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Genes encoding lipid A modifying phosphoethanolamine transferases (PETs) are genetically diverse and can confer resistance to colistin and antimicrobial peptides. To better understand the functional diversity of PETs, we characterized three canonical mobile colistin resistance ( mcr ) alleles ( mcr-1 , -3 , -9 ), one intrinsic pet ( eptA ), and two mcr -like genes ( petB , petC ) in Escherichia coli . Using an isogenic expression system, we show that mcr-1 and mcr-3 confer similar phenotypes of decreased colistin susceptibility with low fitness costs. mcr-9 , which is phylogenetically closely related to mcr-3 , and eptA only provide fitness advantages in the presence of sub-inhibitory concentrations of colistin and significantly reduce fitness in media without colistin. PET-B and PET-C were phenotypically distinct from bonafide PETs; neither impacted colistin susceptibility nor caused considerable fitness cost. Strikingly, we found for the first time that different PETs selectively modify different phosphates of lipid A; MCR-1, MCR-3, and PET-C selectively modify the 4'-phosphate, whereas MCR-9 and EptA modify the 1-phosphate. However, 4'-phosphate modifications facilitated by MCR-1 and -3 are associated with lowered colistin susceptibility and low toxicity. Our results suggest that PETs have a wide phenotypic diversity and that increased colistin resistance is associated with specific lipid A modification patterns that have been largely unexplored thus far., Importance: Rising levels of resistance to increasing numbers of antimicrobials have led to the revival of last resort antibiotic colistin. Unfortunately, resistance to colistin is also spreading in the form of mcr genes, making it essential to (i) improve the identification of resistant bacteria to allow clinicians to prescribe effective drug regimens and (ii) develop new combination therapies effective at targeting resistant bacteria. Our results demonstrate that PETs, including MCR variants, are site-selective in Escherichia coli and that site-selectivity correlates with the level of susceptibility and fitness costs conferred by certain PETs. Site selectivity associated with a given PET may not only help predict colistin resistance phenotypes but may also provide an avenue to (i) improve drug regimens and (ii) develop new combination therapies to better combat colistin-resistant bacteria., Competing Interests: M.W. holds stocks for Neogen and is a paid consultant for BioMerieux and Neogen.

SUMMARYLipopolysaccharides (LPS) are an integral part of the outer membrane of Gram-negative bacteria and play essential structural and functional roles in maintaining membrane integrity as well as in stress response and virulence. LPS comprises a membrane-anchored lipid A group, a sugar-based core region, and an O-antigen formed by repeating oligosaccharide units. 3-Deoxy-D- manno -octulosonic acid-lipid A (Kdo 2 -lipid A) is the minimum LPS component required for bacterial survival. While LPS modifications are not essential, they play multifaceted roles in stress response and host-pathogen interactions. Gram-negative bacteria encode several distinct LPS-modifying phosphoethanolamine transferases (PET) that add phosphoethanolamine (pEtN) to lipid A or the core region of LPS. The pet genes differ in their genomic locations, regulation mechanisms, and modification targets of the encoded enzyme, consistent with their various roles in different growth niches and under varied stress conditions. The discovery of mobile colistin resistance genes, which represent lipid A-modifying pet genes that are encoded on mobile elements and associated with resistance to the last-resort antibiotic colistin, has led to substantial interest in PETs and pEtN-modified LPS over the last decade. Here, we will review the current knowledge of the functional diversity of pEtN-based LPS modifications, including possible roles in niche-specific fitness advantages and resistance to host-produced antimicrobial peptides, and discuss how the genetic and structural diversities of PETs may impact their function. An improved understanding of the PET group will further enhance our comprehension of the stress response and virulence of Gram-negative bacteria and help contextualize host-pathogen interactions., Competing Interests: The authors declare no conflict of interest.

The phosphoethanolamine transferase EptA utilizes phosphatidylethanolamine (PE) in the bacterial cell membrane to modify the structure of lipopolysaccharide, thereby conferring antimicrobial resistance on Gram-negative pathogens. Previous studies have indicated that excessive consumption of PE can disrupt the cell membrane, leading to cell death. This implies the presence of a regulatory mechanism for EptA catalysis to maintain a balance between antimicrobial resistance and bacterial growth. Through microsecond-scale all-atom molecular dynamics simulations, we demonstrate that membrane lipid homeostasis modulates the conformational transition and catalytic activation of EptA. The conformation of EptA oscillates between closed and open states, ensuring the precise spatiotemporal sequence of substrates binding. Interestingly, the conformation of EptA is significantly influenced by its surrounding lipid microenvironment, particularly the PE proportion in the membrane. PE-rich membrane conditions initiate and stabilize the open conformation of EptA through both orthosteric and allosteric effects. Importantly, the reaction mediated by EptA gradually depletes PE in the membrane, ultimately hindering its conformational transition and catalytic activation. These findings collectively establish a self-promoted model, illustrating the regulatory mechanism of EptA during the development of antibiotic resistance., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The emergence of plasmid-encoded colistin resistance mechanisms, MCR-1, a phosphoethanolamine transferase, rendered colistin ineffective as last resort antibiotic against severe infections caused by clinical Gram-negative bacterial pathogens. Through screening FDA-approved drug library, we identified two structurally similar compounds, namely cetylpyridinium chloride (CET) and domiphen bromide (DOM), which potentiated colistin activity in both colistin-resistant and susceptible Enterobacterales. These compounds were found to insert their long carbon chain to a hydrophobic pocket of bacterial phosphoethanolamine transferases including MCR-1, competitively blocking the binding of lipid A tail for substrate recognition and modification, resulting in the increase of bacterial sensitivity to colistin. In addition, these compounds were also found to dissipate bacterial membrane potential leading to the increase of bacterial sensitivity to colistin. Importantly, combinational use of DOM with colistin exhibited remarkable protection of test animals against infections by colistin-resistant bacteria in both mouse thigh infection and sepsis models. For mice infected by colistin-susceptible bacteria, the combinational use of DOM and colistin enable us to use lower dose of colistin to for efficient treatment. These properties render DOM excellent adjuvant candidates that help transform colistin into a highly potent antimicrobial agent for treatment of colistin-resistant Gram-negative bacterial infections and allowed us to use of a much lower dosage of colistin to reduce its toxicity against colistin-susceptible bacterial infection such as carbapenem-resistant Enterobacterales., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier GmbH.)

