Salmonella enterica subps. enterica serovar Heidelberg (SH) is one of the most common serovars isolated from poultry and associated with severe infections in humans. Commonly considered multidrug-resistant, it represents a risk to public health. We analyzed 317 SH genomes, including 314 from the Enterobase database from Brazil and the United States (US), and added three recently sequenced Brazilian isolates. In genomes from both countries, the main identified resistance genes were: aac(6')-Iaa, fosA7, sul2, tet(A), and blaCMY-2. Mutations in GyrA (S83Y only from US genomes and S83F and D87N from Brazilian genomes) were observed in 17 % and 90.62 % of genomes from US and Brazil, respectively, and ParC mutation (T57S), was identified in all genomes. The plasmid replicons most identified in both countries were ColpVC, IncC, IncI1-I(Gamma), and IncX1. The core and soft-core genes were utilized as the basis for conducting a phylogenetic analysis, showing seven clusters of strains, of which only one was shared between strains from the US and Brazil. Overall, this study highlights the variation in genomic profiles of SH circulating in poultry production in both countries, emphasizing the need for improved surveillance measures to protect human and animal populations from potential outbreaks worldwide.

To determine the molecular characteristics and mechanisms of colistin-resistant (COLr) Salmonella isolates from 1224 chicken samples in Shanghai, China, between January 2021 and January 2022.

Antimicrobial susceptibility testing, site-directed mutagenesis, RT-qPCR, and lipid A analysis was conducted to investigate the mechanisms of colistin resistance in Salmonella.

A total of 268 Salmonella isolates were obtained from chicken samples and 41 isolates were resistant to colistin. The uncommon extensively antimicrobial-resistant Salmonella Muenster was the predominant serotype, accounting for 87.8% (36/41) of the COLr isolates. Most (95.1%, 39/41) of the COLr isolates exhibited amino acid substitutions in the PmrAB. Ten different substitutions in PmrAB were identified, with Val161Gly (n = 14) and Gly206Glu (n = 10) in PmrB being the most prevalent. Four substitutions (Thr147Ser, Phe203Ser, Gly206Glu, and Asp250Tyr) in PmrB have not been reported before and were considered novel mutations. Additionally, it was demonstrated that these substitutions upregulated pmrE and pmrK expression, resulting in the production of L-Ara4N, which modified the lipid A and resulted in an 8-64-fold increase in the colistin MIC (2-8 mg/L). Finally, the deletion of pmrE or pmrK in mutants showed MIC values comparable to parental strains (0.25 mg/L), which suggested that colistin resistance may be conferred through the pmrE and pmrK pathways.

These findings illustrate the complex molecular mechanisms of colistin resistance in Salmonella, and the amino acid substitutions in PmrAB are the predominant molecular mechanisms. It is essential to implement monitoring and control measures for colistin resistance.

Human activity is accelerating the emergence of fungal pathogens, prompting substantial efforts to discover novel fungicides. Meanwhile, the runoff and spray drift from agricultural fields adversely affect aquatic and terrestrial nontarget organisms. However, few studies have examined the effects of co-contamination by agrochemical fungicides and pharmaceutical antibiotics on microorganisms and antibiotic resistance genes (ARGs) in the soil-animal-plant system. To further explore the mechanisms, an investigation was conducted into the individual and combined effects of a widely used fungicide (cyazofamid, CZF) and a last-resort antibiotic (colistin, polymyxin E, PME) in the soil-earthworm-tomato system. This study revealed that CZF and PME co-contamination exerted synergistic toxicity, significantly reducing earthworm survival and inhibiting tomato growth. This study found that the structure of microbial communities was more severely disturbed by the fungicide CZF than by the antibiotic PME, with the most severe impact being that of CZF + PME co-contamination. Fungicides and antibiotics had significantly distinct effects on bacterial functional pathways: CZF and CZF + PME treatments enhanced compound degradation, whereas PME treatments promoted biological nitrogen cycling. Moreover, co-contamination significantly increased the abundance of insertional and plasmid-associated genes and number of total ARGs in bulk and rhizosphere soil. In addition, the relationships between bacterial communities and the antibiotic resistome were investigated. The analysis revealed that Gram-positive bacteria (Sporosarcina, Bacillus, and Rhodococcus) capable of resistance and degradation, as well as the genes MexB (multidrug) and aadA2 (aminoglycoside) were enriched. Taken together, interactions between co-pollutants can significantly increase toxicity levels and the risk of ARG proliferation. The findings provide new insights into the potential impacts of co-contamination in complex real-life environments, such as soil-animal-plant systems.

