Characterization of NMCR-3, NMCR-4 and NMCR-5, three novel non-mobile colistin resistance determinants: Implications for MCR-3, MCR-7, and MCR-5 progenitors, respectively.

In this study, the progenitors of MCR-3, MCR-7 and MCR-5, namely NMCR-3, NMCR-4 and NMCR-5, were firstly discovered and indicating Aeromonas was a natural reservoir for MCR-3 and MCR-7. Furthermore, different evolutionary models for MCR-3, MCR-7 and MCR-5 were proposed.

Phage-mediated colistin resistance in Acinetobacter baumannii.

AIMS: Antimicrobial resistance is a global threat to human health, and Acinetobacter baumannii is a paradigmatic example of how rapidly bacteria become resistant to clinically relevant antimicrobials. The emergence of multidrug-resistant A. baumannii strains has forced the revival of colistin as a last-resort drug, suddenly leading to the emergence of colistin resistance. We investigated the genetic and molecular basis of colistin resistance in A. baumannii, and the mechanisms implicated in its regulation and dissemination. METHODS: Comparative genomic analysis was combined with genetic, biochemical, and phenotypic assays to characterize Φ19606, an A. baumannii temperate bacteriophage that carries a colistin resistance gene. RESULTS: Ф19606 was detected in 41% of 523 A. baumannii complete genomes and demonstrated to act as a mobile vehicle of the colistin resistance gene eptA1, encoding a functional lipid A phosphoethanolamine transferase. The eptA1 gene is coregulated with its chromosomal homolog pmrC via the PmrAB two-component system and confers colistin resistance when induced by low calcium and magnesium levels. Resistance selection assays showed that the eptA1-harbouring phage Ф19606 promotes the emergence of spontaneous colistin-resistant mutants. CONCLUSIONS: Φ19606 is an unprecedented example of a self-transmissible phage vector implicated in the dissemination of colistin resistance.

Re-sensitization of mcr carrying multidrug resistant bacteria to colistin by silver.

Colistin is considered the last-line antimicrobial for the treatment of multidrug-resistant gram-negative bacterial infections. The emergence and spread of superbugs carrying the mobile colistin resistance gene (mcr) have become the most serious and urgent threat to healthcare. Here, we discover that silver (Ag+), including silver nanoparticles, could restore colistin efficacy against mcr-positive bacteria. We show that Ag+ inhibits the activity of the MCR-1 enzyme via substitution of Zn2+ in the active site. Unexpectedly, a tetra-silver center was found in the active-site pocket of MCR-1 as revealed by the X-ray structure of the Ag-bound MCR-1, resulting in the prevention of substrate binding. Moreover, Ag+effectively slows down the development of higher-level resistance and reduces mutation frequency. Importantly, the combined use of Ag+ at a low concentration with colistin could relieve dermonecrotic lesions and reduce the bacterial load of mice infected with mcr-1­carrying pathogens. This study depicts a mechanism of Ag+ inhibition of MCR enzymes and demonstrates the potentials of Ag+ as broad-spectrum inhibitors for the treatment of mcr-positive bacterial infection in combination with colistin.

PmrAB, the two-component system of Acinetobacter baumannii, controls the phosphoethanolamine modification of lipooligosaccharide in response to metal ions.

