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Escherichia coli and Klebsiella pneumonia; along with other Gram-negative bacteria (GNB), are known for their resilience to a broad spectrum of antibiotics, including last-resort options such as colistin, which is regarded as the last line of defense in treating multidrugresistant (MDR) infections. This elevated level of resistance presents a substantial threat to the healthcare system, paving the way for a post-antibiotic era. In the present study, a checkerboard assay was employed to pinpoint the optimal combinations of Origanum majorana (OM) essential oil and colistin (CS) for the purpose of enhancing colistin's antibacterial and antibiofilm activities (FICI ≤ 0.5). The investigation focused on eight colistin-resistant (CS_R) bacterial isolates, with a particular focus on two strains carrying the mobile colistin resistance mcr-1 gene. The CS@OM combination resulted in a 2- to 16-fold improvement of colistin efficacy, resulting in a biofilm eradication rate ranging from ∼ 52 % to 82 %. Furthermore, it evinced a complete eradication of the free-floating cells of the isolates, inclusive of those harboring the mcr-1 gene. The bactericidal activity of the CS@OM combination was achieved through various mechanisms, including bacterial membrane disruption and leakage of internal bacterial constituents, leading to bacterial death. Consequently, the CS@OM combination evinced a high capacity to prevent the development of drug resistance in the examined isolates. To the best of our knowledge, this is the first study to appraise the combination of colistin and Origanum majorana essential oil against colistinresistant isolates, including those carrying mcr-1 gene with the aim of regaining colistin susceptibility.

1Laboratorio de Investigaciones en Mecanismos de Resistencia a Antibio´ ticos, Instituto de Investigaciones en Microbiologı ´a y Parasitologı ´a Me´ dica, Facultad de Medicina, Universidad de Buenos Aires - Consejo Nacional de Investigaciones Cientı ´ficas y Tecnolo´ gicas (IMPaM, UBACONICET), Buenos Aires, Argentina, [ABSTRACT FROM AUTHOR]

ObjectiveTo provide technical support for the molecular surveillance of pathogenic bacteria strains carrying mobile colistin resistance-1 （mcr⁃1） gene isolate from inlet of wastewater treatment plants （WWTP）.MethodsThe Enterobacteriaceae strains carrying mcr⁃1 resistance gene isolate from inlet of WWTP during April 1 to June 30， 2023 in Shanghai were cultured on blood-rich and SS culture medium and were identified using a mass spectrometry analyzer. The mcr⁃1 gene and copy number were detected by real-time fluorescence quantitative PCR. Drug susceptibility test was performed by microbroth dilution method. The copy numbers of Escherichia coli carrying mcr⁃1 gene isolated from wastewater and human fecel were statistically analyzed by SPSS 25.0.ResultsA total of 14 strains carrying the mcr⁃1 gene were isolated from 49 WWTP samples， and the positive isolation rate was 28.6%， including 12 non-diarrheal E. coli strains and 2 Klebsiella pneumoniae strains. The drug susceptibility results showed that all 14 strains were multi-drug resistant bacteria. They were all sensitive to imipenem and tigecycline， but were ampicillin- and cefazolin-resistant. There was no significant difference in the copy number between human-sourced diarrheal E. coli and wastewater-sourced non-diarrheal E. coli （t=0.647， P>0.05）.ConclusionThe isolation and identification of strains carrying the mcr⁃1 gene from inlet of WWTP samples were firstly established in Shanghai. The multi-drug resistance among the isolated strains is severe. To effectively prevent and control the spread of colistin-resistant bacteria， more attention should be paid to the surveillance of mcr⁃1 gene.

