Overcoming Klebsiella pneumoniae antibiotic resistance: new insights into mechanisms and drug discovery

Background Due to antibiotic overuse, numerous organisms have evolved multidrug resistance, a global health crisis. Klebsiella pneumoniae ( K. pneumoniae ) causes respiratory, biliary, and urinary infections. It initially impacts individuals with impaired immune systems. Main body of abstract The treatment of K. pneumoniae clones poses significant challenges, highlighting the need for new, more effective pathogen control methods. Our understanding of the natural or acquired antibiotic resistance mechanisms in K. pneumoniae is inadequate. Therefore, it is crucial to continue searching for novel compounds to fight against K. pneumoniae and to understand its antibiotic resistance processes fully . Short conclusion This review covers K. pneumoniae antibiotic resistance in detail. The review also identifies the molecular components of natural or acquired K. pneumoniae antibiotic resistance mechanisms. This study further digs into the novel pharmacological targets to offer therapy insights.


Background
Klebsiella pneumoniae is an ubiquitous gram-negative facultative anaerobic bacteria [1].K. pneumoniae lives in many different environments, including the respiratory and gastrointestinal systems of humans and animals [1].K. pneumoniae infection accounts for over 95% of Klebsiella infection.K. pneumoniae infection can trigger various infections and mostly affect the individuals with weakened immune systems.This particular infection is highly prevalent in medical facilities across the globe [1].The emergence of genetically modified strains of K. pneumoniae that exhibit heightened virulence or demonstrate resistance to antimicrobial agents has become a subject of significant apprehension [1].
The global phenomenon concerning the emergence and progression of multidrug resistance (MDR) in human diseases holds significant significance within the realm of scientific investigation.MDR K. pneumoniae has become a significant concern within the healthcare system due to its ability to restrict treatment options.K. pneumoniae has two main antibiotic resistance types.The expression of extended-spectrum β-lactamase (ESBL) makes bacteria resistant to cephalosporins and monobactams [1].In addition, the presence of carbapenemases makes bacteria resistant to most β-lactams, including carbapenems [1].The aforementioned strains have resulted in an excess of 90.000 infections and 7.000 fatalities within the European region [2].
Colistin and tigecycline, both of which are used as a last-line drugs, are another option for treating CRKp [3].Last-line drugs have only been effective when used in combination with other medications, not alone.However, colistin and tigecycline use has several adverse effects, including nephrotoxicity [4].New ESBL-producing and CRKp strains are also developing resistance to the last-line antibiotics.Conventional medicines have proved to be ineffective in eradicating these pathogens.So, multidrug resistant infections have posed a significant challenge to public health and the global economy.More seriously, the quantity of antimicrobial drug development initiatives has significantly diminished since the era of remarkable antibiotic discovery due to many factors, including antibiotic resistance, lower profit margins, etc.Therefore, it is necessary to identify new drugs in order to address this pressing public health crisis.Furthermore, our comprehension of the natural or acquired antibiotic resistance of K. pneumoniae remains limited.Hence, it is of outmost significance to continuously explore novel methodologies in combating K. pneumoniae, while concurrently attaining a comprehensive comprehension of the mechanisms underlying K. pneumoniae resistance to currently available pharmaceutical interventions.This review aims to give a comprehensive overview of how K. pneumoniae resists antibiotics.The review also identifies the molecular components contributing to natural or acquired antibiotic resistance mechanism in K. pneumoniae.This research delves deeper into the new pharmacological targets, which can lead to new insights into how to treat K. pneumoniae infections.

Intrinsic resistance
Since K. pneumoniae is antibiotic-resistant, standard treatments are less effective.K. pneumoniae generates intrinsic resistance to antibiotics through the following mechanisms: enzymatic antibiotic inactivation and modification, porin loss, enhanced antibiotic efflux pump expression, and biofilm development (see Fig. 1) [5,6].Table 1 gives a summary of natural resistance mechanisms in K. pneumoniae.