Polymyxins, including colistin and polymyxin B, serve as crucial last-resort antibiotics for managing infections caused by carbapenem-resistant Enterobacterales (CRE). However, the rapid spread of the mobilized colistin resistance gene (mcr-1) challenged the efficacy of treatment by polymyxins. The mcr-1 gene encoded a transmembrane phosphoethanolamine (PEA) transferase enzyme, MCR-1. MCR-1 could catalyze the transfer of PEA moiety of phosphatidylethanolamine (PE) to the 1' (or 4')-phosphate group of the lipid A. Despite the determination of several structures of the soluble domain of MCR-1, the structural and biochemical mechanisms of integral MCR-1 remain less understood. In this study, we utilized an alanine scanning mutagenesis approach to systematically investigate the functional attributes of distinct regions within MCR-1. We identified fifteen critical residues that are indispensable for the enzymatic activity of MCR-1 and are pivotal for its ability to confer resistance to colistin. Furthermore, molecular docking of MCR-1 complexed with the phosphoethanolamine (PE) substrate revealed the presence of a channel-shaped cavity, a characteristic feature shared with other phosphoethanolamine transferases. Despite MCR-1 exhibiting a low sequence identity with both MCR homologues and other phosphoethanolamine (PEA) transferases, several conserved sites were identified, including Y 97 , M 105 , K 333 , H 395 , L 477 , and H 478 , suggesting a potentially shared catalytic mechanism among them for modifying LPS-lipid A. Overall, these findings provide a deep understanding of the catalytic mechanism of MCR-1 for colistin resistance. Moreover, these findings provide a robust structural and functional foundation, enabling the rational design of targeted inhibitors and restoring colistin activity against serious infections with carbapenem-resistant Enterobacterales (CRE)., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Selenoprotein I (Selenoi) is highly expressed in liver and plays a key role in lipid metabolism as a phosphatidylethanolamine (PE) synthase. However, the precise function of Selenoi in the liver remains elusive. In the study, we generated hepatocyte-specific Selenoi conditional knockout (cKO) mice on a high-fat diet to identify the physiological function of Selenoi. The cKO group exhibited a significant increase in body weight, with a 15.6% and 13.7% increase in fat accumulation in white adipose tissue (WAT) and the liver, respectively. Downregulation of the lipolysis-related protein (p-Hsl) and upregulation of the adipogenesis-related protein (Fasn) were observed in the liver of cKO mice. The cKO group also showed decreased oxygen consumption (VO 2 ), carbon dioxide production (VCO 2 ), and energy expenditure (p < .05). Moreover, various metabolites of the steroid hormone synthesis pathway were affected in the liver of cKO mice. A potential cascade of Selenoi-phosphatidylethanolamine-steroid hormone synthesis might serve as a core mechanism that links hepatocyte-specific Selenoi cKO to biochemical and molecular reactions. In conclusion, we revealed that Selenoi inhibits body fat accumulation and hepatic steatosis and elevates energy consumption; this protein could also be considered a therapeutic target for such related diseases., (© 2024 Federation of American Societies for Experimental Biology.)

Objectives: The rapid dissemination of the mcr-1 gene via plasmid-mediated transfer has raised concerns regarding the efficacy of colistin as a last-resort treatment for multidrug-resistant Gram-negative bacterial infections. Current mcr-1 gene detection methods mainly focus on cultured bacteria, which is a complex and time-consuming process requiring skilled personnel, making it unsuitable for field analysis., Methods: A rapid detection technique combining recombinase polymerase amplification with a lateral flow dipstick targeting uncultured clinical samples was developed., Results: This new method targeting the mcr-1 gene region (23 232-23 642 bp, no. KP347127.1) achieved a low detection limit of 10 copies/μL. The whole process was carried out with high specificity and was completed within 20 min. The evaluation assay was conducted using 45 human faecal samples; 16 strains yielded a 98% accuracy, closely matching antimicrobial susceptibility outcomes., Conclusions: The novel method integrates nucleic acid extraction, isothermal amplification, and a test assay, suggesting the potential for timely colistin resistance surveillance in frontline disease control and healthcare settings, supporting future prevention and clinical standardization efforts., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Hereditary spastic paraplegia 81 is a recently identified, rare autosomal recessive disease, caused by biallelic pathogenic variants in the SELENOI gene, with only two families reported to date. The features documented in the two previous affected families include sensorineural deafness, blindness, cleft palate, delayed motor development, regression of motor skills, impaired intellectual development, poor speech and language acquisition, spasticity, hyperreflexia, white matter abnormalities and cerebral and cerebellar atrophy., Methods: In the present study, we performed exome sequencing analysis in a single family with two affected siblings to identify the genetic cause of complicated hereditary spastic paraplegia. The results were further confirmed by Sanger sequencing, cDNA analysis and 3D protein modelling., Results: Exome sequencing identified a homozygous, synonymous variant in the SELENOI gene (NM&#95;033505.4:c.126G>A:p.(Lys42Lys)) in both of the siblings. Sanger sequencing confirmed the heterozygous status in both parents consistent with the autosomal recessive inheritance. This variant has been found to disrupt normal splicing and lead to skipping of exon 2, causing in-frame deletion of SELENOI N-terminal 23 amino acids [NM&#95;033505.4:c.57&#95;126del:p.(Tyr20&#95;Lys42del)] and further leading to structural changes in the protein., Conclusions: We report a novel homozygous synonymous variant in the SELENOI gene causing abnormal splicing in two patients affected with hereditary spastic paraplegia 81. This report further expands the phenotypic and genotypic spectrum of hereditary spastic paraplegia 81., (© 2023 John Wiley & Sons Ltd.)

Objectives: Colistin-resistant Gram-negative pathogens have become a serious worldwide medical problem. This study was designed to reveal the effects of an intrinsic phosphoethanolamine transferase from Acinetobacter modestus on Enterobacterales., Methods: A strain of colistin-resistant A. modestus was isolated from a sample of nasal secretions taken in 2019 from a hospitalised pet cat in Japan. The whole genome was sequenced by next generation sequencing, and transformants of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae harbouring the phosphoethanolamine transferase-encoding gene from A. modestus were constructed. Lipid A modification in E. coli transformants was analysed using electrospray ionization mass spectrometry., Results: Sequencing of the entire genome revealed that the isolate harboured a phosphoethanolamine transferase-encoding gene, eptA&#95;AM, on its chromosome. Transformants of E. coli, K. pneumoniae, and E. cloacae harbouring both the promoter and eptA&#95;AM gene from A. modestus had 32-fold, 8-fold, and 4-fold higher minimum inhibitory concentrations (MICs) for colistin, respectively, than transformants harbouring a control vector. The genetic environment surrounding eptA&#95;AM in A. modestus was similar to that surrounding eptA&#95;AM in Acinetobacter junii and Acinetobacter venetianus. Electrospray ionization mass spectrometry analysis revealed that EptA&#95;AM modified lipid A in Enterobacterales., Conclusion: This is the first report to describe the isolation of an A. modestus strain in Japan and show that its intrinsic phosphoethanolamine transferase, EptA&#95;AM, contributes to colistin resistance in Enterobacterales and A. modestus., Competing Interests: Competing interests None declared., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: This study aimed to describe a clinical isolate of Aeromonas jandaei (A. jandaei) in Nepal that harboured four types of genes encoding phosphoethanolamine transferases., Methods: An isolate of colistin-resistant A. jandaei was obtained from a blood sample of an inpatient in a hospital in Nepal, and its complete genome sequence was determined. Escherichia coli (E. coli) and Aeromonas hydrophila (A. hydrophila) transformants expressing genes encoding novel phosphoethanolamine transferase variants were constructed and colistin-susceptibility profiles were determined., Results: The isolate harboured four genes encoding phosphoethanolamine transferases on the chromosome, which were designated eptAv3.2, eptAv3.3, eptAv3.4 and eptAv7.2. The amino acid sequences of EptAv3.2, 3.3 and 3.4 were > 80% identical to MCR-3.1, and that of EptAv7.2 was > 79% identical to MCR-7.1. E. coli expressing eptAv3.2, 3.3 and 3.4 showed reduced susceptibility to colistin, whereas E. coli expressing eptAv7.2 did not. In contrast, A. hydrophila expressing eptAv7.2 showed reduced susceptibility to colistin, whereas A. hydrophila expressing eptAv3.2, 3.3 and 3.4 did not; eptAv3.3 and 3.4 formed a tandem structure. The genomic environments surrounding eptAv3.2, 3.3 and 3.4 were similar to Aeromonas veronii obtained from the effluent of a treatment plant in Japan in 2018. The genomic environment surrounding eptAv7.2 was similar to that of A. jandaei obtained from a chicken in the USA in 2019., Conclusions: The highly colistin-resistant A. jandaei clinical isolate harboured four chromosomal genes encoding phosphoethanolamine transferases, suggesting that Aeromonas spp. harbouring eptAv genes with strong similarities to mcr-3 and mcr-7 are emerging in medical settings as well as environments., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Expression of mobile colistin resistance gene mcr-1 results in the addition of phosphoethanolamine (pEtN) to the lipid A headgroup in the bacterial outer membrane (OM) of Gram-negative bacteria, increasing the resistance to the last-line polymyxins. However, the potential biological consequences of such modification remain unclear. Using coarse-grained molecular simulations with quantitative lipidomics models, we discovered pEtN modification of the lipid A headgroup caused substantial changes to the morphology and physicochemical properties of the OM. Single-lipid level structural and energetic analyses revealed that this modification resulted in lipid A-pEtN adopting an abnormally twisted and slanted conformation with a closer packing state because of strengthened inter-lipid attraction. The consequent accumulation of lipid A-pEtN produced a negative curvature of the OM and altered the membrane's tension, fluidity, and rigidity. Our results provide a key mechanistic connection between mcr-1 expression and biophysical changes in the bacterial OM.

