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Restoring colistin's efficacy is crucial in addressing the resistance crisis of colistin. This study utilized a high-throughput screening method to identify 43 compounds from 800 FDA-approved drugs that exhibited significant antibacterial effects when combined with colistin. Among these, cinacalcet hydrochloride (CH) was selected for its potential synergistic effect with colistin against multidrug-resistant (MDR) E. coli strains, including mcr-1-positive strains. A series of experiments revealed that the combination of CH and colistin showed strong synergy, especially in mcr-1-positive strains, restoring colistin sensitivity. The combination significantly inhibited bacterial growth and reduced CFU counts more effectively than either drug alone. Additionally, CH and colistin together significantly inhibited biofilm formation and eradicated existing biofilms, as visualized through confocal microscopy. Mechanistic studies showed that the combination increased bacterial membrane permeability and disrupted membrane integrity. The treatment also elevated extracellular ATP release and ROS production, indicating oxidative stress-induced bacterial death. Safety evaluations showed that the combination did not increase toxicity in host cells. Finally, animal models further validated the combination's efficacy. Overall, this study showed that the combination of colistin and CH significantly restored colistin sensitivity in mcr-1-positive E. coli, revealing their synergistic antibacterial mechanism involving membrane damage and oxidative stress, with promising clinical applications. [ABSTRACT FROM AUTHOR]

Escherichia coli, a rod-shaped Gram-negative bacterium, is a significant causative agent of severe clinical bacterial infections. This study aimed to analyze the epidemiology of extended-spectrum β-lactamase (ESBL)-producing mcr-1 -positive E. coli in Shandong, China. We collected 668 non-duplicate ESBL-producing E. coli strains from clinical samples at Shandong Provincial Hospital between January and December 2018, and estimated their minimum inhibitory concentrations (MICs) using a VITEK® 2 compact system and broth microdilution. Next-generation sequencing and bioinformatic analyses identified the mcr-1 gene and other resistance genes in the polymyxin B-resistant strains. The conjugation experiment assessed the horizontal transfer capacity of the mcr-1 gene. Of the strains collected, 24 polymyxin B-resistant strains were isolated with a positivity rate of 3.59% and among the 668 strains, 19 clinical strains carried the mobile colistin resistance gene mcr-1, with a positivity rate of approximately 2.8%. All 19 clinical strains were resistant to ampicillin, cefazolin, ceftriaxone, ciprofloxacin, levofloxacin, and polymyxin B. Seventeen strains successfully transferred the mcr-1 gene into E. coli J53. All transconjugants were resistant to polymyxin B, and carried the drug resistance gene mcr-1. The 19 clinical strains had 14 sequence types (STs), with ST155 (n = 4) being the most common. The whole-genome sequencing results of pECO-POL-29_mcr1 revealed that no ISApl1 insertion sequences were found on either side of the mcr-1 gene. Our study uncovered the molecular epidemiology of mcr-1-carrying ESBL-producing E. coli in the region and suggested horizontal transmission mediated by plasmids as the main mode of mcr-1 transmission. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Faculty of Pharmacy of Ankara University / Ankara Üniversitesi Eczacilik Fakültesi Dergisi is the property of Ankara University and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Objectives: Colistin is known as the last resort antibiotic to treat the infections caused by multidrug resistant foodborne pathogens. The emergence and widespread dissemination of plasmid-mediated colistin resistance gene mcr-1 in the Escherichia coli (E. coli) incurs potential threat to public health. Here, we investigated the epidemiology, transmission dynamics, and genetic characterization of mcr-1 harbouring E. coli isolates from poultry originated in Hebei Province, China. Methods: A total of 297 faecal samples were collected from the two large poultry farms in Hebei Province, China. The samples were processed for E. coli identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry and 16S rDNA sequencing. Then, the mcr-1 gene harbouring E. coli strains were identified by polymerase chain reaction and subjected to antimicrobial susceptibility testing by broth microdilution assay. The genomic characterization of the isolates was done by whole genome sequencing using the various bioinformatics tools, and multi-locus sequence typing was done by sequence analysis of the seven housekeeping genes. The conjugation experiment was done to check the transferability of mcr-1 along with the plasmid stability testing. Results: A total of six mcr-1 E. coli isolates with minimum inhibitory concentration of 4 μg/mL were identified from 297 samples (2.02%). The mcr-1 harbouring E. coli were identified as multidrug resistant and belonged to ST101 (n = 4) and ST410 (n = 2). The genetic environment of mcr-1 presented its position on IncHI2 plasmid in 4 isolates and p0111 in 2 isolates, which is a rarely reported plasmid type for mcr-1. Moreover, both type of plasmids was transferable to recipient J53, and mcr-1 was flanked by 3 mobile elements ISApl1, Tn3, and IS26 forming a novel backbone Tn3–IS26-mcr-1– pap2-ISApl1 on the p0111 plasmid. The phylogenetic analysis shared a common lineage with mcr-1 harbouring isolates from the environment, humans, and animals, which indicate its horizontal spread among the diverse sources, species, and hosts. Conclusion: This study recommends the one health approach for future surveillance across multiple sources and bacterial species to adopt relevant measures and reduce global resistance crises.

Colistin is considered the last resort for treating infections caused by multidrug-resistant bacteria. However, the spread of the plasmid-borne colistin-resistance gene mcr-1 has become a public health threat. In this study, we identified mcr-1-harboring Leclercia adecarboxylata strain (WWCOL-134) isolated from wastewater in Seoul. The strain had a colistin MIC value of 2 µg/ml and was resistant to cefotaxime, gentamicin, tetracycline, trimethoprim and sulfamethoxazole. The mcr-1 gene, along with an array of resistance genes, was located on a 236-kb plasmid (pCOL134-1), which contained the typical IncHI2 backbone of reported mcr-1-carrying plasmids, and was transferred to an Escherichia coli strain by conjugation. To the best of our knowledge, this is the first study to report the emergence of mcr-1-harboring Leclercia sp. isolate. Our findings demonstrate the ongoing spread of colistin resistance among Enterobacterales species, emphasizing the need for surveillance of antimicrobial resistance in wastewater environments.

Abstract Background Carbapenem-resistant E. coli (CREco) pose a significant public health threat due to their multidrug resistance. Colistin is often a last-resort treatment against CREco; however, the emergence of colistin resistance gene mcr-1 complicates treatment options. Methods Two E. coli strains (ECO20 and ECO21), recovered from hospitalized patients in distinct wards, exhibited resistance to carbapenems and colistin. Whole-genome sequencing and phenotypic characterization were employed to study resistance patterns, plasmid profiles, transferability of resistance and virulence genes, and siderophore production capabilities. Comparative genome analysis was used to investigate the genetic environment of mcr-1, bla NDM−7, and virulence clusters. Results Both E. coli strains exhibited thr presence of both mcr-1 and bla NDM−7 genes, showing high resistance to multiple antibiotics. Genomic analysis revealed the clonal transmission of these strains, possessing identical plasmid profiles (pMCR, pNDM, and pVir) associated with colistin resistance, carbapenem resistance, and virulence factors. Conjugation experiments confirmed the transferability of these plasmids, indicating their potential to disseminate resistance and virulence traits to other strains. Comparative genomic analyses unveiled the distribution of mcr-1 (IncX4-type) and bla NDM (IncX3-type) plasmids across diverse bacterial species, emphasizing their adaptability and threat. The novelty of pVir indicates its potential role in driving the evolution of highly adaptable and pathogenic strains. Conclusions Our findings underscore the co-occurrence of mcr-1, bla NDM−7, and siderophore-producing plasmids in E. coli, which poses a significant concern for global health. This research is crucial to unravel the complex mechanisms governing plasmid transfer and recombination and to devise robust strategies to control their spread in healthcare settings.

Objectives: The emergence of pathogens co-harbouring multiple mobile resistance and virulence elements is of great concern in clinical settings. Herein, we report an O101: H10-ST167 Escherichia coli Hu106 strain isolated from the urinary tract of a female in China. Methods: Antibiotic susceptibility testing was used to present the antimicrobial resistance spectrum. Whole-genome sequencing (WGS) and bioinformatic analysis were used to clarify the virulent and resistance mechanisms. Furthermore, the virulence of this strain was tested by the Greater wax moth larvae and siderophore production experiment. Results: The strain E. coli Hu106 was resistant to almost all antimicrobials tested, and only susceptible to aztreonam, amikacin, and tigecycline. WGS analysis revealed that the strain Hu106 co-harboured blaNDM-9 and mcr-1 on p2-Hu106, belonging to IncHI2/IncHI2A (256,000 bp). The co-existence of both resistance genes, blaNDM-9 and mcr-1, on the plasmid p2-Hu106 was mainly acquired by transposition recombination of mobile antibiotic elements mediated by IS26 and/or IS1 on IncHI2/IncHI2A type plasmid. In addition, the virulence clusters aerobactin (iutA-iucABCD) and salmochelin (iroBCDEN) were identified on an IncFIB/IncFIC(IncFII) type plasmid p1-Hu106, flanked by small mobile elements such as IS1A, ISkpn28, and IS3, respectively. After performing genomic comparison of p1-Hu106 with the WGS in NCBI, we identified that the virulent plasmid p1-Hu106-like could spread in different clones of E. coli and Klebsiella pneumoniae, revealing its underlying dissemination mechanism between Enterobacterales. Furthermore, the strain caused a decreased survival rate of larvae and produced high siderophore units (62.33%), similar to hypervirulent K. pneumoniae NTUH-K2044. Conclusions: The strains co-carrying the multidrug-resistant plasmid p2-Hu106 and virulent plasmid p1-Hu106 should be closely monitored to prevent its further spreading.