Antibiotic modifying or inactivating enzyme
K. pneumoniae often develops resistance to antibiotics due to medication modification.Currently, the most usual way to treat K. pneumoniae infection in the clinic is β-lactam antibiotics.β-lactam antibiotics kill bacteria by covalently attaching to Penicillin-binding proteins (PBPs), which are necessary for peptidoglycan crosslinking formation in bacteria [9].This stops the bacteria from making cell walls.K. pneumoniae makes an enzyme called β-lactamase to get around this. β-lactamase hydrolyzes the β-loop of β-lactam.This is a key resistance mechanism.Cephalosporinases (AmpC), ESBLs, and carbapenemases are the three subtypes of β-lactamases [10][11][12][13].ESBLs are plasmid-based antibiotic resistance mechanisms.The blaSHV-2 and blaTEM-3 were found in K. Pneumoniae [14,15].They are viral carrier ESBL mutant genes [15].Clavulanic acid stops ESBL genes from working against carbapenems in a way that is called "extended-spectrum".Since plasmids and transposons that create bla CTX-M-type ESBLs are simple to obtain, it slowly superseded TEM and SHV as the primary genotype.Horizontal gene transfer also happened with bla PER, bla SFO, bla TLA, bla OXA, bla GES, bla VEB, and bla KLUC-5 [16].For ESBL-producing bacterium infections, carbapenems have long been the preferred choice [14].From first to third-generation cephalosporins, cephalomycin, and enzyme inhibitors, AmpC is resistant [12].The bacteria contains over 40 AmpC genotypes, which can swiftly disseminate among various strains via plasmid-mediated mechanisms [14].

Bacterial influx
A group of outer membrane proteins called porins is highly prevalent in the outer membrane [17].The regulation of flux is accomplished through the formation of channels that traverse the outer membrane, facilitating the transport of molecules across the lipid bilayer membranes [17].Antibiotics with paramount therapeutic importance, such as β-lactams and fluoroquinolones, can diffuse across porins thanks to the small hydrophilic molecules (<600 Da) [17].They protect bacterial cells by acting as receptors for bacteriophages and bacteriocins [18].Pore-forming proteins, which comprise the bulk of the outer membrane, contribute to bacterial pathogenicity by aiding in processes including adhesion, invasion, and resistance to serum [18].Loss of porins in ESBL-generating bacteria may contribute to antibiotic resistance and enable the selection of additional resistance mechanisms [19].Porins expressed by K. pneumoniae under hostbody conditions aid in outer membrane stability and have a role in bacterial protection against defensins and other antimicrobial peptides produced by the immune system [20].Altering the porin type, expression level, or function may limit cell drug uptake [20].The porins LamB, OmpK26, PhoE, OmpK35, OmpK36 and KpnO contributes significantly to innate drug resistance [7].

Efflux pump
Bacteria heavily depend on their efflux capability as a means of defense to adapt and ensure survival within their environment.The defense mechanism in question assumes a pivotal function in the restriction of drug buildup within bacterial cells.Membrane efflux pumps release antimicrobials from the cell into the surrounding medium, reducing the concentration of medicines inside the cell.Efflux pumps is critical to how drug resistance develops [21][22][23].Overexpression of efflux pumps to deal with a rising drug concentration within the cell and/or the accumulation of mutations in the pumps that improve their ability to expel the drug are two possible ways [24].Efflux pumps become ready to rid the body of harmful substances.Inferred from whole genome sequencing data, over 30 genes or operons for MDR efflux pumps are present on the Klebsiella chromosome [25].Klebsiella has many MDR efflux pumps comprising the major facilitator superfamily, resistance-nodulation-division, small multidrug resistance, and toxin extrusion families.[20].Bacterial innate and acquired antibiotic resistance depends on resistance-nodulation-division efflux pumps.Both TolC and AcrA are known to interact with various resistance-nodulation-division and significant facilitator superfamily pharmacological efflux pumps, demonstrating that they are multi-compatible compounds [26].TolC, an outer membrane component, is connected with the AcrAB pump, the most thoroughly investigated efflux mechanism to date.Clinically significant efflux systems in Klebsiella include the AcrAB-TolC pump complex, which is responsible for the energy-dependent expulsion of drugs [20].Since AcrAB substrates can have negative or positive charges, the protein only needs a hydrophobic region to enter the phospholipid bilayer to transport the medication [20].The active efflux system AcrAB-TolC can exocytose several medicines, such as β-lactams, macrolides, fluoroquinolones, and tetracycline [20].