Acinetobacter baumannii is highly resistant to antimicrobial agents, and XDR strains have become widespread. A. baumannii has developed resistance to colistin, which is considered the last resort against XDR Gram-negative bacteria, mainly caused by lipooligosaccharide (LOS) phosphoethanolamine (pEtN) and/or galactosamine (GalN) modifications induced by mutations that activate the two-component system (TCS) pmrAB. Although PmrAB of A. baumannii has been recognized as a drug resistance factor, its function as TCS, including its regulatory genes and response factors, has not been fully elucidated. In this study, to clarify the function of PmrAB as TCS, we elucidated the regulatory genes (regulon) of PmrAB via transcriptome analysis using pmrAB-activated mutant strains. We discovered that PmrAB responds to low pH, Fe2+, Zn2+, and Al3+. A. baumannii selectively recognizes Fe2+ rather than Fe3+, and a novel region ExxxE, in addition to the ExxE motif sequence, is involved in the environmental response. Furthermore, PmrAB participates in the phosphoethanolamine modification of LOS on the bacterial surface in response to metal ions such as Al3+, contributing to the attenuation of Al3+ toxicity and development of resistance to colistin and polymyxin B in A. baumannii. This study demonstrates that PmrAB in A. baumannii not only regulates genes that play an important role in drug resistance but is also involved in responses to environmental stimuli such as metal ions and pH, and this stimulation induces LOS modification. This study reveals the importance of PmrAB in the environmental adaptation and antibacterial resistance emergence mechanisms of A. baumannii. IMPORTANCE: Antimicrobial resistance (AMR) is a pressing global issue in human health. Acinetobacter baumannii is notably high on the World Health Organization's list of bacteria for which new antimicrobial agents are urgently needed. Colistin is one of the last-resort drugs used against extensively drug-resistant (XDR) Gram-negative bacteria. However, A. baumannii has become increasingly resistant to colistin, primarily by modifying its lipooligosaccharide (LOS) via activating mutations in the two-component system (TCS) PmrAB. This study comprehensively elucidates the detailed mechanism of drug resistance of PmrAB in A. baumannii as well as its biological functions. Understanding the molecular biology of these molecules, which serve as drug resistance factors and are involved in environmental recognition mechanisms in bacteria, is crucial for developing fundamental solutions to the AMR problem.

Anti-inflammatory and immunoregulatory effects of colistin sulphate on human PBMCs.

In previous studies, CST has been identified as having an immunostimulatory effect on Caenorhabditis elegans and macrophage of rats. Here, we further investigated its immunomodulatory effects on human peripheral blood mononuclear cells (PBMCs). LPS-stimulated PBMCs inflammatory model was established. Flow cytometry was applied to measure phagocytosis of PBMCs. Cytokine mRNA and protein expression levels of LPS-stimulated PBMCs with or without CST were measured by qRT-PCR and ELISA. The transcriptomic profile of CST-treated PBMCs was investigated by RNA-sequencing. Gene Ontology (GO) and Kyoto Encylopedia of Genes and Genomes (KEGG) were applied to find potential signalling pathways. PBMCs showed a significant increase in phagocytic activity at 6 h after being incubated with CST at the concentration of 10 µg/mL. In the presence of LPS, CST maintained and promoted the expression of TNF-α and chemokine CCL24. The content of pro-inflammatory cytokines, such as IL-1ß, IL-6 and IFN-γ, which were released from LPS-stimulated PBMCs, was reduced by CST at 6 h. Anti-inflammatory cytokines, such as IL-4, IL-13 and TGF-ß1, were significantly increased by CST at 24 h. A total of 277 differentially expressed immune-related genes (DEIRGs) were detected and cytokine-cytokine receptor interaction was highly enriched. CST presented obvious anti-inflammatory and immunoregulatory effects in LPS-induced PBMCs inflammatory model not only by improving the ability of PBMCs to clear pathogens but also by decreasing pro-inflammatory cytokines and increasing anti-inflammatory cytokines. And the mechanism may be related to cytokine-cytokine receptor interaction.

Lipid A modification of colistin-resistant Klebsiella pneumoniae does not alter innate immune response in a mouse model of pneumonia.

Polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae bacteria is associated with high morbidity and mortality in vulnerable populations throughout the world. Ineffective antimicrobial activity by these last resort therapeutics can occur by transfer of mcr-1, a plasmid-mediated resistance gene, causing modification of the lipid A portion of lipopolysaccharide (LPS) and disruption of the interactions between polymyxins and lipid A. Whether this modification alters the innate host immune response or carries a high fitness cost in the bacteria is not well established. To investigate this, we studied infection with K. pneumoniae (KP) ATCC 13883 harboring either the mcr-1 plasmid (pmcr-1) or the vector control (pBCSK) ATCC 13883. Bacterial fitness characteristics of mcr-1 acquisition were evaluated. Differentiated human monocytes (THP-1s) were stimulated with KP bacterial strains or purified LPS from both parent isolates and isolates harboring mcr-1. Cell culture supernatants were analyzed for cytokine production. A bacterial pneumonia model in WT C57/BL6J mice was used to monitor immune cell recruitment, cytokine induction, and bacterial clearance in the bronchoalveolar lavage fluid (BALF). Isolates harboring mcr-1 had increased colistin MIC compared to the parent isolates but did not alter bacterial fitness. Few differences in cytokines were observed with purified LPS from mcr-1 expressing bacteria in vitro. However, in a mouse pneumonia model, no bacterial clearance defect was observed between pmcr-1-harboring KP and parent isolates. Consistently, no differences in cytokine production or immune cell recruitment in the BALF were observed, suggesting that other mechanisms outweigh the effect of these lipid A mutations in LPS.