Introduction: Hospitals are breeding grounds for multidrug-resistant (MDR) bacteria, posing treatment challenges and increasing the risk of spreading "superbugs." This study investigates the prevalence of colistinresistant bacteria, a last-resort antibiotic, in wastewater from tertiary hospitals in Enugu, Nigeria. Methods: Twenty wastewater samples were collected over three months from two tertiary hospitals in Enugu. A standardized protocol by the American Public Health Association (APHA) was followed. Samples were collected aseptically from key drainage points and transported to the lab within 2 hours. Bacteria were isolated using the pour-plate method and characterized by morphological and biochemical tests, including Catalase, Oxidase, and Glucose Fermentation. Antibiotic susceptibility was assessed using Kirby-Bauer disc diffusion, and colistin resistance was confirmed via broth microdilution. Multiplex PCR detected mcr genes indicating plasmid-mediated resistance. Data were analyzed using SPSS version 23 with Chi-Square and ANOVA tests at a significance level of P < 0.05. Results: Gram-negative bacteria were isolated from 63.1% of samples, with Klebsiella spp. being the most prevalent, accounting for 24.6%. Colistin resistance was phenotypically observed in E. coli (83%), Klebsiella spp. (75%), and Pseudomonas aeruginosa (100%). Genotypically, E. coli harbored mcr-1 (17%) and mcr-3 (83%), while all Klebsiella and Pseudomonas isolates carried multiple mcr genes. Additionally, these bacteria showed resistance to multiple antibiotics, including Septrin, Gentamycin, and Ceftriaxone. Conclusion: The significant presence of colistin-resistant bacteria, especially E. coli and Klebsiella, poses a public health concern, potentially leading to treatment failures and spreading resistance genes. Stricter monitoring of hospital wastewater is necessary to identify emerging resistance trends and improve antibiotic practices in hospitals. [ABSTRACT FROM AUTHOR]

Addressing the emergence of antimicrobial resistance (AMR) poses a significant challenge in veterinary and public health. In this study, we focused on determining the presence, phenotypic background, and genetic epidemiology of plasmid-mediated colistin resistance (mcr) in Escherichia coli bacteria isolated from camels farmed in the United Arab Emirates (UAE). Fecal samples were collected from 50 camels at a Dubai-based farm in the UAE and colistin-resistant Gram-negative bacilli were isolated using selective culture. Subsequently, a multiplex PCR targeting a range of mcr-genes, plasmid profiling, and whole-genome sequencing (WGS) were conducted. Eleven of fifty camel fecal samples (22%) yielded colonies positive for E. coli isolates carrying the mcr-1 gene on mobile genetic elements. No other mcr-gene variants and no chromosomally located colistin resistance genes were detected. Following plasmid profiling and WGS, nine E. coli isolates from eight camels were selected for in-depth analysis. E. coli sequence types (STs) identified included ST7, ST21, ST24, ST399, ST649, ST999, and STdaa2. Seven IncI2(delta) and two IncX4 plasmids were found to be associated with mcr-1 carriage in these isolates. These findings represent the first identification of mcr-1-carrying plasmids associated with camels in the Gulf region. The presence of mcr-1 in camels from this region was previously unreported and serves as a novel finding in the field of AMR surveillance. [ABSTRACT FROM AUTHOR]

There is scarce information concerning the role of sporadic clones in the dissemination of antimicrobial resistance genes (ARGs) within the nosocomial niche. We confirmed that the clinical Escherichia coli M19736 ST615 strain, one of the first isolates of Latin America that harbors a plasmid with an mcr-1 gene, could receive crucial ARG by transformation and conjugation using as donors critical plasmids that harbor blaCTX-M-15, blaKPC-2, blaNDM-5, blaNDM-1, or aadB genes. Escherichia coli M19736 acquired blaCTX-M-15, blaKPC-2, blaNDM-5, blaNDM-1, and aadB genes, being only blaNDM-1 maintained at 100% on the 10th day of subculture. In addition, when the evolved MDR-E. coli M19736 acquired sequentially blaCTX-M-15 and blaNDM-1 genes, the maintenance pattern of the plasmids changed. In addition, when the evolved XDR-E. coli M19736 acquired in an ulterior step the paadB plasmid, a different pattern of the plasmid’s maintenance was found. Interestingly, the evolved E. coli M19736 strains disseminated simultaneously the acquired conjugative plasmids in different combinations though selection was ceftazidime in all cases. Finally, we isolated and characterized the extracellular vesicles (EVs) from the native and evolved XDR-E. coli M19736 strains. Interestingly, EVs from the evolved XDR-E. coli M19736 harbored blaCTX-M-15 though the pDCAG1-CTX-M-15 was previously lost as shown by WGS and experiments, suggesting that EV could be a relevant reservoir of ARG for susceptible bacteria. These results evidenced the genetic plasticity of a sporadic clone of E. coli such as ST615 that could play a relevant transitional link in the clinical dynamics and evolution to multidrug/extensively/pandrug-resistant phenotypes of superbugs within the nosocomial niche by acting simultaneously as a vector and reservoir of multiple ARGs which later could be disseminated.

