Investigating the Transcriptome of Imipenem-Sensitive A. baumannii

Methods

I just need help to pharaphrase this discussion part for my data analysis report.

In order to investigate the transcriptome of Imipenem-sensitive A. baumannii an RNA-seq data analysis was performed. The initial part of analysis involved generating a gene count table and the next part involved using this given gene count to analyze the expression of genes in imipenem sensitive A. baumannii under control and treatment conditions for comparison. The main purpose is to identify which genes are expressed as a response to antibiotic treatment in imipenem-sensitive A. baumannii and if these genes are responsible for A. baumannii being sensitive.

Imipenem is a beta-lactam antibiotic. The class of Beta-lactam antibiotics work by inhibiting the production of peptidoglycan which disrupts the cell wall synthesis. The mechanism is by binding to the penicillin-binding proteins and acylates the transpeptidase which results in the bacterial cell wall becoming weak and lose its stability (Hellinger, W.C, 1991).

One of the major findings, in this data analysis is the data obtained from the Comprehensive Antibiotic Resistance Database (CARD). The result from this analysis is displayed in figure 10 and 11. The figure 10 summarizes the genes which were discovered, with 15 perfect hits indicating sensitive genes and 14 strict hits which were the resistant genes. Some of the perfect hits indicating the sensitive genes were The data in figure 9, highlights the main mechanisms exhibited for A. baumannii were antibiotic inactivation and antibiotic efflux. The FHGFAHKG_01216 and the FHGFAHKG_03388 were the only two genes which displayed different mechanisms as antibiotic target inactivation and reduced permeability to antibiotic.

The results give a clear indication that these genes are the reason contributing to the antimicrobial resistance in A. baumannii compared to the sensitive strain of A. baumannii. Moreover, the CARD analysis also provided data on the class of drugs which were responsible for the 14 strict genes providing resistant to which are fluoroquinolone antibiotic, sulfonamide antibiotic, tetracycline antibiotic and glycylcycline. The strict hit-resistant gene provided resistance against tetracycline antibiotic, glycylcycline, aminoglycoside antibiotic, and cephalosporin. The study conducted by Amiri et al in 2019, concluded that Imipenem resistance for A. baumannii is obtained from the efflux pump and this mechanism is a major factor for drug resistant. The antibiotics such as glycylcycline and tetracycline are eliminated by reducing the entry of antibiotics into the periplasmic place of the bacterium by the genes that code for the antibiotic efflux pump. 

Results

According to the heatmap in figure 9, it denoted that A. baumannii expressed its resistance using the upregulation of the resistant genes. The antibiotic resistance for the efflux pump was seen by upregulating the efflux transporter which emphasizes our resistant strain is upregulated.

Another major finding from this data analysis was obtained using the EggNOG-mapper. There were 635 genes which were differentially expressed between resistant and the sensitive strain with 0.1 MIC imipenem. The EggNOG-mapper is a feature that aids in annotating the sources and the taxonomic ranges are classified based on the genes against its updated databases (Tatusov, R.L, 2000).  In graph 1, it highlights the most expressed genes are under S category with 152 genes and least expressed genes under the F category with just 3 genes. The S category with 152 genes has an unknown function. This unknown function is the reason for the resistance in A. baumannii. Among these 152 genes there could be the presence of AMR genes and with proceeding CARD analysis it showed there are 14 resistant strains present. The expression of genes in the COG data were led by the resistant strains that were identified in CARD analysis (Huerta-Cepas, J, 2017). One of the main reasons for the genes in the S category could be due to the stress response caused by the 0.1 MIC Imipenem antibiotic (Wachino,J, 2019).

Other studies conducted, identified the bacteria Francisella tularensis has the upregulation of the dehydrogenase enzymes. In situations of stress, there will be an increase in the occurrence of the TCA cycle which is the mechanism used to protect themselves from stress and survive (Dieppedale et al., 2013).

The CARD analysis provides a great insight on the AMR gene family which include sulfonamide resistant major facilitator superfamily, antibiotic efflux pump, resistance-nodulation-cell division (RND) antibiotic efflux pump, AAC (3)-la, ANT (3''), and ADC beta-lactamase without carbapenems activity (Vila, J, 2007). The β-lactamases are encoded using the ADC-25 gene which has the ability to hydrolyze the β-lactam ring in antibiotic such as cephalosporin and inactivate its activity. Other antibiotics such as aminoglycoside is inactivated by the AAC (3)-la gene which encodes for the acetyltransferases aminoglycoside modifying enzyme.

Following the data analysis there are some limitations in the study which can be improved. The bacteria A. baumannii is only treated with one antibiotic Imipenem for both the resistant and sensitive strains. Thus, it suggests this antibiotic cannot be used to generalize the antibiotic resistance of this bacteria. This could have been improved by using a wider array of antibiotics to be tested to investigate the A. baumannii multidrug resistance. Moreover, the length of data analysis procedure was quite long and included many steps so could be prone to many errors. The EggNOG mapper had some functions which were blank and unknown, but these genes would have been relevant and used in research. Moreover, the volcano plot may not display all genes which could have been present with the low, borderline, P-adjusted value.

The implication of this study include A. baumannii is a soil pathogen which is adapted to survive and reflects on to its resistance mechanism. The resistance from A. baumannii is obtained from mutation and DNA transfer from bacterial cells which is incorporated into itself for survival. The findings from this study also suggest that this pathogen has the ability to be resistant against various antibiotics and can live in harsh environments.  Furthermore, other suggestions in order to improve the study is to do further study with a wider array of antibiotics. Other studies could be directed in the study of rlpA gene which is responsible for coding for endolytic peptidoglycan transglucosylase.