The insertion sequence excision enhancer: A PrimPol-based primer invasion system for immobilizing transposon-transmitted antibiotic resistance genes.

Evolutionary studies often identify genes that have been exchanged between different organisms and the phrase Lateral or Horizontal Gene Transfer is often used in this context. However, they rarely provide any mechanistic information concerning how these gene transfers might have occurred. With the astonishing increase in the number of sequences in public databases over the past two or three decades, identical antibiotic resistance genes have been identified in many different sequence contexts. One explanation for this would be that genes are initially transmitted by transposons which have subsequently decayed and can no longer be detected. Here, we provide an overview of a protein, IEE (Insertion Sequence Excision Enhancer) observed to facilitate high-frequency excision of IS629 from clinically important Escherichia coli O157:H7 and subsequently shown to affect a large class of bacterial insertion sequences which all transpose using the copy-out-paste-in transposition mechanism. Excision depends on both IEE and transposase indicating association with the transposition process itself. We review genetic and biochemical data and propose that IEE immobilizes genes carried by compound transposons by removing the flanking insertion sequence (IS) copies. The biochemical activities of IEE as a primase with the capacity to recognize DNA microhomologies and the observation that its effect appears restricted to IS families which use copy-out-paste-in transposition, suggests IS deletion occurs by abortive transposition involving strand switching (primer invasion) during the copy-out step. This reinforces the proposal made for understanding the widespread phenomenon loss of ISApl1 flanking mcr-1 in the compound transposon Tn6330 which we illustrate with a detailed model. This model also provides a convincing way to explain the high levels of IEE-induced precise IS excision.

Metabolic engineering of "last-line antibiotic" colistin in Paenibacillus polymyxa.

Colistin, also known as polymyxin E, is a lipopeptide antibiotic used to treat infections caused by multidrug-resistant gram-negative bacteria. It is considered a "last-line antibiotic", but its clinical development is hindered by low titer and impurities resulting from the presence of diverse homologs in microbial fermentation. To ensure consistent pharmaceutical activity and kinetics, it is crucial to have high-purity colistin active pharmaceutical ingredient (API) in the pharmaceutical industry. This study focused on the metabolic engineering of a natural colistin producer strain to produce colistin with a high titer and purity. Guided by genome mining, we identified Paenibacillus polymyxa ATCC 842 as a natural colistin producer capable of generating a high proportion of colistin A. By systematically inactivating seven non-essential biosynthetic gene clusters (BGCs) of peptide metabolites that might compete precursors with colistin or inhibit colistin production, we created an engineered strain, P14, which exhibited an 82% increase in colistin titer and effectively eliminated metabolite impurities such as tridecaptin, paenibacillin, and paenilan. Additionally, we engineered the L-2,4-diaminobutyric acid (L-2,4-DABA) pathway to further enhance colistin production, resulting in the engineered strain P19, which boosted a remarkable colistin titer of 649.3 mg/L - a 269% improvement compared to the original strain. By concurrently feeding L-isoleucine and L-leucine, we successfully produced high-purity colistin A, constituting 88% of the total colistin products. This study highlights the potential of metabolic engineering in improving the titer and purity of lipopeptide antibiotics in the non-model strain, making them more suitable for clinical use. These findings indicate that efficiently producing colistin API in high purity directly from fermentation can now be achieved in a straightforward manner.

Prevalence and molecular characterization of colistin resistance in Pseudomonas aeruginosa isolates: insights from a study in Ardabil hospitals.

