Circumventing colistin resistance by combining colistin and antimicrobial peptides to kill colistin-resistant and multidrug-resistant Gram-negative bacteria

ABSTRACTThe recent emergence of a transferable colistin resistance mechanism, MCR-1, has gained global attention because of its threat to clinical treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, the possible transmission route ofmcr-1amongEnterobacteriaceaespecies in clinical settings is largely unknown. Here, we present a comprehensive genomic analysis ofEscherichia coliisolates collected in a hospital in Hangzhou, China. We found thatmcr-1-carrying isolates from clinical infections and feces of inpatients and healthy volunteers were genetically diverse and were not closely related phylogenetically, suggesting that clonal expansion is not involved in the spread ofmcr-1. Themcr-1gene was found on either chromosomes or plasmids, but in most of theE. coliisolates,mcr-1was carried on plasmids. The genetic context of the plasmids showed considerable diversity as evidenced by the different functional insertion sequence (IS) elements, toxin-antitoxin (TA) systems, heavy metal resistance determinants, and Rep proteins of broad-host-range plasmids. Additionally, the genomic analysis revealed nosocomial transmission ofmcr-1and the coexistence ofmcr-1with other genes encoding β-lactamases and fluoroquinolone resistance in theE. coliisolates. These findings indicate thatmcr-1is heterogeneously disseminated in both commensal and pathogenic strains ofE. coli, suggest the high flexibility of this gene in its association with diverse genetic backgrounds of the hosts, and provide new insights into the genome epidemiology ofmcr-1among hospital-associatedE. colistrains.IMPORTANCEColistin represents one of the very few available drugs for treating infections caused by extensively multidrug-resistant Gram-negative bacteria. The recently emergentmcr-1colistin resistance gene threatens the clinical utility of colistin and has gained global attention. Howmcr-1spreads in hospital settings remains unknown and was investigated by whole-genome sequencing ofmcr-1-carryingEscherichia coliin this study. The findings revealed extraordinary flexibility ofmcr-1in its spread among genetically diverseE. colihosts and plasmids, nosocomial transmission ofmcr-1-carryingE. coli, and the continuous emergence of novel Inc types of plasmids carryingmcr-1and newmcr-1variants. Additionally,mcr-1was found to be frequently associated with other genes encoding β-lactams and fluoroquinolone resistance. These findings provide important information on the transmission and epidemiology ofmcr-1and are of significant public health importance as the information is expected to facilitate the control of this significant antibiotic resistance threat.

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Antimicrobial Peptides from Rat-Tailed Maggots of the Drone Fly Eristalis tenax Show Potent Activity against Multidrug-Resistant Gram-Negative Bacteria

Microorganisms ◽

10.3390/microorganisms8050626 ◽

2020 ◽

Vol 8 (5) ◽

pp. 626 ◽

Cited By ~ 1

Author(s):

Rolf Hirsch ◽

Jochen Wiesner ◽

Armin Bauer ◽

Alexander Marker ◽

Heiko Vogel ◽

...

Keyword(s):

Antimicrobial Peptides ◽

Multidrug Resistant ◽

Therapeutic Window ◽

Gram Negative Bacteria ◽

Strong Activity ◽

Gram Negative ◽

Physiological Conditions ◽

Manure Storage ◽

Defense Molecules ◽

Generation Sequencing

The spread of multidrug-resistant Gram-negative bacteria is an increasing threat to human health, because novel compound classes for the development of antibiotics have not been discovered for decades. Antimicrobial peptides (AMPs) may provide a much-needed breakthrough because these immunity-related defense molecules protect many eukaryotes against Gram-negative pathogens. Recent concepts in evolutionary immunology predict the presence of potent AMPs in insects that have adapted to survive in habitats with extreme microbial contamination. For example, the saprophagous and coprophagous maggots of the drone fly Eristalis tenax (Diptera) can flourish in polluted aquatic habitats, such as sewage tanks and farmyard liquid manure storage pits. We used next-generation sequencing to screen the E. tenax immunity-related transcriptome for AMPs that are synthesized in response to the injection of bacterial lipopolysaccharide. We identified 22 AMPs and selected nine for larger-scale synthesis to test their activity against a broad spectrum of pathogens, including multidrug-resistant Gram-negative bacteria. Two cecropin-like peptides (EtCec1-a and EtCec2-a) and a diptericin-like peptide (EtDip) displayed strong activity against the pathogens, even under simulated physiological conditions, and also achieved a good therapeutic window. Therefore, these AMPs could be used as leads for the development of novel antibiotics.

