Alterations in Outer Membrane Permeability Favor Drug-Resistant Phenotype of Klebsiella pneumoniae

Abstract

The increasing number of infections caused by multidrug-resistant and pandrug-resistant bacteria represents a serious worlwide problem. Drug resistance limits available antimicrobials and can lead to suboptimal treatment of bacterial infections. It can be predicted that resistance to more antimicrobial drugs will be acquired by even more bacteria in the future. Among the distinct resistance strategies, preventing drug entrance to intracellular compartment through modification of membrane permeability (bacterial influx) and active export of compounds to the external environment (bacterial efflux) are of particular importance as they limit the interaction of the drug with its intracellular targets and, consequently, its deleterious effects on the cell. Several current studies have extended our understanding of drug resistance mechanisms associated with altered membrane permeability in gram-negative bacteria. In this study, we propose a summary of resistance mechanisms associated with transport of drugs across bacterial cell envelope exploited by Klebsiella pneumoniae, one of the most common nosocomial infection-causing pathogens. The better understanding of molecular bases of drug transport in/out of the single cell may have consequence for success in antimicrobial therapy of infection caused by drug-resistant Klebsiella.

Introduction

The outbreaks of infections due to gram-negative bacteria resistant to many antibiotic classes have increased in recent years, and several definitions of multidrug-resistant (MDR), extensively drug-resistant, and pandrug-resistant (PDR) bacteria have been formulated, which complicated reliable collection and comparison of epidemiological surveillance data. The harmonized definition of MDR in Enterobacteriacae for public health use and epidemiological studies has been formulated by a group of international scientists through the European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC). MDR can be defined as resistance to at least one agent in three or more antimicrobial categories.¹

Gram-negative bacteria are more likely to become drug resistant due to their sophisticated multilayered cell envelope. Alterations in membrane transport affecting the drug influx and efflux limit the interaction between the drug and its intracellular target molecules and represent the intrinsic mechanism of drug resistance (Fig. 1).

FIG. 1.

Membrane-associated resistance patterns in gram-negative bacteria. Prevention of interaction between the drug and its target molecules in cytoplasm, usually periplasm, through downregulation of porins or loss-of-function mutations in porin genes (A) and/or reduction of inhibitory concentration of the drug through rapid removal of drug from the cytoplasm or periplasm by efflux pumps (B). Color images available online at www.liebertpub.com/mdr

From the gram-negative bacteria, detoxifying enzymes producing Klebsiella pneumoniae is of special notion due to multidrug resistance phenotype.² K. pneumoniae is a nonmotile facultative anaerobe belonging to the Enterobacteriaceae family (phylum Proteobacteria). This opportunistic pathogen causes about 8% of nosocomial infections, including community-acquired pneumonia and pyogenic liver abscesses, diarrhea, urinary tract infections, and soft tissue infections.³ K. pneumoniae isolates have been reported to be resistant to almost all classes of antibiotics through progressive mutations in chromosomally encoded genes as well as through the acquisition of genes from mobile plasmids and integrons.⁴

The significance of Klebsiella infections has arisen with outbreaks of various plasmid encoded beta-lactamases and/or carbapenemases. The resistance to beta-lactam class of antibiotics was associated with increased morbidity and mortality.⁵ Treatment of infections caused by K. pneumoniae carbapenemase (KPC)-producing Klebsiella is complicated because this organism is frequently resistant to various other families of antibiotics. Although there is no consensus as to the most effective drug intervention program for KPC-associated infections, the combination of two or more active drugs (colistin, tigecycline, gentamicin, and meropenem with MIC of less than equal to 4 mg/L) was found to decrease mortality rates.^6,7 The combination of colistin with drug chosen according to its MIC significantly improves survival compared with colistin monotherapy.⁸

Several mechanisms of drug resistance are known in bacteria: (1) drug inactivation by enzymatic degradation/modification; (2) alteration of the drug target (mutations); (3) emergence of a bypass pathway, which is not inhibited by the drug; (4) alteration of membrane influx function (reduced membrane permeability for the drug); (5) drug efflux, especially multidrug efflux from cells, or (6) promotion of resistant growth modes (biofilm formation). These mechanisms can act simultaneously in a pathogen, producing a high level of resistance.