This study aimed to analyze Escherichia coli from marketed meat samples in Peru. Sixty-six E. coli isolates were recovered from 21 meat samples (14 chicken, 7 beef), and antimicrobial resistance levels and the presence of mechanisms of antibiotic resistance, as well as clonal relationships and phylogeny of colistin-resistant isolates, were established. High levels of antimicrobial resistance were detected, with 93.9% of isolates being multi-drug resistant (MDR) and 76.2% of samples possessing colistin-resistant E. coli; of these, 6 samples from 6 chicken samples presenting mcr-1-producer E. coli. Colistin-resistant isolates were classified into 22 clonal groups, while phylogroup A (15 isolates) was the most common. Extended-spectrum β-lactamase- and pAmpC-producing E. coli were found in 18 and 8 samples respectively, with blaCTX-M-55 (28 isolates; 16 samples) and blaCIT (8 isolates; 7 samples) being the most common of each type. Additionally, blaCTX-M-15, blaCTX-M-65, blaSHV-27, blaOXA-5/10-like, blaDHA, blaEBC and narrow-spectrum blaTEM were detected. In addition, 5 blaCTX-M remained unidentified, and no sought ESBL-encoding gene was detected in other 6 ESBL-producer isolates. The tetA, tetE and tetX genes were found in tigecycline-resistant isolates. This study highlights the presence of MDR E. coli in Peruvian food-chain. The high relevance of CTX-M-55, the dissemination through the food-chain of pAmpC, as well as the high frequency of unrelated colistin-resistant isolates is reported.

Colistin, a last-resort antimicrobial, is often used in combination with other drugs to treat multidrug-resistant infections, but its nephrotoxic potential raises concerns, especially in combination therapies.

A retrospective analysis was performed on 29,163,222 reports from the USFDA Adverse Event Reporting System (AERS), identifying 125 reports meeting inclusion criteria for acute renal failure (ARF) associated with colistin-antimicrobial combinations. Disproportionality analysis using frequentists and Bayesian techniques were employed to evaluate ARF risk. Drug interaction and clinical outcomes were assessed using the Interaction-Adjusted SignalScores (INTSS) and mortality data, respectively.

The results showed significantly higher ARF risk for colistin combinations with ceftazidime, vancomycin, meropenem, and tigecycline, with particularly strong signals observed for colistin-ceftazidime combinations, corroborated by INTSS. Mortality rates were paradoxically higher in patients receiving colistin without nephrotoxic antimicrobials. Comprehensive signal detection metrics highlighted a consistent ARF risk for several other antimicrobial combinations. Clinical outcomes analysis revealed a complex relationship between combination therapy choices and patient mortality.

Colistin combinations with certain antimicrobials, especially ceftazidime, vancomycin, and tigecycline, carry significantly higher nephrotoxicity risks, warranting careful clinical consideration. Rigorous renal monitoring protocols are essential in managing these therapies, particularly in critically ill patients.

Antibiotic resistance poses a formidable challenge, especially with the emergence of multidrug-resistant Gram-negative bacteria. Colistin serves as a last-resort antibiotic to combat multidrug-resistance, but it is limited by its nephrotoxicity and rising resistance. This study introduces menadione, a synthetic form of vitamin K, as a potential adjuvant to enhance colistin's efficacy against both susceptible and resistant strains of Gram-negative bacteria.