Biofilm formation
K. pneumoniae exhibits a propensity for biofilm formation, with structures such as capsules and pili playing Table 1 The intrinsic antibiotic resistance mechanisms in K. pneumoniae

Resistance mechanism Antibiotic class References
Antibiotic modifying or inactivating enzyme β-Lactam [9-12, 15, 16] Bacterial influx β-lactam and fluoroquinolones [7] Efflux pump β-lactam, macrolides, fluoroquinolones, and tetracycline [7] Biofilm formation Aminoglycoside, penicillin, and quinolone [8] crucial roles in facilitating this process [21].Antibiotic penetration is hindered by biofilms because they slow bacterial growth, promote the development of persister cells, and allow for the dissemination of genetic material [21].Microorganisms that produce an exo-polymeric matrix cling to an inanimate or living surface to form a biofilm.This matrix contains polysaccharides, proteins, and extracellular DNA [22].Biofilms are characterized by osmotic barrier properties and antimicrobial drug resistance.Biofilm formation in K. pneumoniae leads to a decreasing in the gentamicin, ampicillin, and ciprofloxacin susceptibility [8].The development of biofilms and resistance to colistin have been linked [23].

Acquired resistance
The β-lactam antibiotic class is commonly used to treat K. pneumoniae infection.Other antibiotics must be used when patients are infected with multidrug-resistant or extended-duration K. pneumoniae.However, drug resistance can develop when these antibiotics are administered clinically.Table 2 gives a summary of acquired resistance mechanisms in K. pneumoniae.Instead of relying on chromosomal gene alterations, horizontally acquired accessory antimicrobial resistance (AMR) genes appear responsible for most K. pneumoniae resistance.These are often carried by plasmids, though they can be integrated into the chromosome.

Carbapenem resistance
The ESBL-producing K. pneumoniae has increased carbapenem use dramatically.They may have resulted from carbapenem selective pressure.Plasmid-regulated carbapenem enzymes remain the most worrisome multidrug resistance route.The serine-based class β-lactamase (KPC) is the most prevalent and potentially lethal carbapenemase.KPCs is associated with CG258, a clonal subgroup [24,25].Both ST258 and ST11 are widespread in Asia, but ST258 is more common in Europe and the Americas [25][26][27][28][29]. Bla KPC genes use a unique Tn4401 transposition form to transfer onto other plasmids and spread clonally [30,31].High mortality rates and difficulty treating carbapenemase-producing Enterobacteriaceae are consequences of the widespread of resistance genes of this sort.Most β-lactamase inhibitors are ineffective against KPCs, making treatment difficult.The chromosomal translocation of carbapenemase-encoding genes from plasmids complicates these resistances [32].CRKP presents significant challenges in clinical practice [14].K. pneumoniae adaptability in attaching β-lactamase genes to transportable plasmids aids in the dissemination of AmpC-like cephalosporinase in these species [33,34].The presence of bla AmpC in K. pneumoniae and gene expressing deletions or improved efflux, comparable to bla ACT-1, lead to β-lactam antibiotic resistance.Increased copies or promoter strength can easily express genes on plasmids, resulting in carbapenem resistance [33].Some K. pneumoniae strains may carry many lactamase genes, such as SHV, AmpC, KPC, and β-lactamase inhibitors.The effects of many resistance genes present in the same strain are additive.While Vim, NDM, and IMP are impossible to exhibit resistance to aztreonam, their presence can facilitate the development of such resistance in the presence of ESBL or AmpC.

