Phytochrome-interacting factors orchestrate hypocotyl adventitious root initiation in Arabidopsis.

Adventitious roots (ARs) are an important type of plant root and display high phenotypic plasticity in response to different environmental stimuli. It is known that photoreceptors inhibit darkness-induced hypocotyl adventitious root (HAR) formation by directly stabilizing Aux/IAA proteins. In this study, we further report that phytochrome-interacting factors (PIFs) plays a central role in HAR initiation by simultaneously inducing the expression of genes involved in auxin biosynthesis, auxin transport and the transcriptional control of root primordium initiation. We found that, on the basis of their activity downstream of phytochrome, PIFs are required for darkness-induced HAR formation. Specifically, PIFs directly bind to the promoters of some genes involved in root formation, including auxin biosynthesis genes YUCCA2 (YUC2) and YUC6, the auxin influx carrier genes AUX1 and LAX3, and the transcription factors WOX5/7 and LBD16/29, to activate their expression. These findings reveal a previously uncharacterized transcriptional regulatory network underlying HAR formation.

A genome-wide association study identifies a transporter for zinc uploading to maize kernels.

The Zn content in cereal seeds is an important trait for crop production as well as for human health. However, little is known about how Zn is loaded to plant seeds. Here, through a genome-wide association study (GWAS), we identify the Zn-NA (nicotianamine) transporter gene ZmYSL2 that is responsible for loading Zn to maize kernels. High promoter sequence variation in ZmYSL2 most likely drives the natural variation in Zn concentrations in maize kernels. ZmYSL2 is specifically localized on the plasma membrane facing the maternal tissue of the basal endosperm transfer cell layer (BETL) and functions in loading Zn-NA into the BETL. Overexpression of ZmYSL2 increases the Zn concentration in the kernels by 31.6%, which achieves the goal of Zn biofortification of maize. These findings resolve the mystery underlying the loading of Zn into plant seeds, providing an efficient strategy for breeding or engineering maize varieties with enriched Zn nutrition.

Polysaccharide-Targeting Lipid Nanoparticles to Kill Gram-Negative Bacteria.

The rapid increase and spread of Gram-negative bacteria resistant to many or all existing treatments threaten a return to the preantibiotic era. The presence of bacterial polysaccharides that impede the penetration of many antimicrobials and protect them from the innate immune system contributes to resistance and pathogenicity. No currently approved antibiotics target the polysaccharide regions of microbes. Here, describe monolaurin-based niosomes, the first lipid nanoparticles that can eliminate bacterial polysaccharides from hypervirulent Klebsiella pneumoniae, are described. Their combination with polymyxin B shows no cytotoxicity in vitro and is highly effective in combating K. pneumoniae infection in vivo. Comprehensive mechanistic studies have revealed that antimicrobial activity proceeds via a multimodal mechanism. Initially, lipid nanoparticles disrupt polysaccharides, then outer and inner membranes are destabilized and destroyed by polymyxin B, resulting in synergistic cell lysis. This novel lipidic nanoparticle system shows tremendous promise as a highly effective antimicrobial treatment targeting multidrug-resistant Gram-negative pathogens.

Correlative proteomics identify the key roles of stress tolerance strategies in Acinetobacter baumannii in response to polymyxin and human macrophages.

The opportunistic pathogen Acinetobacter baumannii possesses stress tolerance strategies against host innate immunity and antibiotic killing. However, how the host-pathogen-antibiotic interaction affects the overall molecular regulation of bacterial pathogenesis and host response remains unexplored. Here, we simultaneously investigate proteomic changes in A. baumannii and macrophages following infection in the absence or presence of the polymyxins. We discover that macrophages and polymyxins exhibit complementary effects to disarm several stress tolerance and survival strategies in A. baumannii, including oxidative stress resistance, copper tolerance, bacterial iron acquisition and stringent response regulation systems. Using the spoT mutant strains, we demonstrate that bacterial cells with defects in stringent response exhibit enhanced susceptibility to polymyxin killing and reduced survival in infected mice, compared to the wild-type strain. Together, our findings highlight that better understanding of host-pathogen-antibiotic interplay is critical for optimization of antibiotic use in patients and the discovery of new antimicrobial strategy to tackle multidrug-resistant bacterial infections.