As for most bacterial pathogens, drug resistance in Klebsiella is multifactorial and depends on the differential regulation of many resistance factors. The whole genome analysis of KPC-producing K. pneumoniae revealed the genes coding for beta-lactamases, efflux pumps (EPs), and evidenced mutations affecting the porin expression in the single isolate.⁹ Among resistance mechanisms, membrane-associated resistance patterns (decreased influx by porin loss, increased efflux by EPs) are considered to be the one of the most important contributors to antibiotic resistance of Klebsiella. Membrane transport may be responsible for resistance to one specific class of antibiotics or a large number of unrelated antimicrobial agents (cross-resistance).¹⁰

Bacterial Influx Associated with Drug Resistance in K. pneumoniae

The outer membrane (OM) is involved in a variety of functions: it regulates passive transport of extracellular solutes into the cell, expels the toxic compounds from intracellular space, and interacts with the life environment. Influx is largely controlled by a family of outer membrane proteins (OMPs) or porins that are represented in large amounts in the OM, where they form channels spanning the OM and allow the transport of molecules across the lipid bilayer membranes. In gram-negative bacteria, different types of porins have been characterized and classified according to their activity, their functional structure, and their regulation and expression.¹¹

Porins allow the diffusion of small hydrophilic molecules (<600 Da) and clinically significant antibiotics (beta-lactams and fluoroquinolones).¹² They serve as receptors for bacteriophages and bacteriocins and maintain the integrity of bacterial cells. As the major components of the OM, pore-forming proteins play a role in bacterial pathogenesis, such as adherence, invasion, and serum resistance.¹³ The loss of porins seems to be one of the factors contributing to antimicrobial resistance in extended-spectrum beta-lactamase (ESBL)-producing bacteria and may favor the selection of additional mechanisms of resistance.¹⁴

Porins expressed by Klebsiella under conditions present in the host body contribute to the stability of the OM¹⁵ and participate in the bacterial defense against antimicrobial substances produced by the immune system, for example, defensins and other antimicrobial peptides.¹⁶ The change in the type of expressed porin; the change in the expression level and/or modification that impairs the porin function may be the strategy to limit drug uptake by the cell (summarized in Table 1).

Table 1.

Porins of Klebsiella pneumoniae Associated with Drug Resistance

Porin loss	Increased resistance/decreased susceptibility	Reference
OmpK35	Cephamycins, oxyimino-cephalosporins, zwitterionic cephalosporins, and imipenem	¹⁴
OmpK36	Cefoxitin, oxyimino-cephalosporins, zwitterionic cephalosporins, carbapenem, and fluoroquinolones	^22,23
LamB	Cefepime, piperacillin–tazobactam, cefotaxime, imipenem, meropenem, and ertapenem	²⁶
PhoE	Carbapenems	⁴
KpnO	Ceftazidime, cefepime, ceftriaxone, tobramycin, streptomycin, spectinomycin, and nalidixic acid tetracycline	²⁸

Klebsiella produces two major nonspecific porins, OmpK35 and OmpK36, and the quiescent porin, OmpK37. OmpK35 is the homolog of OmpF porin (slightly larger functional pore) and Ompk36 is homologous to OmpC porin (smaller functional pore) of Escherichia coli. Both are termed as classical porins and their molecular mass may vary among different Klebsiella isolates.¹⁷ OmpK37 is a small porin related to OmpN of E. coli and is not normally expressed. Its absence in isolated membranes has never been associated with antibiotic resistance.¹⁸