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Combining Colistin with Furanone C-30 Rescues Colistin Resistance of Gram-Negative Bacteria in Vitro and in Vivo

Microbiology Spectrum ◽

10.1128/spectrum.01231-21 ◽

2021 ◽

Author(s):

Ying Zhang ◽

Yishuai Lin ◽

Xiaodong Zhang ◽

Liqiong Chen ◽

Chunyan Xu ◽

...

Keyword(s):

Multidrug Resistant ◽

Clinical Isolates ◽

Gram Negative Bacteria ◽

Colistin Resistance ◽

Gram Negative ◽

C 30

Colistin is among the few antibiotics effective against multidrug-resistant Gram-negative bacteria (GNB) clinical isolates. However, colistin-resistant GNB strains have emerged in recent years.

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A Molecular Perspective on Colistin and Klebsiella pneumoniae: Mode of Action, Resistance Genetics, and Phenotypic Susceptibility

Diagnostics ◽

10.3390/diagnostics11071165 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1165

Author(s):

Rita Elias ◽

Aida Duarte ◽

João Perdigão

Keyword(s):

Klebsiella Pneumoniae ◽

Mode Of Action ◽

Lipid A ◽

Drug Susceptibility ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Drug Susceptibility Testing ◽

Gram Negative Bacteria ◽

Colistin Resistance ◽

Gram Negative

Klebsiella pneumoniae is a rod-shaped, encapsulated, Gram-negative bacteria associated with multiple nosocomial infections. Multidrug-resistant (MDR) K. pneumoniae strains have been increasing and the therapeutic options are increasingly limited. Colistin is a long-used, polycationic, heptapeptide that has regained attention due to its activity against Gram-negative bacteria, including the MDR K. pneumoniae strains. However, this antibiotic has a complex mode of action that is still under research along with numerous side-effects. The acquisition of colistin resistance is mainly associated with alteration of lipid A net charge through the addition of cationic groups synthesized by the gene products of a multi-genic regulatory network. Besides mutations in these chromosomal genes, colistin resistance can also be achieved through the acquisition of plasmid-encoded genes. Nevertheless, the diversity of molecular markers for colistin resistance along with some adverse colistin properties compromises the reliability of colistin-resistance monitorization methods. The present review is focused on the colistin action and molecular resistance mechanisms, along with specific limitations on drug susceptibility testing for K. pneumoniae.

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Emerging Opportunity and Destiny of mcr-1 - and tet (X4)-Coharboring Plasmids in Escherichia coli

Microbiology Spectrum ◽

10.1128/spectrum.01520-21 ◽

2021 ◽

Author(s):

Xiaoyu Lu ◽

Xia Xiao ◽

Yuan Liu ◽

Ruichao Li ◽

Zhiqiang Wang

Keyword(s):

Escherichia Coli ◽

Human Health ◽

Resistance Gene ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Colistin Resistance ◽

Gram Negative

Tigecycline and colistin are used as last-resort therapies to treat infections caused by multidrug-resistant (MDR) Gram-negative bacteria. However, the emergence of the plasmid-mediated tigecycline resistance gene tet (X4) and the plasmid-mediated colistin resistance gene mcr-1 represents a significant threat to human health.

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Evaluation of the rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test for rapid colistin resistance detection in lactose non-fermenting Gram-negative bacteria

Journal of Medical Microbiology ◽

10.1099/jmm.0.001373 ◽

2021 ◽

Vol 70 (6) ◽

Author(s):

Hyunsul Jung ◽

Johann D. D. Pitout ◽

Barend C. Mitton ◽

Kathy-Anne Strydom ◽

Chanel Kingsburgh ◽

...

