Jingxia Lin

Dissemination and Mechanism for the MCR-1 Colistin Resistance

Polymyxins are the last line of defense against lethal infections caused by multidrug resistant Gram-negative pathogens. Very recently, the use of polymyxins has been greatly challenged by the emergence of the plasmid-borne mobile colistin resistance gene (mcr-1). However, the mechanistic aspects of the MCR-1 colistin resistance are still poorly understood. Here we report the comparative genomics of two new mcr-1-harbouring plasmids isolated from the human gut microbiota, highlighting the diversity in plasmid transfer of the mcr-1 gene. Further genetic dissection delineated that both the trans-membrane region and a substrate-binding motif are required for the MCR-1-mediated colistin resistance. The soluble form of the membrane protein MCR-1 was successfully prepared and verified. Phylogenetic analyses revealed that MCR-1 is highly homologous to its counterpart PEA lipid A transferase in Paenibacili, a known producer of polymyxins. The fact that the plasmid-borne MCR-1 is placed in a subclade neighboring the chromosome-encoded colistin-resistant Neisseria LptA (EptA) potentially implies parallel evolutionary paths for the two genes. In conclusion, our finding provids a first glimpse of mechanism for the MCR-1-mediated colistin resistance.

Deciphering MCR-2 Colistin Resistance

ABSTRACT Antibiotic resistance is a prevalent problem in public health worldwide. In general, the carbapenem β-lactam antibiotics are considered a final resort against lethal infections by multidrug-resistant bacteria. Colistin is a cationic polypeptide antibiotic and acts as the last line of defense for treatment of carbapenem-resistant bacteria. Very recently, a new plasmid-borne colistin resistance gene, mcr-2, was revealed soon after the discovery of the paradigm gene mcr-1, which has disseminated globally. However, the molecular mechanisms for MCR-2 colistin resistance are poorly understood. Here we show a unique transposon unit that facilitates the acquisition and transfer of mcr-2. Evolutionary analyses suggested that both MCR-2 and MCR-1 might be traced to their cousin phosphoethanolamine (PEA) lipid A transferase from a known polymyxin producer, Paenibacillus. Transcriptional analyses showed that the level of mcr-2 transcripts is relatively higher than that of mcr-1. Genetic deletions revealed that the transmembrane regions (TM1 and TM2) of both MCR-1 and MCR-2 are critical for their location and function in bacterial periplasm, and domain swapping indicated that the TM2 is more efficient than TM1. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) confirmed that all four MCR proteins (MCR-1, MCR-2, and two chimeric versions [TM1-MCR-2 and TM2-MCR-1]) can catalyze chemical modification of lipid A moiety anchored on lipopolysaccharide (LPS) with the addition of phosphoethanolamine to the phosphate group at the 4′ position of the sugar. Structure-guided site-directed mutagenesis defined an essential 6-residue-requiring zinc-binding/catalytic motif for MCR-2 colistin resistance. The results further our mechanistic understanding of transferable colistin resistance, providing clues to improve clinical therapeutics targeting severe infections by MCR-2-containing pathogens. IMPORTANCE Carbapenem and colistin are the last line of refuge in fighting multidrug-resistant Gram-negative pathogens. MCR-2 is a newly emerging variant of the mobilized colistin resistance protein MCR-1, posing a potential challenge to public health. Here we report transfer of the mcr-2 gene by a unique transposal event and its possible origin. Distribution of MCR-2 in bacterial periplasm is proposed to be a prerequisite for its role in the context of biochemistry and the colistin resistance. We also define the genetic requirement of a zinc-binding/catalytic motif for MCR-2 colistin resistance. This represents a glimpse of transferable colistin resistance by MCR-2. IMPORTANCE Carbapenem and colistin are the last line of refuge in fighting multidrug-resistant Gram-negative pathogens. MCR-2 is a newly emerging variant of the mobilized colistin resistance protein MCR-1, posing a potential challenge to public health. Here we report transfer of the mcr-2 gene by a unique transposal event and its possible origin. Distribution of MCR-2 in bacterial periplasm is proposed to be a prerequisite for its role in the context of biochemistry and the colistin resistance. We also define the genetic requirement of a zinc-binding/catalytic motif for MCR-2 colistin resistance. This represents a glimpse of transferable colistin resistance by MCR-2.