Antimicrobial resistance (AMR) is a significant global health threat, with multidrug-resistant (MDR) bacterial clones becoming a major concern. Polymyxins, especially colistin, have reemerged as last-resort treatments for MDR Gram-negative infections. However, colistin use in livestock has spread mobile colistin resistance (mcr) genes, notably mcr-1, impacting human health. In consequence, its livestock use was banned in 2017, originating a natural experiment to study bacterial adaptation. The aim of this work was to analyse the changes in the mcr-1 genetic background after colistin restriction across the world. This study analyses 3163 Escherichia coli genomes with the mcr-1 gene from human and livestock hosts, mainly from Asia (n = 2621) and Europe (n = 359). Genetic characterisation identifies IncI2 (40.4%), IncX4 (26.7%), and multidrug-resistant IncHI2 (18.8%) as the most common plasmids carrying mcr-1. There were differences in plasmids between continents, with IncX4 (56.6%) being the most common in Europe, while IncI2 (44.8%) was predominant in Asia. Promoter variants related to reduced fitness costs and ISApl1 showed a distinct pattern of association that appears to be associated with adaptation to colistin restriction, which differed between continents. Thus, after the colistin ban, Europe saw a shift to specialised mcr-1 plasmids as IncX4, while ISApl1 decreased in Asia due to changes in the prevalence of the distinct promoter variants. These analyses illustrate the evolution of mcr-1 adaptation following colistin use restrictions and the need for region-specific strategies against AMR following colistin restrictions. [ABSTRACT FROM AUTHOR]

Resistance to last-resort antibiotics is a global threat to public health. Therefore, surveillance and monitoring systems for antimicrobial resistance should be established on a national and international scale. For the development of a One Health surveillance system, we collected exemplary data on carbapenem and colistin-resistant bacterial isolates from human, animal, food, and environmental sources. We pooled secondary data from routine screenings, hospital outbreak investigations, and studies on antimicrobial resistance. For a joint One Health evaluation, this study incorporates epidemiological metadata with phenotypic resistance information and molecular data on the isolate level. To harmonise the heterogeneous original information for the intended use, we developed a generic strategy. By defining and categorising variables, followed by plausibility checks, we created a catalogue for prospective data collections and applied it to our dataset, enabling us to perform preliminary descriptive statistical analyses. This study shows the complexity of data management using heterogeneous secondary data pools and gives an insight into the early stages of the development of an AMR surveillance programme using secondary data. [ABSTRACT FROM AUTHOR]

Background: Carbapenem-resistant E. coli (CREco) pose a significant public health threat due to their multidrug resistance. Colistin is often a last-resort treatment against CREco; however, the emergence of colistin resistance gene mcr-1 complicates treatment options. Methods: Two E. coli strains (ECO20 and ECO21), recovered from hospitalized patients in distinct wards, exhibited resistance to carbapenems and colistin. Whole-genome sequencing and phenotypic characterization were employed to study resistance patterns, plasmid profiles, transferability of resistance and virulence genes, and siderophore production capabilities. Comparative genome analysis was used to investigate the genetic environment of mcr-1, blaNDM−7, and virulence clusters. Results: Both E. coli strains exhibited thr presence of both mcr-1 and blaNDM−7 genes, showing high resistance to multiple antibiotics. Genomic analysis revealed the clonal transmission of these strains, possessing identical plasmid profiles (pMCR, pNDM, and pVir) associated with colistin resistance, carbapenem resistance, and virulence factors. Conjugation experiments confirmed the transferability of these plasmids, indicating their potential to disseminate resistance and virulence traits to other strains. Comparative genomic analyses unveiled the distribution of mcr-1 (IncX4-type) and blaNDM (IncX3-type) plasmids across diverse bacterial species, emphasizing their adaptability and threat. The novelty of pVir indicates its potential role in driving the evolution of highly adaptable and pathogenic strains. Conclusions: Our findings underscore the co-occurrence of mcr-1, blaNDM−7, and siderophore-producing plasmids in E. coli, which poses a significant concern for global health. This research is crucial to unravel the complex mechanisms governing plasmid transfer and recombination and to devise robust strategies to control their spread in healthcare settings. [ABSTRACT FROM AUTHOR]

Copyright of Modern Food Science & Technology is the property of Editorial Office of Modern Food Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The rising incidence of colistin (COL) resistance poses a significant challenge, undermining the therapeutic efficacy of COL against life-threatening bacterial infections. Therefore, the urgent identification and development of new therapeutics are imperative. It has been proven that combinations of antibiotics and promising non-antibiotic agents could be a potential strategy to combat infections caused by MDR pathogens. Due to various antimicrobial properties, medicinal plants have attracted significant attention, which could be promising adjuvant. In this study, we investigated the synergistic effects of combining COL with resveratrol (RST) and baicalin (BAI) against mcr-1-positive Escherichia coli through antibiotic susceptibility testing, checkerboard method and time-killing assays. The mechanisms of combination treatment were analyzed using SEM, fluorometric assays and transcriptome analysis. The molecular docking assay was conducted to elucidate potential interactions between RST, BAI and the MCR-1 protein. Finally, we assessed the in vivo efficacy of combination against mcr-1-positive Escherichia coli. The results demonstrated that the combination of RST, BAI and COL showed significant synergistic activity both in vitro and in vivo. Further mechanistic study revealed that the combination could increase the membrane-damaging ability of COL, disrupt the homeostasis of proton motive force (PMF), inhibit the activity of efflux pumps and impair ATP supply. The molecular docking revealed that RST and BAI could bind to MCR-1 stably, indicating the combination of RST and BAI may be an effective MCR-1 inhibitor. Our findings demonstrated that the combination of RST and BAI might be potential COL adjuvant, providing an alternative approach to address mcr-1-positive Escherichia coli infections.

ABSTRACT: Carbapenems and colistin are vital antimicrobials used to treat Enterobacteriaceae-caused infections. The present study aimed to characterize the coexistence mechanism of carbapenem and colistin resistance in an Escherichia coli isolated from retail chicken meat. A total of 4 E. coli isolates co-harboring carbapenem resistance gene blaNDM (2 E. coli isolates with blaNDM-5 and 2 with blaNDM-9) and colistin resistance gene mcr-1. Antimicrobial susceptibility testing exhibited that all the 4 E. coli strains had multidrug resistance profile and consistent with the resistance genes they carried. MLST showed that 3 E. coli isolates belonged to a pathogenic E. coli lineage ST354, which is closely associated with human infections and pose a serious threat to public health. Whole genome sequencing (WGS) showed that 4 mcr-1-positive plasmids with sizes of 60.4 kb to 67.4 kb all belonged to the IncI2 type. A total of 5 blaNDM-harboring plasmids ranged from 99.0 kb to 138.3 kb, among which 4 plasmids belonged to unknow type and only pCS5L-NDM belonged to IncFIA/IncFIB group of hybrid plasmids, a novel carrier for blaNDM. Comparative analysis exhibited that the mcr-1 or blaNDM-carrying plasmids of E. coli strains from chicken meat showed high identity with that from Enterobacteriaceae of human origin, which indicated the risk of mcr-1 or blaNDM dissemination from retail meat to human. The simultaneous occurrence of mcr-1 and blaNDM in E. coli emphasizes the significant of antimicrobial resistance surveillance in retail meat.

ABSTRACT The plasmid-mediated gene mcr-1 that makes bacteria resistant to the antibiotic colistin is spreading quickly, which means that colistin is no longer working well to treat Gram-negative bacterial infections. Herein, we utilized a computer-aided high-throughput screening drugs method to identify the natural product apigenin, a potential mcr-protein inhibitor, which effectively enhanced the antimicrobial activity of colistin. Several assays, including a checkerboard minimum inhibitory concentration assay, a time-kill assay, the combined disk test, molecular simulation dynamics, and animal infection models assay, were conducted to verify whether apigenin enhanced the ability of colistin to fight Gram-negative bacterial infections. The results showed that apigenin improved the antimicrobial activity of colistin against multidrug-resistant Enterobacteriaceae infection. Moreover, apigenin not only did not increase the toxic effect of colistin but also had the ability to effectively inhibit the frequency of bacterial resistance mutations to colistin. Studies clearly elucidated that apigenin could interfere with the thermal stability of the protein by binding to the mcr-1 protein. Additionally, the combination of apigenin and colistin could exert multiple effects, including disrupting bacterial membranes, the generation of bacterial nitric oxide and reactive oxygen species, as well as inhibiting bacterial adenosine triphosphate production. Furthermore, the addition of apigenin was able to significantly inhibit colistin-stimulated high expression levels of the bacterial mcr-1 gene. Finally, apigenin exhibited a characteristic anti-inflammatory effect while enhancing the antimicrobial activity of colistin against mcr-1-positive Escherichia coli (E. coli) infected animals. In conclusion, as a potential lead compound, apigenin is promising in combination with colistin in the future treatment of mcr-1-positive E. coli infections.IMPORTANCEThis study found that apigenin was able to inhibit the activity of the mcr-1 protein using a high-throughput virtual screening method. Apigenin effectively enhanced the antimicrobial activity of colistin against multidrug-resistant Enterobacteriaceae, including mcr-1-positive strains, in vitro and in vivo. This study will provide new options and strategies for the future treatment of multidrug-resistant pathogen infections.

