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Team:IIT Roorkee/ML Results

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Results

We have utilized a machine learning algorithm over the strain-gene/allele dataset of A. baumannii available from the PATRIC database that can predict the resistance phenotype of strains. In nutshell, we have used the presence or absence of particular genes or alleles as features in predicting the phenotype of strain. We have utilized the data of 1360 A. baumannii strains for 10 different antibiotics.








The results of machine learning can be summarized in the following two points

  1. Detection of Genes conferring Antibiotic resistance
  2. Correlation and Mutational analysis of gene-gene pair


Detection of Genes conferring Antibiotic resistance


The machine-learning algorithm has helped in the identification of genes that are either specific to the mechanism of the particular antibiotic or involved in the novel pathway/target. There are few genes that are involved in basic cellular processes that strongly relate to the survival and growth of A. baumannii . The genes corresponding to the particular antibiotic are listed below.

Genes Antibiotic Gene Ontology
Annotations
Mechanism of Action References
esiB Ciprofloxacin
Levofloxacin
GO_esiB Secretory immunoglobulin
A-binding protein
Ref1_esiB
aroP Ciprofloxacin
Levofloxacin
GO_aroP Aromatic amino acid
transport protein
-
tnsB Ciprofloxacin GO_tnsB Transposon Tn7 transposition protein Ref1_tnsB
Ref2_tnsB
xerC Ciprofloxacin GO_xerC Tyrosine recombinase Ref1_xerC
Ref2_xerC
asnC Levofloxacin GO_asnC Regulatory protein, AsnC Ref1_asnC
puuP Levofloxacin GO_puuP Putrescine importer Ref1_puuP
Ref2_puuP


esiB (Ciprofloxacin and Levofloxacin)

esiB encodes for Secretory immunoglobulin A-binding protein (UP_esiB).

GO Molecular function: IgA binding and Metal ion binding

GO Biological function: Negative regulation of immune response and neutrophil activation, and pathogenesis

Complete GO annotation: GO_esiB

According to the study of Pastorello et al. (Ref1_esiB), esiB helps in secretion of the protein which binds with immunoglobulins in the blood or antibodies helping bacteria escape from neutrophil (cell eating bacteria). The neutrophil is the most common White Blood Cells (WBC) in the human body, so these proteins help the bacterial pathogen in escaping the immune system pathway in the patients of Urinary Tract Infections. The study also concluded that esiB is preferentially associated with extraintestinal strains, while the gene is rarely found in either intestinal or nonpathogenic strains of E. coli.

Importance: The presence and importance of this gene in the case of patients with Urinary Tract Infection (one of the major Hospital Acquired Infections) make it an important target/gene to explore using wet-lab experiments in the case of A. baumannii.



aroP (Ciprofloxacin and Levofloxacin)

aroP encodes for Aromatic amino acid transport protein. It is a permease that is involved in the transport across the cytoplasmic membrane of the aromatic amino acids (phenylalanine, tyrosine, and tryptophan), (UP_aroP).

GO Molecular function: Transmembrane transporter activity of aromatic amino acids

GO Biological function: Amino acids transport

GO Cellular Component: Integral component of Plasma membrane

Complete GO annotation: GO_aroP

Since aroP helps in encoding protein responsible for transportation aromatic amino acids, therefore it is related to very basic cellular functions. Amino acids are important for the process of transcription and translation, their transportation plays an important role in these functions.

Importance: There is a lack of studies conducted for exploring the functioning of aroP in the context of A. baumannii, which makes it a novel and important target pathway to be explored for using wet-lab experiments, especially because it is involved in the basic cellular process i.e. amino acid transport.



tnsB (Ciprofloxacin)

tnsB encodes for Transposon Tn7 transposition protein, which are very special proteins helping in cutting, pasting, and making copies of DNA in the chromosome (UP_tnsB).

GO Molecular function: DNA Binding, and Transposase activity

GO Biological function: DNA Integration and DNA-mediated transposition

GO Cellular Component: Cytoplasmic membrane

Complete GO annotation: GO_tnsB

Ciprofloxacin acts by inhibition of DNA replication by inhibiting bacterial DNA topoisomerase and DNA-gyrase. Transposons help in DNA strand breakage which is also carried out by topoisomerase. These facts make it very clear that tnsB is an important gene and target in the context of DNA replication and is involved in a similar mechanism as that of Ciprofloxacin.