Most clinical isolates of K. pneumoniae lacking ESBL express both OmpK35 and OmpK36 porins, while most K. pneumoniae ESBL producers express only OmpK36 ¹⁸ or lack both porins.¹⁹ Insertional disruption of probable promoter in the region upstream ompK35 sequence was associated with porin loss in the highly virulent strain, Kp13.⁹ Lower expression of OmpK35 in K. pneumoniae increases the resistance to cephamycins, oxyimino-cephalosporins, zwitterionic cephalosporins, and meropenem. Significant elevation in resistance was also described for imipenem, ciprofloxacin, and chloramphenicol.¹⁴

Two ompK36 insertional mutations encoding glycine and aspartic acid at aa134 and aa135 detected in KPC-producing K. pneumoniae with doripenem MIC >8 μg/ml can predict carbapenem–colistin therapy failures.^20,21 Deficiency in OmpK36 expression is related to cefoxitin resistance, increased resistance to oxyimino-cephalosporins and zwitterionic cephalosporins in ESBL-producing strains, and to carbapenem resistance in strains producing plasmid-mediated AmpC-type beta-lactamase. Lack of OmpK36 expression also results in a moderate increase in fluoroquinolone resistance in strains with altered topoisomerases and/or active efflux of quinolones.^22,23 The absence or downregulation of porins, OmpK35 and OmpK36, accompanied with production of various beta-lactamases has been implicated in carbapenem resistance.

Derivatives of ertapenem have a pleiotropic negative effect on K. pneumoniae porin expression (lack of OmpK35 and OmpK36),²⁴ which may cause phenotypic resistance to several antimicrobials. The loss of OmpK36 expression results in higher antimicrobial resistance in Klebsiella cultivated in a high-osmolarity medium (similar to body fluid) compared with the OmpK35, suggesting an important role of OmpK36 in clinical drug resistance.²⁵ The lack of OmpK35/36 expression was associated with a decrease in the virulence even in K. pneumoniae overproducing EPs.²⁴ Deletion of ompK35 and ompK36 showed, in a mouse peritonitis model, decreased virulence and slower growth rate.²⁵

A possible strategy for Klebsiella to maintain fitness following the loss of OmpK35/36 may involve a further exchange to other porins, such as OmpK37, LamB, OmpK26, PhoE, and KpnO, although their contribution to antimicrobial resistance has been poorly investigated so far.^4,26,27 The OmpK37 is small quiescent porin and its plasmid-encoded overexpression has no significant impact on antibiotic susceptibility.⁴

The LamB is involved in the transport of maltose and maltodextrins and its overexpression is associated with the deficiency of the OmpK36. LamB and OmpK36 deficiency slightly decreases the susceptibility to cefepime, piperacillin–tazobactam, cefotaxime, imipenem, meropenem, and ertapenem, suggesting that LamB may contribute to the penetration of these agents into the porin-deficient strains.²⁶ The loss of LamB expression increases carbapenem resistance.

The absence of LamB expression is associated with the expression of a 26-kDa protein further designated as OmpK26.²⁶ The OmpK26 and OmpK36 exhibit structural difference that may lead to a lower penetration of carbapenem, causing reduced susceptibility of OmpK26-producing OmpK36-deficient strains. OmpK26 is the first porin of Klebsiella that may form an alternative pore for the uptake of nutrients and allows the resistance to carbapenems in the absence of major nonspecific porins and thus could maintain the fitness of pathogen.²⁷

The physiological function of alternative porin PhoE is uptake of inorganic phosphate, phosphorylated compounds, and some other negatively charged solutes. The analysis of OMPs of carbapenem-susceptible Klebsiella strain showed the presence of phosphate-regulated porin PhoE. It can be concluded that the loss of PhoE expression may act as one of the carbapenem resistance mechanisms.⁴ KpnO protein is inevitable for export of high-molecular-weight polysaccharides to the bacterial surface to form the capsule.²⁸ Loss of PhoBR-regulated porin KpnO resulted in increased antimicrobial resistance not only to beta-lactams (ceftazidime, cefepime, ceftriaxone) but also to aminoglycosides (tobramycin, streptomycin, spectinomycin), nalidixic acid, and tetracycline.²⁸

Klebsiella may express additional porins, such as OmpN, OmpW, OmpK17, and OprD, which may be essential for normal cellular function in the absence of major porins OmpK36/35.