Tetracycline resistance
The novel tetracycline antibiotic tigecycline shows broadspectrum efficacy against ESBL-producing bacteria [37].Antibiotic resistance is mostly caused by the Oqx-AB, Ade-ABC efflux pumps, Tet(A) mutant, KpgABC, and ribosomal protein.This antibiotic's resistance gene lies on the chromosome and alters cell permeability and ribosomal targets [14].The rpsJ gene encodes ribosomal protein S10, a 30S subunit component.It is close to the main binding site for tetracycline and tigecycline in the 30S subunit of the ribosome.Among three discovered K. pneumoniae-resistant tigecycline strains, one of them showed a point mutation in the rpsJ gene which closes the tigecycline target in the 30S subunit [38].Thus, the S10 ribosomal protein alternation may be the new mechanism.Ribosomal proteins S3, S13, and S10 lie near the tetracycline-ribosomal subunit binding domain, and S3 maintains the site's structural integrity [39].The S3 protein structural mutation may also produce tigecycline resistance.gene mutation can cause tigecycline resistance without efflux pump participation, according to studies [14].

Quinolone resistance
Quinolone antibiotics inhibit topoisomerases, which prevent bacterial DNA replication [40].Alterations in the target gene boosted MDR efflux production, and enzyme and protein alterations made K. pneumoniae fluoroquinolone-tolerant [40].With chromosomal resistance mechanisms, topoisomerase IV and DNA gyrase bind quinolones.OqxAB is present in numerous bacteria and is connected to plasmid-mediated quinolone resistance [41].Other quinolone resistance genes include the plasmid-mediated determinant in Enterobacteriaceae species.These gene protein family protects DNA gyrase and topoisomerase IV from quinolones.Quinolone modification is expected to only occur in K. pneumoniae via plasmid-mediated quinolone resistance gene aa(6')-Ibcr [42].Other antibiotics and restricted quinolones with the enzyme's substrate can also be inactivated by aa(6')-Ib-cr [42].It was also found on chromosomes recently.Plasmid-mediated quinolone resistance gene expression promotes chromosomal genetic alterations and mild to moderate quinolone resistance [41].

Other antibiotic mechanisms
Polymyxin resistance in K. pneumoniae is usually caused by mutations in regulative genes such as mgrB, which regulates bacterial lipid A, a polymyxin antibiotic target, reducing polymyxin interaction [43][44][45].K. pneumoniae BSI isolates in China rarely include mcr-1, although E. coli does.America had its first mcr-1 case in 2016 [46].
Fosfomycin, an ancient antibiotic, is now used to treat MDR bacterial infections [47].With increased fosfomycin use, resistant strains are emerging.Amino acid replacement or overexpression of MurA, deficient or reduced expression of GlpT and UhpT, and the fos gene, which encodes a glutathione S-transferase-activated enzyme that inactivates fosfomycin, constitute fosfomycin resistance mechanisms [48].The fosA3 gene has been identified as the primary determinant responsible for conferring fosfomycin resistance in carbapenem-resistant K. pneumoniae (CRKp).The horizontal transferability of this resistance mechanism observed through plasmids that are frequently encountered within hospital settings [49].The CRKp strains that exhibited resistance to fosfomycin and lacked the FosA3 gene were found to possess mutations in the MurA gene and the glpT transporter [49].

