Role of Novel Multidrug Efflux Pump Involved in Drug Resistance in Klebsiella pneumoniae

Background

Multidrug resistant Klebsiella pneumoniae have caused major therapeutic problems worldwide due to the emergence of the extended-spectrum β-lactamase producing strains. Although there are >10 major facilitator super family (MFS) efflux pumps annotated in the genome sequence of the K. pneumoniae bacillus, apparently less is known about their physiological relevance.

Principal Findings

Insertional inactivation of kpnGH resulting in increased susceptibility to antibiotics such as azithromycin, ceftazidime, ciprofloxacin, ertapenem, erythromycin, gentamicin, imipenem, ticarcillin, norfloxacin, polymyxin-B, piperacillin, spectinomycin, tobramycin and streptomycin, including dyes and detergents such as ethidium bromide, acriflavine, deoxycholate, sodium dodecyl sulphate, and disinfectants benzalkonium chloride, chlorhexidine and triclosan signifies the wide substrate specificity of the transporter in K. pneumoniae. Growth inactivation and direct fluorimetric efflux assays provide evidence that kpnGH mediates antimicrobial resistance by active extrusion in K. pneumoniae. The kpnGH isogenic mutant displayed decreased tolerance to cell envelope stressors emphasizing its added role in K. pneumoniae physiology.

Conclusions and Significance

The MFS efflux pump KpnGH involves in crucial physiological functions besides being an intrinsic resistance determinant in K. pneumoniae.     

Sequencing and Genetic Variation of Multidrug Resistance Plasmids in Klebsiella pneumoniae

Background

The development of multidrug resistance is a major problem in the treatment of pathogenic microorganisms by distinct antimicrobial agents. Characterizing the genetic variation among plasmids from different bacterial species or strains is a key step towards understanding the mechanism of virulence and their evolution.

Results

We applied a deep sequencing approach to 206 clinical strains of Klebsiella pneumoniae collected from 2002 to 2008 to understand the genetic variation of multidrug resistance plasmids, and to reveal the dynamic change of drug resistance over time. First, we sequenced three plasmids (70 Kb, 94 Kb, and 147 Kb) from a clonal strain of K. pneumoniae using Sanger sequencing. Using the Illumina sequencing technology, we obtained more than 17 million of short reads from two pooled plasmid samples. We mapped these short reads to the three reference plasmid sequences, and identified a large number of single nucleotide polymorphisms (SNPs) in these pooled plasmids. Many of these SNPs are present in drug-resistance genes. We also found that a significant fraction of short reads could not be mapped to the reference sequences, indicating a high degree of genetic variation among the collection of K. pneumoniae isolates. Moreover, we identified that plasmid conjugative transfer genes and antibiotic resistance genes are more likely to suffer from positive selection, as indicated by the elevated rates of nonsynonymous substitution.

Conclusion

These data represent the first large-scale study of genetic variation in multidrug resistance plasmids and provide insight into the mechanisms of plasmid diversification and the genetic basis of antibiotic resistance.     

Identification of Antigenic Proteins of the Nosocomial Pathogen Klebsiella pneumoniae