Expanding landscapes of the diversified mcr-1 -bearing plasmid reservoirs

BackgroundPolymyxin is a cationic polypeptide antibiotic that can disrupt bacterial cell membrane by interacting with its lipopolysaccharide molecules and is used as a last resort drug against lethal infections by the carbapenem-resistant superbugs (like NDM-1). However, global discovery of the MCR-1 colistin resistance dramatically challenges the newly renewed interest in colistin for clinical use.MethodsThe mcr-1-harboring plasmids were acquired from swine and human Escherichia coli isolated in China, from 2015 to 2016, and subjected to Illumina PacBio RSII and Hi-Seq2000 for full genome sequencing. PCR was applied to close the gap of the assembled contigs. Ori-Finder was employed to predict the replication origin (oriC) in plasmids. The phenotype of MCR-1-producing isolates was evaluated on the LBA plates with various level of colistin. Genetic deletion was used to test the requirement of the initial “ATG” codon for the MCR-1 function.ResultsHere, we report full genomes of over 10 mcr-1-harboring plasmids with diversified replication incompatibilities. A novel hybrid IncI2/IncFIB plasmid pGD17-2 was discovered and characterized from a swine isolate with colistin resistance. Intriguingly, co-occurrence of two unique mcr-1-bearing plasmids (pGD65-3, IncI2, and pGD65-5, IncX4) was detected in a single isolate GD65, which might accelerate dissemination of the mcr-1 under environmental selection pressure. Genetic analyses of these plasmids mapped mobile elements in the context of antibiotic resistance and determined two insertion sequences (ISEcp1 and ISApl1) that are responsible for the mobilization of mcr-1. Gene deletion also proved that the first ATG codon is redundant in the mcr-1 gene.ConclusionsCollectively, our results extend landscapes of the diversified mcr-1-bearing plasmid reservoirs.

Unexpected complexity of multidrug resistance in the mcr-1-harbouring Escherichia coli.

The discovery that the mobile colistin resistance gene (mcr-1) is encoded by plasmids and is prevalent in food animals and human beings worldwide (Hasman et al., 2015; Liu et al., 2015) has challenged greatly our traditional idea that polymyxin (consisting of two isoforms：polymyxin B and polymyxin E (colistin)) acts as an ultimate line of refuge in the clinical treatment against the severe infections by the multidrug-resistant Gram-negative pathogens (Nation et al., 2015; Paterson and Harris, 2015). To make matters worse, the mcr-1 gene was recently found to be co-localized with other drug resistance genes in the plasmid pKH457-3-BE with an IncP backbone from a bovine isolate in Belgium (Surbi Malhotra-Kumar, 2016), which is far different from the pig-isolated plasmid, pHNSHP45 with an IncI2 backbone (Liu et al., 2015). It raised the possibility that super-bugs with pan-drug resistance might be emerging. Given the fact that (1) genomic sequences of the mcr-1-harbouring plasmids are extremely limited right now, and (2) co-occurrence of MCR-1-mediated colistin resistance with other multidrug resistance remains unclear, we screened a collection of antibiotic-resistant isolates (no., 102) from swine tissues in China. In particular, only 6 of the 16 colistin-resistant isolates (namely WH01, WH02, …, WH16) are verified to be mcr-1-positive Escherichia coli (Figure 1A, 1B and S1). They are namely WH03, WH07, WH09, WH12, WH13, and WH15. In contrast, the majority of the isolates we checked is mcr-1-negative, but remains appreciable level of the colistin resistance (not shown), implying the possibility that some mystical machiner-ies/vectors claim for this antibiotic resistance. Subsequently, we tested the sensitivity of the six mcr-1-positive E. coli isolates to a dozen of various antibiotics (Figure 1C and S1). The antibiotics (15 in total) we used here are categorized into eight groups 1: β-lactams antibiotics including ampicillin (AMP), cefotaxime (CTX); 2: quinolone antibiotic such as ciprofloxacin (CIP), norfloxacin (NOR) and levofloxacin (LEV); 3: tetracycline antibiotics (tetracycline (TET) and doxycycline (DOX)); 4: aminoglycoside antibiotics like amikacin (AMK), gentamycin (GEN) and kana-mycin (KAN); 5: amino alcohol antibiotic, chloramphenicol (CHL); 6: sulfonamide antibiotic, trimethoprim (TMP); 7: nitrofuran antibiotic, macrodantin (NFT); and 8: cationic polypeptide antibiotic, colistin (COL)). To our surprise, the unexpected complexity of the multi-drug resistance was observed in the mcr-1-harbouring isolates. First, the mcr-1-poitive isolate WH13 exhibited the mostly-broad-spectrum antibiotic resistance in that it can be tolerant with nearly all the 15 antibiotics with an exception of amikacin (Figure 1C). Second to the WH13, the mcr-1-carrying …

Diversified variants of the mcr-1-carrying plasmid reservoir in the swine lung microbiota.