This study aims to describe trends of mcr-positive Enterobacterales in humans based on laboratory surveillance with a defined catchment population. The data source is the Micro-RER surveillance system, established in Emilia-Romagna region (Italy), to monitor the trend of mcr resistance. Enterobacterales isolates from human clinical samples with minimum inhibitory concentration (MIC) ≥ 2 mg/L for colistin were sent to the study reference laboratory for the detection of mcr genes. Isolates prospectively collected in the period 2018-2020 were considered for the assessment of population rates and trends; further analyses were carried out for the evaluation of clonality and horizontal mcr gene transfer. Previous isolates from local laboratory collection were also described. In the period 2018-2020, 1164 isolates were sent to the reference laboratory, and 51 (4.4%) were confirmed as mcr-positive: 50 mcr-1 (42 Escherichia coli, 6 Klebsiella pneumoniae, 2 Salmonella enterica) and 1 mcr-4 (Enterobacter cloacae). The number of mcr-positive isolates dropped from 24 in the first half of 2018 to 3 in the whole of 2020 (trend p value < 0.001). Genomic analyses showed the predominant role of the horizontal transfer of mcr genes through plasmids or dissemination of transposable elements compared to clonal dissemination of mcr-positive microorganisms. The study results demonstrate a substantial decrease in the circulation of mcr-1 plasmid genes in Emilia-Romagna Region., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Among the 10 reported mcr genes, mcr-9 was first identified in Salmonella enterica serotype Typhimurium, which is a leading cause of foodborne illness worldwide. However, information about the prevalence and genetic features of mcr-9 is still lacking, especially among food samples. This study reports the presence of mcr-9 in raw milk samples from China; the prevalence rate was low (0.83%, 1/120). mcr-9 was located on a transferable plasmid, and was stable in wild-type S. enterica. However, it had a biological fitness cost when transferred to an Escherichia coli recipient. Whole-genome sequencing revealed that mcr-9 was located on the IncHI2A-type plasmid, and was surrounded by IS903B and IS26 in its flanking regions. The mcr-9-carrying S. enterica 19SE belonged to ST26 and had a multi-drug-resistant phenotype. It was confirmed that mcr-9 did not mediate colistin resistance in this study, indicating that its transfer may not facilitate the dissemination of colistin resistance., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Polymyxins, especially polymyxin B and polymyxin E (colistin), are considered to be the last line of defence against infections caused by multi-drug-resistant (MDR) gram-negative bacteria such as carbapenem-resistant Enterobacteriaceae (CRE). However, the recent emergence and dissemination of the plasmid-mediated colistin resistance gene mcr-1 and its variants pose a serious challenge to public health and the livestock industry. This review describes the prevalence and dissemination of mcr-1 -positive isolates from different sources, including animals (food animals, pet animals and wildlife), humans (healthy populations and patients) and the environment (farms, urban and rural communities and natural environments) based on existing epidemiological studies of mcr-1 and MCR-1-producing Enterobacteriaceae bacteria around the world. The major mechanisms of mcr-1 transmission across humans, animals and the environment are discussed.

A wide variety of antibiotics are targeted to the bacterial membrane due to its unique arrangement and composition relative to the host mammalian membranes. By modification of their membranes, some gram-negative pathogens resist the action of antibiotics. Lipid A phosphoethanolamine transferase (EptA) is an intramembrane enzyme that modifies the lipid A portion of lipopolysaccharide/lipooligosaccharide by the addition of phosphoethanolamine. This modification reduces the overall net-negative charge of the outer membrane of some gram-negative bacteria, conferring resistance to polymyxin. This resistance mechanism has resulted in a global public health issue due to the increased use of polymyxin as last-resort antibiotic treatments against multi-drug-resistant pathogens. Studies show that, without EptA, pathogenic bacteria become more sensitive to polymyxin and to clearance by the host immune system, suggesting the importance of this target enzyme for the development of novel therapeutic agents. In this review, EptA will be discussed comprehensively. Specifically, this review will cover the regulation of eptA expression by the two component systems PmrA/PmrB and PhoP/PhoQ, the site of modification on lipid A, the structure and catalytic mechanism of EptA in comparison to MCR-1 and Escherichia coli alkaline phosphatase, and the host immune system's response to lipid A modification by EptA. The overarching aim of this review is to provide a comprehensive overview of polymyxin resistance mediated by EptA., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Two novel phosphoethanolamine transferase genes, eptAv7 and eptAv3, were identified in the chromosome of an Aeromonas jandaei isolate from retail fish. The variants showed 79.9% and 80.0% amino acid identity to MCR-7.1 and MCR-3.1, respectively, and increased colistin resistance 128- to 256-fold in Aeromonas salmonicida. The two variants with no mobile genetic element in the flanking regions were also observed in other Aeromonas species. This finding supports the view that Aeromonas is a reservoir for MCR-3 and MCR-7 mobile colistin resistance., (Copyright © 2019 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