The continuous expansion of nosocomial infections around the globe has become a precarious situation. Key challenges include mounting dissemination of multiple resistances to antibiotics, the easy transmission and the growing mortality rates of hospital-acquired bacterial diseases. Thus, new ways to rapidly detect these infections are vital. Consequently, researchers around the globe pursue innovative approaches for point-of-care devices. In many cases the specific interaction of an antigen and a corresponding antibody is pivotal. However, the knowledge about suitable antigens is lacking. The aim of this study was to identify novel antigens as specific diagnostic markers. Additionally, these proteins might be aptly used for the generation of vaccines to improve current treatment options. Hence, a cDNA-based expression library was constructed and screened via microarrays to detect novel antigens of Klebsiella pneumoniae, a prominent agent of nosocomial infections well-known for its extensive antibiotics resistance, especially by extended-spectrum beta-lactamases (ESBL). After screening 1536 clones, 14 previously unknown immunogenic proteins were identified. Subsequently, each protein was expressed in full-length and its immunodominant character examined by ELISA and microarray analyses. Consequently, six proteins were selected for epitope mapping and three thereof possessed linear epitopes. After specificity analysis, homology survey and 3d structural modelling, one epitope sequence GAVVALSTTFA of KPN_00363, an ion channel protein, was identified harboring specificity for K. pneumoniae. The remaining epitopes showed ambiguous results regarding the specificity for K. pneumoniae. The approach adopted herein has been successfully utilized to discover novel antigens of Campylobacter jejuni and Salmonella enterica antigens before. Now, we have transferred this knowledge to the key nosocomial agent, K. pneumoniae. By identifying several novel antigens and their linear epitope sites, we have paved the way for crucial future research and applications including the design of point-of-care devices, vaccine development and serological screenings for a highly relevant nosocomial pathogen.     

Glycerol Dehydrogenase Plays a Dual Role in Glycerol Metabolism and 2,3-Butanediol Formation in Klebsiella pneumoniae

Glycerol dehydrogenase (GDH) is an important polyol dehydrogenase for glycerol metabolism in diverse microorganisms and for value-added utilization of glycerol in the industry. Two GDHs from Klebsiella pneumoniae, DhaD and GldA, were expressed in Escherichia coli, purified and characterized for substrate specificity and kinetic parameters. Both DhaD and GldA could catalyze the interconversion of (3R)-acetoin/(2R,3R)-2,3-butanediol or (3S)-acetoin/meso-2,3-butanediol, in addition to glycerol oxidation. Although purified GldA appeared more active than DhaD, in vivo inactivation and quantitation of their respective mRNAs indicate that dhaD is highly induced by glycerol and plays a dual role in glycerol metabolism and 2,3-butanediol formation. Complementation in K. pneumoniae further confirmed the dual role of DhaD. Promiscuity of DhaD may have vital physiological consequences for K. pneumoniae growing on glycerol, which include balancing the intracellular NADH/NAD⁺ ratio, preventing acidification, and storing carbon and energy. According to the kinetic response of DhaD to modified NADH concentrations, DhaD appears to show positive homotropic interaction with NADH, suggesting that the physiological role could be regulated by intracellular NADH levels. The co-existence of two functional GDH enzymes might be due to a gene duplication event. We propose that whereas DhaD is specialized for glycerol utilization, GldA plays a role in backup compensation and can turn into a more proficient catalyst to promote a survival advantage to the organism. Revelation of the dual role of DhaD could further the understanding of mechanisms responsible for enzyme evolution through promiscuity, and guide metabolic engineering methods of glycerol metabolism.     

Role of Efflux Pumps in Adaptation and Resistance of Listeria monocytogenes to Benzalkonium Chloride

In this study, potential mechanisms underlying resistance and adaptation to benzalkonium chloride (BC) in Listeria monocytogenes were investigated. Two groups of strains were studied. The first group consisted of strains naturally sensitive to BC which could be adapted to BC. The second group consisted of naturally resistant strains. For all adapted isolates, there was a correlation between the resistance to BC and ethidium bromide, but this was not the case for the naturally resistant isolates. To investigate the role of efflux pumps in adaptation or resistance, reserpine, an efflux pump inhibitor, was added to the strains. Addition of reserpine to the sensitive and adapted strains resulted in a decrease in the MIC for BC, whereas no such decrease was observed for the resistant strains, indicating that efflux pumps played no role in the innate resistance of certain strains of L. monocytogenes to this compound. Two efflux pumps (MdrL and Lde) have been described in L. monocytogenes. Studies showed low and intermediate levels of expression of the genes encoding the efflux pumps for two selected resistant strains, H7764 and H7962, respectively. Adaptation to BC of sensitive isolates of L. monocytogenes resulted in significant increases in expression of mdrl (P < 0.05), but no such increase was observed for lde for two adapted strains of L. monocytogenes, LJH 381 (P = 0.91) and C719 (P = 0.11). This indicates that the efflux pump Mdrl is at least partly responsible for the adaptation to BC.     