Tn7 class transposon proteins are associated with carbapenem-resistance in A. baumannii (Ref1_tnsB). The study by Rose (Ref2_tnsB), discovered a novel Tn7-related transposon, TnAbaR1 which contributes to the accumulation and dissemination of antibiotic resistance genes. According to their study, Tn7 is a well-studied, highly promiscuous cut-and-paste transposon, found in a variety of bacteria and mainly important for resistance to antibiotics such as trimethoprim and streptomycin.

Importance: The involvement of tnsB in being a cause of resistance to several antibiotics makes it an important target and pathway to be explored especially in the context of our novel protein-based therapeutic and our pathogen of interest, A. baumannii.



xerC (Ciprofloxacin)

xerC encodes for tyrosine recombinase, which acts by catalyzing the cutting and rejoining of the recombining DNA molecules (UP_xerC).

GO Molecular function: DNA binding, Site-specific recombinase activity

GO Biological function: Cell cycle, Cell division, and Chromosome segregation

GO Cellular Component: Cytoplasm

Complete GO annotation: GO_xerC

It binds cooperatively to specific DNA consensus sequences that are separated from XerD binding sites by a short central region, forming the heterotetrameric XerC-XerD complex is essential to convert dimers of the bacterial chromosome into monomers to permit their segregation at cell division. It also contributes to the segregational stability of plasmid (UP_xerC).

During the recombination phase, this complex catalyzes two consecutive pairs of strand exchanges, implying that specific pairs of active sites are sequentially switched on and off in the recombinase tetramer to ensure that appropriate DNA strands will be exchanged at both reaction steps. These findings have been made for E. coli and it would be interesting to check for the same in the case of A. baumannii.

According to the study related to A. baumannii conducted by Lin et al. (Ref1_xerC), they concluded that XerC and XerD are functional proteins and participate in horizontal dissemination of resistant genes among bacteria. The horizontal dissemination or transfer of resistance genes is a major cause of the increase in Antibiotic resistance. Furthermore, the study conducted by Merino et al. (Ref2_xerC), found that DNA recombination through the Xer system in plasmids requires XerC and XerD (recombinases). DNA recombination helps in the natural editing of the bacterial genome and makes the natural process of evolution faster.

Importance: Since A. baumannii is an opportunistic pathogen that is evolving at a faster rate, and as mentioned above, xerC helps in DNA recombination which leads to natural editing of the genome, becomes an important target or pathway to be explored using wet-lab experiments.



asnC (Levofloxacin)

asnC encodes for a regulatory protein called AsnC (UP_asnC).

GO Molecular function: Amino acid-binding, DNA-binding transcription activity, and Sequence-specific DNA binding

GO Biological function: Positive and negative regulation of transcription, Response to amino acid

Complete annotation: GO_asnC

The study conducted by Gebhardt et al. (Ref1_asnC), finds the list of around 300 genes which are important for the survival and growth of A. baumannii, and find two AsnC/Lrp family regulators as putative transcriptional regulators.

Importance: The involvement of asnC in amino acid binding and impacting the process of transcription makes it an interesting pathway to be explored using wet-lab experiments. Like, aroP, it helps bacteria in performing basic cellular functions which are essential for survival and growth.



puuP (Levofloxacin)

puuP encodes for putrescine importer PuuP (UP_puuP)

GO Molecular function: Putrescine transmembrane transporter activity

GO Biological function: Amino acid transport and cellular response to DNA damage stimulus

Complete annotation: GO_puuP

It is involved in the uptake of Putrescine, and according to a study conducted by Terui et al. (Ref1_puuP) it helps in the import of putrescine to be utilized as an energy resource in absence of glucose. Further, it has a biological process of helping in the cellular response to DNA damage, and given the fact that Levofloxacin is involved in the mechanism of DNA replication which makes it a very interesting and prospective pathway to be explored.

According to the study by Hassan et al. (Ref2_puuP), A. baumannii encodes for the transport protein AceI, which confers resistance to chlorhexidine, a widely used antiseptic. They also concluded that several gene expression studies have revealed that the aceI gene responsible for encoding AceI protein is induced in A. baumannii by the short-chain diamines cadaverine and putrescine. It helps us in understanding the indirect involvement of putrescine imported by puuP in antibiotic resistance.