BACKGROUND: Pseudomonas aeruginosa is a common cause of nosocomial infections. However, the emergence of multidrug-resistant strains has complicated the treatment of P. aeruginosa infections. While polymyxins have been the mainstay for treatment, there is a global increase in resistance to these antibiotics. Therefore, our study aimed to determine the prevalence and molecular details of colistin resistance in P. aeruginosa clinical isolates collected between June 2019 and May 2023, as well as the genetic linkage of colistin-resistant P. aeruginosa isolates. RESULTS: The resistance rate to colistin was 9% (n = 18) among P. aeruginosa isolates. All 18 colistin-resistant isolates were biofilm producers and carried genes associated with biofilm formation. Furthermore, the presence of genes encoding efflux pumps, TCSs, and outer membrane porin was observed in all colistin-resistant P. aeruginosa strains, while the mcr-1 gene was not detected. Amino acid substitutions were identified only in the PmrB protein of multidrug- and colistin-resistant strains. The expression levels of mexA, mexC, mexE, mexY, phoP, and pmrA genes in the 18 colistin-resistant P. aeruginosa strains were as follows: 88.8%, 94.4%, 11.1%, 83.3%, 83.3%, and 38.8%, respectively. Additionally, down-regulation of the oprD gene was observed in 44.4% of colistin-resistant P. aeruginosa strains. CONCLUSION: This study reports the emergence of colistin resistance with various mechanisms among P. aeruginosa strains in Ardabil hospitals. We recommend avoiding unnecessary use of colistin to prevent potential future increases in colistin resistance.

Influence of PhoPQ and PmrAB two component system alternations on colistin resistance from non-mcr colistin resistant clinical E. Coli strains.

BACKGROUND: The current understanding of acquired chromosomal colistin resistance mechanisms in Enterobacterales primarily involves the disruption of the upstream PmrAB and PhoPQ two-component system (TCS) control caused by mutations in the regulatory genes. Interestingly, previous studies have yielded conflicting results regarding the interaction of regulatory genes related to colistin resistance in Escherichia coli, specifically those surrounding PhoPQ and PmrAB TCS. RESULTS: In our study, we focused on two clinical non-mcr colistin-resistant strains of E. coli, TSAREC02 and TSAREC03, to gain a better understanding of their resistance mechanisms. Upon analysis, we discovered that TSAREC02 had a deletion (Δ27-45) in MgrB, as well as substitutions (G206R, Y222H) in PmrB. On the other hand, TSAREC03 exhibited a long deletion (Δ84-224) in PhoP, along with substitutions (M1I, L14P, P178S, T235N) in PmrB. We employed recombinant DNA techniques to explore the interaction between the PhoPQ and PmrAB two-component systems (TCSs) and examine the impact of the mutated phoPQ and pmrB genes on the minimum inhibitory concentrations (MICs) of colistin. We observed significant changes in the expression of the pmrD gene, which encodes a connector protein regulated by the PhoPQ TCS, in the TSAREC02 wild-type (WT)-mgrB replacement mutant and the TSAREC03 WT-phoP replacement mutant, compared to their respective parental strains. However, the expressions of pmrB/pmrA, which reflect PmrAB TCS activity, and the colistin MICs remained unchanged. In contrast, the colistin MICs and pmrB/pmrA expression levels were significantly reduced in the pmrB deletion mutants from both TSAREC02 and TSAREC03, compared to their parental strains. Moreover, we were able to restore colistin resistance and the expressions of pmrB/pmrA by transforming a plasmid containing the parental mutated pmrB back into the TSAREC02 and TSAREC03 mutants, respectively. CONCLUSION: While additional data from clinical E. coli isolates are necessary to validate whether our findings could be broadly applied to the E. coli population, our study illuminates distinct regulatory pathway interactions involving colistin resistance in E. coli compared to other species of Enterobacterales. The added information provided by our study contribute to a deeper understanding of the complex pathway interactions within Enterobacterales.

Molecular characterization and epidemiological investigation of colistin resistance in carbapenem-resistant Klebsiella pneumoniae in a tertiary care hospital in Tehran, Iran.