Characterization of MATE-Type Multidrug Efflux Pumps from Klebsiella pneumoniae MGH78578

We previously described the cloning of genes related to drug resistance from Klebsiella pneumoniae MGH78578. Of these, we identified a putative gene encoding a MATE-type multidrug efflux pump, and named it ketM. Escherichia coli KAM32 possessing ketM on a plasmid showed increased minimum inhibitory concentrations for norfloxacin, ciprofloxacin, cefotaxime, acriflavine, Hoechst 33342, and 4'',6-diamidino-2-phenyl indole (DAPI). The active efflux of DAPI was observed in E. coli KAM32 possessing ketM on a plasmid. The expression of mRNA for ketM was observed in K. pneumoniae cells, and we subsequently disrupted ketM in K. pneumoniae ATCC10031. However, no significant changes were observed in drug resistance levels between the parental strain ATCC10031 and ketM disruptant, SKYM. Therefore, we concluded that KetM was a multidrug efflux pump, that did not significantly contribute to intrinsic resistance to antimicrobial chemicals in K. pneumoniae. MATE-type transporters are considered to be secondary transporters; therefore, we investigated the coupling cations of KetM. DAPI efflux by KetM was observed when lactate was added to produce a proton motive force, indicating that KetM effluxed substrates using a proton motive force. However, the weak efflux of DAPI by KetM was also noted when NaCl was added to the assay mixture without lactate. This result suggests that KetM may utilize proton and sodium motive forces.     

Interplay between Three RND Efflux Pumps in Doxycycline-Selected Strains of Burkholderia thailandensis

Background

Efflux systems are involved in multidrug resistance in most Gram-negative non-fermentative bacteria. We have chosen Burkholderia thailandensis to dissect the development of multidrug resistance phenotypes under antibiotic pressure.

Methodology/Principal Findings

We used doxycycline selection to obtain several resistant B. thailandensis variants. The minimal inhibitory concentrations of a large panel of structurally unrelated antibiotics were determined ± the efflux pump inhibitor phenylalanine-arginine ß-naphthylamide (PAßN). Membrane proteins were identified by proteomic method and the expressions of major efflux pumps in the doxycycline selected variants were compared to those of the parental strains by a quantitative RT-PCR analysis. Doxycycline selected variants showed a multidrug resistance in two major levels corresponding to the overproduction of two efflux pumps depending on its concentration: AmrAB-OprA and BpeEF-OprC. The study of two mutants, each lacking one of these pumps, indicated that a third pump, BpeAB-OprB, could substitute for the defective pump. Surprisingly, we observed antagonistic effects between PAßN and aminoglycosides or some ß-lactams. PAßN induced the overexpression of AmrAB-OprA and BpeAB-OprB pump genes, generating this unexpected effect.

Conclusions/Significance

These results may account for the weak activity of PAßN in some Gram-negative species. We clearly demonstrated two antagonistic effects of this molecule on bacterial cells: the blocking of antibiotic efflux and an increase in efflux pump gene expression. Thus, doxycycline is a very efficient RND efflux pump inducer and PAßN may promote the production of some efflux pumps. These results should be taken into account when considering antibiotic treatments and in future studies on efflux pump inhibitors.     