Importance: puuP helps in conferring resistance to chlorhexidine, it would be very important to check for the same in the case of Levofloxacin especially for A. baumannii.




Genes Antibiotic Gene Ontology
Annotations
Mechanism of Action References
aadB Gentamicin
Tobramycin
Amikacin
GO_aadB Antibiotic inactivation CARD_aadB
Ref1_aadB
Ref2_aadB
Ref3_aadB
Ref4_aadB
neo Gentamicin
Tobramycin
Amikacin
GO_neo Kanamycin kinase activity -
msr(E) Gentamicin
Tobramycin
Amikacin
GO_msrE Plasmid DNA Ref1_msrE
Ref2_msrE
Ref3_msrE
Ref4_msrE
emrE Gentamicin GO_emrE Antibiotic efflux CARD_emrE
cysL Tobramycin GO_cysL DNA binding -
rmtB Amikacin GO_rmtB Antibiotic target alteration CARD_rmtB
Ref1_rmtB
Ref2_rmtB
Ref3_rmtB
Ref4_rmtB


aadB (Gentamicin, Tobramycin and Amikacin)

aadB encodes for 2''-aminoglycoside nucleotidyltransferase (UP_aadB)

It helps in mediating bacterial resistance to kanamycin, gentamicin, dibekacin, sisomicin, and tobramycin by adenylate the 2''-hydroxyl group of these antibiotics in K. pneumoniae (UP_Kp_aadB) and kanamycin, gentamicin, and tobramycin in E. coli (UP_aadB).

GO Molecular function: Aminoglycoside 2''-nucleotidyltransferase activity, and Metal ion binding

GO Biological function: Response to antibiotic and Antibiotic resistance

Complete annotation: GO_aadB

aadB is a resistance-conferring gene which is confirmed by one of the most essential and relevant databases i.e. The Comprehensive Antibiotic Resistance Database (CARD). It works by the mechanism of antibiotic inactivation and confers resistance to aminoglycoside antibiotics (CARD_aadB).

The study conducted by Rizk et al. (Ref1_aadB) involved the collection of clinical samples of A. baumannii strains from patients in Intensive Care Units (ICUs) with suspected hospital-acquired infections followed by checking them for resistance to aminoglycoside antibiotics. The study concluded that the most common prevalent resistant genes among A. baumannii resistance to aminoglycosides was aadB with a contribution towards antibiotic resistance as high as 42%. Since this study involves strains taken from hospitals with suspected infections, it makes it essential for our novel protein-based drug to be checked for its efficacy against aadB.

As per the study by Anderson et al. (Ref2_aadB), in A. baumannii AB5075, a large plasmid (p1AB5075) carries aadB, a 2″-nucleotidyltransferase that confers resistance to both tobramycin and gentamicin but not amikacin. It is very important in the case of our machine learning approach since our approach ranks aadB as the most important feature (gene/allele) in the case of Gentamicin and Tobramycin but not in the case of Amikacin.

The study conducted by Chan et al. (Ref3_aadB), found a novel antibiotic resistance island in A. baumannii by analyzing genomes of several isolates collected from the US hospital system. They further concluded that after sequencing the genomes to completion, they found tobramycin-resistance gene aadB.

Si-Tuan et al., (Ref4_aadB, Ref4_msrE) characterized the genome of the A. baumannii strain DMS06669 which was isolated from the sputum of a male patient with hospital-acquired pneumonia, and identified genes related to antibiotic resistance. They find aadB which is majorly resistant to gentamicin, as one of the genes responsible for conferring resistance in the strain.