Colistin used to be regarded as the last-resort treatment for the infection caused by multidrug-resistant gram-negative bacteria. However, the emergence and widespread of mcr-1 seriously reduce the clinical effectiveness of colistin, constituting a serious threat to global public health. Due to the difficulties in the development of new antibiotics, restoring antibiotic susceptibility with adjuvants is undoubtedly a rational option. As naturally occurring compounds, resveratrol and capsaicin could be extracted from many natural plants, due to various antimicrobial properties, they have received significant attention. Herein, the synergistic activity of colistin combined with resveratrol and capsaicin against mcr-1-positive Escherichia coli were investigated by checkerboard method and time-killing assays. The in-depth molecular mechanisms were elucidated by scanning electron microscopy, fluorescent probe experiments, transcriptome and metabolome analysis. Molecular docking assay was taken to analyse potential interactions between resveratrol/capsaicin and MCR-1. Finally, the in vivo efficacy of combined therapy against mcr-1-positive Escherichia coli was assessed. Our results demonstrated that colistin combined with resveratrol and capsaicin acted synergistic activity against mcr-1-positive Escherichia coli both in vivo and in vitro. Further mechanistic studies showed that the combined therapy could exacerbate cell membrane damage, increase membrane permeability, disrupt the homeostasis of PMF, inhibit ATP synthesis, and efflux pump activity. In addition, the combined therapy could inhibit central carbon metabolism, and reduce tricarboxylic acid cycle and oxidative phosphorylation. Moreover, molecular docking assay revealed resveratrol/capsaicin could bind to MCR-1 stably. Our study indicated that colistin in combination with resveratrol and capsaicin as a novel therapy could provide a trustworthy foundation to establish the treatment plan for mcr-1-positive Escherichia coli.

Plasmid-mediated conjugative transfer of antibiotic resistance genes (ARGs) within the human and animal intestine represents a substantial global health concern. linoleic acid (LA) has shown promise in inhibiting conjugation in vitro, but its in vivo effectiveness in the mammalian intestinal tract is constrained by challenges in efficiently reaching the target site. Recent advancements have led to the development of waterborne polyurethane nanoparticles for improved drug delivery. In this study, we synthesized four waterborne polyurethane nanoparticles incorporating LA (WPU@LA) using primary raw materials, including N-methyldiethanolamine, 2,2'-(piperazine-1,4-diyl) diethanol, isophorone diisocyanate, castor oil, and acetic acid. These nanoparticles, identified as WPU0.89@LA, WPU0.99@LA, WPU1.09@LA, and WPU1.19@LA, underwent assessment for their pH-responsive release property and biocompatibility. Among these, WPU0.99@LA displayed superior pH-responsive release properties and biocompatibility towards Caco-2 and IPEC-J2 cells. In a mouse model, a dosage of 10 mg/kg/day WPU0.99@LA effectively reduced the conjugation of IncX4 plasmids carrying the mobile colistin resistance gene (mcr-1) by more than 45.1-fold. In vivo toxicity assessment demonstrated that 10 mg/kg/day WPU0.99@LA maintains desirable biosafety and effectively preserves gut microbiota homeostasis. In conclusion, our study provides crucial proof-of-concept support, demonstrating that WPU0.99@LA holds significant potential in controlling the spread of antibiotic resistance within the mammalian intestine.

ABSTRACT: Colistin (CST) is considered as “agent of last resort” against gram-negative bacteria as feed additive. Its clinical effectiveness has reduced since the emergence of mcr-1 gene in ducks. Isopropoxy benzene guanidine (IBG), a new guanidine derivative, showed positive effects on improving animal weights and alleviating intestinal pathogens, therefore, the objective of this study was to evaluate the effect of this compound supplement with CST in ducks and explore the possibilities in feed additive. A total of fifteen duck-origin Escherichia coli carrying the mcr-1 gene were included in this study. A checkerboard microdilution assay was used to evaluate the in vitro antibacterial activity of IBG combined with CST against mcr-1-positive E. coli. A 3-by-2 time–kill array of IBG (16, 32, and 64 μg/mL) and CST (1/2 MIC and 1/4 MIC) over 24 hours was utilized to characterize the activity of the agents alone and in combination against E. coli strain 1 in vitro. The intestinal colonization model was used to evaluate the in vivo effect of IBG combined with CST. These results indicated that the combination of IBG plus CST showed a synergistic effect against all clinical isolates (FICI < 0.5). The bacterial burden was reduced by more than 2 log10 CFU/mL when E. coli strain 1 was tested with 1/2 MIC CST plus 64 μg/mL IBG for 24 h. Further experiments in vivo demonstrated that the CST combined with IBG was able to increase duck weights, reduced intestinal pathogenic E. coli and showed a synergistic antibacterial effect. Combination of CST (4 mg/kg b.w.) plus IBG (32 or 64 mg/kg b.w.) achieved 1.84 to 3.29 log10 CFU/g killing after 7 d of therapy, which was significantly different from that in the challenge control group (p

ABSTRACT: Objectives: Klebsiella pneumoniae is a major opportunistic pathogen that is a member of the Enterobacteriaceae. Klebsiella pneumoniae causes pneumonia in mink and has become the primary infectious disease that limits mink farming. In this study, we report the draft genome sequence of a multidrug-resistant (MDR) strain of K. pneumoniae that harbours the mcr-1 gene isolated from a mink in China. Methods: The agar microdilution method was used to determine the minimum inhibitory concentration of the strain. The entire genomic DNA was sequenced using an Illumina MiSeq platform. A multilocus sequence type (MLST) and a core genome SNP phylogenetic tree analysis with a heatmap of the resistance genes and virulence genes were performed. Results: The size of the genome was 5451.826 kb, and it included one chromosome and one plasmid. The draft genome of K. pneumoniae indicated that the isolate was a member of MLST 661. Four types of virulence genes were detected. The results of antimicrobial susceptibility testing showed multiple drug resistance, and 17 resistance genes were identified. Conclusion: The genome sequence reported in this study will help to reveal the key role of antibiotic resistance and pathogenic mechanisms. It will provide useful information for the role of mobile genetic elements in the adaptive translocation and spread of antimicrobial resistance.

Polymyxins are the last line of defense against multidrug-resistant (MDR) Gram-negative bacterial infections. However, this last resort has been threatened by the emergence of superbugs carrying the mobile colistin resistance gene-1 (mcr-1). Given the high concentration of matrix metalloproteinase 3 (MMP-3) in bacterial pneumonia, limited plasma accumulation of colistin (CST) in the lung, and potential toxicity of ionic silver (Ag+), we designed a feasible clinical transformation platform, an MMP-3 high-performance lung-targeted bio-responsive delivery system, which we named “CST&Ag@CNMS”. This system exhibited excellent lung-targeting ability (>80% in lungs), MMP-3 bio-responsive release property (95% release on demand), and synergistic bactericidal activity in vitro (2–4-fold minimum inhibitory concentration reduction). In the mcr-1+ CST-resistant murine pneumonia model, treatment with CST&Ag@CNMS improved survival rates (70% vs. 20%), reduced bacteria burden (2–3 log colony-forming unit [CFU]/g tissue), and considerably mitigated inflammatory response. In this study, CST&Ag@CNMS performed better than the combination of free CST and AgNO3. We also demonstrated the superior biosafety and biodegradability of CST&Ag@CNMS both in vitro and in vivo. These findings indicate the clinical translational potential of CST&Ag@CNMS for the treatment of lung infections caused by CST-resistant bacteria carrying mcr-1.

ABSTRACT: IncX4 plasmids are one of the most epidemiologically successful vehicles for mcr-1 spread. Here we found that the IncX4 plasmids carried two different replication proteins encoded by genes pir-1 and pir-2, respectively, but mcr-1 was only carried by IncX4 plasmid encoding pir-1. The copy number of pir-2 encoding plasmids (3.15 ± 0.9 copies) are higher than that of pir-1 encoding plasmids (0.85 ± 0.5 copies). When mcr-1 was cloned into IncX4 plasmid encoding pir-2, the higher copy number of these plasmids resulted in increased expression of mcr-1 and a greater fitness burden on their host cells. However, these plasmids exhibited a lower rate of invasion into the bacterial population compared with mcr-1-positive plasmids encoding the pir-1 gene. These findings collectively explain the absence of mcr-1 in all IncX4 plasmids encoding pir-2. Our results further confirmed that low-copy numbers are important for the spread of mcr-1 plasmid from the perspective of natural evolution.

ABSTRACT: Objectives: Plasmid genes, termed mobile colistin resistance-1 (mcr-1) and mobile colistin resistance-2 (mcr-2), are associated with resistance to colistin in Escherichia coli (E. coli). These mcr genes result in a range of protein modifications contributing to colistin resistance. This study aims to discern the proteomic characteristics of E. coli–carrying mcr-1 and mcr-2 genes. Furthermore, it evaluates the expression levels of various proteins under different conditions (with and without colistin). Methods: Plasmid extraction was performed using an alkaline lysis–based plasmid extraction kit, whereas polymerase chain reaction was used to detect the presence of mcr-1 and mcr-2 plasmids. The E. coli DH5α strain served as the competent cell for accepting and transforming mcr-1 and mcr-2 plasmids. We assessed proteomic alterations in the E. coli DH5α strain both with and without colistin in the growth medium. Proteomic data were analysed using mass spectrometry. Results: The findings revealed significant protein changes in the E. coli DH5α strain following cloning of mcr-1 and mcr-2 plasmids. Of the 20 proteins in the DH5α strain, expression in 8 was suppressed following transformation. In the presence of colistin in the culture medium, 39 new proteins were expressed following transformation with mcr-1 and mcr-2 plasmids. The proteins with altered expression play various roles. Conclusion: The results of this study highlight numerous protein alterations in E. coli resulting from mcr-1 and mcr-2–mediated resistance to colistin. This understanding can shed light on the resistance mechanism. Additionally, the proteomic variations observed in the presence and absence of colistin might indicate potential adverse effects of indiscriminate antibiotic exposure on treatment efficacy and heightened pathogenicity of microorganisms.