Characterization of outer membrane vesicles released by clinical isolates of Neisseria gonorrhoeae.

The sexually transmitted pathogen Neisseria gonorrhoeae releases membrane vesicles including outer membrane vesicles (OMVs) during infections. OMVs traffic outer membrane molecules, such as the porin PorB and lipo-oligosaccharide (LOS), into host innate immune cells, eliciting programmed cell death pathways, and inflammation. Little is known, however, about the proteome and LOS content of OMVs released by clinical strains isolated from different infection sites, and whether these vesicles similarly activate immune responses. Here, we characterized OMVs from four N. gonorrhoeae isolates and determined their size, abundance, proteome, LOS content, and activation of inflammatory responses in macrophages. The overall proteome of the OMVs was conserved between the four different isolates, which included major outer membrane and periplasm proteins. Despite this, we observed differences in the rate of OMV biogenesis and the relative abundance of membrane proteins and LOS. Consequently, OMVs from clinical isolates induced varying rates of macrophage cell death and the secretion of interleukin-1 family members, such as IL-1α and IL-1ß. Overall, these findings demonstrate that clinical isolates of N. gonorrhoeae utilize membrane vesicles to release proteins and lipids, which affects innate immune responses.

Phytochrome B inhibits darkness-induced hypocotyl adventitious root formation by stabilizing IAA14 and suppressing ARF7 and ARF19.

Adventitious roots (ARs) are an important root type for plants and display a high phenotypic plasticity in response to different environmental stimuli. Previous studies found that dark-light transition can trigger AR formation from the hypocotyl of etiolated Arabidopsis thaliana, which was used as a model for the identification of regulators of AR biogenesis. However, the central regulatory machinery for darkness-induced hypocotyl AR (HAR) remains elusive. Here, we report that photoreceptors suppress HAR biogenesis through regulating the molecular module essential for lateral roots. We found that hypocotyls embedded in soil or in continuous darkness are able to develop HARs, wherein photoreceptors act as negative regulators. Distinct from wound-induced ARs that require WOX11 and WOX12, darkness-induced HARs are fully dependent on ARF7, ARF19, WOX5/7, and LBD16. Further studies established that PHYB interacts with IAA14, ARF7, and ARF9. The interactions stabilize IAA14 and inhibit the transcriptional activities of ARF7 and ARF19 and thus suppress biogenesis of darkness-induced HARs. This finding not only revealed the central machinery controlling HAR biogenesis but also illustrated that AR formation could be initiated by multiple pathways.

Sec24C mediates a Golgi-independent trafficking pathway that is required for tonoplast localisation of ABCC1 and ABCC2.

Protein sorting is an essential biological process in all organisms. Trafficking membrane proteins generally relies on the sorting machinery of the Golgi apparatus. However, many proteins have been found to be delivered to target locations via Golgi-independent pathways, but the mechanisms underlying this delivery system remain unknown. Here, we report that Sec24C mediates the direct secretory trafficking of the phytochelatin transporters ABCC1 and ABCC2 from the endoplasmic reticulum (ER) to prevacuolar compartments (PVCs) in Arabidopsis thaliana. Genetic analysis showed that the sec24c mutants are hypersensitive to cadmium (Cd) and arsenic (As) treatments due to mislocalisation of ABCC1 and ABCC2, which results in defects in the vacuole compartmentalisation of the toxic metals. Furthermore, we found that Sec24C recognises ABCC1 and ABCC2 through direct interactions to mediate their exit from the ER to PVCs, which is independent of brefeldin A-sensitive post-Golgi trafficking pathway. These findings expand our understanding of Golgi-independent trafficking, which also provide key insights regarding the mechanism of tonoplast protein sorting and open a new perspective on the function of Sec24 proteins.

Lipid A profiling and metabolomics analysis of paired polymyxin-susceptible and -resistant MDR Klebsiella pneumoniae clinical isolates from the same patients before and after colistin treatment.