Importance: These several studies make it very clear that aadB is an important gene especially considering resistance towards aminoglycoside antibiotics. Moreover, the presence of resistance by aadB to majorly gentamicin and tobramycin but not amikacin further validates the effectiveness of our machine learning analysis as aadB was the top feature in the first two antibiotics but not in the latter.



neo (Gentamicin, Tobramycin and Amikacin)

neo encodes Aminoglycoside 3'-phosphotransferase (UP_neo)

GO Molecular function: ATP binding, Kanamycin kinase activity

GO Biological function: Response to antibiotic and Antibiotic resistance

Complete GO annotation: GO_neo

It helps in providing resistance to kanamycin, neomycin, paromomycin, ribostamycin, butirosin, and gentamicin B in the case of K. pneumoniae. This enzyme is encoded by the kanamycin and neomycin resistance transposon Tn5. Tn5 was originally isolated from K. pneumoniae, but has been transferred to a number of bacteria including E. coli. Since it has been transferred to E. coli, it is quite important to check for its relevance in the case of A. baumannii.

Importance: There has been a lack of literature studies conducted for neo in the context of A. baumannii, but it targets using protein pathway which is similar in mechanism to aminoglycoside antibiotics. Moreover, as mentioned before, it would be interesting to check for its relevance in A. baumannii in context of our novel protein-based drug.



msr(E) (Gentamicin, Tobramycin and Amikacin)

msr(E) encodes for ABC-F type ribosomal protection protein (UP_msrE).

GO Molecular function: ATPase binding and ATP binding

Complete annotation: GO_msrE

msr(E) is a resistant conferring gene as per the Comprehensive Antibiotic Resistance Database (CARD) and provides resistance through antibiotic target alteration (CARD_msrE). Furthermore, as per CARD, Msr(E) is an ABC-F subfamily protein expressed to K. pneumoniae that confers resistance to erythromycin and streptogramin B antibiotics. It is associated with plasmid DNA. It is also 100% identical to ABC-F type ribosomal protection protein Msr(E) which is in multiple species. Since it is associated with plasmid DNA, it becomes an important factor in horizontal transfer and dissemination of antibiotic resistance.

Blackwell and Hall (Ref1_msrE) find in their study that macrolide resistance genes msrE and mphE were present in an 18.2-kb plasmid of A. baumannii isolate from Singapore which confers resistance to erythromycin and tetracycline, both of which follow protein synthesis mechanism.

A study conducted by Karah et al. (Ref2_msrE) concluded that msr(E) is one of the resistance genes present in clinical isolates of A. baumannii in Pakistan. The study by Kumburu et al. (Ref3_msrE) utilized Whole Genome Sequencing (WGS) to identify resistance-conferring genes in MDR A. baumannii in Tanzania. They found several antibiotic resistance genes some of which were present in chromosomes while some on plasmids. msr(E) was detected as an antibiotic resistance gene that is present on plasmids and playing an important role in the spreading of the resistance.

Similar to the case of aadB, the study conducted by Si-Tuan et al., (Ref4_aadB, Ref4_msrE) identified msr(E) which is majorly resistant to streptogramin, which follows the similar mechanism as of aminoglycoside antibiotics.

Importance: As shown by several studies, msr(E) is responsible for resistance to several antibiotics like macrolide, streptogramin, etc, which follow a similar mechanism to those of aminoglycosides, it becomes exciting and interesting to check for its relevance to Gentamicin, Tobramycin, and Amikacin in case of A. baumannii.



emrE (Gentamicin)

emrE encodes for Multidrug transporter EmrE (UP_emrE).

GO Molecular function: Antiporter activity, Identical protein binding, etc.

GO Biological function: Cellular response to DNA damage stimulus, Response to drug, etc.

Complete GO annotation: GO_emrE

It is a multidrug efflux protein that confers resistance to a wide range of toxic compounds, including ethidium, methyl viologen, acriflavine, tetraphenylphosphonium (TPP+), benzalkonium, propidium, dequalinium, and the aminoglycoside antibiotics streptomycin and tobramycin (UP_emrE).

Further, as per the Comprehensive Antibiotic Resistance Database (CARD), EmrE is a small multidrug transporter and works by antibiotic efflux mechanism to confer antibiotic resistance (CARD_emrE).

Importance: emrE is majorly found in P. aeruginosa and E. coli, which makes it quite interesting to check for existence on A. baumannii genomes. Moreover, it has been shown to be causing resistance to Tobramycin while our machine learning detected it to be an important gene in the case of Gentamicin, so it would also be exciting to validate and confirm by wet-lab experiments.



cysL (Tobramycin)

cysL encodes for HTH-type transcriptional regulator CysL (UP_cysL).