Through checkerboard dilution assays, we demonstrate that menadione significantly lowers the MICs of colistin, with fractional inhibitory concentration indices ranging from 0.031 to 0.375. Furthermore, synergistic effects were confirmed via time-kill kinetics, indicating effective bacterial growth inhibition. The study also explores the mechanism underlying this synergy, revealing that menadione in combination with colistin disrupts the bacterial outer membrane, reduces the proton motive force and adenosine triphosphate content, and amplify the production of reactive oxygen species, contributing to bacterial cell death.

Menadione was shown to prevent the evolution of colistin resistance.

This research highlights the potential of using menadione as a colistin adjuvant to combat antibiotic-resistant Gram-negative bacteria, providing a promising approach to extend the utility of existing antibiotics in clinical settings.

Bacteria are becoming progressively more resistant to available antimicrobials. The increased ease and availability of genome sequencing has made it possible to identify putative, novel antimicrobial resistance (AMR) genes bioinformatically. However, no standardized system is available to phenotypically characterize the ability of novel AMR genes in Enterobacteriaceae to confer resistance and impact bacterial physiology and pathogenicity in relation to expression levels. We previously used plasmid pBAD24, which allows for arabinose-inducible expression of heterologous genes, and Escherichia coli Top10 to characterize mobile colistin resistance genes. Based on the pBAD24 backbone, we constructed a new plasmid (pBAD25) that carries a kanamycin resistance gene (instead of an ampicillin resistance gene). We show that our expression system allows for the characterization of five different blaOXA genes, which differ in their ability to confer susceptibility to β-lactams, detected protein levels, and impact on bacterial growth. We characterized blaOXA-48b, a close relative of blaOXA-48, previously uncharacterized in E. coli, to be phenotypically similar to blaOXA-48, and blaOXA-549, a previously uncharacterized gene of the blaOXA-548 family, as encoding a β-lactamase that is detected intra- but not extracellularly, has moderate growth defects, and decreases susceptibility to carbapenems and ampicillin. Additionally, we found that, in blaOXA expressing strains, (i) levels of intracellular proteins and bacterial growth negatively correlate and (ii) susceptibility to 2nd and 3rd generation cephalosporins and susceptibility to different carbapenems positively correlate. Our results demonstrate that the expression of AMR genes, specifically blaOXA genes, through pBAD25 allows for easy characterization of putative, novel AMR genes.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a global concern owing to its difficult treatment. This study aimed to determine the impact of colistin resistance on susceptibility to cefiderocol.

The colistin-susceptible clinical strain CRKP12-130 (colistin minimum inhibitory concentration [MIC] 0.5 mg/L) was cultured in medium containing 4× and 8× the MIC of colistin. Eight colistin-resistant derivatives were randomly selected for susceptibility testing of cefiderocol and zeta potential changes. To compare the impact of colistin resistance on bacterial uptake of iron, growth curve experiments were conducted in cation-adjusted Mueller-Hinton broth (CAMHB) and iron-depleted CAMHB (ID-CAMHB). Resistant strains and the original strain CRKP12-130 were subjected to next-generation sequencing.

Colistin MICs ranged from 16 to 128 mg/L for the eight colistin-resistant derivatives. The key genetic variants identified in colistin-resistant strains involved insertions and deletions in mgrB, and missense mutations in pmrB and phoQ. The colistin-resistant derivatives also exhibited reduced susceptibility to cefiderocol, with MICs increasing from 1 mg/L to 2-8 mg/L. Additionally, colistin-resistant strains demonstrated higher zeta potentials, ranging from -45.2 mV to levels between -32.8 mV and -14.2 mV. Resistant strains showed a more significant decrease in growth rate when cultivated in ID-CAMHB medium.

This study investigated the phenomenon of co-resistance to colistin and cefiderocol in CRKP under pressure of colistin. The simultaneous decrease in susceptibility poses a potential threat to the efficacy of clinical treatment of CRKP infections.