Emergence of bacteria with multiple drug resistance is a major problem in global public health. Polymyxin comprising polymyxin E (colistin) and polymyxin B is generally recognized as the last-resort antibiotics with broad-spectrum activity against the lethal infections by the multi-drug resistant gram-negative bacteria (Ye et al., 2016). Very recently , Yi-Yun Liu and colleagues reported, for the first time, that the plasmid-encoded mobilized colistin resistance gene (mcr-1) confers the transmissible colistin resistance in Enterobacteriaceae (Liu et al., 2015), which attracted much attention /debate from scientific society and triggered extensive public worrisome. Given the fact that the pHNSHP45 (isolated from swine Escherichia coli) is only one mcr-1-harbouring plasmid with full genome reported, after screening thousands of samples from five provinces of China, from 2011 to 2014 (Liu et al., 2015), we speculated that diversified plasmids and/or pHNSHP45 variants carrying the mcr-1 colistin resistance gene might be present in the microbiota of swine populations where the mcr-1-harbouring mobile elements are transmissible by bacterial conjugation-aided gene horizontal transfer. Similar scenarios were found in the human gut microbiota (Zhang et al., 2016; Ye et al., 2016). Driven by this hypothesis, we re-analyzed our bacterial samples collected from six provinces of China, from 2011 to 2013 (Table S1). Apart from the three provinces (Guangdong, Hunan, and Zhejiang) investigated by Liu and coworkers (Liu et al., 2015), we sampled three more provinces (Jiangsu, Henan, and Hubei, in Figure 1A). Among hundreds of bacteria isolated from the different tissues (like lung, liver, and throat trachea) of pigs, totally 16 bacterial strains were determined to be of colistin resistance where the minimum inhibitory concentrations (MIC) of polymyxin B varied from 4 mg L –1 to 32 mg L –1 (Table S1). The routine biochemical assays combined with 16S rDNA se-quencing (Figure 1C) validated that all these col-istin-resistant bacteria belong to E. coli. Following the PCR screening, six of the above 16 isolates (namely WH03, WH07, WH09, WH12, WH13 and WH15) were totally revealed to be positive for mcr-1 (Figure 1B, 1C and S1). The six strains consistently exhibited the colistin resistance at appreciable level up to 32 mg mL –1 (Figure S2). In contrast, the colistin resistance in the mcr-1-negative E. coli isolates might indicate a different/unknown mechanism. Direct DNA sequencing showed that all the six mcr-1 genes we screened can match 100% to the counterpart from the paradigm mcr-1-harbouring plasmid pHNSHP45 (Figure 1B). We also confirmed the …

Mechanistic insights into transferable polymyxin resistance among gut bacteria

Polymyxins such as colistin are antibiotics used as a final line of defense in the management of infections with multidrug-resistant Gram-negative bacteria. Although natural resistance to polymyxins is rare, the discovery of a mobilized colistin resistance gene (mcr-1) in gut bacteria has raised significant concern. As an intramembrane enzyme, MCR-1 catalyzes the transfer of phosphoethanolamine (PEA) to the 1 (or 4′)-phosphate group of the lipid A moiety of lipopolysaccharide, thereby conferring colistin resistance. However, the structural and biochemical mechanisms used by this integral membrane enzyme remain poorly understood. Here, we report the modeled structure of the full-length MCR-1 membrane protein. Together with molecular docking, our structural and functional dissection of the complex of MCR-1 with its phosphatidylethanolamine (PE) substrate suggested the presence of a 12 residue–containing cavity for substrate entry, which is critical for both enzymatic activity and its resultant phenotypic resistance to colistin. More importantly, two periplasm-facing helices (PH2 and PH2′) of the trans-membrane domain were essential for MCR-1 activity. MALDI-TOF MS and thin-layer chromatography assays provide both in vivo and in vitro evidence that MCR-1 catalyzes the transfer of PEA from the PE donor substrate to its recipient substrate lipid A. Also, the chemical modification of lipid A species was detected in clinical species of bacteria carrying mcr-1. Our results provide mechanistic insights into transferable MCR-1 polymyxin resistance, raising the prospect of rational design of small molecules that reverse bacterial polymyxin resistance, as a last-resort clinical option to combat pathogens with carbapenem resistance.

Complex dissemination of the diversified mcr-1 -harbouring plasmids in Escherichia coli of different sequence types

The emergence of the mobilized colistin resistance gene, representing a novel mechanism for bacterial drug resistance, challenges the last resort against the severe infections by Gram-negative bacteria with multi-drug resistances. Very recently, we showed the diversity in the mcr-1-carrying plasmid reservoirs from the gut microbiota. Here, we reported that a similar but more complex scenario is present in the healthy swine populations, Southern China, 2016. Amongst the 1026 pieces of Escherichia coli isolates from 3 different pig farms, 302 E. coli isolates were determined to be positive for the mcr-1 gene (30%, 302/1026). Multi-locus sequence typing assigned no less than 11 kinds of sequence types including one novel Sequence Type to these mcr-1-positive strains. PCR analyses combined with the direct DNA sequencing revealed unexpected complexity of the mcr-1-harbouring plasmids whose backbones are at least grouped into 6 types four of which are new. Transcriptional analyses showed that the mcr-1 promoter of different origins exhibits similar activity. It seems likely that complex dissemination of the diversified mcr-1-bearing plasmids occurs amongst the various ST E. coli inhabiting the healthy swine populations, in Southern China.