GO Molecular function: DNA-binding transcription factor activity

GO Biological function: DNA-templated regulation of transcription

Complete GO annotation: GO_cysL

There is a lack of literature evidence in the case of cysL, but it has been identified as one of the topmost features in the case of Tobramycin, and given the fact our machine learning has identified several genes confirming to literature evidence, cysL is one of the novel genes uncovered by our algorithm responsible for antibiotic resistance.

Importance: It would be interesting to check the relevance and importance of cysL in the context of A. baumannii and our novel protein-based drug as well, as it has been detected as the topmost feature by machine learning.



rmtB (Amikacin)

rmtB encodes for 16S rRNA (guanine(1405)-N(7))-methyltransferase (UP_rmtB).

GO Molecular function: rRNA methyltransferase activity

GO Biological function: Response to antibiotic and Antibiotic resistance

Complete GO annotation: GO_rmtB

rmtB encoding protein specifically methylated the N7 position of guanine 1405 in 16S rRNA, and conferring resistance to various aminoglycosides (UP_rmtB).

It is a resistance gene as per the Comprehensive Antibiotic Resistance Database (CARD), which works with the mechanism of antibiotic target alteration and belongs to the drug class of aminoglycoside antibiotics (CARD_rmtB).

Tada et al. (Ref1_rmtB) conducted a study on strains of A. baumannii and P. aeruginosa isolated from patients in intensive care units (ICUs) in two medical settings in Vietnam and out of which 71.3% strains were highly resistant to amikacin and gentamicin. They further concluded that, 16S rRNA methylase RmtB was produced by 9 strains (of 101) of A. baumannii and 2 (of 15) strains of P. aeruginosa .

The study by Lee et al. (Ref2_rmtB) analyzed amikacin resistant strains of gram-negative bacteria in Korea and concluded that armA and rmtB were genes predominantly responsible for the resistance.

Wachino et al. (Ref3_rmtB) and Wang et al. (Ref4_rmtB) concluded that 16S rRNA methylases, which lead to the high-level resistance of various aminoglycosides, can easily transfer to other bacteria since their genes are typically present on plasmids. The transfer of genes plays an important role in horizontal gene transfer and the dissemination of antibiotic resistance.

Importance: Several studies have indicated the spread of aminoglycoside resistance in A. baumannii which is a major cause of worry for the researchers. Since our novel drug is protein-based therapeutic, it becomes apparent to test our drug for its efficacy against such antibiotic-resistant genes.




Genes Antibiotic Gene Ontology
Annotations
Mechanism of Action References
glmM Ceftriaxone GO_glmM Phosphoglucosamine mutase protein Ref1_glmM
Ref2_glmM
Ref3_glmM
mshA Ceftriaxone GO_mshA D-inositol 3-phosphate glycosyltransferase CARD1_mshA
CARD2_mshA
CARD3_mshA
relE Imipenem GO_relE mRNA interferase toxin Ref1_relE
Ref2_relE
CARD_relE
tufA Imipenem GO_tufA Elongation factor Tu 1 Ref1_tufA
yafQ Ceftazidime GO_yafQ mRNA interferase toxin -
eptA Ceftazidime GO_eptA Phosphoethanolamine transferase Ref1_eptA
Ref2_eptA
CARD_eptA


glmM (Ceftriaxone)

glmM encodes for Phosphoglucosamine mutase protein (UP_glmM).

GO Molecular function: Magnesium ion binding, Phosphoglucosamine mutase activity, and Phosphomannomutase activity

GO Biological function: Carbohydrate metabolic process, Protein autophosphorylation, and UDP-N-acetylglucosamine biosynthetic process

Complete GO annotation: GO_glmM

Li et al. (Ref1_glmM) analyzed carbapenem-resistant clinical A. baumannii strains. They identified several AbaR resistance islands for a better understanding of evolutionary processes contributing to the emergence of carbapenem-resistant A. baumannii. As per their analysis, phosphoglucosamine mutase (GlmM) was detected in type 2, 7, and 10 AbaR islands. It is important to note that GlmM can catalyze the conversion of glucosamine-6-phosphate to glucosamine-1-phosphate, which is an essential step in the formation of the cell wall precursor UDP-N-acetylglucosamine (Ref2_glmM).