BACKGROUND: Carbapenemase-producing Klebsiella pneumoniae (CRKP) presents a significant challenge to antimicrobial therapy, especially when compounded by resistance to colistin. The objective of this study was to explore molecular epidemiological insights into strains of clinical K. pneumoniae that produce carbapenemases and exhibit resistance to colistin. Eighty clinical isolates of CRKP were obtained from Milad Hospital in Tehran, Iran. Antimicrobial susceptibility and colistin broth disk elution were determined. PCR assays were conducted to examine the prevalence of resistance-associated genes, including blaKPC, blaIMP, blaVIM, blaOXA-48, blaNDM and mcr-1 to -10. Molecular typing (PFGE) was used to assess their spread. RESULTS: Colistin resistance was observed in 27 isolates (33.7%) using the Broth Disk Elution method. Among positive isolates for carbapenemase genes, the most frequent gene was blaOXA-48, identified in 36 strains (45%). The mcr-1 gene was detected in 3.7% of the obtained isolates, with none of the other of the other mcr genes detected in the studied isolates. CONCLUSION: To stop the spread of resistant K. pneumoniae and prevent the evolution of mcr genes, it is imperative to enhance surveillance, adhere rigorously to infection prevention protocols, and implement antibiotic stewardship practices.

Outbreak of colistin and carbapenem-resistant Klebsiella pneumoniae ST16 co-producing NDM-1 and OXA-48 isolates in an Iranian hospital.

BACKGROUND: Colistin and carbapenem-resistant Klebsiella pneumoniae (Col-CRKP) represent a significant and constantly growing threat to global public health. We report here an outbreak of Col-CRKP infections during the fifth wave of COVID-19 pandemic. METHODS: The outbreak occurred in an intensive care unit with 22 beds at a teaching university hospital, Isfahan, Iran. We collected eight Col-CRKP strains from seven patients and characterized these strains for their antimicrobial susceptibility, determination of hypermucoviscous phenotype, capsular serotyping, molecular detection of virulence and resistance genes. Clonal relatedness of the isolates was performed using MLST. RESULTS: The COVID-19 patients were aged 24-75 years with at least 50% pulmonary involvement and were admitted to the intensive care unit. They all had superinfection caused by Col-CRKP, and poor responses to antibiotic treatment and died. With the exception of one isolate that belonged to the ST11, all seven representative Col-CRKP strains belonged to the ST16. Of these eight isolates, one ST16 isolate carried the iucA and ybtS genes was identified as serotype K20 hypervirulent Col-CRKP. The blaSHV and blaNDM-1 genes were the most prevalent resistance genes, followed by blaOXA-48 and blaCTX-M-15 and blaTEM genes. Mobilized colistin-resistance genes were not detected in the isolates. CONCLUSIONS: The continual emergence of ST16 Col-CRKP strains is a major threat to public health worldwide due to multidrug-resistant and highly transmissible characteristics. It seems that the potential dissemination of these clones highlights the importance of appropriate monitoring and strict infection control measures to prevent the spread of resistant bacteria in hospitals.

Genome analyses of colistin-resistant high-risk blaNDM-5 producing Klebsiella pneumoniae ST147 and Pseudomonas aeruginosa ST235 and ST357 in clinical settings.