ABSTRACT: Objectives: Aquatic ecosystems serve as a dissemination pathway and a reservoir of both antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). This study aimed to determine the prevalence of colistin-resistant mcr-like genes in Enterobacteriales in aquatic products, which may be contribute to the transfer of ARGs in water environments. Methods: The mcr-1-positive Escherichia coli were recovered from 123 freshwater fish and 34 cultured crocodile cecum samples from 10 farmers’ markets in Guangdong, China. Minimum inhibitory concentration (MIC) was determined using the agar dilution method. Genotyping was performed using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Conjugation assay was carried out to investigate the transferability of mcr-1. Genomic information was obtained by whole genome sequencing (WGS) and bioinformatic analysis. Results: Forty-four mcr-1 positive isolates showed co-resistance to tetracycline, trimethoprim/sulfamethoxazole, and gentamicin, while they were all sensitive to tigecycline, meropenem, and amikacin. They were typed into sixteen PFGE clusters. ST10 and ST117 were the most popular sequence types, followed by ST1114. S1-PFGE verified the presence of the mcr-1 gene on plasmids in sizes of ∼60 kb (n = 1) and ∼240 kb (n = 3). Whole genome sequencing-based analysis identified mcr-1 integrated in IncHI2 plasmid (n = 3), IncI2 plasmid (n = 2), and bacterial chromosome in two copies (n = 1). In addition to mcr-1, they carried several other antibiotic resistance genes, such as blaCTX-M-14, fosA3, and aac(6’)-Ib-cr. Conclusion: These data suggest that aquatic products are an important antibiotic resistance reservoir and highlight possible risks regarding food safety.

Food contamination with plasmid-mediated antibiotic-resistant Escherichia coli has not been thoroughly investigated. This study aimed to clarify the prevalence of colistin-resistant AmpC/extended-spectrum β-lactamase (ESBL)-producing E. coli harbouring mcr (COL-ESBL-EC) and to determine antibiotic resistance by comparison with AmpC/ESBL-producing E. coli (ESBL-EC) and colistin-resistant E. coli harbouring mcr (COL-EC). Sixty chicken meats were tested for COL-ESBL-EC contamination. The result showed that 64 COL-ESBL-EC were isolated from 66.7% of the samples, and compared with ESBL-EC and COL-EC were isolated before. The genotypes of the COL-ESBL-EC showed blaCTX-M-55/TEM, blaCTX-M-55, blaCMY-2, and blaCTX-M-65 were predominant. The COL-ESBL-EC results showed a similar trend for blaCTX-M identification to that of ESBL-EC. The mcr was investigated, and mcr-1 was detected in COL-ESBL-EC and COL-EC. Antibiotic susceptibility testing revealed that many strains of COL-ESBL-EC and ESBL-EC were resistant to quinolones, and fosfomycin (FOS). These results suggest that COL-ESBL-EC has simultaneously acquired AmpC/ESBL-related, quinolone, and FOS resistance genes in COL-EC. [ABSTRACT FROM AUTHOR]

The global spread of plasmid-mediated mobile colistin resistance (mcr) genes threatens the vital role of colistin as a drug of last resort. We investigated whether the recurrent occurrence of specific E. coli pathotypes and plasmids in individual pig farms resulted from the continued presence or repeated reintroduction of distinct E. coli strains. E. coli isolates (n = 154) obtained from three pig farms with at least four consecutive years of mcr detection positive for virulence-associated genes (VAGs) predicting an intestinal pathogenic pathotype via polymerase chain reaction were analyzed. Detailed investigation of VAGs, antimicrobial resistance genes and plasmid Inc types was conducted using whole genome sequencing for 87 selected isolates. Sixty-one E. coli isolates harbored mcr-1, and one isolate carried mcr-4. On Farm 1, mcr-positive isolates were either edema disease E. coli (EDEC; 77.3%) or enterotoxigenic E. coli (ETEC; 22.7%). On Farm 2, all mcr-positive strains were ETEC, while mcr-positive isolates from Farm 3 showed a wider range of pathotypes. The mcr-1.1 gene was located on IncHI2 (Farm 1), IncX4 (Farm 2) or IncX4 and IncI2 plasmids (Farm 3). These findings suggest that various pathogenic E. coli strains play an important role in maintaining plasmid-encoded colistin resistance genes in the pig environment over time. [ABSTRACT FROM AUTHOR]

Abstract Background mcr-1-positive Escherichia coli has emerged as a significant threat to human health, veterinary health, and food safety in recent years. After the prohibition of colistin as a feed additive in animal husbandry in China, a noticeable reduction in both colistin resistance and the prevalence of mcr-1 was observed in E. coli from animals and humans. Objectives To assess the prevalence of the colistin resistance gene mcr-1 and characterize its genetic context in E. coli strains derived from fecal and meat samples from food-producing animals in China. Methods A total of 1,353 fecal samples and 836 food samples were collected between 2019 and 2020 in China. E. coli isolates were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and their susceptibility to colistin were determined using the broth microdilution method. The colistin-resistant E. coli isolates were screened for the presence of mcr by PCR analysis and sequencing. The minimal inhibitory concentrations (MICs) of 15 antimicrobial agents against the mcr-1-positive strains were further tested using the agar dilution method, conjugation assays were performed, and whole genome sequencing was performed using Illumina HiSeq. Results In total, 1,403 E. coli strains were isolated. Thirteen isolates from chicken meat (n = 7), chickens (n = 3), and pigs (n = 3) were resistant to colistin with MIC values of 4 to 16 mg/L, and carried mcr-1. All mcr-1-positive strains, except for isolate AH20PE105, contained multiple resistance genes and exhibited multidrug-resistant phenotypes. They belonged to 10 sequence types (STs), including a novel ST (ST14521). mcr-1 was located on IncI2 (n = 9), IncX4 (n = 2), and IncHI2 (n = 2) plasmids, which were highly similar to other mcr-1-carrying plasmids sharing the same incompatibility type. Seven mcr-1-carrying plasmids could be successfully conjugally transferred to E. coli C600. Conclusions While the low prevalence of mcr-1 (0.93%) identified in this study may not immediately seem alarming, the very emergence of this gene merits attention given its implications for colistin resistance and public health. Hence, ongoing surveillance of mcr-1 in E. coli remains crucial.

The emergence and spread of the mobile colistin-resistance gene, mcr-1, and its variants pose a challenge to the use of colistin, a last-resort antibiotic used to treat severe infections caused by extensively drug-resistant (XDR) Gram-negative pathogens. Antibiotic adjuvants are a promising strategy to enhance the efficacy of colistin against colistin-resistant pathogens; however, few studies have considered the effects of adjuvants on limiting resistance-gene transmission. We found that chelerythrine (4 mg∙L−1) derived from Macleaya cordata extract, which is used as an animal feed additive, reduced the minimal inhibitory concentration (MIC) of colistin against an mcr-1 positive Escherichia coli (E. coli) strain by 16-fold (from 2.000 to 0.125 mg∙L−1), eliminated approximately 104 colony-forming units (CFUs) of an mcr-1-carrying strain in a murine intestinal infection model, and inhibited the conjugation of an mcr-1-bearing plasmid in vitro (by > 100-fold) and in a mouse model (by up to 5-fold). A detailed analysis revealed that chelerythrine binds to phospholipids on bacterial membranes and increases cytoplasmic membrane fluidity, thereby impairing respiration, disrupting proton motive force (PMF), generating reactive oxygen species (ROS), and decreasing intracellular adenosine triphosphate (ATP) levels, which subsequently downregulates mcr-1 and conjugation-associated genes. These dual effects of chelerythrine can expand the use of antibiotic adjuvants and may provide a new strategy for circumventing mobile colistin resistance.

Abstract Objectives Pseudomonas aeruginosa (P. aeruginosa) is one of the most serious pathogens implicated in antimicrobial resistance, and it has been identified as an ESKAPE along with other extremely significant multidrug resistance pathogens. The present study was carried out to explore prevalence, antibiotic susceptibility phenotypes, virulence-associated genes, integron (int1), colistin (mcr-1), and β-lactamase resistance' genes (ESBls), as well as biofilm profiling of P. aeruginosa isolated from broiler chicks and dead in-shell chicks. Design A total of 300 samples from broiler chicks (n = 200) and dead in-shell chicks (n = 100) collected from different farms and hatcheries located at Mansoura, Dakahlia Governorate, Egypt were included in this study. Bacteriological examination was performed by cultivation of the samples on the surface of both Cetrimide and MacConkey’s agar. Presumptive colonies were then subjected to biochemical tests and Polymerase Chain Reaction (PCR) targeting 16S rRNA. The recovered isolates were tested for the presence of three selected virulence-associated genes (lasB, toxA, and exoS). Furthermore, the retrieved isolates were subjected to phenotypic antimicrobial susceptibility testing by Kirby–Bauer disc diffusion method as well as phenotypic detection of ESBLs by both Double Disc Synergy Test (DDST) and the Phenotypic Confirmatory Disc Diffusion Test (PCDDT). P. aeruginosa isolates were then tested for the presence of antibiotic resistance genes (ARGs): int1, mcr-1, and ESBL genes (OXA-10, OXA-2, VEB-1, SHV, TEM, and CTX-M). Additionally, biofilm production was examined by the Tube Adherent method (TA) and Microtiter Plate assay (MTP). Results Fifty –five isolates were confirmed to be P. aeruginosa, including 35 isolates from broiler chicks and 20 isolates from dead in-shell chicks. The three tested virulence genes (lasB, toxA, and exoS) were detected in all isolates. Antibiogram results showed complete resistance against penicillin, amoxicillin, ceftriaxone, ceftazidime, streptomycin, erythromycin, spectinomycin, and doxycycline, while a higher sensitivity was observed against meropenem, imipenem, colistin sulfate, ciprofloxacin, and gentamicin. ESBL production was confirmed in 12 (21.8%) and 15 (27.3%) isolates by DDST and PCDDT, respectively. Antibiotic resistance genes (ARGs): int1, mcr-1, and ESBL genes (OXA-10, SHV, TEM, and CTX-M), were detected in 87.3%, 18.2%, 16.4%, 69.1%, 72.7%, and 54.5% of the examined isolates respectively, whereas no isolate harbored the OXA-2 or VEB-1 genes. Based on the results of both methods used for detection of biofilm formation, Kappa statistics [kappa 0.324] revealed a poor agreement between both methods. Conclusions the emergence of mcr-1 and its coexistence with other resistance genes such as β-lactamase genes, particularly bla OXA-10, for the first time in P. aeruginosa from young broiler chicks and dead in-shell chicks in Egypt pose a risk not only to the poultry industry but also to public health. Graphical Abstract