Keyword(s):

Type Species ◽

Susceptibility Testing ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Major Error ◽

Colistin Resistance ◽

Gram Negative ◽

Content Type ◽

Link Type ◽

Extensively Drug Resistant

Introduction. Colistin is one of the last-resort antibiotics for treating multidrug-resistant (MDR) or extensively drug-resistant (XDR) lactose non-fermenting Gram-negative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii . Gap Statement. As the rate of colistin resistance is steadily rising, there is a need for rapid and accurate antimicrobial susceptibility testing methods for colistin. The Rapid ResaPolymyxin Acinetobacter / Pseudomonas NP test has recently been developed for rapid detection of colistin resistance in P. aeruginosa and A. baumannii . Aim. The present study aimed to evaluate the performance of the Rapid ResaPolymyxin Acinetobacter / Pseudomonas NP test in comparison with the reference broth microdilution (BMD) method. Methodology. The Rapid ResaPolymyxin Acinetobacter / Pseudomonas NP test was performed using a total of 135 P . aeruginosa (17 colistin-resistant and 118 colistin-susceptible) and 66 A. baumannii isolates (32 colistin-resistant and 34 colistin-susceptible), in comparison with the reference BMD method. Results. The categorical agreement of the Rapid ResaPolymyxin Acinetobacter / Pseudomonas NP test with the reference BMD method was 97.5 % with a major error rate of 0 % (0/152) and a very major error (VME) rate of 10.2 %. The VME rate was higher (23.5 %) when calculated separately for P. aeruginosa isolates. The overall sensitivity and specificity were 89.8 and 100 %, respectively. Conclusion. The Rapid ResaPolymyxin Acinetobacter / Pseudomonas NP test performed better for A. baumannii than for P. aeruginosa .

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Identification of Ly2 members as antimicrobial peptides from zebrafish Danio rerio

Bioscience Reports ◽

10.1042/bsr20160265 ◽

2017 ◽

Vol 37 (1) ◽

Cited By ~ 6

Author(s):

Xuemin Liu ◽

Xuwen Cao ◽

Su Wang ◽

Guangdong Ji ◽

Shicui Zhang ◽

...

Keyword(s):

Antimicrobial Peptides ◽

Recombinant Proteins ◽

Mammalian Cells ◽

Multidrug Resistant ◽

Scatchard Analysis ◽

Gram Negative Bacteria ◽

Gram Positive ◽

Gram Negative ◽

E Coli ◽

Gram Positive Bacteria

The emergence of multidrug-resistant (MDR) microbes caused by overuse of antibiotics leads to urgent demands for novel antibiotics exploration. Our recent data showed that Ly2.1–3 (a novel lymphocyte antigen 6 (Ly6) gene cluster) were proteins with cationic nature and rich in cysteine content, that are characteristic of antimicrobial peptides (AMPs) and their expression were all significantly up-regulated after challenge with lipopolysaccharide (LPS). These strongly suggested that Ly2.1–3 are potential AMPs, but firm evidence are lacking. Here, we clearly showed that the recombinant proteins of Ly2.1–3 were capable of killing Gram-negative bacteria Aeromonas hydrophila and Escherichia coli, while they had little bactericidal activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis. We also showed that recombinant proteins Ly2.1–3 (rLy2.1–3) were able to bind to the Gram-negative bacteria A. hydrophila, E. coli and the microbial signature molecule LPS, but not to the Gram-positive bacteria S. aureus and B. subtilis as well as the microbial signature molecule LTA. Moreover, the Scatchard analysis revealed that rLy2.1–3 could specifically bind to LPS. Finally, we found that Ly2.1–3 were not cytotoxic to mammalian cells. All these together indicate that Ly2.1–3 can function as AMPs.

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In vitro evolution of colistin resistance in the Klebsiella pneumoniae complex follows multiple evolutionary trajectories with variable effects on fitness and virulence characteristics

10.1101/2020.05.24.112334 ◽

2020 ◽

Author(s):