BACKGROUND: The increased incidence of polymyxin-resistant MDR Klebsiella pneumoniae has become a major global health concern. OBJECTIVES: To characterize the lipid A profiles and metabolome differences between paired polymyxin-susceptible and -resistant MDR K. pneumoniae clinical isolates. METHODS: Three pairs of K. pneumoniae clinical isolates from the same patients were examined [ATH 7 (polymyxin B MIC 0.25 mg/L) versus ATH 8 (64 mg/L); ATH 15 (0.5 mg/L) versus ATH 16 (32 mg/L); and ATH 17 (0.5 mg/L) versus ATH 18 (64 mg/L)]. Lipid A and metabolomes were analysed using LC-MS and bioinformatic analysis was conducted. RESULTS: The predominant species of lipid A in all three paired isolates were hexa-acylated and 4-amino-4-deoxy-l-arabinose-modified lipid A species were detected in the three polymyxin-resistant isolates. Significant metabolic differences were evident between the paired isolates. Compared with their corresponding polymyxin-susceptible isolates, the levels of metabolites in amino sugar metabolism (UDP-N-acetyl-α-d-glucosamine and UDP-N-α-acetyl-d-mannosaminuronate) and central carbon metabolism (e.g. pentose phosphate pathway and tricarboxylic acid cycle) were significantly reduced in all polymyxin-resistant isolates [fold change (FC) > 1.5, P < 0.05]. Similarly, nucleotides, amino acids and key metabolites in glycerophospholipid metabolism, namely sn-glycerol-3-phosphate and sn-glycero-3-phosphoethanolamine, were significantly reduced across all polymyxin-resistant isolates (FC > 1.5, P < 0.05) compared with polymyxin-susceptible isolates. However, higher glycerophospholipid levels were evident in polymyxin-resistant ATH 8 and ATH 16 (FC > 1.5, P < 0.05) compared with their corresponding susceptible isolates. CONCLUSIONS: To our knowledge, this study is the first to reveal significant metabolic perturbations associated with polymyxin resistance in K. pneumoniae.

A new vesicle trafficking regulator CTL1 plays a crucial role in ion homeostasis.

Ion homeostasis is essential for plant growth and environmental adaptation, and maintaining ion homeostasis requires the precise regulation of various ion transporters, as well as correct root patterning. However, the mechanisms underlying these processes remain largely elusive. Here, we reported that a choline transporter gene, CTL1, controls ionome homeostasis by regulating the secretory trafficking of proteins required for plasmodesmata (PD) development, as well as the transport of some ion transporters. Map-based cloning studies revealed that CTL1 mutations alter the ion profile of Arabidopsis thaliana. We found that the phenotypes associated with these mutations are caused by a combination of PD defects and ion transporter misregulation. We also established that CTL1 is involved in regulating vesicle trafficking and is thus required for the trafficking of proteins essential for ion transport and PD development. Characterizing choline transporter-like 1 (CTL1) as a new regulator of protein sorting may enable researchers to understand not only ion homeostasis in plants but also vesicle trafficking in general.

AtHKT1 drives adaptation of Arabidopsis thaliana to salinity by reducing floral sodium content.

Arabidopsis thaliana high-affinity potassium transporter 1 (AtHKT1) limits the root-to-shoot sodium transportation and is believed to be essential for salt tolerance in A. thaliana. Nevertheless, natural accessions with 'weak allele' of AtHKT1, e.g. Tsu-1, are mainly distributed in saline areas and are more tolerant to salinity. These findings challenge the role of AtHKT1 in salt tolerance and call into question the involvement of AtHKT1 in salinity adaptation in A. thaliana. Here, we report that AtHKT1 indeed drives natural variation in the salt tolerance of A. thaliana and the coastal AtHKT1, so-called weak allele, is actually hyper-functional in reducing flowers sodium content upon salt stress. Our data showed that AtHKT1 positively contributes to saline adaptation in a linear manner. Forward and reverse genetics analysis established that the single AtHKT1 locus is responsible for the variation in the salinity adaptation between Col-0 and Tsu-1. Reciprocal grafting experiments revealed that shoot AtHKT1 determines the salt tolerance of Tsu-1, whereas root AtHKT1 primarily drives the salt tolerance of Col-0. Furthermore, evidence indicated that Tsu-1 AtHKT1 is highly expressed in stems and is more effective compared to Col-0 AtHKT1 at limiting sodium flow to the flowers. Such efficient retrieval of sodium to the reproductive organ endows Tsu-1 with stronger fertility compared to Col-0 upon salt stress, thus improving Tsu-1 adaptation to a coastal environment. To conclude, our data not only confirm the role of AtHKT1 in saline adaptation, but also sheds light on our understanding of the salt tolerance mechanisms in plants.

