Keywords efflux pump systems -
K. pneumoniae
- efflux pump inhibitors - antisense oligonucleotides
Introduction
Enterobacteriaceae , in particular, Klebsiella , is one of the outstanding etiological agents of significant importance in causing
nosocomial and community-associated infections in humans.[1 ] Bacteria belonging to the genus Klebsiella cause a wide range of infections such as community-acquired infections including
pneumonia, wound infections, urinary tract infections, septicemia, and gastrointestinal
diseases.[2 ] Recognized species of the genus Klebsiella include K. pneumoniae , K. oxytoca , K. terrigena , and K. planticola .[3 ]
K. pneumoniae is the principal species of the genus, gaining importance as it is developing multidrug
resistance in hospital settings similar to Acinetobacter and Pseudomonas .[4 ] The source of isolation of K. oxytoca from clinical specimens includes blood and respiratory secretions. It causes infections
in the immunocompromised individuals admitted in intensive critical care units. In
the history of therapeutics, one of the major milestones was the discovery of antibiotics,
which no doubt was effective in controlling infectious agents and considered a wonder
drug. Yet, extensive use of antibiotics in the medical, agricultural, and veterinary
sectors has led to the emergence of drug-resistant strains. Listed as one of the ESKAPE
(Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomonas aeruginosa , and Enterobacter species) pathogens, Klebsiella has gained resistance to almost all the drugs.[5 ] The possible reasons for frequent infections caused by Klebsiella in comparison to other Gram-negatives could be (a) the ability to naturally resist
the antibiotics, (b) the ability to outcompete other bacteria, (c) the potential to
withstand starvation, (d) the capability to exchange genetic material readily with
other members of the human microbiome, and (e) mobile genetic elements that encode
broad-spectrum of virulence and antibiotic genes.[6 ]
[7 ] Utmost strains of K. pneumoniae produce the capsular polysaccharide, conferring resistance to antimicrobial peptides
and phagocytosis of the host immune response. Mobile genetic elements play a crucial
role in the dissemination of several factors that play an essential role in the spread
of drug-resistant Klebsiella .[8 ]
K. pneumoniae surpasses the host immune responses via several virulence genes harbored such as
siderophores, fimbriae, adhesins, lipopolysaccharide (LPS), and outer membrane proteins
for the survival and immune evasion during infection.[5 ] The potential of the resistant bacteria and its genes to survive the hospital sewage
water treatments contribute to the spread and act as a reservoir of antimicrobial
resistance causing constant risks to humans and animals.[9 ] The bacteria can be directly transmitted via direct contact, contaminated environments,
and medical equipment are in patients affected with Klebsiella infections.
Drug Resistance
Under constant antibiotic pressure, the bacteria acquire antimicrobial resistance
genes via mutations, plasmids, and transferable genetic elements resulting in the
multidrug-resistant and extremely drug-resistant strains (XDR) possessing a “super
resistome.”[5 ] This evolution over the years has enabled the bacteria to gain resistance to all
possible antibiotics with no treatment options left behind.[10 ] The likelihood of dissemination of these drug-resistant pathogens is recognized
as a global threat. In the era of drug resistance, K. pneumoniae is one of the most alarming nosocomial pathogens associated with antibiotic resistance,
labeled as a significantly important MDR strain and listed under the ESKAPE groups
of pathogens.[11 ]
Klebsiella species harbor plasmids responsible for resistance to β-lactams, specifically extended-spectrum
cephalosporins and also carbapenems.[12 ] All these make the treatment options limited and end up in the usage of last line
of drugs like fluoroquinolones. Unfortunately, K. pneumoniae resistance to fluoroquinolones has drastically increased and has been reported in
the recent past. Resistance toward fluoroquinolones occurs due to specific mutations
in the DNA gyrase, topoisomerase IV, and overexpression of the multidrug efflux system.[13 ] The European Antimicrobial Resistance Surveillance Network data for the years 2005
to 2015 demonstrate the K. pneumoniae nonsusceptibility toward four major classes of drugs: (a) carbapenems, (b) third-generation
cephalosporins, and (c) fluoroquinolones and aminoglycosides (http://atlas.ecdc.europa.eu/public/index.aspx?Instance ). Multifactorial dissemination of the resistance genes increases the occurrence of
the MDR and XDR strains of K . pneumoniae . In this regard knowledge on various mechanisms of resistance conferred by the bacteria
to various drugs has been described in this review. However, the main focus is on
efflux-mediated resistance and alternatives to combat this particular mode of resistance
in Klebsiella.