Kenyon and Hall (Ref3_glmM) analyzed the biosynthesis of extracellular polysaccharides which are major immunogenic components of the bacterial cell envelope. They further mentioned that GlmM is required for the synthesis of UDP-D-GlcpNAc.

Importance: There are several studies stating that glmM encodes for the formation of cell wall precursors, and it has been detected as one of the top features in the case of Ceftriaxone which also works with the mechanism of bacterial cell wall synthesis. So, our machine learning algorithm has identified genes involved in the pathway of antibiotics.



mshA (Ceftriaxone)

mshA encodes for D-inositol 3-phosphate glycosyltransferase (UP_mshA).

GO Molecular function: Acetylglucosaminyltransferase activity, transferring glycosyl groups

GO Biological function: Mycothiol biosynthetic process

Complete annotation: GO_mshA

It is involved in the mechanism of Acetylglucosaminyltransferase which is important for cell wall mechanism as mentioned in the case of glmM.

The Comprehensive Antibiotic Resistance Database (CARD) provides several evidences for the involvement of mshA in antibiotics targeting cell wall mechanisms. Mutations in mshA result in the inactivation of antibiotics and it works by the mechanism of antibiotic target alteration (CARD1_mshA).

As mentioned above, mshA is glycosyltransferase and is involved in the first step of mycothiol biosynthesis. This is a step that is required for growth in M. tuberculosis and resistance has been in the gene to isoniazid, which is antibiotic inhibiting mycobacterial cell wall (CARD2_mshA). Further, the mutations in mshA confer resistance to isoniazid in M. tuberculosis (CARD3_mshA).

Importance: Our machine learning approach identifies allele of mshA as one of the most important features in predicting resistance phenotype of strain, which in accordance with literature evidence related to mutations in mshA causing antibiotic resistance. Moreover, it has been detected as one of the most important genes in the case of Ceftriaxone, which also works with mechanisms of bacterial cell wall synthesis.



relE (Imipenem)

relE encodes for mRNA interferase toxin RelE (UP_relE).

GO Molecular function: DNA-binding transcription repressor activity, ribosome binding, rRNA binding

GO Biological function: Cellular response to amino acid starvation, mRNA catabolic process, negative regulation of translation

GO Cellular Component: Protein-DNA complex

Complete GO annotation: GO_relE

relE encodes for mRNA interferase, and mRNA interferases play a role in bacterial persistence to antibiotics; overexpression of this protein induces persisters resistant to ciprofloxacin and ampicillin (UP_relE, Ref1_relE).

relE is a part of type II toxin-antitoxin system relBE wherein it is toxin and relB is anti-toxin. In presence of unfavorable conditions, toxin relE sharply increases persisters (cells that neither grow nor die in the presence of bactericidal agents) and are largely responsible for high levels of biofilm tolerance to antimicrobials (CARD_relE). So it blocks the process of mRNA to protein conversion inhibiting cell growth. The increase in biofilm tolerance makes it difficult for antibiotics to reach the bacteria for necessary action.

Pourhajibagher et al. (Ref2_relE) utilized the concept of this toxin-antitoxin system, relBE, for designing Antimicrobial Photodynamic Therapy as an alternative to conventional antibiotic therapy using in-silico modeling and bioinformatics analysis.

Importance: There are several studies indicating the involvement of relE in being responsible for antibiotic resistance, so it makes it interesting to look for its relevance in the case of A. baumannii using wet-lab experiments.



tufA (Imipenem)

tufA encodes for the Elongation factor Tu 1 (UP_tufA).

GO Molecular function: GTPase activity, GTP binding

GO Biological function: Translational elongation, Response to antibiotic and Antibiotic Resistance.

Complete GO annotation: GO_tufA

The study conducted by Koenigs et al. (Ref1_tufA) showed for the first time that A. baumannii binds to host-derived plasminogen with help of the translation elongation factor Tuf as a moonlighting plasminogen-binding protein that is exposed on the outer surface of A. baumannii. This binding phenomenon is at least partly dependent on lysine residues and ionic interactions. Once bound to Tuf, plasminogen can be converted to active plasmin and proteolytically degrade fibrinogen as well as the key complement component C3b. Therefore they concluded that Tuf acts as a multifunctional protein that may contribute to the virulence of A. baumannii by aiding in dissemination and evasion of the complement system.