Metabolomics Study of the Synergistic Killing of Polymyxin B in Combination with Amikacin against Polymyxin-Susceptible and -Resistant Pseudomonas aeruginosa.

In the present study, we employed untargeted metabolomics to investigate the synergistic killing mechanism of polymyxin B in combination with an aminoglycoside, amikacin, against a polymyxin-susceptible isolate of Pseudomonas aeruginosa, FADDI-PA111 (MIC = 2 mg/liter for both polymyxin B and amikacin), and a polymyxin-resistant Liverpool epidemic strain (LES), LESB58 (the corresponding MIC for both polymyxin B and amikacin is 16 mg/liter). The metabolites were extracted 15 min, 1 h, and 4 h following treatment with polymyxin B alone (2 mg/liter for FADDI-PA111; 4 mg/liter for LESB58), amikacin alone (2 mg/liter), and both in combination and analyzed using liquid chromatography-mass spectrometry (LC-MS). At 15 min and 1 h, polymyxin B alone induced significant perturbations in glycerophospholipid and fatty acid metabolism pathways in FADDI-PA111 and, to a lesser extent, in LESB58. Amikacin alone at 1 and 4 h induced significant perturbations in peptide and amino acid metabolism, which is in line with the mode of action of aminoglycosides. Pathway analysis of FADDI-PA111 revealed that the synergistic effect of the combination was largely due to the inhibition of cell envelope biogenesis, which was driven initially by polymyxin B via suppression of key metabolites involved in lipopolysaccharide, peptidoglycan, and membrane lipids (15 min and 1 h) and later by amikacin (4 h). Overall, these novel findings demonstrate that the disruption of cell envelope biogenesis and central carbohydrate metabolism, decreased levels of amino sugars, and a downregulated nucleotide pool are the metabolic pathways associated with the synergistic killing of the polymyxin-amikacin combination against P. aeruginosa This mechanistic study might help optimize synergistic polymyxin B combinations in the clinical setting.

Novel Polymyxin Combination with the Antiretroviral Zidovudine Exerts Synergistic Killing against NDM-Producing Multidrug-Resistant Klebsiella pneumoniae.

Polymyxins are used as a last-line therapy against multidrug-resistant (MDR) New Delhi metallo-ß-lactamase (NDM)-producing Klebsiella pneumoniae However, polymyxin resistance can emerge with monotherapy; therefore, novel strategies are urgently needed to minimize the resistance and maintain their clinical utility. This study aimed to investigate the pharmacodynamics of polymyxin B in combination with the antiretroviral drug zidovudine against K. pneumoniae Three isolates were evaluated in static time-kill studies (0 to 64 mg/liter) over 48 h. An in vitro one-compartment pharmacokinetic/pharmacodynamic (PK/PD) model (IVM) was used to simulate humanized dosage regimens of polymyxin B (4 mg/liter as continuous infusion) and zidovudine (as bolus dose thrice daily to achieve maximum concentration of drug in broth [Cmax] of 6 mg/liter) against K. pneumoniae BM1 over 72 h. The antimicrobial synergy of the combination was further evaluated in a murine thigh infection model against K. pneumoniae 02. In the static time-kill studies, polymyxin B monotherapy produced rapid and extensive killing against all three isolates followed by extensive regrowth, whereas zidovudine produced modest killing followed by significant regrowth at 24 h. Polymyxin B in combination with zidovudine significantly enhanced the antimicrobial activity (≥4 log10 CFU/ml) and minimized bacterial regrowth. In the IVM, the combination was synergistic and the total bacterial loads were below the limit of detection for up to 72 h. In the murine thigh infection model, the bacterial burden at 24 h in the combination group was ≥3 log10 CFU/thigh lower than each monotherapy against K. pneumoniae 02. Overall, the polymyxin B-zidovudine combination demonstrates superior antimicrobial efficacy and minimized emergence of resistance to polymyxins.

Polymyxin resistance in Klebsiella pneumoniae: multifaceted mechanisms utilized in the presence and absence of the plasmid-encoded phosphoethanolamine transferase gene mcr-1.