As stated earlier, development of resistance in bacteria is an evolutionary process.
Resistance to antibiotics could be developed either by spontaneous mutations or through
the acquisition of resistance genes. Drug resistance can evolve through several mechanisms.
Two broadly categorized mechanisms are the intrinsic (innate) and the acquired resistance.
The acquired resistance may be due to the (a) acquisition of exogenous genes by conjugation
(transposons) or transformation (plasmids) and transduction (integrons and bacteriophages),
(b) mutation of cellular genes, and (c) combination of these mechanisms.[14 ]
Plasmid-Mediated Resistance
Plasmid-Mediated Resistance
In general, the plasmids are double-stranded deoxyribonucleic acid (DNA) encoding
almost 10% of the host cell chromosome.[15 ] In conferring resistance to toxic heavy metals, antimicrobial agents, and virulence
determinants, transfer of genes is very efficient in helping the bacterial cell survive
in the environment regardless of the lethal antibiotic dose.[14 ]
Transposons and Insertion Elements
Transposons and Insertion Elements
The multidrug resistance genes located in a DNA sequence can be transferred from one
plasmid to the other, or between genomes via transposon (jumping gene) systems.[16 ] The insertion (IS) elements have terminal repeat sequences and can recognize a protein
necessary to insert or remove a transposon from a specific region.[17 ] The integrons (gene capture) systems are also responsible for conferring resistance
and drive the expression of resistance genes by the use of a specific recombination
mechanism. The three main components of the integrons encoded in the conserved 5’
segment are (i) enzyme integrase (int ) that serves as a specific recombination system to insert or remove a new gene cassette,
(ii) a specific recombination site (attI site), and (iii) a promoter to initiate gene transcription. Nearly all the integrons
of class I in the 3’ conserved segment have a supplementary gene (sulI ) responsible for conferring resistance to sulphonamide.[18 ]
Mutations are the genetic changes occurring naturally, yet influence the bacteria
to sustain and grow under extreme environmental pressure (antimicrobials).[19 ] The selection of mutants involves factors such as the size of the bacterial population,
immune status of the host, presence of other microbiota.[20 ] Frequently, random mutations of the genes that encode antibiotic lytic enzymes generate
modified catalysts resulting in an increased spectrum of antibiotic resistance enzymes.
Efflux Pumps and Outer Membrane Permeability
Efflux Pumps and Outer Membrane Permeability
Besides enzymatic modifications, overexpression of the MDR efflux systems substantially
contribute to drug resistance. Efflux pumps, specifically the multidrug efflux, is
of major concern to the drug therapy as they expel a wide range of substrates and
clinically significant drugs.[21 ]
[22 ] Efflux pumps are dominant in mediating resistance to diverse classes of drugs and
biofilm structures.[23 ] The efflux pumps not only extrude the given antibiotics out of the bacterial cell
but also act as virulence factors and adaptive responses that bestow antimicrobial
resistance during infections. In brief, these efflux systems allow the microorganisms
in balancing the internal environment by extruding the toxic substances, such as metabolites,
antimicrobial agents, and quorum-sensing-regulated expression of virulence determinant.
