speci cation may be due random or naturally transfer of genes among plasmids. Although there is correlation between genome size and guanine-cytosine (GC) content in bacteria, however, the this correlation not well explained [14].
The prevalence of AMR genes was screened in ResFinder database. Plasmids sequences were analyzed to detect acquired antimicrobial resistance genes. Disadvantage of this way is that the ResFinder database cannot nd mutation, therefore it only detects acquired resistance genes. There is need to qualify resistance genotype with phenotypic identi cation. Investigation showed that there is very high correlation between phenotypic detection and AMR gene detection in ResFinder database, therefore, whole-genome sequences alignment is an alternative way to nd drug resistance patterns [15]. In overall 71.28% of plasmid were containing AMR genes, of which, 38, 5, 2 isolates were MDR, XDR and PDR respectively. The Sul1 gene located in plasmids and products of this gene can degrade sulfonamides and trimethoprim antibiotics. Those are cheap and e cient antibiotics that have been used for a long time to treatment of human and animals' gram-negative infections [17]. Three genes sul1, sul2 and sul3 encode dihydropteroate synthase enzyme that inactivate sulfonamides and sul1 gene is mostly transferred in associated with class 1 integrons and others by plasmids [18]. Quinolone resistance has been occur mostly by plasmids by tree mechanisms including (i) qnr genes that produce a quinolone-protective proteins, (ii) aac(6′)-Ib gene that produce a double class antibiotic-modifying enzyme which acetylates cipro oxacin and nor oxacin and (iii) qepA gene that produce an e ux pump proteins [19].
Analyzing the Information in annotated sequence is reveal the mechanism of spreading of resistance gene in plasmids. Detection the trace of AMR genes in world papulation can help to nd more response to question in regards to spread of infection and analysis of AMR gene sequences give more insight to scientist to control of Pseudomonas aeruginosa infections. MEGA X software were used for analyzing phylogenetic relationship between 67 plasmid containing AMR genes. Due to some restriction test were accomplished in three group and representative genomic sequences from each clade were more analyzed. Finally, three clades were revealed from analyzing representative sequences. There was no correlation of MDR with clades (p-value = 0.682)

Conclusion
This study results showed that there is no correlation between size and GC content of Pseudomonas aeruginosa plasmids. Most of plasmid carrying AMR genes that acquired horizontally. Three phylogenetically clade was revealed by molecular epidemiology software but there were not associated with drug resistance.

Declarations
Ethical Approval and Consent to participate Not applicable

Consent for publication
Not applicable Availability of data and materials Not applicable

Competing interests
The authors have no con ict of interest.

Not applicable
Authors' contributions RR did all process including doing project and manuscript writing, editing and submitting.

Figure 1
Prevalence of 74 AMR genes found in 40 Aeromonas plasmids after in silico analysis Page 18/19

Figure 2
Frequency of Antibiotics family in AMS containing plasmids