OBJECTIVES: Until plasmid-mediated mcr-1 was discovered, it was believed that polymyxin resistance in Gram-negative bacteria was mainly mediated by the chromosomally-encoded EptA and ArnT, which modify lipid A with phosphoethanolamine (pEtN) and 4-amino-4-deoxy-l-arabinose (l-Ara4N), respectively. This study aimed to construct a markerless mcr-1 deletion mutant in Klebsiella pneumoniae, validate a reliable reference gene for reverse transcription quantitative PCR (RT-qPCR) and investigate the interactions among mcr-1, arnT and eptA, in response to polymyxin treatments using pharmacokinetics/pharmacodynamics (PK/PD). METHODS: An isogenic markerless mcr-1 deletion mutant (II-503Δmcr-1) was generated from a clinical K. pneumoniae II-503 isolate. The efficacy of different polymyxin B dosage regimens was examined using an in vitro one-compartment PK/PD model and polymyxin resistance was assessed using population analysis profiles. The expression of mcr-1, eptA and arnT was examined using RT-qPCR with a reference gene pepQ, and lipid A was profiled using LC-MS. In vivo polymyxin B efficacy was investigated in a mouse thigh infection model. RESULTS: In K. pneumoniae II-503, mcr-1 was constitutively expressed, irrespective of polymyxin exposure. Against II-503Δmcr-1, an initial bactericidal effect was observed within 4 h with polymyxin B at average steady-state concentrations of 1 and 3 mg/L, mimicking patient PK. However, substantial regrowth and concomitantly increased expression of eptA and arnT were detected. Predominant l-Ara4N-modified lipid A species were detected in II-503Δmcr-1 following polymyxin B treatment. CONCLUSIONS: This is the first study demonstrating a unique markerless deletion of mcr-1 in a clinical polymyxin-resistant K. pneumoniae. The current polymyxin B dosage regimens are suboptimal against K. pneumoniae, regardless of mcr, and can lead to the emergence of resistance.

[Cloning and expression of SmDXS2 gene in Swertia mussotii].

1-deoxy-D-xylulose-5-phosphate synthase2(DXS2) is the first key enzyme of the MEP pathway,which plays an important role in terpene biosynthesis of plants. According to the data of Swertia mussotii transcriptome, DXS2 gene(Gen Bank number MH535905) was cloned and named as Sm DXS2. The bioinformatics results showed that Sm DXS2 has no intron,with a 2 145 bp open reading frame encoding a polypeptide of 714 amino acids. They are belonging to 20 kinds of amino acids,and the most abundant amino acids include Ala,Gly and Trp. The predicted protein molecular weight was 76. 91 k Da and its theoretical isoelectric point(p I) was6. 5,which belonging to a hydrophilic protein. α-Helix and loop were the major motifs of predicted secondary structure of DXS2. The three function domains are TPP＿superfamily,Transket＿pyr＿ superfamily and Transketolase＿C superfamily,respectively. The Sm DXS2 protein shared high identity with other DXS2 proteins of plants. Phylogenetic analysis showed that Sm DXS2 protein is grouped with the gentian DXS2 protein. The recombinant protein of Sm DXS2 gene in Escherichia coli was approximately 92. 00 k Da(containing sumo-His tag protein 13 k Da),which was consistent with the anticipated size.This work will provide a foundation for further functional research of Sm DXS2 protein and increasing the product of iridoid compound by genetic engineering in S. mussotii.

Mechanism-Based Pharmacokinetic/Pharmacodynamic Modeling of Aerosolized Colistin in a Mouse Lung Infection Model.

Optimized dosage regimens of aerosolized colistin (as colistin methanesulfonate [CMS]) are urgently required to maximize bacterial killing against multidrug-resistant Gram-negative bacteria while minimizing toxicity. This study aimed to develop a mechanism-based pharmacokinetic (PK)/pharmacodynamic (PD) model (MBM) for aerosolized colistin based upon PK/PD data in neutropenic infected mice and to perform a deterministic simulation with the PK of aerosolized colistin (as CMS) in critically ill patients. In vivo time-kill experiments were carried out with three different strains of Pseudomonas aeruginosa An MBM was developed in S-ADAPT and evaluated by assessing its ability to predict the PK/PD index associated with efficacy in mice. A deterministic simulation with human PK data was undertaken to predict the efficacy of current dosage regimens of aerosolized colistin in critically ill patients. In the final MBM, the total bacterial population for each isolate consisted of colistin-susceptible and -resistant subpopulations. The antimicrobial efficacy of aerosolized colistin was best described by a sigmoidal Emax model whereby colistin enhanced the rate of bacterial death. Deterministic simulation with human PK data predicted that an inhalational dosage regimen of 60 mg colistin base activity (CBA) every 12 h is needed to achieve a ≥2-log10 bacterial reduction (as the number of CFU per lung) in critically ill patients at 24 h after commencement of inhaled therapy. In conclusion, the developed MBM is a useful tool for optimizing inhalational dosage regimens of colistin. Clinical studies are warranted to validate and refine our MBM for aerosolized colistin.