Potential drug targets of K. pneumoniae
As multidrug-resistant Klebsiella cases multiply and antimicrobial medication activity decreases, global clinical issues arise.Klebsiella infections were treated with β-lactam antibiotics, but the advent of hypervirulent and MDR forms has rendered these treatments ineffective [50].Identifying new medicines and medication combinations to treat these fatal illnesses is urgent.
Identifying pharmacological targets requires microorganism genomic data.Pathogenic microorganism entire genomes and virulence-causing genes can be sequenced first.Next, subtractive genomic methods can exclude homologous proteins and identify the pathogenic nonhomologous protein lacking in the host [51].Researchers searched for K. pneumoniae-host homologous proteins using in silico methods like Blastp and BLAT [52].Selecting non-homologous proteins not found in humans reduces cross-reactivity and adverse effects.The database of essential genes can identify pathogen-survival genes.Use the KEGG pathway database to analyze the shortlisted genes and proteins pathways [53].Most identified therapeutic target candidates with promising drug development prospects participate in important biosynthetic activities such as peptidoglycan, fatty acids, pyrimidine deoxyribonucleotides, pyrimidine deoxyribonucleotides, LPS, and purine nucleotides.
In addition to druggable, essential, and conserved enzymes, the metabolic enzymes FabB, FabI, and FabH which are involved in fatty acid biosynthesis, are interesting targets.FabI, a crucial focal point, exhibits immense potential as a target for pioneering therapeutic interventions against bacterial pathogens [54].By facilitating the incorporation of two carbon atoms derived from malonyl-acyl carrier protein (ACP) into an acyl acceptor, the enzyme FabB effectively mediates a diverse range of acyl-ACP elongation reactions within the type II fatty acid elongation cycle [55].The enzyme FabH initiates the process of fatty acid synthesis by incorporating a twocarbon acyl acceptor from malonyl-ACP into the initial condensation step [56].FabH controls total fatty acid synthesis.This enzyme acts as a synthase and transacylase for acetyl-CoA.The FabH enzyme's acetyl-CoA substrate selectivity controls straight-chain fatty acid production [57].It can utilise propionyl-CoA less efficiently.In the subsequent step, FabI enzymatically catalyzes the reduction of a carbon-carbon double bond within an enoyl moiety that is covalently linked to an acyl carrier protein [54].This process occurs within the fatty acid elongation cycle, which is crucial for lipid metabolism and biotin synthesis [54].LPS production is essential for bacterial cell wall lipid development.LpxA, LpxB, LpxC, and LpxD are potential drug targets [58,59].
MurG is a crucial component in the process of cell wall formation [60].Simultaneously, MurF actively participates in the process of cell wall biogenesis [61].Important targets, including aspartate semialdehyde dehydrogenase, synthesize L-lysine, L-threonine, L-methionine, and L-homoserine.An important membrane ATPase, SecA, powers Sec-dependent protein translocation.YajC and SecYEG form an oligomer.By helping bacteria secrete proteins, poisons, and virulence factors, SecA was crucial to their survival [62].Thus, making it an ideal antibacterial target.Since SecA is a membrane protein, developing SecA inhibitors allows easy access without entering the cytoplasm [63].
Targeting histidine kinase Bacterial two-component system receptor includes EvgS.Acid and drug resistance are linked to EvgS/EvgA bacterial two-component system receptor in E. coli [64].A slight acidity increase activates EvgS, which phosphorylates EvgA.EvgS/EvgA activation promotes several acid-resistance genes and gives bacteria acid resistance [65].Secretion of hazardous chemicals such antibiotics, dyes, disinfectants, host-produced bile, hormones, and defense compounds depends on TolC [65].TolC is involved in exporting extracellular proteins [66].QseC, a receptor belonging to the bacterial twocomponent system, presents itself as an alluring subject for further investigation.Epinephrine and norepinephrine activate this QseC kinase [67].

Conclusion
Global drug-resistant K. pneumoniae infections have increased.Previously, nothing was known about K. pneumoniae, which infects the brain, liver, circulation, lungs, and bladder.Despite its century-old discovery, the pathogen's virulence mechanisms are unknown.The host immune responses against K. pneumoniae infection is also poorly defined.Current antibiotic regimens are nearly untreatable due to the widespread of MDR and hypervirulent clones.Numerous studies have explored ESBLs, carbapenemase, or AmpC target change, porin loss and mutation, efflux pump overexpression, and horizontal diffusion of mobile gene elements in resistance mechanisms.So far, nothing is known about how biofilm development governs antibiotic resistance in K. pneumoniae.Fatty acids, LPS, peptidoglycan, pyrimidine deoxyribonucleotides, and purine nucleotides are promising targets against K. pneumonia via high-throughput analysis.These targets meet most pharmacological targeting criteria, including a host gene absence for their homolog.The chosen targets require numerous steps to design and produce inhibitors.

Table 2
The acquired antibiotic resistance mechanisms in K. pneumoniae