Distinct but Spatially Overlapping Intestinal Niches for Vancomycin-Resistant Enterococcus faecium and Carbapenem-Resistant Klebsiella pneumoniae

Antibiotic resistance among enterococci and γ-proteobacteria is an increasing problem in healthcare settings. Dense colonization of the gut by antibiotic-resistant bacteria facilitates their spread between patients and also leads to bloodstream and other systemic infections. Antibiotic-mediated destruction of the intestinal microbiota and consequent loss of colonization resistance are critical factors leading to persistence and spread of antibiotic-resistant bacteria. The mechanisms underlying microbiota-mediated colonization resistance remain incompletely defined and are likely distinct for different antibiotic-resistant bacterial species. It is unclear whether enterococci or γ-proteobacteria, upon expanding to high density in the gut, confer colonization resistance against competing bacterial species. Herein, we demonstrate that dense intestinal colonization with vancomycin-resistant Enterococcus faecium (VRE) does not reduce in vivo growth of carbapenem-resistant Klebsiella pneumoniae. Reciprocally, K. pneumoniae does not impair intestinal colonization by VRE. In contrast, transplantation of a diverse fecal microbiota eliminates both VRE and K. pneumoniae from the gut. Fluorescence in situ hybridization demonstrates that VRE and K. pneumoniae localize to the same regions in the colon but differ with respect to stimulation and invasion of the colonic mucus layer. While VRE and K. pneumoniae occupy the same three-dimensional space within the gut lumen, their independent growth and persistence in the gut suggests that they reside in distinct niches that satisfy their specific in vivo metabolic needs.     

Identification of Outer Membrane and Exoproteins of Carbapenem-Resistant Multilocus Sequence Type 258 Klebsiella pneumoniae

Carbapenem-resistant Klebsiella pneumoniae strains have emerged as a cause of life-threatening infections in susceptible individuals (e.g., transplant recipients and critically ill patients). Strains classified as multilocus sequence type (ST) 258 are among the most prominent causes of carbapenem-resistant K. pneumoniae infections worldwide, but the basis for the success of this lineage remains incompletely determined. To gain a more comprehensive view of the molecules potentially involved in the success of ST258, we used a proteomics approach to identify surface-associated and culture supernatant proteins produced by ST258. Protein samples were prepared from varied culture conditions in vitro, and were analyzed by a combination of two-dimensional electrophoresis and liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). We identified a total of 193 proteins in outer membrane preparations from bacteria cultured in Luria-Bertani broth (LB) or RPMI 1640 tissue culture media (RPMI). Compared with LB, several iron-acquisition proteins, including IutA, HmuR, HmuS, CirA, FepA, FitA, FoxA, FhuD, and YfeX, were more highly expressed in RPMI. Of the 177 proteins identified in spent media, only the fimbrial subunit, MrkA, was predicted to be extracellular, a finding that suggests few proteins (or a limited quantity) are freely secreted by ST258. Notably, we discovered 203 proteins not reported in previous K. pneumoniae proteome studies. In silico modeling of proteins with unknown function revealed several proteins with beta-barrel transmembrane structures typical of porins, as well as possible host-interacting proteins. Taken together, these findings contribute several new targets for the mechanistic study of drug-resistance and pathogenesis by ST258 K. pneumoniae isolates.     

The Role of Efflux Pumps in Schistosoma mansoni Praziquantel Resistant Phenotype

Background

Schistosomiasis is a neglected disease caused by a trematode of the genus Schistosoma that is second only to malaria in public health significance in Africa, South America, and Asia. Praziquantel (PZQ) is the drug of choice to treat this disease due to its high cure rates and no significant side effects. However, in the last years increasingly cases of tolerance to PZQ have been reported, which has caused growing concerns regarding the emergency of resistance to this drug.

Methodology/Principal Findings

Here we describe the selection of a parasitic strain that has a stable resistance phenotype to PZQ. It has been reported that drug resistance in helminths might involve efflux pumps such as members of ATP-binding cassette transport proteins, including P-glycoprotein and multidrug resistance-associated protein families. Here we evaluate the role of efflux pumps in Schistosoma mansoni resistance to PZQ, by comparing the efflux pumps activity in susceptible and resistant strains. The evaluation of the efflux activity was performed by an ethidium bromide accumulation assay in presence and absence of Verapamil. The role of efflux pumps in resistance to PZQ was further investigated comparing the response of susceptible and resistant parasites in the absence and presence of different doses of Verapamil, in an ex vivo assay, and these results were further reinforced through the comparison of the expression levels of SmMDR2 RNA by RT-PCR.