BACKGROUND: Colistin is a last-resort antibiotic used in extreme cases of multi-drug resistant (MDR) Gram-negative bacterial infections. Colistin resistance has increased in recent years and often goes undetected due to the inefficiency of predominantly used standard antibiotic susceptibility tests (AST). To address this challenge, we aimed to detect the prevalence of colistin resistance strains through both Vitek®2 and broth micro-dilution. We investigated 1748 blood, tracheal aspirate, and pleural fluid samples from the Intensive Care Unit (ICU), Neonatal Intensive Care Unit (NICU), and Tuberculosis and Respiratory Disease centre (TBRD) in an India hospital. Whole-genome sequencing (WGS) of extremely drug-resitant (XDR) and pan-drug resistant (PDR) strains revealed the resistance mechanisms through the Resistance Gene Identifier (RGI.v6.0.0) and Snippy.v4.6.0. Abricate.v1.0.1, PlasmidFinder.v2.1, MobileElementFinder.v1.0.3 etc. detected virulence factors, and mobile genetic elements associated to uncover the pathogenecity and the role of horizontal gene transfer (HGT). RESULTS: This study reveals compelling insights into colistin resistance among global high-risk clinical isolates: Klebsiella pneumoniae ST147 (16/20), Pseudomonas aeruginosa ST235 (3/20), and ST357 (1/20). Vitek®2 found 6 colistin-resistant strains (minimum inhibitory concentrations, MIC = 4 µg/mL), while broth microdilution identified 48 (MIC = 32-128 µg/mL), adhering to CLSI guidelines. Despite the absence of mobile colistin resistance (mcr) genes, mechanisms underlying colistin resistance included mgrB deletion, phosphoethanolamine transferases arnT, eptB, ompA, and mutations in pmrB (T246A, R256G) and eptA (V50L, A135P, I138V, C27F) in K. pneumoniae. P. aeruginosa harbored phosphoethanolamine transferases basS/pmrb, basR, arnA, cprR, cprS, alongside pmrB (G362S), and parS (H398R) mutations. Both strains carried diverse clinically relevant antimicrobial resistance genes (ARGs), including plasmid-mediated blaNDM-5 (K. pneumoniae ST147) and chromosomally mediated blaNDM-1 (P. aeruginosa ST357). CONCLUSION: The global surge in MDR, XDR and PDR bacteria necessitates last-resort antibiotics such as colistin. However, escalating resistance, particularly to colistin, presents a critical challenge. Inefficient colistin resistance detection methods, including Vitek2, alongside limited surveillance resources, accentuate the need for improved strategies. Whole-genome sequencing revealed alarming colistin resistance among K. pneumoniae and P. aeruginosa in an Indian hospital. The identification of XDR and PDR strains underscores urgency for enhanced surveillance and infection control. SNP analysis elucidated resistance mechanisms, highlighting the complexity of combatting resistance.

Nanoemulsion of cinnamon oil to combat colistin-resistant Klebsiella pneumoniae and cancer cells.

This study aimed to investigate the potential of cinnamon oil nanoemulsion (CONE) as an antibacterial agent against clinical strains of colistin-resistant Klebsiella pneumoniae and its anticancer activity. The prepared and characterized CONE was found to have a spherical shape with an average size of 70.6 ± 28.3 nm under TEM and a PDI value of 0.076 and zeta potential value of 6.9 mV using DLS analysis. The antibacterial activity of CONE against Klebsiella pneumoniae strains was investigated, and it was found to have higher inhibitory activity (18.3 ± 1.2-30.3 ± 0.8 mm) against the tested bacteria compared to bulk cinnamon oil (14.6 ± 0.88-20.6 ± 1.2) with MIC values ranging from 0.077 to 0.31 % v/v which equivalent to 0.2-0.82 ng/ml of CONE. CONE inhibited the growth of bacteria in a dose and time-dependent manner based on the time-kill assay in which Klebsiella pneumoniae B-9 was used as a model among the bacterial strains under investigation. The study also investigated the expression of the mcr-1 gene in the Klebsiella pneumoniae strains and found that all strains were positive for the gene expression and subsequently its presence. The level of mcr-1 gene expression among the B-2, B-4, B-9, and B-11 control strains and that treated with colistin was similar, but it was different in both B-5 and B-2. However, all strains exhibited a significant downregulation in gene expression (ranging from 3.97 to 8.7-fold) after their treatment with CONE. Additionally, the CONE-treated bacterial cells appeared with a great deformation compared with control cells under TEM. Finally, CONE exhibited selective toxicity against different cancer cell lines depending on comparison with the normal cell lines.

EptA of Riemerella anatipestifer mediates phenotypes involved in colistin resistance and virulence.