In the present study, we provide the results of a detailed genomic analysis and the growth characteristics of a colistin-resistant KPC-3-producing Klebsiella pneumoniae sequence type 512 (ST512) isolate (the colR-KPC3-KP isolate) with a mutated pmrB and isogenic isolates of colR-KPC3-KP with mcr-1.2 isolated from an immunocompromised patient. From 2014 to 2017, four colR-KPC3-KP isolates were detected in rectal swab samples collected from a pediatric hematology patient at the Azienda Ospedaliero-Universitaria Pisana in Pisa, Italy. Whole-genome sequencing was performed by MiSeq sequencing (Illumina). Growth experiments were performed using different concentrations of colistin. The growth lag phases both of an isolate harboring a deletion in pmrB and of clonal variants with mcr-1.2 were assessed by the use of real-time light-scattering measurements. In the first isolate (isolate 1000- pmrB Δ, recovered in September 2014), a 17-nucleotide deletion in pmrB was detected. In subsequent isolates, the mcr-1.2 gene associated with the plasmid pIncX4-AOUP was found, while pmrB was intact. Additionally, plasmid pIncQ-AOUP, harboring aminoglycoside resistance genes, was detected. The growth curves of the first three isolates were identical without colistin exposure; however, at higher concentrations of colistin, the growth curves of the isolate with a deletion in pmrB showed longer lag phases. We observed the replacement of mutated colR-KPC3-KP pmrB by isogenic isolates with multiple resistance plasmids, including mcr-1.2- carrying pIncX4, probably due to coselection under gentamicin treatment in a patient with prolonged colR-KPC3-KP carriage. The carriage of these isolates persisted in follow-up cultures. Coselection and the advantages in growth characteristics suggest that the plasmid-mediated resistance conferred by mcr has fewer fitness costs in colR-KPC3-KP than mutations in chromosomal pmrB , contributing to the success of this highly resistant hospital-adapted epidemiological lineage. IMPORTANCE Our study shows a successful prolonged human colonization by a colistin-resistant Klebsiella pneumoniae isolate harboring mcr-1.2 An intense antibiotic therapy contributed to the maintenance of this microorganism through the acquisition of new resistance genes. The isolates carrying mcr-1.2 showed fewer fitness costs than isogenic isolates with a pmrB mutation in the chromosome. Coselection and reduced fitness costs may explain the replacement of isolates with the pmrB mutation by other isolates and the ability of the microorganism to persist despite antibiotic treatment., (Copyright © 2019 Giordano et al.)

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The plasmid-located mcr-9 gene, encoding a putative phosphoethanolamine transferase, was identified in a colistin-resistant human fecal Escherichia coli strain belonging to a very rare phylogroup, the D-ST69-O15:H6 clone. This MCR-9 protein shares 33% to 65% identity with the other plasmid-encoded MCR-type enzymes identified (MCR-1 to -8) that have been found as sources of acquired resistance to polymyxins in Enterobacteriaceae Analysis of the lipopolysaccharide of the MCR-9-producing isolate revealed a function similar to that of MCR-1 by adding a phosphoethanolamine group to lipid A and subsequently modifying the structure of the lipopolysaccharide. However, a minor impact on susceptibility to polymyxins was noticed once the mcr-9 gene was cloned and produced in an E. coli K-12-derived strain. Nevertheless, we showed here that subinhibitory concentrations of colistin induced the expression of the mcr-9 gene, leading to increased MIC levels. This inducible expression was mediated by a two-component regulatory system encoded by the qseC and qseB genes located downstream of mcr-9 Genetic analysis showed that the mcr-9 gene was carried by an IncHI2 plasmid. In silico analysis revealed that the plasmid-encoded MCR-9 shared significant amino acid identity (ca. 80%) with the chromosomally encoded MCR-like proteins from Buttiauxella spp. In particular, Buttiauxella gaviniae was found to harbor a gene encoding MCR-BG, sharing 84% identity with MCR-9. That gene was neither expressed nor inducible in its original host, which was fully susceptible to polymyxins. This work showed that mcr genes may circulate silently and remain undetected unless induced by colistin., (Copyright © 2019 American Society for Microbiology.)

Adipogenesis is an organized process of cellular differentiation by which pre-adipocytes differentiate towards mature adipocytes. miR-16-5p has been reported to be involved in cell proliferation, apoptosis, differentiation and angiogenesis. However little is known about miR-16-5p functional role in 3T3-L1 adipocyte differentiation. In this study, we found that miRNA-16-5p was significantly upregulated during 3T3-L1 preadipocytes differentiation towards mature adipocytes. Over-expression of miRNA-16-5p promoted mature adipocytes specific genes expression and fat droplet accumulation in vitro and in vivo. Meanwhile we have identified EPT1 as the target gene of miRNA-16-5p. Taken together, our data provided evidence to support that miRNA-16-5p promotes adipocyte differentiation by suppressing EPT1., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The presence of mobilized colistin resistance (mcr) genes is a global concern. However, data concerning mcr in fresh vegetables, a reservoir for antibiotic resistance genes, are still rare. In this study, mcr genes were analysed in 528 vegetable samples from 53 supermarkets or farmer's markets in 23 cities of 9 provinces in China, and the mcr-positive Enterobacteriaceae were characterized. Nineteen (3.6%) samples carried one or more mcr-positive isolates, and the highest three detection rates were found in carrot, pak choi and green pepper. Twenty-four mcr-1-positive isolates (23 Escherichia coli and one Enterobacter cloacae) were obtained, and E. coli isolates showed high genetic diversity. Different multilocus sequence type (MLST) isolates were also observed within the same sample. All 24 isolates showed multidrug resistance, and 14 carried bla CTX-M genes. Most isolates harbored similarly conjugative IncX4-type (∼33 kb) or IncI2-type (∼60 kb) mcr-1-bearing plasmids. The sequenced prevalent IncX4 plasmid and IncI2 plasmid from tomato were similar to the relevant plasmids from animals and clinical isolates in various countries. mcr-1-bearing IncHI2/ST3 plasmid highly similar to that carrying 14 resistance genes from E. coli of chicken was also observed. In conclusion, a high prevalence of mcr-1 in fresh vegetables was found in China, and the dissemination of mcr-1 was mediated by similar IncX4 or IncI2 plasmids. The plasmids from vegetables showed high similarity to plasmids from clinical isolates, indicating MCR-1-producers in ready-to-eat vegetables may pose a huge threat to public health and measures need to be taken to ensure food safety., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Introduction: Carbapenem-resistant Klebsiella pneumoniae are a major problem. We aimed to investigate carbapenemase-encoding genes and transferable mcr-1 genes among 57 carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates from hospitalized patients., Methodology: Antibiotic susceptibility tests were performed by Phoenix (BD). Results for ertapenem and colistin were confirmed by gradient diffusion and microdilution methods. Carbapenemase and mcr-1 genes were investigated by Polymerase Chain Reaction (PCR)., Results: Thirty-two (56.14%) isolates were from intensive care units (ICU). Antibiotic resistance rates by Phoenix: 52.63% for amikacin; 73.69% trimethoprim sulfamethoxazole; 91.23% cefepime; 82.46% tigecycline; 59.65% colistin. Carbapenemases positivity: 82.45% (47) for blaOXA-48, 40.35% (23) blaOXA-55, 3.50% (2) blaOXA-51, 1.75% (1) blaOXA-23, 1.75% (1) blaOXA-24, 1.75% (1) blaIMP. blaOXA-58, blaKPC, blaNDM-1, and blaVIM were not detected. Twenty (35.08%) isolates had both blaOXA-48 and blaOXA-55. Three isolates were mcr-1 (+) and blaOXA-48 (+). One mcr-1 (+) isolates was blaOXA-51 (+). One colistin sensitive isolate determined by Phoenix, was found colistin resistant by microdilution., Conclusion: OXA-48 and OXA-55 co-harboring isolates and mcr-1 gene (+) isolates were spreading. Automated colistin susceptibility results should be confirmed by microdilution method. Resistance mechanisms in Enterobacteriaceae should be determined and the isolates should be monitored by molecular epidemiological methods. Effective infection control measures will contribute to reduce risk of antibiotic resistant bacterial infections and dissemination of antibiotic resistance., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2019 Cigdem Arabaci, Tuba Dal, Tugcan Basyigit, Neslihan Genisel, Riza Durmaz.)