In the European Union, gastrointestinal disease in pigs is the main indication for the use of colistin, but large-scale epidemiologic data concerning the frequency of mobile colistin resistance (mcr) genes in pig-associated pathotypes of Escherichia coli (E. coli) are lacking. Multiplex polymerase chain reactions were used to detect virulence-associated genes (VAGs) and mcr-1–mcr-10 genes in 10,573 porcine E. coli isolates collected in Germany from July 2000 to December 2021. Whole genome sequencing was performed on 220 representative mcr-positive E. coli strains. The total frequency of mcr genes was 10.2%, the most frequent being mcr-1 (8.4%) and mcr-4 (1.6%). All other mcr genes were rarely identified (mcr-2, mcr-3, mcr-5) or absent (mcr-6 to mcr-10). The highest frequencies of mcr genes were found in enterotoxigenic and shiga toxin-encoding E. coli (ETEC/STEC hybrid) and in edema disease E. coli (EDEC) strains (21.9% and 17.7%, respectively). We report three novel mcr variants, mcr-1.36, mcr-4.8, and mcr-5.5. In 39 attaching and effacing E. coli (AEEC) isolates analyzed in our study, the eae subtype β1 was the most prevalent (71.8%). Constant surveillance for the presence of mcr genes in various sectors should consider the different frequency of mcr-positive isolates in pathogenic E. coli.

Background: The United Arab Emirates (UAE) has witnessed rapid urbanization and a surge in pet ownership, sparking concerns about the possible transfer of antimicrobial resistance (AMR) from pets to humans and the environment. This study delves into the whole-genome sequencing analysis of ESBL-producing E. coli strains from healthy cats and dogs in the UAE, which exhibit multidrug resistance (MDR). Additionally, it provides a genomic exploration of the mobile colistin resistance gene mcr-1.1, marking the first instance of its detection in Middle Eastern pets. Methods: We investigate 17 ESBL-producing E. coli strains from healthy UAE pets using WGS and bioinformatics analysis to identify genes encoding virulence factors, assign diverse typing schemes to the isolates, and scrutinize the presence of AMR genes. Furthermore, we characterized plasmid contigs housing the mcr-1.1 gene and conducted phylogenomic analysis to evaluate their relatedness to previously identified UAE isolates. Results: Our study unveiled a variety of virulence factor-encoding genes within the isolates, with fimH emerging as the most prevalent. Regarding β-lactamase resistance genes, the blaCTX group 1 gene family predominated, with CTX-M-15 found in 52.9% (9/17) of the isolates, followed by CTX-M-55 in 29.4% (5/17). These isolates were categorized into multiple sequence types (STs), with the epidemic ST131 being the most frequent. The presence of the mcr-1.1 gene, linked to colistin resistance, was confirmed in two isolates. These isolates belonged to ST1011 and displayed distinct profiles of β-lactamase resistance genes. Phylogenomic analysis revealed close connections between the isolates and those from chicken meat in the UAE. Conclusion: Our study underscores the presence of MDR ESBL-producing E. coli in UAE pets. The identification of mcr-1.1-carrying isolates warrants the urgency of comprehensive AMR surveillance and highlights the role of companion animals in AMR epidemiology. These findings underscore the significance of adopting a One Health approach to mitigate AMR transmission risks effectively.

Postweaning diarrhoea (PWD) is a frequent multifactorial disease occurring in swine stocks worldwide. Since pathogenic Escherichia (E.) coli play a pivotal role in the pathogenesis of PWD and porcine E. coli are often resistant to different antibiotics, colistin is frequently applied to treat piglets with PWD. However, the application of colistin to livestock has been associated with the emergence of colistin resistance. This case report describes the detection of the colistin resistance gene mcr-1-1 in two E. coli isolated from piglets with PWD in an Austrian organic piglet-producing farm, which was managed by two farmers working as nurses in a hospital. Both mcr-1-positive E. coli were further analysed by Illumina short-read-sequencing, including assemblies and gene prediction. Both isolates belonged to the same clonal type and were positive for eaeH and espX5, which are both virulence genes associated with enteropathogenic E. coli (EPEC). Due to the detection of mcr-1-positive EPEC and based on the results of the antimicrobial resistance testing, the veterinarian decided to apply gentamicin for treatment instead of colistin, leading to improved clinical signs. In addition, after replacing faba beans with whey, PWD was solely observed in 2/10 weaned batches in the consecutive months. [ABSTRACT FROM AUTHOR]

Background: mcr-1-positive Escherichia coli has emerged as a significant threat to human health, veterinary health, and food safety in recent years. After the prohibition of colistin as a feed additive in animal husbandry in China, a noticeable reduction in both colistin resistance and the prevalence of mcr-1 was observed in E. coli from animals and humans. Objectives: To assess the prevalence of the colistin resistance gene mcr-1 and characterize its genetic context in E. coli strains derived from fecal and meat samples from food-producing animals in China. Methods: A total of 1,353 fecal samples and 836 food samples were collected between 2019 and 2020 in China. E. coli isolates were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and their susceptibility to colistin were determined using the broth microdilution method. The colistin-resistant E. coli isolates were screened for the presence of mcr by PCR analysis and sequencing. The minimal inhibitory concentrations (MICs) of 15 antimicrobial agents against the mcr-1-positive strains were further tested using the agar dilution method, conjugation assays were performed, and whole genome sequencing was performed using Illumina HiSeq. Results: In total, 1,403 E. coli strains were isolated. Thirteen isolates from chicken meat (n = 7), chickens (n = 3), and pigs (n = 3) were resistant to colistin with MIC values of 4 to 16 mg/L, and carried mcr-1. All mcr-1-positive strains, except for isolate AH20PE105, contained multiple resistance genes and exhibited multidrug-resistant phenotypes. They belonged to 10 sequence types (STs), including a novel ST (ST14521). mcr-1 was located on IncI2 (n = 9), IncX4 (n = 2), and IncHI2 (n = 2) plasmids, which were highly similar to other mcr-1-carrying plasmids sharing the same incompatibility type. Seven mcr-1-carrying plasmids could be successfully conjugally transferred to E. coli C600. Conclusions: While the low prevalence of mcr-1 (0.93%) identified in this study may not immediately seem alarming, the very emergence of this gene merits attention given its implications for colistin resistance and public health. Hence, ongoing surveillance of mcr-1 in E. coli remains crucial. [ABSTRACT FROM AUTHOR]

Objectives: Pseudomonas aeruginosa (P. aeruginosa) is one of the most serious pathogens implicated in antimicrobial resistance, and it has been identified as an ESKAPE along with other extremely significant multidrug resistance pathogens. The present study was carried out to explore prevalence, antibiotic susceptibility phenotypes, virulence-associated genes, integron (int1), colistin (mcr-1), and β-lactamase resistance' genes (ESBls), as well as biofilm profiling of P. aeruginosa isolated from broiler chicks and dead in-shell chicks. Design: A total of 300 samples from broiler chicks (n = 200) and dead in-shell chicks (n = 100) collected from different farms and hatcheries located at Mansoura, Dakahlia Governorate, Egypt were included in this study. Bacteriological examination was performed by cultivation of the samples on the surface of both Cetrimide and MacConkey's agar. Presumptive colonies were then subjected to biochemical tests and Polymerase Chain Reaction (PCR) targeting 16S rRNA. The recovered isolates were tested for the presence of three selected virulence-associated genes (lasB, toxA, and exoS). Furthermore, the retrieved isolates were subjected to phenotypic antimicrobial susceptibility testing by Kirby–Bauer disc diffusion method as well as phenotypic detection of ESBLs by both Double Disc Synergy Test (DDST) and the Phenotypic Confirmatory Disc Diffusion Test (PCDDT). P. aeruginosa isolates were then tested for the presence of antibiotic resistance genes (ARGs): int1, mcr-1, and ESBL genes (OXA-10, OXA-2, VEB-1, SHV, TEM, and CTX-M). Additionally, biofilm production was examined by the Tube Adherent method (TA) and Microtiter Plate assay (MTP). Results: Fifty –five isolates were confirmed to be P. aeruginosa, including 35 isolates from broiler chicks and 20 isolates from dead in-shell chicks. The three tested virulence genes (lasB, toxA, and exoS) were detected in all isolates. Antibiogram results showed complete resistance against penicillin, amoxicillin, ceftriaxone, ceftazidime, streptomycin, erythromycin, spectinomycin, and doxycycline, while a higher sensitivity was observed against meropenem, imipenem, colistin sulfate, ciprofloxacin, and gentamicin. ESBL production was confirmed in 12 (21.8%) and 15 (27.3%) isolates by DDST and PCDDT, respectively. Antibiotic resistance genes (ARGs): int1, mcr-1, and ESBL genes (OXA-10, SHV, TEM, and CTX-M), were detected in 87.3%, 18.2%, 16.4%, 69.1%, 72.7%, and 54.5% of the examined isolates respectively, whereas no isolate harbored the OXA-2 or VEB-1 genes. Based on the results of both methods used for detection of biofilm formation, Kappa statistics [kappa 0.324] revealed a poor agreement between both methods. Conclusions: the emergence of mcr-1 and its coexistence with other resistance genes such as β-lactamase genes, particularly blaOXA-10, for the first time in P. aeruginosa from young broiler chicks and dead in-shell chicks in Egypt pose a risk not only to the poultry industry but also to public health. [ABSTRACT FROM AUTHOR]