The emergence of multidrug-resistant pathogens, particularly β-lactam, colistin, and fosfomycin-resistant Escherichia coli and Salmonella, is a significant public health concern. This study evaluated the in vitro synergistic effects of antimicrobial combinations against these resistant isolates. Ten isolates that originated from retail chicken meat, including five E. coli and five Salmonella isolates, were tested against cefotaxime (CTA), fosfomycin (FOS), and colistin (COL), both individually and in combinations. Antimicrobial susceptibility was assessed using the broth microdilution method, and synergistic interactions were evaluated using checkerboard and time-killing assays. All isolates were multidrug-resistant (MDR) and were resistant to CTA, COL, and FOS. The checkerboard assay showed varying levels of synergy: two out of five E. coli isolates exhibited synergy with FOS-COL, while one E. coli isolates out of four isolates showed synergy with CTA-COL. No E. coli isolates showed synergy with FOS-CTA. For Salmonella, two out of five isolates exhibited synergy with both FOS-CTA and FOS-COL, while three out of four isolates showed synergy with CTA-COL. The time-killing assay confirmed these results, with the FOS-COL combinations showing synergy against both E. coli and Salmonella strains. Notably, the FOS-COL combination demonstrated bactericidal effects against E. coli, and all three combinations were bactericidal against Salmonella. The study highlights the potential of antimicrobial combinations, particularly FOS-COL, in combating MDR E. coli and Salmonella. These findings support the use of combination therapy as a promising strategy to in effectively treating multi-drug-resistant foodborne infections, ensuring better medical outcomes and enhanced food safety, warranting further investigation into their mechanisms and clinical applications.

Colistin is a last-resort antibiotic used to treat multidrug-resistant Gram-negative bacterial infections. The global emergence of colistin resistance has been attributed to plasmid-mediated mobile colistin resistance (mcr) genes. In Lebanon, bacteria carrying the mcr-1 gene have increasingly been identified in food animal sources. This study is aimed at detecting colistin-resistant Shiga toxigenic Escherichia coli O157:H7 in raw meat samples from local markets in the suburbs of Beirut and evaluating their antimicrobial resistance profiles. A total of 50 meat samples, including 25 minced beef and 25 burger samples, were collected and analyzed. Antimicrobial resistance patterns were determined using the Kirby-Bauer method, while colistin resistance and the presence of mcr-2 and mcr-3 genes were assessed using broth microdilution and PCR amplification techniques. Among these samples, 23 (46%) tested positive for E. coli O157:H7. Resistance to ampicillin and amoxicillin/clavulanic acid was observed in 96% of the samples, while 61% were resistant to trimethoprim/sulfamethoxazole, and 43% to chloramphenicol. Notably, 87% of the samples displayed colistin resistance, with a minimum inhibitory concentration (MIC) of ≥ 4 μg/mL. The mcr-2 gene was present in four isolates (17.4%), and the mcr-3 gene was identified in 10 isolates (43.4%). This study is the first to document the presence of plasmid-mediated colistin resistance genes, mcr-2 and mcr-3, in E. coli O157:H7 strains in Lebanon. These findings highlight a serious public health concern for the Lebanese community. Therefore, the responsible use of antibiotics across all healthcare sectors, combined with strict hygiene measures in food handling, is essential to control the spread of colistin-resistant genes.

This study aimed to investigate the epidemiology and dissemination of mcr-positive Enterobacteriaceae in urban sewage in Yangzhou, China.

A total of 366 sewage samples were collected from the Yangzhou Wastewater Treatment Plant in Jiangsu Province. Colistin-resistant Enterobacteriaceae was identified through PCR targeting mcr-1 to mcr-10 genes. The isolates underwent antimicrobial susceptibility testing, and whole-genome sequencing was performed to analyze their genomic features. Additionally, conjugation experiments were conducted to assess the transferability of mcr-positive plasmids.