We characterized eight mcr-5 -positive Salmonella enterica subsp. enterica serovar Typhimurium sequence type 34 (ST34) isolates obtained from pigs and meat in Germany. Five plasmid types were identified harboring mcr-5 on Tn 6452 or putative mobile insertion cassettes. The mobility of mcr-5 was confirmed by integration of Tn 6452 into the bacterial chromosomes of two strains and the detection of conjugative mcr-5 plasmids. The association with mobile genetic elements might further enhance mcr-5 distribution., (Copyright © 2019 American Society for Microbiology.)

Two hundred isolates of Enterobacterales were tested by Rapid Polymyxins NP for the detection of polymyxin resistance and compared to the reference test broth microdilution (BMD). The sensitivity and specificity of the NP test were 98% and the results are faster than the BMD, decreasing from approximately 24 to 2 h.

Outer membrane vesicles (OMVs) are naturally produced by Gram-negative bacteria by a bulging of the outer membrane (OM) and subsequent release into the environment. By serving as vehicles for various cargos, including proteins, nucleic acids and small metabolites, OMVs are central to interbacterial interactions and both symbiotic and pathogenic host bacterial interactions. However, despite their importance, the mechanism of OMV formation remains unclear. Recent evidence indicates that covalent modifications of lipopolysaccharides (LPS) influence OMV biogenesis. Several enteric bacteria modify LPS with phosphoethanolamine (pEtN) using the iron-regulated PmrC (EptA) and CptA pEtN transferases. In wild-type Citrobacter rodentium , the presence of increasing subtoxic concentrations of iron was found to stimulate OMV production 4- to 9-fold above baseline. C. rodentium uses the two-component system PmrAB to sense and adapt to environmental iron. Compared to the wild type, the C. rodentium Δ pmrAB strain exhibited heightened OMV production at similar iron concentrations. PmrAB regulates transcription of pmrC (also known as eptA ) and cptA OMV production in strains lacking either pmrC ( eptA ) or cptA was similarly increased in comparison to that of the wild type. Importantly, plasmid complementation of C. rodentium strains with either pmrC ( eptA ) or cptA resulted in a drastic inhibition of OMV production. Finally, we showed that β-lactamase and CroP, two enzymes found in the C. rodentium periplasm and outer membrane (OM), respectively, are associated with OMVs. These data suggest a novel mechanism by which C. rodentium and possibly other Gram-negative bacteria can negatively affect OMV production through the PmrAB-regulated genes pmrC ( eptA ) and cptA IMPORTANCE Although OMVs secreted by Gram-negative bacteria fulfill multiple functions, the molecular mechanism of OMV biogenesis remains ill defined. Our group has previously shown that PmrC (also known as EptA) and CptA maintain OM integrity and provide resistance to iron toxicity and antibiotics in the murine pathogen Citrobacter rodentium In several enteric bacteria, these proteins modify the lipid A and core regions of lipopolysaccharide with phosphoethanolamine moieties. Here, we show that these proteins also repress OMV production in response to environmental iron in C. rodentium These data support the emerging understanding that lipopolysaccharide modifications are important regulators of OMV biogenesis in Gram-negative bacteria., (Copyright © 2019 Sinha et al.)

Objectives: Historically, colistin has been considered a last-line therapeutic option against multidrug-resistant Gram-negative bacterial infections. However, chromosomally-encoded and plasmid-mediated colistin resistance is increasingly being reported worldwide. Spread of the plasmid-borne colistin resistance gene mcr-1 is of great concern since it can be transferred between bacteria. The aim of this study was to investigate the prevalence of mcr-1 in Escherichia coli and Klebsiella pneumoniae collected from human clinical specimens in Thailand during 2014-2017., Methods: Minimum inhibitory concentrations (MICs) of colistin were determined by the broth microdilution method for 317 non-duplicate Enterobacteriaceae clinical isolates (37 E. coli and 280 K. pneumoniae). All isolates were screened for the mcr-1 gene by PCR., Results: The colistin MIC 50 , MIC 90 and MIC range for the 37 E. coli isolates were 0.5, 8 and 0.5-32mg/L, respectively. The mcr-1 gene was detected in 11 E. coli isolates (29.7%). Escherichia coli harbouring the mcr-1 gene had a colistin MIC range of 4-32mg/L. The colistin MIC 50 , MIC 90 , and MIC range for the 280 K. pneumoniae isolates were 32, >128, and 0.25 to >128mg/L, respectively. The mcr-1 gene was detected in 4 K. pneumoniae isolates (1.4%). Klebsiella pneumoniae harbouring the mcr-1 gene had a colistin MIC range of 4-64mg/L., Conclusions: This is the first report on the prevalence of the mcr-1 gene in colistin-resistant E. coli and K. pneumoniae isolated from humans in Thailand. These data provide added insight into the mechanism of colistin resistance among Enterobacteriaceae pathogens., (Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

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 IS Aba125 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., (Copyright © 2018 American Society for Microbiology.)

The plasmid-mediated colistin resistance gene mcr-1 has been found worldwide, but the diversity of organisms harbouring this gene is unknown. In this study, 12 colistin-resistant Citrobacter spp. isolates were obtained from diseased or dead chickens in China, and PCR analysis indicated that five were positive for mcr-1. One Citrobacter braakii strain (SCC4) with a multidrug-resistant phenotype was chosen for further analysis. SCC4 was resistant or intermediate-resistant to ten of the tested antibiotics, and the colistin minimum inhibitory concentration (MIC) was >4 µg/mL. A conjugation assay demonstrated successful transfer of colistin resistance to Escherichia coli strain J53 at a frequency of 10 -7 cells per recipient cell. Whole-genome sequencing revealed that SCC4 contained 13 antibiotic resistance genes in its genome, and the mcr-1 gene resided on a 44-kb self-transmissible IncP-type plasmid of a recently discovered IncP-1 clade. In addition, the mcr-1 gene was part of an insertion element (ISApl1-mcr-1-orf-ISApl1) that was excised from the plasmid as a circular intermediate form. This is the first report of mcr-1-posiitve C. braakii of animal origin and these findings highlight the fact that the mcr-1 gene can be found in normal enteric flora as part of broad-host-range plasmids., (Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