Colistin (polymyxin E) is a group of cationic antimicrobial cyclic peptides and is recognized as a last-resort defense against lethal infections with carbapenem-resistant pathogens. In addition to the plasmid-borne mobilized phosphoethanolamine (PEA) transferases, the functional expression of lipid A-modifying enzymes encoded on chromosomes has been attributed to intrinsic bacterial colistin resistance. However, the mechanisms of colistin resistance in Riemerella anatipestifer remain unknown. Herein, the GE296_RS09715 gene-encoded Lipid A PEA transferases (RaEptA) was identified in R. anatipestifer. Genetic and structural analyses revealed that the amino acid sequence of RaEptA shared 26.6%-33.1% similarities with the family of Lipid A PEA transferases (EptA) and MCR-like proteins and have defined 12 residues that contribute to the formation of phosphatidylethanolamine (PE)-recognizable cavities. Comparative analyses of colistin resistance in RA-LZ01 and RA-LZ01ΔRaEptA showed the level of colistin has fallen from 96 µg mL-1 down to 24 ~ 32 µg mL-1 . Site-directed mutagenesis assay of the PE-binding cavity and expression of the mutants reveals that K309-rRaEptA can remodel the surface of Escherichia coli and rendering it resistant to colistin, suggesting this point-mutation of P309K is necessary for EptA-mediated lipid A modification. Moreover, the virulence of RA-LZ01ΔRaEptA was attenuated compared with RA-LZ01 both in vivo and vitro. Taken together, the results represent the RaEptA involved in the colistin resistance and pathogenicity, and the P309K mutation might alter bacterial adaptation and increase the spread of colistin resistance from R. anatipestifer to other gram-negative bacteria. The findings of this study suggest another scenario for the spread of colistin resistance genes and should be considered by a wide audience.

Disruption of the primocolonizing microbiota alters epithelial homeostasis and imprints stem cells in the colon of neonatal piglets.

The gut microbiota plays a key role in the postnatal development of the intestinal epithelium. However, the bacterial members of the primocolonizing microbiota driving these effects are not fully identified and the mechanisms underlying their long-term influence on epithelial homeostasis remain poorly described. Here, we used a model of newborn piglets treated during the first week of life with the antibiotic colistin in order to deplete specific gram-negative bacteria that are transiently dominant in the neonatal gut microbiota. Colistin depleted Proteobacteria and Fusobacteriota from the neonatal colon microbiota, reduced the bacterial predicted capacity to synthetize lipopolysaccharide (LPS), and increased the concentration of succinate in the colon. The colistin-induced disruption of the primocolonizing microbiota was associated with altered gene expression in the colon epithelium including a reduction of toll-like receptor 4 (TLR4) and lysozyme (LYZ). Our data obtained in porcine colonic organoid cell monolayers suggested that these effects were not driven by the variation of succinate or LPS levels nor by a direct effect of colistin on epithelial cells. The disruption of the primocolonizing microbiota imprinted colon epithelial stem cells since the expression of TLR4 and LYZ remained lower in organoids derived from colistin-treated piglet colonic crypts after several passages when compared to control piglets. Finally, the stable imprinting of LYZ in colon organoids was independent of the H3K4me3 level in its transcription start site. Altogether, our results show that disruption of the primocolonizing gut microbiota alters epithelial innate immunity in the colon and imprints stem cells, which could have long-term consequences for gut health.

Coacervation in Slow Motion: Kinetics of Complex Micelle Formation Induced by the Hydrolysis of an Antibiotic Prodrug.

Colistin methanesulfonate (CMS) is the less-toxic prodrug of highly nephrotoxic colistin. To develop and understand highly necessary new antibiotic formulations, the hydrolysis of CMS to colistin must be better understood. Herein, with the addition of poly(ethylene oxide)-b-poly(methacrylic acid) (PEO-b-PMAA) to CMS, we show that we can follow the hydrolysis kinetics, employing small-angle X-ray scattering (SAXS) through complex coacervation. During this hydrolysis, hydroxy methanesulfonate (HMS) groups from CMS are cleaved, while the newly formed cationic amino groups complex with the anionic charge from the PMAA block. As the hydrolysis of HMS groups is slow, we can follow the complex coacervation process by the gradual formation of complex micelles containing activated antibiotics. Combining mass spectrometry (MS) with SAXS, we quantify the hydrolysis as a function of pH. Upon modeling the kinetic pathways, we found that complexation only happens after complete hydrolysis into colistin and that the process is accelerated under acidic conditions. At pH = 5.0, effective charge switching was identified as the slowest step in the CMS conversion, constituting the rate-limiting step in colistin formation.