Three mcr-positive Enterobacteriaceae isolates were identified, representing an isolation rate of 0.82%. These included one mcr-1-positive Escherichia coli (ST167) and two mcr-10-positive Klebsiella pneumoniae complex strains with novel sequence types ST6801 and ST6825. The mcr-1 gene was located on an IncI2 plasmid (pYZ22WS208_3) and successfully transferred to recipient strains. In contrast, the mcr-10 gene was carried on IncF plasmids (pYZ22WS067_1 and pYZ22WS223_1) but was not transferable in this study. Phylogenetic analysis revealed that the mcr-1-positive E. coli strain clustered within Clade II, alongside strains from various countries and sources. Phylogenomic analysis of mcr-10-positive isolates showed their sporadic distribution across 13 countries, with associations to diverse hosts and environments, indicating potential for widespread transmission.

This study demonstrates the presence of mcr-1 and mcr-10-positive Enterobacteriaceae in wastewater, emphasizing the importance of wastewater surveillance for tracking antibiotic resistance. The horizontal transfer of mcr-1 and potential spread of mcr-10 across various hosts underscore the need for ongoing monitoring and preventive measures.

Urinary tract infections caused by Gram-negative bacteria (GNB) are among the most common infections and a significant cause of sepsis. The increasing prevalence of multidrug-resistant (MDR) bacteria poses challenges to empirical treatment. Colistin may be used a last-resort antibiotic for treating MDR infections, but this requires the rapid determination of susceptibility to colistin. Traditional susceptibility testing methods can take up to 48 h, and there are specific challenges in determining colistin susceptibility. This study evaluates a novel, rapid method for determining colistin susceptibility directly from positive urine samples using the FASTcolistin MIC kit from FASTinov®. A total of 100 urine samples positive for Gram-negative bacilli when screened by the UF-1000i system were included in this study. After a simple sample prep, the same bacterial suspension was used for identification on MALDI-TOF and inoculated in the FASTcolistin MIC panel for our AST; after incubation at 37 °C for 1 h, it was analyzed via flow cytometry using a CytoFLEX cytometer (Beckman Coulter, Brea, CA, USA). The categorical susceptibility to colistin according to EUCAST or CLSI standards as well as the MIC values were given by bioFAST software (bioFAST 2.0). The essential agreement (EA) and bias were calculated. Different species of Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter spp. were correctly identified by MALDI-TOF directly from the FASTcolistin MIC sample prep. The essential agreement between the two methods was 99%, with a bias of -17%. Both identification and susceptibility were obtained in less than 2 h. This study presents a rapid and accurate method for determining colistin MIC directly from urine samples. The shortness of time required to produce a result, 2 h versus 48 h with the conventional methods, will significantly impact treatment decisions, especially in urinary tract infections difficult to treat.

Antibiotic resistance is frequently observed shortly after the clinical introduction of an antibiotic. Whether and how frequently that resistance occurred before the introduction is harder to determine, as isolates could not have been tested for resistance before an antibiotic was discovered. Historical collections, like the British National Collection of Type Cultures (NCTC), stretching back to 1885, provide a window into this history. Here we match 1,817 sequenced high-quality genomes from the NCTC collection to their respective year of isolation to study resistance genes before and concurrent with the age of antibiotics. Concordant with previous work, we find resistance genes in both pathogens and environmental samples before the age of antibiotics. While generally rare before the introduction of an antibiotic, we find an associated increase in frequency with antibiotic introduction. Finally, we observe a trend of resistance elements becoming both increasingly mobile and nested within multiple mobile elements as time goes on. More broadly, our findings suggest that likely-functional antibiotic resistance genes were circulating in clinically relevant isolates before the age of antibiotics, but human usage is associated with increasing both their overall prevalence and mobility.

Colistin is an antibiotic used as a last resort to treat multidrug-resistant Gram-negative bacterial infections. Plasmid-mediated mobile colistin-resistant (mcr) genes in Escherichia coli (E. coli) are disseminated globally and are considered to be a major public health threat. This study aimed to determine the molecular characteristics of colistin-resistant Escherichia coli isolates in clinical settings in Pakistan.