Ethanolamine phosphotransferase (EPT)1, also known as selenoprotein 1 (SELENOI), is an enzyme that transfers phosphoethanolamine from cytidine diphosphate-ethanolamine to lipid acceptors to produce ethanolamine glycerophospholipids, such as diacyl-linked phosphatidylethanolamine (PE) and ether-linked plasmalogen [1-alkenyl-2-acyl-glycerophosphoethanolamine (plasmenyl-PE)]. However, to date there has been no analysis of the metabolomic consequences of the mutation of EPT1 on the concentration of ethanolamine glycerophospholipids in mammalian cells. We studied a patient with severe complicated hereditary spastic paraplegia, sensorineural-deafness, blindness, and seizures. Neuroimaging revealed hypomyelination, followed by brain atrophy mainly in the cerebellum and brainstem. Using whole exome sequencing, we identified a novel EPT1 mutation (exon skipping). In vitro EPT activity, as well as the rate of biosynthesis of ethanolamine glycerophospholipids, was markedly reduced in cultures of the patient's skin fibroblasts. Quantification of phospholipids by LC-MS/MS demonstrated reduced levels of several PE species with polyunsaturated fatty acids, such as 38:6, 38:4, 40:6, 40:5, and 40:4. Notably, most plasmenyl-PE species were significantly decreased in the patient's cells, whereas most plasmanylcholine [1-alkyl-2-acyl-glycerophosphocholine (plasmanyl-PC)] species were increased. Similar findings regarding decreased plasmenyl-PE and increased plasmanyl-PC were obtained using EPT1 -KO HeLa cells. Our data demonstrate for the first time the indispensable role of EPT1 in the myelination process and neurodevelopment, and in the maintenance of normal homeostasis of ether-linked phospholipids in humans., (Copyright © 2018 by the American Society for Biochemistry and Molecular Biology, Inc.)

Polymyxins, a family of cationic antimicrobial cyclic peptides, act as a last line of defense against severe infections by Gram-negative pathogens with carbapenem resistance. In addition to the intrinsic resistance to polymyxin E (colistin) conferred by Neisseria eptA , the plasmid-borne mobilized colistin resistance gene mcr-1 has been disseminated globally since the first discovery in Southern China, in late 2015. However, the molecular mechanisms for both intrinsic and transferable resistance to colistin remain largely unknown. Here, we aim to address this gap in the knowledge of these proteins. Structural and functional analyses of EptA and MCR-1 and -2 have defined a conserved 12-residue cavity that is required for the entry of the lipid substrate, phosphatidylethanolamine (PE). The in vitro and in vivo data together have allowed us to visualize the similarities in catalytic activity shared by EptA and MCR-1 and -2. The expression of either EptA or MCR-1 or -2 is shown to remodel the surface of enteric bacteria (e.g., Escherichia coli , Salmonella enterica , Klebsiella pneumoniae , etc.), rendering them resistant to colistin. The parallels in the PE substrate-binding cavities among EptA, MCR-1, and MCR-2 provide a comprehensive understanding of both intrinsic and transferable colistin resistance. Domain swapping between EptA and MCR-1 and -2 reveals that the two domains (transmembrane [TM] region and p hospho e thanol a mine [PEA] transferase) are not functionally exchangeable. Taken together, the results represent a common mechanism for intrinsic and transferable PEA resistance to polymyxin, a last-resort antibiotic against multidrug-resistant pathogens. IMPORTANCE EptA and MCR-1 and -2 remodel the outer membrane, rendering bacteria resistant to colistin, a final resort against carbapenem-resistant pathogens. Structural and functional analyses of EptA and MCR-1 and -2 reveal parallel PE lipid substrate-recognizing cavities, which explains intrinsic and transferable colistin resistance in gut bacteria. A similar mechanism is proposed for the catalytic activities of EptA and MCR-1 and -2. Together, they constitute a common mechanism for intrinsic and transferable polymyxin resistance., (Copyright © 2018 Xu et al.)

The rapid emergence of Gram-negative 'superbugs' has become a significant threat to human health globally, and polymyxins have become a last-line therapy for these very problematic pathogens. Polymyxins exhibit their antibacterial killing by initial interaction with lipid A in Gram-negative bacteria. Polymyxin resistance can be mediated by phosphoethanolamine (PEA) modification of lipid A, which abolishes the initial electrostatic interaction with polymyxins. Both chromosome-encoded (e.g. EptA, EptB and EptC) and plasmid-encoded (e.g. MCR-1 and MCR-2) PEA transferases have been reported in Gram-negative bacteria; however, their sequence and functional heterogeneity remain unclear. This article reports a comparative analysis of PEA transferases across 10 clinically relevant Gram-negative bacterial species using multiple sequence alignment and phylogenetic analysis. The results show that the pairwise identities among chromosome-mediated EptA, EptB and EptC from Escherichia coli are low, and EptA shows the greatest similarity with MCR-1 and MCR-2. Among PEA transferases from representative strains of 10 clinically relevant species, the catalytic domain is more conserved compared with the transmembrane domain. In particular, PEA acceptor sites and zinc-binding pockets show high conservation between different species, indicating their potential importance for the function of PEA transferases. The evolutionary relationship of MCR-1, MCR-2 and EptA from the 10 selected bacterial species was evaluated by phylogenetic analysis. Cluster analysis illustrates that 325 EptA from 275 strains of 10 species within each individual species are highly conserved, whereas interspecies conservation is low. This comparative analysis provides key bioinformatic information to better understand the mechanism of polymyxin resistance via PEA modification of lipid A., (Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

Plasmid-borne colistin resistance mediated by mcr-1 may contribute to the dissemination of pan-resistant Gram-negative bacteria. Here, we show that mcr-1 confers resistance to colistin-induced lysis and bacterial cell death, but provides minimal protection from the ability of colistin to disrupt the Gram-negative outer membrane. Indeed, for colistin-resistant strains of Enterobacteriaceae expressing plasmid-borne mcr-1, clinically relevant concentrations of colistin potentiate the action of antibiotics that, by themselves, are not active against Gram-negative bacteria. The result is that several antibiotics, in combination with colistin, display growth-inhibition at levels below their corresponding clinical breakpoints. Furthermore, colistin and clarithromycin combination therapy displays efficacy against mcr-1-positive Klebsiella pneumoniae in murine thigh and bacteremia infection models at clinically relevant doses. Altogether, these data suggest that the use of colistin in combination with antibiotics that are typically active against Gram-positive bacteria poses a viable therapeutic alternative for highly drug-resistant Gram-negative pathogens expressing mcr-1.