Objective: The co-occurrence of colistin and tigecycline resistance genes in Klebsiella pneumoniae poses a serious public health problem. This study aimed to characterize a K. pneumoniae strain, K82, co-harboring a colistin resistance gene (CoRG) and tigecycline resistance gene (TRG), and, importantly, investigate the genetic characteristics of the plasmid with CoRG or TRG in GenBank. Methods: K. pneumoniae strain K82 was subjected to antimicrobial susceptibility testing, conjugation assay, and whole-genome sequencing (WGS). In addition, comparative genomic analysis of CoRG or TRG-harboring plasmids from K82 and GenBank was conducted. K. pneumoniae strain K82 was resistant to all the tested antimicrobials including colistin and tigecycline, except for carbapenems. Results: WGS and bioinformatic analysis showed that K82 belonged to the ST656 sequence type and carried multiple drug resistance genes, including mcr-1 and tmexCD1-toprJ1, which located on IncFIA/IncHI2/IncHI2A/IncN/IncRtype plasmid pK82-mcr-1 and IncFIB/IncFII-type plasmid pK82-tmexCD-toprJ, respectively. The pK82-mcr-1 plasmid was capable of conjugation. Analysis of the CoRG/TRG-harboring plasmid showed that mcr-8 and tmexCD1-toprJ1 were the most common CoRG and TRG of Klebsiella spp., respectively. These TRG/CoRGharboring plasmids could be divided into two categories based on mash distance. Moreover, we found an IncFIB/IncHI1B-type plasmid, pSYCC1_tmex_287k, coharboring mcr-1 and tmexCD1-toprJ1. To the best of our knowledge, this is the first report on the co-occurrence of mcr-1 and tmexCD1-toprJ1 on a single plasmid. Conclusion: Our research expands the known diversity of CoRG and TRG-harboring plasmids in K. pneumoniae. Effective surveillance should be implemented to assess the prevalence of co-harboring CoRG and TRG in a single K. pneumoniae isolate or even a single plasmid. [ABSTRACT FROM AUTHOR]

Background: Antimicrobial resistance (AMR) is a global health problem largely due to the overuse of antimicrobials. In recognition of this, the World Health Assembly in 2015 agreed on a global action plan to tackle AMR. Following the global emergence of the mcr‐1‐associated colistin resistance gene in the livestock industry in 2016, several countries including South Africa restricted the veterinary use of colistin as the gene threatens the clinical utility of the drug. This study is a follow‐up to the restriction in place in order to evaluate the impact of such policy adoption. Objective: To assess the prevalence of antibacterial resistance (ABR), and the mcr‐1 colistin resistance gene in broiler chicken over a 2‐year period, as a follow‐up to the veterinary ban on colistin use in South Africa. Methods: A total of 520 swab samples were obtained during 2019 (March–April) and 2020 (February–March), from healthy broiler chicken carcasses (n = 20) and chicken droppings in transport crates (n = 20) at various poultry abattoirs (N = 7) in the Gauteng province of South Africa. Escherichia coli organisms were isolated and subjected to a panel of 24 antibacterials using the MicroScan machine. Screening for mcr‐1 colistin resistance gene was undertaken using PCR. Result: Four hundred and thirty‐eight (438) E. coli strains were recovered and none demonstrated phenotypic resistance towards colistin, amikacin, carbapenems, tigecycline and piperacillin/tazobactam. The mcr‐1 gene was not detected in any of the isolates tested. Resistances to the aminoglycosides (0%–9.8%) and fluoroquinolones (0%–18.9%) were generally low. Resistances to ampicillin (32%–39.3%) and trimethoprim/sulphamethoxazole (30.6%–3.6%) were fairly high. A significant (p < 0.05) increase in cephalosporins and cephamycin resistance was noted in the year 2020 (February–March) when compared with the year 2019 (March–April). Conclusion: The absence of mcr‐1 gene and colistin resistance suggests that mitigation strategies adopted were effective and clearly demonstrated the significance of regulatory interventions in reducing resistance to critical drugs. Despite the drawback in regulatory framework such as free farmers access to antimicrobials OTC and a dual registration system in place, there is a general decline in the prevalence of ABR when the present data are compared with the last national veterinary surveillance on AMR (SANVAD 2007). To further drive resistance down, mitigation strategies should focus on strengthening regulatory framework, the withdrawal of OTC dispensing of antimicrobials, capping volumes of antimicrobials, banning growth promoters and investing on routine surveillance/monitoring of AMR and antimicrobial consumption. [ABSTRACT FROM AUTHOR]

Over the last decade, New Delhi metallo‐beta‐lactamase (NDM) carbapenemase has silently spread in Brazil. In this study, we analyzed a large collection of Enterobacterales other than Klebsiella spp. received in our reference laboratory between 2013 and 2022. A total of 32 clinical isolates displaying different pulsed‐field gel electrophoresis profiles, and represented by 11 species in the families Enterobacteriaceae (Citrobacter freundii, Citrobacter portucalensis, Enterobacter hormaechei, and Escherichia coli), Morganellaceae (Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, Providencia stuartii, and Raoultella ornithinolytica), and Yersiniaceae (Serratia marcescens) had their whole genomes sequenced and further analyzed. Antimicrobial susceptibility was determined by disk diffusion, except for polymyxin B, assessed by broth microdilution. The blaNDM‐1 allele was predominant (n = 29), but blaNDM‐5 was identified in an E. coli specimen with a novel ST, and the blaNDM‐7 allele was found in E. hormaechei ST45 and E. coli ST1049. Polymyxin was active against all but one Enterobacteriaceae isolate: an mcr‐1–producing E. coli presenting minimal inhibitory concentration (4 mg/L). Isolates producing extended‐spectrum β‐lactamases were common: cefotaximase from Munich (CTX‐M)‐15 (n = 10), CTX‐M‐2 (n = 4), and CTX‐M‐8 (n = 3) were detected, and the mcr‐1–producing E. coli was found to co‐produce both CTX‐M‐8 and CTX‐M‐55 β‐lactamases. The mcr‐9 gene was found in 5/8 E. hormaechei isolates, distributed in four different sequence types, all of them presenting susceptibility to polymyxin. This study showed that NDM‐producing Enterobacterales other than Klebsiella are already spread in Brazil, in diversified species, and cocarrying important resistance genes. Prompt detection and effective implementation of measures to prevent further spread are mandatory for mitigating the dissemination of NDM carbapenemase in hospital settings and preserving the already limited antimicrobial therapy options. [ABSTRACT FROM AUTHOR]

The United Arab Emirates (UAE) has witnessed rapid urbanization and a surge in pet ownership, sparking concerns about the possible transfer of antimicrobial resistance (AMR) from pets to humans and the environment. This study delves into the whole-genome sequencing analysis of ESBL-producing E. coli strains from healthy cats and dogs in the UAE, which exhibit multidrug resistance (MDR). Additionally, it provides a genomic exploration of the mobile colistin resistance gene mcr -1.1, marking the first instance of its detection in Middle Eastern pets. We investigate 17 ESBL-producing E. coli strains from healthy UAE pets using WGS and bioinformatics analysis to identify genes encoding virulence factors, assign diverse typing schemes to the isolates, and scrutinize the presence of AMR genes. Furthermore, we characterized plasmid contigs housing the mcr -1.1 gene and conducted phylogenomic analysis to evaluate their relatedness to previously identified UAE isolates. Our study unveiled a variety of virulence factor-encoding genes within the isolates, with fimH emerging as the most prevalent. Regarding β-lactamase resistance genes, the bla CTX group 1 gene family predominated, with CTX-M-15 found in 52.9% (9/17) of the isolates, followed by CTX-M-55 in 29.4% (5/17). These isolates were categorized into multiple sequence types (STs), with the epidemic ST131 being the most frequent. The presence of the mcr -1.1 gene, linked to colistin resistance, was confirmed in two isolates. These isolates belonged to ST1011 and displayed distinct profiles of β-lactamase resistance genes. Phylogenomic analysis revealed close connections between the isolates and those from chicken meat in the UAE. Our study underscores the presence of MDR ESBL-producing E. coli in UAE pets. The identification of mcr -1.1-carrying isolates warrants the urgency of comprehensive AMR surveillance and highlights the role of companion animals in AMR epidemiology. These findings underscore the significance of adopting a One Health approach to mitigate AMR transmission risks effectively. [ABSTRACT FROM AUTHOR]