So far, acr AB, oqx AB, and kex D efflux systems have been associated with the antibiotic resistance of K . pneumoniae. In addition to acrA B, kex D, kde A, kmr A, kpn EF, and oqx AB few other efflux genes such as Eef AB, ket M, and Cep A are also reported in K . pneumoniae .[24 ]
[25 ]
Several efflux systems have so far been functionally characterized—(a) resistance
nodulation division (RND) family (acr AB and kex D), (b) multidrug and toxic compound extrusion family (MATE) (kde A) efflux gene, (c) major facilitator superfamily (MFS) (kmr A) gene, and (d) small MDR family (kpn EF). Schematic representation of different efflux systems in Gram-negative bacteria
is shown in [Fig. 1 ].
Fig. 1 Schematic representation of different efflux systems in Gram-negative bacteria. (1)
MATE: multidrug and toxic compound extrusion superfamily, (2) SMR: small multidrug
resistance protein, (3) MFS: major facilitator superfamily, (4) ABC: ATP- binding
cassette transporter, and (5) RND: resistance nodulation division superfamily.
RND-type Efflux Pump
Reports demonstrate overexpression of the RND-type efflux pump in the clinical isolates
of K. pneumoniae . The RND type of efflux system consists of three components: inner membrane protein,
periplasmic protein, and outer membrane protein. An inner component consists of a
multisite binding pocket and recognizes several chemicals and the respective substrates.[26 ] A trimeric complex formed by the protein component gains access, binds, and finally
expels out the substrates. Besides its role in intrinsic resistance toward a wide
range of drugs, the RND efflux system facilitates the bacteria in colonizing the gastrointestinal
tract and release virulence factors.[27 ] Majority of them describe the overexpression of AcrB and OqxAB increases the opportunity
of multidrug resistance in K. pneumoniae .[28 ] AcrAB efflux system in K. pneumoniae is encoded by acrRAB operon, wherein the acr R encodes the AcrAB repressor, while the acr A and acr B encode a periplasmic lipoprotein attached to the inner membrane connecting the outer,
inner membranes, and an integral membrane protein situated in the cytoplasmic membrane.[29 ] Resistance to quinolones (ciprofloxacin and nalidixic acid) and other antibiotics
(erythromycin, cefoxitin, tigecycline, and chloramphenicol) is associated with the
multidrug efflux system AcrRAB. The efflux system is also associated with conferring
resistance to antimicrobial peptides in the lungs.[27 ]
OqxAB Efflux System
Resistance toward chloramphenicol, nalidixic acid, cefoxitin, and ciprofloxacin is
related to the OqxAB efflux system. It is a part of the rarA-oqxABR locus, wherein
RarA acts as a transcriptional regulator of oqxAB and OqxR acting as a transcriptional
repressor of oqx AB and rar A.[30 ] KpnEF efflux pump in K. pneumoniae confers resistance to several dyes, detergents, and antimicrobial compounds such
as benzalkonium chloride, ceftriaxone, chlorhexidine, acriflavine, cefepime, colistin,
erythromycin, streptomycin, rifampin, triclosan, and tetracycline.[31 ]
Other Related Efflux Systems
Other Related Efflux Systems
A subset of inner membrane proteins concerned with the MFS function as efflux pumps,
decrease the intracellular concentration of a broad spectrum of drugs and chemicals,
thus conferring resistance to the bacteria.[32 ] The efflux pump Eef AB, though not associated with the antimicrobial drug resistance, was observed to
colonize the murine digestive tract[25 ]; ket M was not significantly found related to contributing toward resistance to antibiotics.
In K. pneumoniae loss of outer membrane protein in syncronization with the acquisition of ampC β-lactamase
and new generation carbapenemase A confers resistance to carbapenems and aminoglycosides,
and reduced sensitivity toward fluoroquinolones, tetracycline, chloramphenicol, erythromycin,
trimethoprim, ethidium bromide, and meropenem.[15 ] Mutations in the quinolone resistance-determining region in K . pneumoniae and K. oxytoca are associated with the resistance phenotypes.[33 ]
Efflux Pump Inhibitors
Inhibiting these efflux pumps might seem like an effective strategy at times when
the conventional antibiotics remain no longer effective. Establishment of natural
substrates and efflux pump inhibitors (EPIs), that enable effective accumulation of
drug inside the bacterial cell, enhances the antibacterial activity. Some of the plant-derived
EPIs used are reserpine, pipeline, catechin gallates, flavonoids, and geraniol.[34 ] Frequently used synthetic efflux inhibitors to detect the efflux activity in Klebsiella pneumoniae are carbonyl cyanide-chlorophenylhydrazone (CCCP) and phenylalanine-arginine β-naphthylamide
(PAβN).[35 ] General mechanism of EPIs is depicted in [Fig. 2 ].[36 ] Many studies have demonstrated the efficacy of the efflux inhibitors in inducing
sensitivity to the bacteria, which still restores hope within us.