Importance: The results of the above study clearly indicates the importance of Tuf protein is increasing and contributing to the virulence of A. baumannii. It would be interesting to explore more about the functioning and mechanism of this protein in the context of our novel protein-based drug-using wet-lab experiments.



yafQ (Ceftazidime)

yafQ encodes for mRNA interferase toxin YafQ (UP_yafQ)

GO Molecular function: DNA binding, ribosome binding

GO Biological function: mRNA catabolic process, response to antibiotic

Complete GO annotation: GO_yafQ

yafQ is working in a similar mechanism that of relE i.e. working as a toxin-antitoxin pair. YafQ protein pairs with DinJ. which seems to play a role in biofilm formation. mRNA interferases play a role in bacterial persistence to antibiotics (UP_yafQ). Since it helps in biofilm formation and biofilm can decrease the amount of antibiotics reaching the bacterial cell, therefore it is indirectly responsible for increasing antibiotic resistance.

Importance: It has not been explored much in the literature, and it would be really interesting to explore its working in the context of A. baumannii along with relE as well.



eptA (Ceftazidime)

eptA encodes Phosphoethanolamine transferase EptA (UP_eptA).

GO Molecular function: phosphotransferase activity, sulfuric ester hydrolase activity

GO Biological function: Lipid A biosynthesis, Antibiotic Resistance

Complete GO annotation: GO_eptA

As per the Comprehensive Antibiotic Resistance Database (CARD), eptA mediates the modification Lipid A by the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) and phosphoethanolamine which results in a less negative cell membrane and decreased binding of polymyxin B. It works by the mechanism of antibiotic target alteration (CARD_eptA).

The study conducted by Gerson et al. (Ref1_eptA) concluded that mutations in eptA were associated with colistin resistance in A. baumannii. Trebosc et al. (Ref2_eptA) suggested that direct targeting of the homologous PetN transferases PmrC/EptA may have the potential to overcome colistin resistance in A. baumannii.

Importance: eptA has been known to provide resistance to polymyxin B which works with the mechanism of membrane disruption. It has been identified as one of the top genes for Ceftazidime which also works with the mechanism of cell wall synthesis. Further, it has been studied to play a role in colistin resistance which makes it very important and interesting to check for the efficacy of our novel protein-based drug against eptA.




Mechanism: Sulfamethoxazole inhibits bacterial synthesis of dihydrofolic acid by competing with para-aminobenzoic acid (PABA). Trimethoprim blocks the production of tetrahydrofolic acid from dihydrofolic acid by binding to and reversibly inhibiting the required enzyme, dihydrofolate reductase. So, in a nutshell, combinations of these drugs mainly work with folate synthesis.

In bacteria, antibacterial sulfonamides act as competitive inhibitors of the enzyme dihydropteroate synthase (DHPS), an enzyme involved in folate synthesis.



folP

folP encodes for Dihydropteroate synthase (UP_folP)

GO Molecular function: Dihydropteroate synthase activity, and metal ion binding

GO Biological function: Folate biosynthesis and Response to drug

GO Cellular Component: Cytoplasm and Cytosol

Complete GO annotation: GO_folP

The protein Dihydropteroate synthase catalyzes the condensation of para-aminobenzoate (PABA) with 6-hydroxymethyl-7,8-dihydropterin diphosphate (DHPt-PP) to form 7,8-dihydropteroate (H2Pte), the immediate precursor of folate derivatives (UP_folP).

As per the Comprehensive Antibiotic Resistance Database (CARD), point mutations in dihydropteroate synthase, folP prevent sulfonamide antibiotics from inhibiting its role in folate synthesis, thus conferring sulfonamide resistance (CARD_folP). It works with the mechanism of antibiotic target alteration. Our machine learning approach identified folP and its alleles as the topmost important features which further validate the efficacy of our algorithm.

Importance: The detection of folP in the case of antibiotics working with folate disruption by our machine learning algorithm is a very important indication for the efficacy of the approach. It would be very interesting to check for the impact of folP in establishing resistance to our novel protein-based drug.