The exact role of porins in antimicrobial resistance is difficult to determine because other mechanisms (production of beta-lactamases and aminoglycoside-modifying enzymes, modified topoisomerases, or energy-dependent efflux systems) are commonly present in the cell at the same time. It can be concluded that variation in expression levels of porins increases antimicrobial resistance of Klebsiella ²⁹ and contributes to cross-resistance through cooperation with other mechanisms.

Bacterial Efflux

The active bacterial efflux is relevant to adaptation and survival of the cell in the environment and represents an important mechanism to withstand antibiotics through limiting the drug accumulation inside the cell. The EPs are involved in intrinsic and acquired resistance to antibiotics in both gram-positive and gram-negative bacteria.^24,30,31 Contribution of efflux systems to clinically important antibiotic resistance has been described in Campylobacter jejuni (CmeABC),³² E. coli (AcrAB-TolC, AcrEF-TolC, EmrB, and EmrD),^33–35 Pseudomonas aeruginosa (MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM),^31,36 Streptococcus pneumoniae (PmrA and PatAB),^37,38 Salmonella typhimurium (AcrAB, D, and F),³⁹ Staphylococcus aureus (NorA),⁴⁰ and Acinetobacter baumannii (AdeABC, AdeFGH, CraA, AmvA, AbeM, and AbeS).⁴¹

There are two mechanisms by which EPs would prepare to expel toxic compounds: (1) an overexpression of these EPs to handle the increasing concentration of drug inside the cell and/or (2) the pumps accumulate mutations for expelling the drug more efficiently.⁴²

Some bacterial EPs may be selective for one substrate or transport antibiotics of different classes, conferring an MDR phenotype. Moreover, exposure to any compound belonging to a substrate profile can cause overexpression of specific EPs, which leads to cross-resistance to all other EP substrates (efflux-related resistance).⁴²

Antimicrobials are not only substrates but can also act as EP inductor, thus allowing the cell to respond quickly to deleterious environmental substances. An apparently susceptible strain can overexpress a pump, decrease concentration of toxic compound in intracellular compartment, and become resistant. Overexpression of EPs alone often does not confer high-level, clinically significant antibiotic resistance. However, such bacteria are better equipped to survive antibiotic pressure for a longer time period and evolve further mutations in genes encoding the target proteins (e.g., gyrase and topoisomerase).⁴³ Significant increase in antimicrobial resistance in E. coli has also been seen after parallel overexpression of more than one EP.⁴⁴

Efflux Associated with Drug Resistance in K. pneumoniae

Based on the results of whole genome sequencing, it can be estimated that there would be more than 30 genes or operons for MDR EPs in the chromosome of Klebsiella.⁴⁵ MDR EPs in Klebsiella belong to the resistance–nodulation–division (RND), major facilitator superfamily (MFS), small multidrug resistance (SMR), and multidrug and toxin extrusion (MATE) family (summarized in Table 2).

Table 2.

Efflux Pump of K. pneumoniae Associated with Drug Resistance

Family of EPs	Components	Increased resistance/decreased susceptibility	Reference
RND	AcrAB, TolC	Fluoroquinolones, erythromycin, tetracycline, chloramphenicol, macrolides, and β-lactams	^50–55
	AcrAB, KocC	Erythromycin	⁵⁶
	KexD	Macrolides	⁵⁷
	OqxAB	Fluoroquinolones	⁶²
SMR	KpnEF	Cefepime, ceftriaxone, colistin, erythromycin, rifampin, tetracycline, and streptomycin	⁶⁶
MFS	KmrA	Norfloxacin, kanamycin	⁶⁷
	KpnGH	Azithromycin, ceftazidime, ciprofloxacin, ertapenem erythromycin, gentamicin, imipenem, ticarcillin, norfloxacin, polymyxin-B, piperacillin, spectinomycin, tobramycin, and streptomycin	⁶⁸

EPs, efflux pumps; MFS, major facilitator superfamily; RND, resistance–nodulation–division; SMR, small multidrug resistance.