Background: Enterobacter cloacae is a major nosocomial pathogen causing bloodstream infections. We retrospectively conducted a study to assess antimicrobial susceptibility and phylogenetic relationships of E. cloacae bloodstream isolates in two tertiary university-affiliated hospitals in Shanghai, in order to facilitate managements of E. cloacae bloodstream infections and highlight some unknowns for future prevention., Methods: Fifty-three non-duplicate E. cloacae bloodstream isolates were consecutively collected from 2013 to 2016. Antimicrobial susceptibility was determined by disk diffusion. PCR was performed to detect extended-spectrum β-lactamase (ESBL), carbapenemase and colistin resistance (MCR-1) gene. Plasmid-mediated AmpC β-lactamase (pAmpC) genes were detected using a multiplex PCR assay targeting MIR/ACT gene (closely related to chromosomal EBC family gene) and other plasmid-mediated genes, including DHA, MOX, CMY, ACC, and FOX. eBURST was applied to analyze multi-locus sequence typing (MLST)., Results: The rates of resistance to all tested antibiotics were <40%. Among 53 E. cloacae isolates, 8(15.1%) were ESBL producers, 3(5.7%) were carbapenemase producers and 18(34.0%) were pAmpC producers. ESBL producers bear significantly higher resistance to cefotaxime (100.0%), ceftazidime (100.0%), aztreonam (100.0%), piperacillin (87.5%), tetracycline (75.0%), and trimethoprim-sulfamethoxazole (62.5%) than non-producers (p<0.05). PAmpC- and non-producers both presented low resistance rates (<40%) to all antibiotics (p>0.05). SHV (6/8, 75.0%) and MIR/ACT (15/18, 83.3%) predominated in ESBL and pAmpC producers respectively. Moreover, 2 isolates co-carried TEM-1, SHV-12, IMP-26 and DHA-1. MLST analysis distinguished the 53 isolates into 51 STs and only ST414 and ST520 were assigned two isolates of each (2/53)., Conclusion: The antimicrobial resistance rates were low among 53 E. cloacae bloodstream isolates in the two hospitals. Multiclonality disclosed no evidence on spread of these isolates in Shanghai. The simultaneous presence of ESBL, carbapenemase and pAmpC detected in 2 isolates was firstly reported in Shanghai, which necessitated active ongoing surveillances and consistent prevention and control of E. cloacae.

Objectives: Plasmid-mediated mobilized colistin resistance is currently known to be caused by phosphoethanolamine transferases termed MCR-1, MCR-2, MCR-3 and MCR-4. However, this study focuses on the dissection of a novel resistance mechanism in mcr-1-, mcr-2- and mcr-3-negative d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B (Salmonella Paratyphi B dTa+) isolates with colistin MIC values >2 mg/L., Methods: A selected isolate from the strain collection of the German National Reference Laboratory for Salmonella was investigated by WGS and bioinformatical analysis to identify novel phosphoethanolamine transferase genes involved in colistin resistance. Subsequently PCR screening, S1-PFGE and DNA-DNA hybridization were performed to analyse the prevalence and location of the identified mcr-5 gene. Cloning and transformation experiments in Escherichia coli DH5α and Salmonella Paratyphi B dTa+ control strains were carried out and the activity of MCR-5 was determined in vitro by MIC testing., Results: In this study, we identified a novel phosphoethanolamine transferase in 14 mcr-1-, mcr-2- and mcr-3-negative Salmonella Paratyphi B dTa+ isolates with colistin MIC values >2 mg/L that were received during 2011-13. The respective gene, further termed as mcr-5 (1644 bp), is part of a 7337 bp transposon of the Tn3 family and usually located on related multi-copy ColE-type plasmids. Interestingly, in one isolate an additional subclone with a chromosomal location of the mcr-5 transposon was observed., Conclusions: Our findings suggest that the transfer of colistin-resistance-mediating phosphoethanolamine transferase genes from bacterial chromosomes to mobile genetic elements has occurred in multiple independent events raising concern regarding their variety, prevalence and impact on public health., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The lipopolysaccharide (LPS) produced by the Gram-negative bacterial pathogen Pasteurella multocida has phosphoethanolamine (PEtn) residues attached to lipid A, 3-deoxy-d-manno-octulosonic acid (Kdo), heptose, and galactose. In this report, we show that PEtn is transferred to lipid A by the P. multocida EptA homologue, PetL, and is transferred to galactose by a novel PEtn transferase that is unique to P. multocida called PetG. Transcriptomic analyses indicated that petL expression was positively regulated by the global regulator Fis and negatively regulated by an Hfq-dependent small RNA. Importantly, we have identified a novel PEtn transferase called PetK that is responsible for PEtn addition to the single Kdo molecule (Kdo 1 ), directly linked to lipid A in the P. multocida glycoform A LPS. In vitro assays showed that the presence of a functional petL and petK , and therefore the presence of PEtn on lipid A and Kdo 1 , was essential for resistance to the cationic, antimicrobial peptide cathelicidin-2. The importance of PEtn on Kdo 1 and the identification of the transferase responsible for this addition have not previously been shown. Phylogenetic analysis revealed that PetK is the first representative of a new family of predicted PEtn transferases. The PetK family consists of uncharacterized proteins from a range of Gram-negative bacteria that produce LPS glycoforms with only one Kdo molecule, including pathogenic species within the genera Vibrio , Bordetella , and Haemophilus We predict that many of these bacteria will require the addition of PEtn to Kdo for maximum protection against host antimicrobial peptides., (Copyright © 2017 American Society for Microbiology.)

We have characterized nine mcr-1-harboring plasmids from clinical Escherichia coli isolates previously described in Argentina and Canada. Three of these plasmids carried a mcr-1-variant called here mcr-1.5. All these E. coli isolates were not clonally related and were recovered in different years and locations. However, their mcr-1-harboring plasmids showed high identity among them and to others characterized in other countries, which strongly suggests that this plasmid-type is playing an important role in spreading this mechanism of resistance to polymyxins.

Plasmid-mediated resistance to polymyxins mediated by the MCR-1/2 determinants has been reported in Enterobacteriaceae worldwide. Using PCR-based and cloning strategies, a series of Moraxella spp. were screened for mcr -like genes. Moraxella spp. that are mainly animal pathogens but may also be human pathogens were identified as potential reservoirs of mcr -like genes., (Copyright © 2017 American Society for Microbiology.)

In this study, we report a novel mcr-1 gene variant, named mcr-1.6 , carried by an IncP plasmid in a colistin-resistant Salmonella enterica serovar Typhimurium isolate from a healthy person. Compared with mcr-1 , the mcr-1.6 gene contains two single-nucleotide polymorphisms, one of which results in an arginine to histidine variation (Arg536→His). The plasmid carrying the mcr-1.6 gene was designated pMCR1.6&#95;P053 and is similar to a recently discovered mcr-1 -bearing plasmid found in Klebsiella pneumoniae ., (Copyright © 2017 American Society for Microbiology.)

The recently discovered colistin resistance element, mcr-1 , adds to the list of antimicrobial resistance genes that rapidly erode the antimicrobial efficacy of not only the commonly used antibiotics but also the last-line agents of carbapenems and colistin. This study investigated the prevalence of the mobile colistin resistance determinant mcr-1 in Salmonella strains recovered from clinical settings in China and the transmission potential of mcr-1 -bearing mobile elements harbored by such isolates. The mcr-1 gene was recoverable in 1.4% of clinical isolates tested, with the majority of them belonging to Salmonella enterica serotype Typhimurium. These isolates exhibited diverse pulsed-field gel electrophoresis (PFGE) profiles and high resistance to antibiotics other than colistin and particularly to cephalosporins. Plasmid analysis showed that mcr-1 was carried on a variety of plasmids with sizes ranging from ∼30 to ∼250 kb, among which there were conjugative plasmids of ∼30 kb, ∼60 kb, and ∼250 kb and nonconjugative plasmids of ∼140 kb, ∼180 kb, and ∼240 kb. Sequencing of representative mcr-1 -carrying plasmids revealed that all conjugative plasmids belonged to the IncX4, IncI2, and IncHI2 types and were highly similar to the corresponding types of plasmids reported previously. Nonconjugative plasmids all belonged to the IncHI2 type, and the nontransferability of these plasmids was attributed to the loss of a region carrying partial or complete tra genes. Our data revealed that, similar to the situation in Escherichia coli , mcr-1 transmission in Salmonella was accelerated by various plasmids, suggesting that transmission of mcr-1 -carrying plasmids between different species of Enterobacteriaceae may be a common event., (Copyright © 2017 American Society for Microbiology.)