Conclusions/Significance

This work strongly suggests the involvement of Pgp-like transporters SMDR2 in Praziquantel drug resistance in S. mansoni. Low doses of Verapamil successfully reverted drug resistance. Our results might give an indication that a combination therapy with PZQ and natural or synthetic Pgp modulators can be an effective strategy for the treatment of confirmed cases of resistance to PZQ in S. mansoni.     

First Emergence of acrAB and oqxAB Mediated Tigecycline Resistance in Clinical Isolates of Klebsiella pneumoniae Pre-Dating the Use of Tigecycline in a Chinese Hospital

Tigecycline is one of the few therapeutic options for treating infections caused by some multi-drug resistant pathogens, such as Klebsiella pneumoniae. However, tigecycline-resistant K. pneumoniae has been discovered recently in China. From 2009 to 2013, nine tigecycline-resistant K. pneumoniae isolates were identified in our hospital. Six of nine strains were identified before using tigecycline. To investigate the efflux-mediated resistance mechanisms of K. pneumoniae, the expression of efflux pump genes (acrA, acrB, tolC, oqxA and oqxB) and pump regulators (acrR, marA, soxS, rarA, rob and ramA) were examined by real-time RT-PCR. Molecular typing of the tigecycline resistant strains was performed. ST11 was the predominant clone of K. pneumoniae strains, while ST1414 and ST1415 were novel STs. Efflux pump inhibitor (EPI)-carbonyl cyanide chlorophenylhydrazone (CCCP) was able to reverse the resistance patterns of 5 resistant K. pneumoniae strains. In comparison with strain A111, a tigecycline-susceptible strain (negative control), we found that the expression levels of efflux pump genes and pump regulators were higher in a majority of resistant strains. Higher expression levels of regulators rarA (2.41-fold, 9.55-fold, 28.44-fold and 18.31-fold, respectively) and pump gene oqxB (3.87-fold, 31.96-fold, 50.61-fold and 29.45-fold, respectively) were observed in four tigecycline resistant strains (A363, A361, A368, A373, respectively). Increased expression of acrB was associated with ramA and marA expression. To our knowledge, studies on tigecycline resistance mechanism in K. pneumoniae are limited especially in China. In our study, we found that both efflux pump AcrAB-TolC and OqxAB contributed to tigecycline resistance in K. pneumoniae isolates.     

Resistance to Mucosal Lysozyme Compensates for the Fitness Deficit of Peptidoglycan Modifications by Streptococcus pneumoniae

The abundance of lysozyme on mucosal surfaces suggests that successful colonizers must be able to evade its antimicrobial effects. Lysozyme has a muramidase activity that hydrolyzes bacterial peptidoglycan and a non-muramidase activity attributable to its function as a cationic antimicrobial peptide. Two enzymes (PgdA, a N-acetylglucosamine deacetylase, and Adr, an O-acetyl transferase) that modify different sites on the peptidoglycan of Streptococcus pneumoniae have been implicated in its resistance to lysozyme in vitro. Here we show that the antimicrobial effect of human lysozyme is due to its muramidase activity and that both peptidoglycan modifications are required for full resistance by pneumococci. To examine the contribution of lysozyme and peptidoglycan modifications during colonization of the upper respiratory tract, competition experiments were performed with wild-type and pgdAadr mutant pneumococci in lysozyme M-sufficient (LysM^+/+) and -deficient (LysM^−/−) mice. The wild-type strain out-competed the double mutant in LysM^+/+, but not LysM^−/− mice, indicating the importance of resistance to the muramidase activity of lysozyme during mucosal colonization. In contrast, strains containing single mutations in either pgdA or adr prevailed over the wild-type strain in both LysM^+/+ and LysM^−/− mice. Our findings demonstrate that individual peptidoglycan modifications diminish fitness during colonization. The competitive advantage of wild-type pneumococci in LysM^+/+ but not LysM^−/− mice suggests that the combination of peptidoglycan modifications reduces overall fitness, but that this is outweighed by the benefits of resistance to the peptidoglycan degrading activity of lysozyme.     