The EPs belonging to RND family are major determinants of intrinsic and acquired antimicrobial resistance in gram-negative bacteria. The most extensively studied example for this kind of pump is the Mex system in P. aeruginosa and Acr system in Enterobacteriaceae. Among EP families, RND-type EPs are recognized to play the most important role in the antibiotic resistance in Klebsiella. RND type EPs function as a complex with an inner membrane protein, an OM channel, and a periplasmic (linker) protein. It is thought that these three components form a tripartite EP spanning both the inner and OMs.⁴⁶ TolC and AcrA are multicompatible molecules and it is known that these proteins are able to function with other RND and MFS drug EPs.^47,48

The most extensively studied efflux system to date is the main multitransporter system AcrAB pump associated with the OM protein TolC. AcrAB pump is present in various bacterial species belonging to the family Enterobacteriaceae with a high degree of homology between pump genes (>70% identity) and amino acid sequences (>80% similarity) of pump proteins both within and across different bacterial species.⁴⁹ The AcrAB-TolC pump complex in Klebsiella is a clinically relevant efflux system, which extrudes compounds in an energy-dependent manner (proton antiporters). The substrates of AcrAB may have very diverse structure (carrying either negative or positive charges) and the only requirement for successful transport of drug is the presence of a hydrophobic domain that inserts into the phospholipid bilayer.

Overexpression of AcrAB system has been associated with fluoroquinolone resistance in clinical isolates of K. pneumoniae⁵⁰ and also plays a role in beta-lactam resistance and resistance to tigecycline.^51–54 A study by Padilla et al.⁵⁵ showed that acrB deficiency increased susceptibility to erythromycin, tetracycline, chloramphenicol and aminoglycoside, bronchoalveolar lavage fluid, and antimicrobial peptides. The lower bacterial loads in the lungs of the mice infected with acrB knockout compared with wild-type strain support the theory that AcrAB-TolC system is involved not only in drug resistance but also in virulence.⁵⁵ It was shown that AcrAB utilize KocC as an OM component to extrude erythromycin from the cell. Protein KocC has an identical amino acid sequence of AcrB interaction site as TolC protein and it is possible that AcrAB-KocC can act as MDR EP.⁵⁶

KexD is RND multidrug EP and can recognize a wide range of substrates. The KexD functions with a periplasmic protein, AcrA, from E. coli and K. pneumoniae, but not with the periplasmic proteins, KexA and KexG, from K. pneumoniae. KexD was able to utilize either TolC of E. coli or KocC of Klebsiella as an OM component.⁵⁷

The newly described OqxAB EP can also contribute to the MDR phenotype. This plasmid-coded OqxAB has been frequently detected in Enterobacteriaceae and has become increasingly prevalent among other gram-negative bacteria.⁵⁸ The oqxAB genes in K. pneumoniae are highly conserved chromosomal genes and may act as a reservoir for plasmid harboring of this resistance determinant through transposition events.^58,59 In K. pneumoniae and Enterobacter spp., the chromosomally encoded rarA regulator, and other regulators belonging to the AraC family lie downstream of the genes coding EP OqxAB.^60,61 It was concluded that OqxAB could contribute to reduced susceptibility to quinolones in K. pneumoniae⁶² and confers resistance to biocides such as triclosan and chlorhexidine.⁶³

The SMR-type pumps are small homo or heterodimers represented by the archetype protein, EmrE.⁶⁴ SMR pumps export molecules only into the periplasmic space from where it can be taken up by membrane-spanning transporters such as AcrAB-TolC.^44,65 The substrate specificity of SMR pumps is not limited to cationic hydrophobic substrates and can extend to antibacterial compounds.