Sub-inhibitory concentrations of colistin and imipenem impact the expression of biofilm-associated genes in Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogen that is responsible for nosocomial infections. Imipenem and colistin are drugs that are commonly used to treat severe infections caused by A. baumannii, such as sepsis, ventilator-associated pneumonia, and bacteremia. However, some strains of A. baumannii have become resistant to these drugs, which is a concern for public health. Biofilms produced by A. baumannii increase their resistance to antibiotics and the cells within the inner layers of biofilm are exposed to sub-inhibitory concentrations (sub-MICs) of antibiotics. There is limited information available regarding how the genes of A. baumannii are linked to biofilm formation when the bacteria are exposed to sub-MICs of imipenem and colistin. Thus, this study's objective was to explore this relationship by examining the genes involved in biofilm formation in A. baumannii when exposed to low levels of imipenem and colistin. The study found that exposing an isolate of A. baumannii to low levels of these drugs caused changes in their drug susceptibility pattern. The relative gene expression profiles of the biofilm-associated genes exhibited a change in their expression profile during short-term and long-term exposure. This study highlights the potential consequences of overuse and misuse of antibiotics, which can help bacteria become resistant to these drugs.

Assessing the synergistic potential of bacteriophage endolysins and antimicrobial peptides for eradicating bacterial biofilms.

Phage-encoded endolysins have emerged as a potential substitute to conventional antibiotics due to their exceptional benefits including host specificity, rapid host killing, least risk of resistance. In addition to their antibacterial potency and biofilm eradication properties, endolysins are reported to exhibit synergism with other antimicrobial agents. In this study, the synergistic potency of endolysins was dissected with antimicrobial peptides to enhance their therapeutic effectiveness. Recombinantly expressed and purified bacteriophage endolysin [T7 endolysin (T7L); and T4 endolysin (T4L)] proteins have been used to evaluate the broad-spectrum antibacterial efficacy using different bacterial strains. Antibacterial/biofilm eradication studies were performed in combination with different antimicrobial peptides (AMPs) such as colistin, nisin, and polymyxin B (PMB) to assess the endolysin's antimicrobial efficacy and their synergy with AMPs. In combination with T7L, polymyxin B and colistin effectively eradicated the biofilm of Pseudomonas aeruginosa and exhibited a synergistic effect. Further, a combination of T4L and nisin displayed a synergistic effect against Staphylococcus aureus biofilms. In summary, the obtained results endorse the theme of combinational therapy consisting of endolysins and AMPs as an effective remedy against the drug-resistant bacterial biofilms that are a serious concern in healthcare settings.

Cinnamaldehyde and baicalin reverse colistin resistance in Enterobacterales and Acinetobacter baumannii strains.

PURPOSE: Colistin is used as a last resort antibiotic against infections caused by multidrug-resistant gram-negative bacteria, especially carbapenem-resistant bacteria. However, colistin-resistance in clinical isolates is becoming more prevalent. Cinnamaldehyde and baicalin, which are the major active constituents of Cinnamomum and Scutellaria, have been reported to exhibit antibacterial properties. The aim of this study was to evaluate the capacity of cinnamaldehyde and baicalin to enhance the antibiotic activity of colistin in Enterobacterales and Acinetobacter baumannii strains. METHODS: The MICs of colistin were determined with and without fixed concentrations of cinnamaldehyde and baicalin by the broth microdilution method. The FIC indices were also calculated. In addition, time-kill assays were performed with colistin alone and in combination with cinnamaldehyde and baicalin to determine the bactericidal action of the combinations. Similarly, the effects of L-arginine, L-glutamic acid and sucrose on the MICs of colistin combined with cinnamaldehyde and baicalin were studied to evaluate the possible effects of these compounds on the charge of the bacterial cell- wall. RESULTS: At nontoxic concentrations, cinnamaldehyde and baicalin partially or fully reversed resistance to colistin in Enterobacterales and A. baumannii. The combinations of the two compounds with colistin had bactericidal or synergistic effects on the most resistant strains. The ability of these agents to reverse colistin resistance could be associated with bacterial cell wall damage and increased permeability. CONCLUSION: Cinnamaldehyde and baicalin are good adjuvants for the antibiotic colistin against Enterobacterales- and A. baumannii-resistant strains.