Mechanism: Ampicillin/Sulbactam is a combination of a β-lactam antibiotic and a β-lactamase inhibitor. Ampicillin works by binding to penicillin-binding proteins (PBPs) to inhibit bacterial cell wall synthesis. Sulbactam blocks the enzyme which breaks down ampicillin and thereby allows ampicillin to attack and kill the bacteria.

Beta-lactam enzymes are produced by some bacteria that are responsible for their resistance to beta-lactam antibiotics like penicillins, cephalosporins, cephamycins, and carbapenems. These antibiotics have a common element in their molecular structure: a four-atom ring known as a beta-lactam.



bla

bla encodes for Beta-lactamase TEM

GO Molecular function: Beta-lactamase activity

GO Biological function: Beta-lactam antibiotic catabolic process, response to antibiotic and Antibiotic resistance

Complete GO annotation: GO_bla

TEM-type is the most prevalent beta-lactamases in Enterobacteriaceae; they hydrolyze the beta-lactam bond in susceptible beta-lactam antibiotics, thus conferring resistance to these antibiotics (UP_bla).

The study conducted by Subramaniyan and Sundaram (Ref1_bla) concluded the presence of bla genes in carbapenem-resistant P. aeruginosa and A. baumannii isolated from clinical settings, Intensive Care Unit (ICU). Further, the study by Kumar et al. (Ref2_bla) analyzed the carbapenem-resistant A. baumannii isolates from two tertiary care hospitals of North India and concluded that bla encoding clones. It is an important discovery especially in the context of hospital settings in India.

Importance: Our machine learning algorithm identifies bla as the most important feature, which is also in the mechanism of Ampicillin and Sulbactam. It shows the efficacy of our approach. Moreover, the above studies clearly indicate the importance of bla gene in A. baumannii and it would be surely interesting to check for its relevance in the case of our novel protein-based drug.






Correlation and mutational analysis of gene-gene pair




xerC - ssuC (Ciprofloxacin)

  1. Resistance increases with mutations in xerC
  2. Resistance increases with mutations in ssuC
  3. The increase in resistance with mutations in both genes confirm a positive correlation between them
  4. Mutations in xerC are accompanied by an increase in resistance for all of the following, ssuC_1, ssuC_2, ssuC_3, ssuC_4 and ssuC_5


puuP - astC (Levofloxacin)

  1. astC is a gene important for resistance but strains became more susceptible in presence of puuP
  2. Mutations in astC causes a decrease in resistance
  3. Mutations in astC increase resistance in presence of puuP but decrease resistance with mutations in puuP which confirms a negative correlation


emrE - folP (Gentamicin)

  1. Resistance increases with mutations in emrE
  2. Resistance increases with mutations in folP
  3. Mutations in both the genes work in tandem and increase the resistance of strains confirming a positive correlation between them


cysL- hcaR (Tobramycin)

  1. Resistance increases with mutations in cysL
  2. Resistance decreases with mutations in hcaR
  3. Mutations in hcaR increases the resistance but not when cysL is present confirming a negative correlation


esiB - cspV (Amikacin)

  1. Resistance decreases with mutations in esiB
  2. Resistance decreases with mutations in cspV
  3. Resistance in strains with cspV decreases or reaches zero with mutations in esiB confirming a positive correlation


ssuA - ssuC (Ceftriaxone)

  1. There is lack of strains having ssuA and ssuC genes without mutations
  2. Resistance increases with mutations in ssuA
  3. Resistance increases with mutations in ssuC which confirms a positive correlation between genes


tufA - tufB (Imipenem)

  1. Resistance decreases with mutations in tufA
  2. Resistance increases with mutations in tufB confirming a negative correlation between genes


aphA - fatA (Ceftazidime)

  1. Resistance increases with mutations in aphA
  2. Resistance increases with mutations in fatA which confirms a positive correlation between genes
  3. There is less number of strains having fatA gene and much more number of strains with mutations in fatA


folP - emrE

  1. Resistance increases with mutations in folP
  2. Resistance increases with mutations in emrE confirming a positive correlation between genes
  3. There is less number of strains with folP and more number of strains with mutations in folP


mobA - yddG

  1. Resistance decreases with mutations in mobA
  2. Resistance increases with mutations in yddG confirming a negative correlation between genes
  3. Increased resistance due to mutations in yddG vanished with mutations in mobA