Genomic Epidemiology of an Endoscope-Associated Outbreak of Klebsiella pneumoniae Carbapenemase (KPC)-Producing K. pneumoniae

Increased incidence of infections due to Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) was noted among patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) at a single hospital. An epidemiologic investigation identified KPC-Kp and non-KPC-producing, extended-spectrum β-lactamase (ESBL)-producing Kp in cultures from 2 endoscopes. Genotyping was performed on patient and endoscope isolates to characterize the microbial genomics of the outbreak. Genetic similarity of 51 Kp isolates from 37 patients and 3 endoscopes was assessed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Five patient and 2 endoscope isolates underwent whole genome sequencing (WGS). Two KPC-encoding plasmids were characterized by single molecule, real-time sequencing. Plasmid diversity was assessed by endonuclease digestion. Genomic and epidemiologic data were used in conjunction to investigate the outbreak source. Two clusters of Kp patient isolates were genetically related to endoscope isolates by PFGE. A subset of patient isolates were collected post-ERCP, suggesting ERCP endoscopes as a possible source. A phylogeny of 7 Kp genomes from patient and endoscope isolates supported ERCP as a potential source of transmission. Differences in gene content defined 5 ST258 subclades and identified 2 of the subclades as outbreak-associated. A novel KPC-encoding plasmid, pKp28 helped define and track one endoscope-associated ST258 subclade. WGS demonstrated high genetic relatedness of patient and ERCP endoscope isolates suggesting ERCP-associated transmission of ST258 KPC-Kp. Gene and plasmid content discriminated the outbreak from endemic ST258 populations and assisted with the molecular epidemiologic investigation of an extended KPC-Kp outbreak.     

Toward Repurposing Ciclopirox as an Antibiotic against Drug-Resistant Acinetobacter baumannii,Escherichia coli,and Klebsiella pneumoniae

Antibiotic-resistant infections caused by gram-negative bacteria are a major healthcare concern. Repurposing drugs circumvents the time and money limitations associated with developing new antimicrobial agents needed to combat these antibiotic-resistant infections. Here we identified the off-patent antifungal agent, ciclopirox, as a candidate to repurpose for antibiotic use. To test the efficacy of ciclopirox against antibiotic-resistant pathogens, we used a curated collection of Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae clinical isolates that are representative of known antibiotic resistance phenotypes. We found that ciclopirox, at 5–15 µg/ml concentrations, inhibited bacterial growth regardless of the antibiotic resistance status. At these same concentrations, ciclopirox reduced growth of Pseudomonas aeruginosa clinical isolates, but some of these pathogens required higher ciclopirox concentrations to completely block growth. To determine how ciclopirox inhibits bacterial growth, we performed an overexpression screen in E. coli. This screen revealed that galE, which encodes UDP-glucose 4-epimerase, rescued bacterial growth at otherwise restrictive ciclopirox concentrations. We found that ciclopirox does not inhibit epimerization of UDP-galactose by purified E. coli GalE; however, ΔgalU, ΔgalE, ΔrfaI, or ΔrfaB mutant strains all have lower ciclopirox minimum inhibitory concentrations than the parent strain. The galU, galE, rfaI, and rfaB genes all encode enzymes that use UDP-galactose or UDP-glucose for galactose metabolism and lipopolysaccharide (LPS) biosynthesis. Indeed, we found that ciclopirox altered LPS composition of an E. coli clinical isolate. Taken together, our data demonstrate that ciclopirox affects galactose metabolism and LPS biosynthesis, two pathways important for bacterial growth and virulence. The lack of any reported fungal resistance to ciclopirox in over twenty years of use in the clinic, its excellent safety profiles, novel target(s), and efficacy, make ciclopirox a promising potential antimicrobial agent to use against multidrug-resistant problematic gram-negative pathogens.