Axel B. Janssen ◽

Dennis J. Doorduijn ◽

Grant Mills ◽

Malbert R.C. Rogers ◽

Marc J.M. Bonten ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Lipid A ◽

Multidrug Resistant ◽

Sensu Stricto ◽

Cross Resistance ◽

Gram Negative Bacteria ◽

Colistin Resistance ◽

Gram Negative ◽

Evolutionary Trajectories

AbstractThe increasing prevalence of multidrug-resistant Gram-negative opportunistic pathogens, including Klebsiella pneumoniae, has led to a resurgence in the use of colistin as a last-resort drug. Colistin is a cationic lipopeptide antibiotic that selectively acts on Gram-negative bacteria through electrostatic interactions with anionic phosphate groups of the lipid A moiety of lipopolysaccharides (LPS). Colistin resistance in K. pneumoniae is mediated through loss of these phosphate groups, or modification with cationic groups (e.g. 4-amino-4-deoxy-L-arabinose (L-Ara4N), or phosphoethanolamine), but also hydroxylation of acyl-groups of lipid A. Here, we study the in vitro evolutionary trajectories towards colistin resistance in clinical K. pneumoniae complex strains (three K. pneumoniae sensu stricto strains and one K. variicola subsp. variicola strain) and their impact on fitness and virulence characteristics.Through population sequencing during the in vitro evolution experiment, we found that resistance develops through a combination of single nucleotide polymorphisms (SNPs), insertion and deletions (indels), and the integration of insertion sequence (IS) elements, affecting genes associated with LPS biosynthesis and modification, and capsule structures. The development of colistin resistance decreased the maximum growth rate of one K. pneumoniae sensu stricto strain, but not in the other three K. pneumoniae sensu lato strains. Colistin-resistant strains had lipid A modified through hydroxylation, palmitoylation, and L-Ara4N addition. Colistin-resistant K. pneumoniae sensu stricto strains exhibited cross-resistance to LL-37, in contrast to the K. variicola subsp. variicola strain that did not change in susceptibility to LL-37. Virulence, as determined in a Caenorhabditis elegans survival assay, was higher in two colistin-resistant strains.Our study suggests that nosocomial K. pneumoniae complex strains can rapidly develop colistin resistance de novo through diverse evolutionary trajectories upon exposure to colistin. This effectively shortens the lifespan of this last-resort antibiotic for the treatment of infections with multidrug-resistant Klebsiella.Author summaryBacteria that frequently cause infections in hospitalised patients are becoming increasingly resistant to antibiotics. Colistin is a positively charged antibiotic that is used for the treatment of infections with multidrug-resistant Gram-negative bacteria. Colistin acts by specifically interacting with the negatively charged LPS molecule in the outer membrane of Gram-negative bacteria. Colistin resistance is mostly mediated through modification of LPS to reduce its negative charge. Here, we use a laboratory evolution experiment to show that strains belonging to the Klebsiella pneumoniae complex, a common cause of multidrug-resistant hospital-acquired infections, can rapidly accumulate mutations that reduce the negative charge of LPS without an appreciable loss of fitness. Colistin resistance can lead to cross-resistance to an antimicrobial peptide of the human innate immune system, but can increase susceptibility to serum, and virulence in a nematode model. These findings show that extensively resistant K. pneumoniae complex strains may rapidly develop resistance to the last-resort antibiotic colistin via different evolutionary trajectories, while retaining their ability to cause infections.

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Effects of Efflux Pump Inhibitors on Colistin Resistance in Multidrug-Resistant Gram-Negative Bacteria

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00248-16 ◽

2016 ◽

Vol 60 (5) ◽

pp. 3215-3218 ◽

Cited By ~ 42

Author(s):

Wentao Ni ◽

Yanjun Li ◽

Jie Guan ◽

Jin Zhao ◽

Junchang Cui ◽

...

Keyword(s):

Stenotrophomonas Maltophilia ◽

Efflux Pump ◽

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Colistin Resistance ◽

Gram Negative ◽

Content Type ◽

Resistant Strains ◽

Efflux Pump Inhibitors ◽

Time Kill

ABSTRACTWe tested the effects of various putative efflux pump inhibitors on colistin resistance in multidrug-resistant Gram-negative bacteria. Addition of 10 mg/liter cyanide 3-chlorophenylhydrazone (CCCP) to the test medium could significantly decrease the MICs of colistin-resistant strains. Time-kill assays showed CCCP could reverse colistin resistance and inhibit the regrowth of the resistant subpopulation, especially inAcinetobacter baumanniiandStenotrophomonas maltophilia. These results suggest colistin resistance in Gram-negative bacteria can be suppressed and reversed by CCCP.

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