An Evolutionarily Conserved Mechanism for Intrinsic and Transferable Polymyxin Resistance

ABSTRACT 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 phosphoethanolamine [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. 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.

Transcriptional Repression of the VC2105 Protein by Vibrio FadR Suggests that It Is a New Auxiliary Member of the fad Regulon

ABSTRACT Recently, our group along with others reported that the Vibrio FadR regulatory protein is unusual in that, unlike the prototypical fadR product of Escherichia coli, which has only one ligand-binding site, Vibrio FadR has two ligand-binding sites and represents a new mechanism for fatty acid sensing. The promoter region of the vc2105 gene, encoding a putative thioesterase, was mapped, and a putative FadR-binding site (AA CTG GTA AGA GCA CTT) was proposed. Different versions of the FadR regulatory proteins were prepared and purified to homogeneity. Both electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) determined the direct interaction of the vc2105 gene with FadR proteins of various origins. Further, EMSAs illustrated that the addition of long-chain acyl-coenzyme A (CoA) species efficiently dissociates the vc2105 promoter from the FadR regulator. The expression level of the Vibrio cholerae vc2105 gene was elevated 2- to 3-fold in a fadR null mutant strain, validating that FadR is a repressor for the vc2105 gene. The β-galactosidase activity of a vc2105-lacZ transcriptional fusion was increased over 2-fold upon supplementation of growth medium with oleic acid. Unlike the fadD gene, a member of the Vibrio fad regulon, the VC2105 protein played no role in bacterial growth and virulence-associated gene expression of ctxAB (cholera toxin A/B) and tcpA (toxin coregulated pilus A). Given that the transcriptional regulation of vc2105 fits the criteria for fatty acid degradation (fad) genes, we suggested that it is a new member of the Vibrio fad regulon. IMPORTANCE The Vibrio FadR regulator is unusual in that it has two ligand-binding sites. Different versions of the FadR regulatory proteins were prepared and characterized in vitro and in vivo. An auxiliary fad gene (vc2105) from Vibrio was proposed that encodes a putative thioesterase and has a predicted FadR-binding site (AAC TGG TA A GAG CAC TT). The function of this putative binding site was proved using both EMSA and SPR. Further in vitro and in vivo experiments revealed that the Vibrio FadR is a repressor for the vc2105 gene. Unlike fadD, a member of the Vibrio fad regulon, VC2105 played no role in bacterial growth and expression of the two virulence-associated genes (ctxAB and tcpA). Therefore, since transcriptional regulation of vc2105 fits the criteria for fad genes, it seems likely that vc2105 acts as a new auxiliary member of the Vibrio fad regulon.

Defining ICR-Mo, an intrinsic colistin resistance determinant from Moraxella osloensis

Polymyxin is the last line of defense against severe infections caused by carbapenem-resistant gram-negative pathogens. The emergence of transferable MCR-1/2 polymyxin resistance greatly challenges the renewed interest in colistin (polymyxin E) for clinical treatments. Recent studies have suggested that Moraxella species are a putative reservoir for MCR-1/2 genetic determinants. Here, we report the functional definition of ICR-Mo from M. osloensis, a chromosomally encoded determinant of colistin resistance, in close relation to current MCR-1/2 family. ICR-Mo transmembrane protein was prepared and purified to homogeneity. Taken along with an in vitro enzymatic detection, MALDI-TOF mass spectrometry of bacterial lipid A pools determined that the ICR-Mo enzyme might exploit a possible “ping-pong” mechanism to accept the phosphoethanolamine (PEA) moiety from its donor phosphatidylethanolamine (PE) and then transfer it to the 1(or 4’)-phosphate position of lipid A via an ICR-Mo-bound PEA adduct. Structural decoration of LPS-lipid A by ICR-Mo renders the recipient strain of E. coli resistant to polymyxin. Domain swapping assays indicate that the two domains of ICR-Mo cannot be functionally-exchanged with its counterparts in MCR-1/2 and EptA, validating its phylogenetic position in a distinct set of MCR-like genes. Structure-guided functional mapping of ICR-Mo reveals a PE lipid substrate recognizing cavity having a role in enzymatic catalysis and the resultant conference of antibiotic resistance. Expression of icr-Mo in E. coli significantly prevents the formation of reactive oxygen species (ROS) induced by colistin. Taken together, our results define a member of a group of intrinsic colistin resistance genes phylogenetically close to the MCR-1/2 family, highlighting the evolution of transferable colistin resistance.