ABSTRACTTigecycline and colistin were referred to as the “last resort” antibiotics in defending against carbapenem-resistant, Gram-negative bacterial infections, and are currently widely used in clinical treatment. However, the emergence and prevalence of plasmid-mediated tet(X4) and mcr-1 genes pose a serious threat to the therapeutic application of tigecycline and colistin, respectively. In this research, a tigecycline- and colistin-resistant bacteria resensitization system was developed based on efficient and specific DNA damage caused by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Associated Protein 9 (Cas9) nucleases. A conjugation method was used to deliver the resensitization system, which harbors two single-guide RNAs targeting tet(X4) and mcr-1 genes and constitutively expressed Cas9. The conjugation efficiency was nearly 100% after conjugation condition optimization in vitro, and the resensitivity efficiency for clinical isolates was over 90%. In addition, when performing resensitization in vivo, the resistance marker was replaced with a glutamate-based, chromosomal, plasmid-balanced lethal system to prevent the introduction of additional resistance genes in clinical settings, making this strategy a therapeutic approach to combat the in vivo spread of antibiotic resistance genes (ARGs) among bacterial pathogens. As a proof of concept, this resensitive system can significantly decrease the counts of tigecycline- and colistin-resistant bacteria to 1% in vivo. Our study demonstrates the efficacy and adaptability of CRISPR-Cas systems as powerful and programmable antimicrobials in resensitizing tet(X4)- and mcr-1-mediated, tigecycline- and colistin-resistant strains, and opens up new pathways for the development of CRISPR-based tools for selective bacterial pathogen elimination and precise microbiome composition change.IMPORTANCEThe emergence of plasmid-encoded tet(X4) and mcr-1 isolated from human and animal sources has affected the treatment of tigecycline and colistin, and has posed a significant threat to public health. Tigecycline and colistin are considered as the “last line of defense” for the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections, so there is an urgent need to find a method that can resensitize tet(X4)-mediated tigecycline-resistant and mcr-1-mediated colistin-resistant bacteria. In this study, we developed a glutamate-based, chromosomal, plasmid-balanced lethal conjugative CRISPR/Cas9 system, which can simultaneously resensitize tet(X4)-mediated tigecycline-resistant and mcr-1-mediated colistin-resistant Escherichia coli. The counts of tigecycline- and colistin-resistant bacteria decreased to 1% in vivo after the resensitization system was administered. This study opens up new pathways for the development of CRISPR-based tools for selective bacterial pathogen elimination and precise microbiome composition change.

ABSTRACTLong-term care facilities (LTCFs) for older people play an important and unique role in multidrug-resistant organism transmission. Herein, we investigated the genetic characteristics of mobile colistin resistance gene (mcr-1)-carrying Escherichia coli strains isolated from wastewater of LTCFs in Shanghai. Antimicrobial susceptibility test was carried out by agar dilution methods. Whole-genome sequencing and plasmid sequencing were conducted, and resistance genes and sequence types of colistin in E. coli isolates were analyzed. Core genome multilocus sequence typing (cgMLST) analysis was performed by the Ridom SeqSphere+ software. Phylogenetic tree through the maximum likelihood method was constructed by MEGA X. Out of 306 isolates, only 1 E. coli named ECSJ33 was found, and the plasmid pECSJ33 from ECSJ33 harbored the mcr-1 gene that was located with 59,080 bp belonging to IncI2 type. The plasmid pECSJ33 was capable of conjugation with an efficiency of 2.9 × 10−2. Bioinformatic analysis indicated pECSJ33 shared backbone with the previously reported mcr-1-harboring pHNGDF93 isolated from fish source. Moreover, the cgMLST analysis revealed that ECSJ33 belongs to different lineages from those reported from previous E. coli strains but shared high similarity to NCTC11129 in cluster 11. The phylogenetic tree revealed MCR-1 of ECSJ33 in this study was mostly of animal food origin and that they were closely related. Our study firstly reports detection of genome sequence of a multidrug-resistant mcr-1-harboring E. coli ST155 from wastewater of LTCF source in China. The data may prove that the plasmid pECSJ33 belongs to food origin and help to understand the antimicrobial resistance mechanisms and genomic features of colistin resistance under One Health approach.IMPORTANCEOne Escherichia coli named ECSJ33 was found from wastewater of a long-term care facility (LTCF) and the plasmid pECSJ33 from ECSJ33 harbored the mobile colistin resistance gene (mcr-1) that was located with 59,080 bp belonging to IncI2 type, which was capable of conjugation with an efficiency of 2.9 × 10-2. This paper firstly reports an mcr-1-carrying E. coli strain ST155 isolated from LTCF in China. Comparative genomics analysis indicated pECSJ33 shared backbone with the previously reported mcr-1-harboring pHNGDF93 isolated from fish source. The phylogenetic tree revealed MCR-1 protein of ECSJ33 in this study was mostly of animal food origin and that they were closely related. Therefore, the pECSJ33 could be considered as food-origin transmission mcr-1-harboring plasmid.

Polymyxin antibiotics are the last resort for treating patients in intensive care units infected with multiple-resistant Gram-negative bacteria. Due to their polycationic structure, their mode of action is based on an ionic interaction with the negatively charged lipid A portion of the lipopolysaccharide (LPS). The most prevalent polymyxin resistance mechanisms involve covalent modifications of lipid A: addition of the cationic sugar 4-amino-L-arabinose (L-Ara4N) and/or phosphoethanolamine (pEtN). The modified structure of lipid A has a lower net negative charge, leading to the repulsion of polymyxins and bacterial resistance to membrane disruption. Genes encoding the enzymatic systems involved in these modifications can be transferred either through chromosomes or mobile genetic elements. Therefore, new approaches to resistance diagnostics have been developed. On another note, interfering with these enzymatic systems might offer new therapeutic targets for drug discovery. This literature review focuses on diagnostic approaches based on structural changes in lipid A and on the therapeutic potential of molecules interfering with these changes.

ABSTRACT: Objectives: The emergence of the plasmid-mediated colistin resistance 1 (mcr-1) of Escherichia coli has become a global health concern. This study reports the prevalence of mcr-1 among E. coli isolates from patients with diarrheal disease in Shanghai and the genetic characterization of mcr-1-harbouring plasmids. Methods: A total of 1723 E. coli strains were collected from the faeces of patients with diarrheal disease in all sentinel hospitals in Shanghai from 2016 to 2021. Antimicrobial susceptibility testing was performed by broth microdilution and plasmid conjunction transfer assay was carried out using E. coli C600 as the recipient. The mcr-1-positive E. coli strains (MCRPEC) were subjected to molecular characterization and bioinformatic analysis of the mcr-1-bearing plasmids that they harboured. Results: Only 5 (0.28%) strains were found to harbour the mcr-1 gene using PCR screening. Plasmid conjugation assay and whole-genome sequencing indicated that EC16500, one MCRPEC strain that co-exhibited mcr-1, blaTEM-1, blaOXA-1, qnrS1, qnrS2, arr-3, and catB3, could be conjugated to EC C600 by horizontal transfer with an average efficiency of 3.2 × 10−5. The plasmid pEC16500 harboured similar backbones as p70_2_15, pECGD-8–33, pNCYU-29–19–1_MCR1, and pIBMC_mcr1, and was shown to be encoded within a type IV secretion system (T4SS)-containing 32.6 kbp IncX4, next to the pap2-like membrane-associated gene, to form a 2.4-kb cassette. Furthermore, sequencing and phylogenetic analyses revealed a similarity between other MCR-1-homolog proteins, indicating that the five E. coli isolates were colistin-resistant. Conclusion: Our data represents a significant snapshot of colistin resistance mcr-1 genes and highlights the need to increase active surveillance, especially among children under five years of age, in Shanghai. Great effort needs to be taken to avoid further dissemination of plasmid-mediated colistin resistance among clinically relevant Gram-negative bacterial pathogens.

Antimicrobial resistance (AMR) is a significant global health threat, with multidrug-resistant (MDR) bacterial clones becoming a major concern. Polymyxins, especially colistin, have reemerged as last-resort treatments for MDR Gram-negative infections. However, colistin use in livestock has spread mobile colistin resistance (mcr) genes, notably mcr-1, impacting human health. In consequence, its livestock use was banned in 2017, originating a natural experiment to study bacterial adaptation. The aim of this work was to analyse the changes in the mcr-1 genetic background after colistin restriction across the world. This study analyses 3163 Escherichia coli genomes with the mcr-1 gene from human and livestock hosts, mainly from Asia (n = 2621) and Europe (n = 359). Genetic characterisation identifies IncI2 (40.4%), IncX4 (26.7%), and multidrug-resistant IncHI2 (18.8%) as the most common plasmids carrying mcr-1. There were differences in plasmids between continents, with IncX4 (56.6%) being the most common in Europe, while IncI2 (44.8%) was predominant in Asia. Promoter variants related to reduced fitness costs and ISApl1 showed a distinct pattern of association that appears to be associated with adaptation to colistin restriction, which differed between continents. Thus, after the colistin ban, Europe saw a shift to specialised mcr-1 plasmids as IncX4, while ISApl1 decreased in Asia due to changes in the prevalence of the distinct promoter variants. These analyses illustrate the evolution of mcr-1 adaptation following colistin use restrictions and the need for region-specific strategies against AMR following colistin restrictions.

Background: The increase of multidrug-resistant Enterobacteriaceae bacteria has led to the reintroduction of colistin for clinical treatments, and colistin has become a last resort for infections caused by multidrug-resistant bacteria. Enterobacteriaceae bacteria carrying the mcr-1 gene are majorly related to colistin resistance, which may be the main reason for the continued increase in the colistin resistance rate of Enterobacteriaceae. The study aimed to investigate the sequence type and prevalence of Escherichia coli (E. coli) harboring the mcr-1 gene in the gut flora of children in southern China. Methods: Fecal samples (n = 2632) of children from three medical centers in Guangzhou were cultured for E. coli. The mcr-1-harboring isolates were screened via polymerase chain reaction (PCR). The colistin resistance transfer frequency was studied by conjugation experiments. DNA sequencing data of seven housekeeping genes were used for multi-locus sequence typing analysis (MLST). Results: PCR indicated that 21 of the 2632 E. coli (0.80%) isolates were positive for mcr-1; these strains were resistant to colistin. Conjugation experiments indicated that 18 mcr-1-harboring isolates could transfer colistin resistance phenotypes to E. coli J53. MLST analysis revealed that the 21 isolates were divided into 18 sequence types (STs); E. coli ST69 was the most common (14.3%), followed by E. coli ST58 (9.5%). Conclusion: These results demonstrate the colonization dynamics and molecular epidemiology of E. coli harboring mcr-1 in the gut flora of children in southern China. The mcr-1 gene can be horizontally transmitted within species; hence, it is necessary to monitor bacteria that harbor mcr-1 in children.