A total of 240 clinical E. coli strains isolated from urine and pus cultures were collected from two hospitals in Faisalabad and analyzed for phenotypic resistance to colistin by cultivation on CHROMagar plates supplemented with colistin 2 ug/ml. Molecular characteristics of colistin-resistant isolates were analyzed using conventional PCR, whole genome sequencing, and bioinformatics analysis.

PCR and whole genome analysis confirmed the presence of the mcr-1 gene in 10 E. coli isolates. The minimum inhibitory concentration for colistin ranged from 4 ug/ml to 32 ug/ml. ResFinder analysis revealed the presence of multiple resistance determinants conferring co-resistance to β-lactams, aminoglycosides, trimethoprim, sulfonamides, tetracycline, quinolones, florfenicol, and macrolides. Hybrid genomic assembly indicated that mcr-1 is carried on IncI2 plasmids. Plasmid replicon typing indicated that IncI2-type plasmids (n = 10) were the most prevalent plasmids in these strains, followed by IncFIB (n = 8), IncFIC (n = 7), IncFIA (n = 6), IncFII (4), IncQ1 (n = 3), IncI1 (n = 1), IncY (n = 1), and IncN (n = 1). The Achtman MLST typing scheme revealed a large diversity of STs among the mcr-1-positive E. coli. VirulenceFinder analysis revealed the presence of numerous virulence factors ranging from 4 to 19.

Our study revealed the emergence and dissemination of colistin-resistant E. coli isolates carrying mcr-1 in hospital settings, posing a potential risk to anti-infective therapy. More efforts should be taken to monitor the prevalence of mcr-1-carrying bacteria in Pakistan.

This study evaluated the phenotypic and genotypic traits of mcr-1.1-harboring Escherichia coli isolates from chickens, pigs, humans, and farm environments. The resistome and the mobile genetic elements associated with the spread of mcr-1.1 in Southern Brazil were also characterized.

The 22 mcr-1.1-harboring E. coli isolates from different origins were selected for antimicrobial susceptibility testing and whole genome sequencing for characterization of the resistome, plasmids, and sequence types. All isolates presented several resistance genes and harbored the mcr-1.1 gene in a highly similar IncX4 plasmid. Furthermore, the mcr-1.1 gene co-occurred with the mcr-3.12 gene in a multidrug-resistant isolate from the farm environment.

These findings demonstrate that the mcr-1.1 gene in E. coli isolates from Brazil is spreading mainly by horizontal transfer of the IncX4 plasmid. The co-occurrence of mcr-1.1 and mcr-3.12 highlights pig farming as an important reservoir of colistin resistance.

The emergence of colistin resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP) is a significant public health concern, as colistin has been the last resort for treating such infections. This study aimed to investigate the prevalence and molecular characteristics of colistin-resistant CRKP isolates in Central South China.

CRKP isolates from twelve hospitals in Central South China were screened for colistin resistance using broth microdilution. The epidemiological characteristics, virulome, resistome, plasmid replicons and two-component systems associated with colistin resistance of colistin-resistant isolates were explored by whole-genome sequencing. The mgrB gene and the relative expression of the pmrC and pmrK genes were analyzed by polymerase chain reaction (PCR) and real-time quantitative PCR, respectively. The bacterial virulence was evaluated through a Galleria mellonella larvae infection model.

Of the 429 nonduplicate CRKP isolates, 26 (6.1%) were colistin-resistant and they included eight clonal clusters. Six distinct sequence type (ST)-capsule loci (KL) types were identified: ST11-KL64, ST11-KL47, ST963-KL16, ST307-KL102, ST751-KL64 and ST5254-KL47. 88.5% (23/26) of them were found to carry at least one carbapenemase gene, including blaKPC-2 (65.4%, 17/26) and blaNDM-1 (7.7%, 2/26), as well as coharbouring blaKPC-2 and blaNDM-1 (15.4%, 4/26). Diverse mutations of colistin resistance-related genes were observed, with mgrB inactivation by insertions and the T157P deleterious mutation in pmrB being detected in 57.7% and 42.3% of the colistin-resistant isolates, respectively. In addition, a novel deleterious mutation, R248P, in the crrB gene was found in two ST11 isolates. 88.5% of the 26 isolates presented an increase in pmrK transcription, and 69.2% of them had an overexpression of the pmrC gene. All the 16 ST11-KL64 isolates and one ST751-KL64 isolate (65.4%, 17/26) carried at least two hypervirulence biomarkers and showed high virulence in vivo.