Mutations in genes involved in lipid metabolism have increasingly been associated with various subtypes of hereditary spastic paraplegia, a highly heterogeneous group of neurodegenerative motor neuron disorders characterized by spastic paraparesis. Here, we report an unusual autosomal recessive neurodegenerative condition, best classified as a complicated form of hereditary spastic paraplegia, associated with mutation in the ethanolaminephosphotransferase 1 (EPT1) gene (now known as SELENOI), responsible for the final step in Kennedy pathway forming phosphatidylethanolamine from CDP-ethanolamine. Phosphatidylethanolamine is a glycerophospholipid that, together with phosphatidylcholine, constitutes more than half of the total phospholipids in eukaryotic cell membranes. We determined that the mutation defined dramatically reduces the enzymatic activity of EPT1, thereby hindering the final step in phosphatidylethanolamine synthesis. Additionally, due to central nervous system inaccessibility we undertook quantification of phosphatidylethanolamine levels and species in patient and control blood samples as an indication of liver phosphatidylethanolamine biosynthesis. Although this revealed alteration to levels of specific phosphatidylethanolamine fatty acyl species in patients, overall phosphatidylethanolamine levels were broadly unaffected indicating that in blood EPT1 inactivity may be compensated for, in part, via alternate biochemical pathways. These studies define the first human disorder arising due to defective CDP-ethanolamine biosynthesis and provide new insight into the role of Kennedy pathway components in human neurological function., (© The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Multidrug-resistant (MDR) gram-negative bacteria have increased the prevalence of fatal sepsis in modern times. Colistin is a cationic antimicrobial peptide (CAMP) antibiotic that permeabilizes the bacterial outer membrane (OM) and has been used to treat these infections. The OM outer leaflet is comprised of endotoxin containing lipid A, which can be modified to increase resistance to CAMPs and prevent clearance by the innate immune response. One type of lipid A modification involves the addition of phosphoethanolamine to the 1 and 4' headgroup positions by phosphoethanolamine transferases. Previous structural work on a truncated form of this enzyme suggested that the full-length protein was required for correct lipid substrate binding and catalysis. We now report the crystal structure of a full-length lipid A phosphoethanolamine transferase from Neisseria meningitidis , determined to 2.75-Å resolution. The structure reveals a previously uncharacterized helical membrane domain and a periplasmic facing soluble domain. The domains are linked by a helix that runs along the membrane surface interacting with the phospholipid head groups. Two helices located in a periplasmic loop between two transmembrane helices contain conserved charged residues and are implicated in substrate binding. Intrinsic fluorescence, limited proteolysis, and molecular dynamics studies suggest the protein may sample different conformational states to enable the binding of two very different- sized lipid substrates. These results provide insights into the mechanism of endotoxin modification and will aid a structure-guided rational drug design approach to treating multidrug-resistant bacterial infections.

Aims: Investigate how Cronobacter sakazakii modify their lipid A structure to avoid recognition by the host immune cells., Methods and Results: Lipid A modification was observed in C. sakazakii BAA894 grown at pH 5·0 but not pH 7·0. Overexpression of C. sakazakii gene ESA&#95;RS09200 in Escherichia coli W3110 caused a phosphoethanolamine (PEA) modification of lipid A; when ESA&#95;RS09200 was deleted in C. sakazakii BAA894, this lipid A modification disappeared. Lipid A modification was observed in BAA894 grown at pH 5·0 when the 1- phosphate residue of lipid A was removed, but disappeared when the 4'- phosphate residue of lipid A was removed. When ESA&#95;RS16430, the orthologous gene of E. coli pmrA, was deleted in C. sakazakii BAA894, this PEA modification of lipid A was still observed, suggesting that this modification was not regulated by the PmrA-PmrB system. Compared to the wild-type BAA894, ESA&#95;RS09200 deletion mutant showed decreased resistance to cationic antimicrobial peptides (CAMP), increased recognition by TLR4/MD2, decreased ability to invade and persist in mammalian cells., Conclusions: ESA&#95;RS09200 in C. sakazakii BAA894 encodes a PEA transferase that specifically adds a PEA to the 4'-phosphate residue of lipid A, but not regulated by the PmrA-PmrB system. PEA modification of lipid A reduces recognition and killing by the host innate immune system., Significance and Impact of the Study: This study showed that modification of the lipid A moiety of C. sakazakii with PEA increased resistance to CAMP and recognition of the immune response although signalling of TLR4/MD2 cascade, suggesting that the organism could not successfully evade the host innate immune system without the transference of PEA to its lipid A moiety., (© 2016 The Society for Applied Microbiology.)

Unlabelled: Colistin is an ultimate line of refuge against multidrug-resistant Gram-negative pathogens. Very recently, the emergence of plasmid-mediatedmcr-1colistin resistance has become a great challenge to global public health, raising the possibility that dissemination of themcr-1gene is underestimated and diversified. Here, we report three cases of plasmid-carried MCR-1 colistin resistance in isolates from gut microbiota of diarrhea patients. Structural and functional analyses determined that the colistin resistance is conferred purely by the singlemcr-1gene. Genetic and sequence mapping revealed thatmcr-1-harbouring plasmid reservoirs are present in diversity. Together, the data represent the first evidence of diversity inmcr-1-harbouring plasmid reservoirs of human gut microbiota., Importance: The plasmid-mediated mobile colistin resistance gene (mcr-1) challenged greatly the conventional idea mentioned above that colistin is an ultimate line of refuge against lethal infections by multidrug-resistant Gram-negative pathogens. It is a possibility that diversified dissemination of themcr-1gene might be greatly underestimated. We report three cases of plasmid-carried MCR-1 colistin resistance in isolates from gut microbiota of diarrhea patients and functionally define the colistin resistance conferred purely by the singlemcr-1gene. Genetic and sequence mapping revealed unexpected diversity among themcr-1-harbouring plasmid reservoirs of human gut microbiota., (Copyright © 2016 Ye et al.)

Shewanella algae MARS 14 is a colistin-resistant clinical isolate retrieved from bronchoalveolar lavage of a hospitalised patient. A functional genomics strategy was employed to discover the molecular support for colistin resistance in S. algae MARS 14. A pZE21 MCS-1 plasmid-based genomic expression library was constructed in Escherichia coli TOP10. The estimated library size was 1.30×10(8) bp. Functional screening of colistin-resistant clones was carried out on Luria-Bertani agar containing 8 mg/L colistin. Five colistin-resistant clones were obtained after complete screening of the genomic expression library. Analysis of DNA sequencing results found a unique gene in all selected clones. Amino acid sequence analysis of this unique gene using the Integrated Microbial Genomes (IMG) and KEGG databases revealed that this gene encodes ethanolamine phosphotransferase (EptA, or so-called PmrC). Reverse transcription PCR analysis indicated that resistance to colistin in S. algae MARS 14 was associated with overexpression of EptA (27-fold increase), which plays a crucial role in the arrangement of outer membrane lipopolysaccharide., (Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)