The putative SMR-type EP KpnEF (EbrAB homolog of Bacillus subtilis) confers the multidrug-resistant phenotype in Klebsiella. The KpnEF plays a role in survival in hyperosmotic environment, resistance to high bile concentrations, and it is involved in capsule synthesis. Functional KpnEF reduces susceptibility to cefepime, ceftriaxone, colistin, erythromycin, rifampin, tetracycline, and streptomycin and toward structurally related compounds such as sodium dodecyl sulfate, deoxycholate, and dyes and disinfectants such as benzalkonium chloride, chlorhexidine, and triclosan. The kpnEF gene is a member of the Cpx regulon and its prevalence in clinical isolates broadens the diversity of antibiotic resistance tools.⁶⁶

Although there are >10 MFS EPs annotated in the genome of the K. pneumoniae, very little is known about their contribution in antimicrobial resistance. KmrA belongs to the MFS family and exhibits 94% similarity with SmvA EP of Salmonella enterica. It is a chromosomally encoded multidrug EP with high extrusion activity and wide substrate specificity.⁶⁷ A novel two-component MFS-type EP KpnGH identified by genome study mediates tolerance to different classes of antimicrobials, disinfectants, and bile salts. The absence of KpnGH reduces the bacterial growth and survival in hyperosmotic and microaerobic conditions in vitro.⁶⁸

MATE pumps have structural similarity with MFS and RND pumps. A key distinguishing feature is that while RND pumps are tripartite, MATE pumps exist as a single efflux protein. Two energy sources have been identified for MATE EPs: the proton motive force and the sodium ion gradient. MATE pumps transport some of those agents also transported by RND pumps. MATE pumps in Klebsiella have not been extensively characterized yet. Protein KetM of K. pneumoniae, belonging to cluster 1 of MATE-type pumps, is very similar to NorM from Vibrio parahaemolyticus and YdhE from E. coli. Cloning of ketM in E. coli increased MIC for norfloxacin, ciprofloxacin, and cefotaxime.⁶⁹

ATP binding cassette (ABC) transporters, also called traffic ATPases, have structural characteristics that differ from RND and MFS pump proteins. This family is of bacteria comprising two major groups: bacterial importers and exporters. ABC transporters were found to be present in the genomes of various pathogenic bacteria, where they are employed in trafficking of molecules synthetized in cytoplasm across the membrane. However, no ABC transporters responsible for clinically relevant MDR in Klebsiella have been identified so far. It may be hypothesized that at least one will respond to antimicrobial resistance in the same way as P glycoprotein that confers resistance to anticancer agents. It has already been shown that Lactococcus lactis LmrA, an ABC transporter, confers MDR in this organism.⁷⁰ In K. pneumoniae, ABC transporters are employed in trafficking O-polysaccharide and the regulation of O-antigen.⁷¹

Conclusion

K. pneumoniae is intrinsically resistant toward many antimicrobial compounds due to highly impermeable OM. Membrane-associated transport plays the important role and it can be altered in response to environmental changes. The deficiency of porins and overexpression of EPs are the most common and a nearly universal mechanism used by bacteria to decrease the concentration of toxic compound in intracellular compartment and survive in an unfavorable environment. The situation is complicated with wide substrate specificity (antibiotics, dyes, disinfectants, and detergents). Antimicrobial compounds do not act as substrate only but they can also induce upregulation of efflux proteins and downregulation of porins.

Changes in membrane permeability alone often do not confer clinically significant antibiotic resistance; however, survival for a longer time period helps to evolve further resistance strategies, which may contribute to MDR or PDR phenotype in clinically relevant K. pneumoniae. Based on this, it can be supposed that antibiotic-sensitive Klebsiella, commonly found in the community and hospitals, might transform to exhibit a resistant phenotype when exposed to selective drug pressure.

Knowledge of molecular basis of membrane-associated transport (influx/efflux) and its specific contribution in antimicrobial resistance in a pathogen such as Klebsiella will help underpin continuing research into effective methods of control and treatment of infections.

Footnotes

Acknowledgments

The study was supported by APVV-14-0218 and VEGA 1/0258/15, 2/0261/15. L.P. was funded by ITMS 26220220185.

Disclosure Statement

No competing financial interests exist.

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