Elucidating the Pharmacokinetics/Pharmacodynamics of Aerosolized Colistin against Multidrug-Resistant Acinetobacter baumannii and Klebsiella pneumoniae in a Mouse Lung Infection Model.

The pharmacokinetics/pharmacodynamics (PK/PD) of aerosolized colistin was investigated against Acinetobacter baumannii and Klebsiella pneumoniae over 24 h in a neutropenic mouse lung infection model. Dose fractionation studies were performed over 2.64 to 23.8 mg/kg/day, and the data were fitted to a sigmoid inhibitory model. The area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC) in the epithelial lining fluid was the most predictive PK/PD index for aerosolized colistin against both pathogens. Our study provides important pharmacological information for optimizing aerosolized colistin.

Alterations of Metabolic and Lipid Profiles in Polymyxin-Resistant Pseudomonas aeruginosa.

Multidrug-resistant Pseudomonas aeruginosa presents a global medical challenge, and polymyxins are a key last-resort therapeutic option. Unfortunately, polymyxin resistance in P. aeruginosa has been increasingly reported. The present study was designed to define metabolic differences between paired polymyxin-susceptible and -resistant P. aeruginosa strains using untargeted metabolomics and lipidomics analyses. The metabolomes of wild-type P. aeruginosa strain K ([PAK] polymyxin B MIC, 1 mg/liter) and its paired pmrB mutant strains, PAKpmrB6 and PAKpmrB12 (polymyxin B MICs of 16 mg/liter and 64 mg/liter, respectively) were characterized using liquid chromatography-mass spectrometry, and metabolic differences were identified through multivariate and univariate statistics. PAKpmrB6 and PAKpmrB12, which displayed lipid A modifications with 4-amino-4-deoxy-l-arabinose, showed significant perturbations in amino acid and carbohydrate metabolism, particularly the intermediate metabolites from 4-amino-4-deoxy-l-arabinose synthesis and the methionine salvage cycle pathways. The genomics result showed a premature termination (Y275stop) in speE (encoding spermidine synthase) in PAKpmrB6, and metabolomics data revealed a decreased intracellular level of spermidine in PAKpmrB6 compared to that in PAKpmrB12 Our results indicate that spermidine may play an important role in high-level polymyxin resistance in P. aeruginosa Interestingly, both pmrB mutants had decreased levels of phospholipids, fatty acids, and acyl-coenzyme A compared to those in the wild-type PAK. Moreover, the more resistant PAKpmrB12 mutant exhibited much lower levels of phospholipids than the PAKpmrB6 mutant, suggesting that the decreased phospholipid level was associated with polymyxin resistance. In summary, this study provides novel mechanistic information on polymyxin resistance in P. aeruginosa and highlights its impacts on bacterial metabolism.

Polymyxin B in Combination with Enrofloxacin Exerts Synergistic Killing against Extensively Drug-Resistant Pseudomonas aeruginosa.