Abstract Background Antimicrobial resistance (AMR) is a global health problem largely due to the overuse of antimicrobials. In recognition of this, the World Health Assembly in 2015 agreed on a global action plan to tackle AMR. Following the global emergence of the mcr‐1‐associated colistin resistance gene in the livestock industry in 2016, several countries including South Africa restricted the veterinary use of colistin as the gene threatens the clinical utility of the drug. This study is a follow‐up to the restriction in place in order to evaluate the impact of such policy adoption. Objective To assess the prevalence of antibacterial resistance (ABR), and the mcr‐1 colistin resistance gene in broiler chicken over a 2‐year period, as a follow‐up to the veterinary ban on colistin use in South Africa. Methods A total of 520 swab samples were obtained during 2019 (March–April) and 2020 (February–March), from healthy broiler chicken carcasses (n = 20) and chicken droppings in transport crates (n = 20) at various poultry abattoirs (N = 7) in the Gauteng province of South Africa. Escherichia coli organisms were isolated and subjected to a panel of 24 antibacterials using the MicroScan machine. Screening for mcr‐1 colistin resistance gene was undertaken using PCR. Result Four hundred and thirty‐eight (438) E. coli strains were recovered and none demonstrated phenotypic resistance towards colistin, amikacin, carbapenems, tigecycline and piperacillin/tazobactam. The mcr‐1 gene was not detected in any of the isolates tested. Resistances to the aminoglycosides (0%–9.8%) and fluoroquinolones (0%–18.9%) were generally low. Resistances to ampicillin (32%–39.3%) and trimethoprim/sulphamethoxazole (30.6%–3.6%) were fairly high. A significant (p

ABSTRACT The treatment of bacterial infections has become more challenging due to the increasing prevalence of colistin-resistant (Col-R) pathogens. Shikonin is a Chinese herbal medicine that has been proven to have antibacterial properties. In recent years, many studies have shown that the combination of traditional antibiotics and non-antibacterial drugs can effectively treat infections caused by multidrug-resistant bacteria. This study evaluated the potential of shikonin in combination with colistin to inhibit colistin-resistant Escherichia coli (Col-R E. coli), and explored potential interaction. In checkerboard analysis, shikonin could significantly reduce the minimum inhibitory concentration (MIC) of colistin by two to eight times, enhancing the susceptibility of Col-R E. coli to colistin. Shikonin also increased the bactericidal effect of colistin against Col-R E. coli in 24 h time-kill assays, resulting in a >3 log-fold decrease in colony-forming units. In vivo tests showed that the combination of colistin and shikonin could significantly improve the survival rate of Galleria mellonella. In addition, confocal laser scanning microscopy and quantitative real-time polymerase chain reaction showed that colistin/shikonin could inhibit the formation of biofilms, and shikonin could down-regulate the transcription level of biofilm-regulated genes (csgA, csgD, flhC, flhD, fliC, fliM, lsrK, and lsrR). The synergistic mechanism of colistin and shikonin was further revealed by detecting membrane integrity, reactive oxygen species production, and mcr-1 gene expression. Our findings indicate that the colistin/shikonin combination may be a promising alternative approach to treat Col-R E. coli infections. IMPORTANCE Infections caused by multidrug-resistant Escherichia coli (MDR E. coli) have become a major global healthcare problem due to the lack of effective antibiotics today. The emergence of colistin-resistant E. coli strains makes the situation even worse. Therefore, new antimicrobial strategies are urgently needed to combat colistin-resistant E. coli. Combining traditional antibiotics with non-antibacterial drugs has proved to be an effective approach of combating MDR bacteria. This study investigated the combination of colistin and shikonin, a Chinese herbal medicine, against colistin-resistant E. coli. This combination showed good synergistic antibacterial both in vivo and in vitro experiments. Under the background of daily increasing colistin resistance in E. coli, this research points to an effective antimicrobial strategy of using colistin and shikonin in combination against colistin-resistant E. coli.

The emergence of colistin resistance in Escherichia coli is a global public health concern. Contaminated food can accelerate the spread of colistin-resistant E. coli to humans. This study aimed to detect and characterize colistin-resistant E. coli from broiler meat in Bangladesh. We analyzed 136 pooled broiler meat samples from 240 carcasses collected from 40 live bird markets in urban and rural areas and 8 metropolitan supermarkets. The mean count of E. coli in broiler meat samples collected from rural retail shops, metropolitan supermarkets, and urban retail shops was 5.3 ± 1.1, 4.1 ± 1.4, and 3.9 ± 0.8 log10 colony-forming unit per gram, respectively. Colistin-resistant E. coli (minimum inhibitory concentration >2 mg/L) was found in 78% (95% confidence interval 70.2–84.1%) of the samples. All colistin-resistant isolates harbored the mcr-1 gene, while the rest of the mcr genes (mcr-2 to mcr-9) were not detected. Most colistin-resistant E. coli isolates (98%) showed coresistance to tetracycline, sulfamethoxazole/trimethoprim followed by ciprofloxacin (95%). Alarmingly, all of the colistin-resistant isolates were found to be multidrug resistant. Phylogenetic analysis showed close similarities of the mcr-1 gene sequences of this study with many strains of Enterobacterales isolated from humans, animals, and the environment. This study detected colistin-resistant E. coli contamination in broiler meat, which can pose a serious public health threat. [ABSTRACT FROM AUTHOR]

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Owing to the emergence of antibiotic resistance, the polymyxin colistin has been recently revived to treat acute, multidrug-resistant Gram-negative bacterial infections. Positively charged colistin binds to negatively charged lipids and damages the outer membrane of Gram-negative bacteria. However, the MCR-1 protein, encoded by the mobile colistin resistance (mcr) gene, is involved in bacterial colistin resistance by catalysing phosphoethanolamine (PEA) transfer onto lipid A, neutralising its negative charge, and thereby reducing its interaction with colistin. Our preliminary results showed that treatment with a reference pyrazolone compound significantly reduced colistin minimal inhibitory concentrations in Escherichia coli expressing mcr-1 mediated colistin resistance (Hanpaibool et al. in ACS Omega, 2023). A docking-MD combination was used in an ensemble-based docking approach to identify further pyrazolone compounds as candidate MCR-1 inhibitors. Docking simulations revealed that 13/28 of the pyrazolone compounds tested are predicted to have lower binding free energies than the reference compound. Four of these were chosen for in vitro testing, with the results demonstrating that all the compounds tested could lower colistin MICs in an E. coli strain carrying the mcr-1 gene. Docking of pyrazolones into the MCR-1 active site reveals residues that are implicated in ligand–protein interactions, particularly E246, T285, H395, H466, and H478, which are located in the MCR-1 active site and which participate in interactions with MCR-1 in ≥ 8/10 of the lowest energy complexes. This study establishes pyrazolone-induced colistin susceptibility in E. coli carrying the mcr-1 gene, providing a method for the development of novel treatments against colistin-resistant bacteria. [ABSTRACT FROM AUTHOR]

The emergence and rapid spread of the plasmid-mediated colistin-resistant mcr-1 gene introduced a serious threat to public health. In 2021, a multi-drug resistant, mcr-1 positive Escherichia coli EC1945 strain, was isolated from pig caecal content in Croatia. Antimicrobial susceptibility testing and whole genome sequencing were performed. Bioinformatics tools were used to determine the presence of resistance genes, plasmid Inc groups, serotype, sequence type, virulence factors, and plasmid reconstruction. The isolated strain showed phenotypic and genotypic resistance to nine antimicrobial classes. It was resistant to colistin, gentamicin, ampicillin, cefepime, cefotaxime, ceftazidime, sulfamethoxazole, chloramphenicol, nalidixic acid, and ciprofloxacin. Antimicrobial resistance genes included mcr-1, blaTEM-1B, blaCTX-M-1, aac(3)-IId, aph(3')-Ia, aadA5, sul2, catA1, gyrA (S83L, D87N), and parC (A56T, S80I). The mcr-1 gene was located within the conjugative IncX4 plasmid. IncI1, IncFIB, and IncFII plasmids were also detected. The isolate also harbored 14 virulence genes and was classified as ST744 and O101:H10. ST744 is a member of the ST10 group which includes commensal, extraintestinal pathogenic E. coli isolates that play a crucial role as a reservoir of genes. Further efforts are needed to identify mcr-1-carrying E. coli isolates in Croatia, especially in food-producing animals to identify such gene reservoirs. [ABSTRACT FROM AUTHOR]

The emergence and dissemination of plasmid-mediated colistin resistance pose a serious threat to global public health. In this study, a total of 454 commensal Escherichia coli strains isolated from cloacal swabs taken from broiler flocks between 2013 and 2014 were first screened for phenotypic colistin resistance by the broth microdilution method. Twelve phenotypically colistin-resistant isolates were screened for mcr-1 to mcr-5 genes by multiplex PCR. Five of these isolates were found to carry the mcr-1 by PCR. Whole-genome sequencing of the mcr-1 carrying isolates revealed the presence of additional antimicrobial resistance genes and chromosomal gyrA and parC mutations conferring resistance to fluoroquinolones. Analysis of virulence gene content revealed 5-20 virulence genes in the isolates and two isolates were defined as avian enteropathogenic E. coli (APEC). Phylogenetic analysis based on whole-genome and multilocus sequence typing revealed that the strains were closely related to mcr-1 carrying isolates from chicken and human clinical isolates previously reported from different parts of the World. The findings further emphasize the urgent need to implement effective control programs to prevent the emergence and dissemination of colistin-resistant bacteria. [ABSTRACT FROM AUTHOR]