Addressing the emergence of antimicrobial resistance (AMR) poses a significant challenge in veterinary and public health. In this study, we focused on determining the presence, phenotypic background, and genetic epidemiology of plasmid-mediated colistin resistance (mcr) in Escherichia coli bacteria isolated from camels farmed in the United Arab Emirates (UAE). Fecal samples were collected from 50 camels at a Dubai-based farm in the UAE and colistin-resistant Gram-negative bacilli were isolated using selective culture. Subsequently, a multiplex PCR targeting a range of mcr-genes, plasmid profiling, and whole-genome sequencing (WGS) were conducted. Eleven of fifty camel fecal samples (22%) yielded colonies positive for E. coli isolates carrying the mcr-1 gene on mobile genetic elements. No other mcr-gene variants and no chromosomally located colistin resistance genes were detected. Following plasmid profiling and WGS, nine E. coli isolates from eight camels were selected for in-depth analysis. E. coli sequence types (STs) identified included ST7, ST21, ST24, ST399, ST649, ST999, and STdaa2. Seven IncI2(delta) and two IncX4 plasmids were found to be associated with mcr-1 carriage in these isolates. These findings represent the first identification of mcr-1-carrying plasmids associated with camels in the Gulf region. The presence of mcr-1 in camels from this region was previously unreported and serves as a novel finding in the field of AMR surveillance.

There is scarce information concerning the role of sporadic clones in the dissemination of antimicrobial resistance genes (ARGs) within the nosocomial niche. We confirmed that the clinical Escherichia coli M19736 ST615 strain, one of the first isolates of Latin America that harbors a plasmid with an mcr-1 gene, could receive crucial ARG by transformation and conjugation using as donors critical plasmids that harbor bla CTX-M-15 , bla KPC-2 , bla NDM-5 , bla NDM-1 , or aadB genes. Escherichia coli M19736 acquired bla CTX-M-15 , bla KPC-2 , bla NDM-5 , bla NDM-1 , and aadB genes, being only blaNDM-1 maintained at 100% on the 10th day of subculture. In addition, when the evolved MDR- E. coli M19736 acquired sequentially bla CTX-M-15 and bla NDM-1 genes, the maintenance pattern of the plasmids changed. In addition, when the evolved XDR- E. coli M19736 acquired in an ulterior step the paadB plasmid, a different pattern of the plasmid's maintenance was found. Interestingly, the evolved E. coli M19736 strains disseminated simultaneously the acquired conjugative plasmids in different combinations though selection was ceftazidime in all cases. Finally, we isolated and characterized the extracellular vesicles (EVs) from the native and evolved XDR- E. coli M19736 strains. Interestingly, EVs from the evolved XDR- E. coli M19736 harbored bla CTX-M-15 though the pDCAG1-CTX-M-15 was previously lost as shown by WGS and experiments, suggesting that EV could be a relevant reservoir of ARG for susceptible bacteria. These results evidenced the genetic plasticity of a sporadic clone of E. coli such as ST615 that could play a relevant transitional link in the clinical dynamics and evolution to multidrug/extensively/pandrug-resistant phenotypes of superbugs within the nosocomial niche by acting simultaneously as a vector and reservoir of multiple ARGs which later could be disseminated., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Carrera Páez, Olivier, Gambino, Poklepovich, Aguilar, Quiroga and Centrón.)