Polymyxins are increasingly used as a last-resort class of antibiotics against extensively drug-resistant (XDR) Gram-negative bacteria. However, resistance to polymyxins can emerge with monotherapy. As nephrotoxicity is the major dose-limiting factor for polymyxin monotherapy, dose escalation to suppress the emergence of polymyxin resistance is not a viable option. Therefore, novel approaches are needed to preserve this last-line class of antibiotics. This study aimed to investigate the antimicrobial synergy of polymyxin B combined with enrofloxacin against Pseudomonas aeruginosa Static time-kill studies were conducted over 24 h with polymyxin B (1 to 4 mg/liter) and enrofloxacin (1 to 4 mg/liter) alone or in combination. Additionally, in vitro one-compartment model (IVM) and hollow-fiber infection model (HFIM) experiments were performed against P. aeruginosa 12196. Polymyxin B and enrofloxacin in monotherapy were ineffective against all of the P. aeruginosa isolates examined, whereas polymyxin B-enrofloxacin in combination was synergistic against P. aeruginosa, with ≥2 to 4 log10 kill at 24 h in the static time-kill studies. In both IVM and HFIM, the combination was synergistic, and the bacterial counting values were below the limit of quantification on day 5 in the HFIM. A population analysis profile indicated that the combination inhibited the emergence of polymyxin resistance in P. aeruginosa 12196. The mechanism-based modeling suggests that the synergistic killing is a result of the combination of mechanistic and subpopulation synergy. Overall, this is the first preclinical study to demonstrate that the polymyxin-enrofloxacin combination is of considerable utility for the treatment of XDR P. aeruginosa infections and warrants future clinical evaluations.

Emergence of High-Level Colistin Resistance in an Acinetobacter baumannii Clinical Isolate Mediated by Inactivation of the Global Regulator H-NS.

Colistin is a crucial last-line drug used for the treatment of life-threatening infections caused by multidrug-resistant strains of the Gram-negative bacterium Acinetobacter baumannii However, colistin-resistant A. baumannii isolates can still be isolated following failed colistin therapy. Resistance is most often mediated by the addition of phosphoethanolamine (pEtN) to lipid A by PmrC, following missense mutations in the pmrCAB operon encoding PmrC and the two-component signal transduction system PmrA/PmrB. We recovered a pair of A. baumannii isolates from a single patient before (6009-1) and after (6009-2) failed colistin treatment. These strains displayed low and very high levels of colistin resistance (MICs, 8 to 16 µg/ml and 128 µg/ml), respectively. To understand how increased colistin resistance arose, we sequenced the genome of each isolate, which revealed that 6009-2 had an extra copy of the insertion sequence element ISAba125 within a gene encoding an H-NS family transcriptional regulator. To confirm the role of H-NS in colistin resistance, we generated an hns deletion mutant in 6009-1 and showed that colistin resistance increased upon the deletion of hns We also provided 6009-2 with an intact copy of hns and showed that the strain was no longer resistant to high concentrations of colistin. Transcriptomic analysis of the clinical isolates identified more than 150 genes as being differentially expressed in the colistin-resistant hns mutant 6009-2. Importantly, the expression of eptA, encoding a second lipid A-specific pEtN transferase but not pmrC, was increased in the hns mutant. This is the first time an H-NS family transcriptional regulator has been associated with a pEtN transferase and colistin resistance.

Fitness cost of mcr-1-mediated polymyxin resistance in Klebsiella pneumoniae.

Objectives: The discovery of mobile colistin resistance mcr-1, a plasmid-borne polymyxin resistance gene, highlights the potential for widespread resistance to the last-line polymyxins. In the present study, we investigated the impact of mcr-1 acquisition on polymyxin resistance and biological fitness in Klebsiella pneumoniae. Methods: K. pneumoniae B5055 was used as the parental strain for the construction of strains carrying vector only (pBBR1MCS-5) and mcr-1 recombinant plasmids (pmcr-1). Plasmid stability was determined by serial passaging for 10 consecutive days in antibiotic-free LB broth, followed by patching on gentamicin-containing and antibiotic-free LB agar plates. Lipid A was analysed using LC-MS. The biological fitness was examined using an in vitro competition assay analysed with flow cytometry. The in vivo fitness cost of mcr-1 was evaluated in a neutropenic mouse thigh infection model. Results: Increased polymyxin resistance was observed following acquisition of mcr-1 in K. pneumoniae B5055. The modification of lipid A with phosphoethanolamine following mcr-1 addition was demonstrated by lipid A profiling. The plasmid stability assay revealed the instability of the plasmid after acquiring mcr-1. Reduced in vitro biological fitness and in vivo growth were observed with the mcr-1-carrying K. pneumoniae strain. Conclusions: Although mcr-1 confers a moderate level of polymyxin resistance, it is associated with a significant biological fitness cost in K. pneumoniae. This indicates that mcr-1-mediated resistance in K. pneumoniae could be attenuated by limiting the usage of polymyxins.