Fig. 2 Representation of mechanism of efflux pump inhibitor (EPI). (Modified from source
Shriram et al. 2018).
Fang et al[37 ] exposed the K. pneumoniae carbapenemase-producing tigecycline-resistant isolates to 1-(1-naphthylmethyl)-piperazine
(NMP). On exposure to EPI K. pneumoniae isolates previously resistant to multiple drugs showed an obvious decrease in the
minimum inhibitory concentration. Several other studies were conducted to evaluate
the efficacy of both natural and synthetic EPIs on MDR strains of K. pneumoniae and other clinically significant nosocomial pathogens and proved it efficacious in
inducing sensitivity toward various classes of drugs.[28 ]
[38 ]
[39 ]
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[60 ] Related results were obtained where PAβN and reserpine tested against commonly used
antimicrobial agents in the case of clinical isolates of Enterobacteriaceae .[61 ]
[62 ] Van Acker and Coenye[63 ] reported the role of PAβN, NMP, CCCP, and thioridazine in significantly reducing
the biofilm formation in E. coli , K. pneumoniae , Pseudomonas aeruginosa , Burkholderia cenocepaci , and Salmonella . Aghayan et al[64 ] studied the potential effects of beberine and palmatine, naturally derived EPIs
on the MexAB-OprM efflux system of P. aeruginosa isolated from burn infections. Rodrigues et al[65 ] suggested the use of EPI as therapeutic adjuvants to increase the efficacy of the
existing antituberculosis drugs and to accelerate the treatment efficacy. Lv et al[66 ] identified a novel RND multidrug–efflux pump gene cluster (TMexCD1-TOprJ1) in K. pneumoniae isolated from animal source. Similar efflux pump sequence was also found in P. aeruginosa , E. coli , and Salmonella , which threatens the possibility of global dissemination. EPIs against the novel
efflux system could restore the antibiotic sensitivity by decreasing the MIC of the
drugs. Grimsey et al[67 ] demonstrated the efficacy of chlorpromazine and amitriptyline as EPIs to restore
the antibacterial activity of the given antibiotics and inhibit the AcrB- mediated
efflux. Abbas et al[68 ] investigated the potential inhibition activity of novel EPI (metformin) on AcrAB
and Mdtk efflux pumps of K. pneumoniae . Their reports suggest the use of metformin over the existing EPIs ascorbic acid
and verapamil in restoring the efficacy of the antibiotics. Reza et al[69 ] reported several EPIs as effective biofilm disruptors in the ESKAPE pathogens. Seukep
et al[70 ] listed the use of numerous plant-derived secondary metabolites as nontoxic source
of EPIs that would allow the use of antibiotics that were clinically ineffective due
to resistance. Adam et al[71 ] developed six new classes of EPIs effective against E. coli AcrAB-TolC, clinical strains of A. baumannii , and K. pneumoniae.
Nevertheless, efflux pumps are a major resistant determinant in the increasing issue
of bacterial infections worldwide. Identification of small-molecule EPI in restoring
the effectiveness of the available antibiotics has restored hope. The greatest challenge
in the generation of active EPI against Gram-negative bacteria, is that the penetration
is controlled by porins, favorable to only zwitterionic and smaller hydrophobic molecules.
On the other hand, major RND efflux pumps prefer large hydrophobic molecules.[72 ] In this instance, penetration into the bacteria could be improved by identification
of suitable position on the EPI, by rational design by which the charge groups could
be attached enhancing the EPI penetration. A group of pyranopyridine derivatives has
been clearly shown to exhibit inhibitory effects against the AcrAB-TolC efflux system
to date. Likewise, a series of pyridopyrimidinones were proved best against MexAB-OprM.
D13–9001 was a well-optimized candidate which served effectively against P. aeruginosa efflux system (MexAB-OprM), but could not continue in clinical trials because it
failed to inhibit MexXY-OprM.[72 ] The PAβN-derived EPIs are currently the most studied and developed families of inhibitors
against P. aeruginosa .[72 ] To date, pyranopyridines, peptidomimetics, and indole derivatives have been patented
as promising compounds. No EPIs have been clinically approved to date, mainly because
of poor pharmacokinetic properties and low in vivo efficacy.[72 ]
Antisense Oligonucleotides
Antisense Oligonucleotides
There is always a quest for an alternative therapeutic strategy in combating drug
resistance conferred by efflux pumps in drug-resistant pathogens. In the quest, the
antisense therapeutics contribute as an alternative antibiotic therapy with the aid
of short single-stranded nucleic acid sequences modified to form stable oligomers.
The molecules are termed antisense oligonucleotides (ASOs) due to their sequence complementarity
to their messenger RNA (mRNA). ASOs modify the gene expression in a sequence-specific
manner by binding to its complementary mRNA and thus inhibit its translation into
protein.[72 ] Zamenick and Stephenson, pioneers in the field of antisense therapy, reported in
Rou’s sarcoma virus in infected chicken embryo fibroblast cells the addition of a
13-mer oligonucleotide complementary to the repeated sequences located at the ends
of the genome inhibited their replication.[73 ] These scientists set a benchmark for the utilization of the antisense technology.
Ever since this pioneering experiment was put forth, various strategies were designed
as therapeutics effects for the treatment of diverse diseases like bacterial, viral,
genetic disorders, and cancer. Toxicity, inability to penetrate the target, and nonspecific
effects were the major challenges during the early years of antisense research which
slowed down the progress. Following which, several antisense drugs of diverse chemical
nature working through different mechanisms, including the utilization of siRNAs have
been approved for trails.
ASOs could be exploited to suppress the bacterial antibiotic resistance determinants
and increase its susceptibility. Increased antibiotic-resistant bacterial pathogens
not only affect the ability to treat infectious diseases and complicate the medical
procedure but also impose an economic burden on the health care system. ASOs technology
permits the generation of antisense drugs that either have antibiotic activity or
help to restore the ability of certain antimicrobials. Several studies have demonstrated
the therapeutic efficacy and applications of the ASOs as mechanisms of inhibition
to design therapies for bacterial infections.[74 ]
[75 ]
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[83 ] A similar strategy of ASOs could be exploited against the MDR efflux system of K. pneumoniae . Versatility and high specificity of complementarity of the antisense oligonucleotides
allow the generation of antisense drugs that disable resistance to antimicrobials
and act as adjuvants to join the armamentarium to fight multidrug resistance.
This technology holds hope in the treatment of a diverse group of diseases including
bacterial, viral, genetic disorder, and cancer. In the years to come we could witness
the antisense adjuvants become a reality.
Conclusion
Bacterial resistance to antibiotics is a critical issue and efflux systems are influential
in this phenomenon. Efflux systems are authoritative for intrinsic resistance in several
pathogens. As they are located on the plasmids, they could be acquired by other microbes.
Diverse efflux systems have been characterized, but the MDR transporters of the RND
and MFS superfamilies are the most implied in the clinical resistance of Gram-negative
bacteria. EPIs for such systems have been identified. Although selectivity and stability
are the concerned drawbacks the results are yet encouraging to prospect in this field.
Future works could consider improvising the inhibitory activity of the molecules.
The antisense oligonucleotide is a promising alternative strategy with potential therapeutic
applications in the field of medicine and industrial microbiology.