This study highlights the presence of different colistin resistance mechanisms in isolates belonging to the same clone and identified multiple clonal transmission clusters in colistin resistant isolates, including the globally high-risk ST11 and ST307 clones, of which a significant proportion exhibited high virulence. Consequently, it is crucial to enforce measures to prevent the ongoing spread of colistin resistance.

Colistin is a last-resort treatment for multidrug-resistant Gram-negative bacterial infections, particularly in critically ill patients. Nevertheless, it remains a major threat to public health. We assessed the proportion of colistin-resistant Gram-negative isolates from intensive care unit (ICU) infections in different years, areas, pathogens, and antimicrobial susceptibility tests (AST). We searched the studies in PubMed, Scopus, Embase, and Web of Science (until November 2021). Statistical analyses were conducted using STATA software (ver. 14.0). The overall rate of colistin resistance was 5.18% (95% CI 2.70%-8.22%). The proportion of colistin resistance was 4% (95% CI 2%-7%) before 2015 and 6% (95% CI 4%-9%) in 2015-2019. The rates of colistin resistance in Europe, America, Asia, and Africa were 8.24%, 3.78%, 3.60%, and 0%, respectively. The proportion of colistin-resistant non-fermenting Gram-negative bacilli isolated from the ICU was 2.25% (Acinetobacter baumannii [1.68%] and Pseudomonas aeruginosa [3.30%]). A 4-fold increase in colistin resistance was observed when comparing EUCAST and CLSI. We described the global epidemiology of colistin resistance over time and shown the distribution of colistin-resistant strains in different countries. Robust antimicrobial stewardship programs can increase the success of ICU physicians in improving patient outcomes.

Carbapenemase-producing Enterobacterales (CPE) have increased in the last decade. In low-income countries, colistin is considered a last resort antimicrobial to treat CPE infections, whose most worrisome mechanism of resistance is MCR-1 production. This study aims to understand the epidemiology of colistin resistance in CPE in the region, through the surveillance of the mcr-1 gene in CPE isolates in Ecuador. A total of 361 CPE isolates were collected across three periods, from 2016 to 2022. Colistin resistance was assessed using the broth microdilution method and the most frequent carbapenemase-encoding genes and the mcr-1 gene were studied. Colistin resistance rate increased from 3.76% to 23.74% during the study period. The mcr-1 gene was not identified in any of the isolates studied and Klebsiella pneumoniaeblaKPC was the most prevalent microorganism (n=322; 89.20%). In conclusion, colistin resistance increased in CPE in Ecuador and was not mediated by the mcr-1 gene. Our results highlight the need to closely monitor national politics on antimicrobial resistance under the One Health Approach.

Colistin resistance significantly constrains available treatment options and results in the emergence of pandrug-resistant (PDR) strains. Treating PDR infections is a major public health issue. A promising solution lies in using colistin-based combinations. Despite the availability of in vitro data evaluating these combinations, the in vivo studies remain limited.

Thirty colistin-resistant Klebsiella pneumoniae (ColRKp) isolates were collected from hospitalized patients. Colistin resistance was detected using broth microdilution, and antimicrobial susceptibility was tested using the Kirby-Bauer method against 18 antibiotics. Extremely high resistance levels were detected, with 17% of the isolates being PDR. Virulence profiling, assessed using Anthony capsule staining, the string test, and the crystal violet assay, indicated the predominance of non-biofilm formers and non-hypermucoid strains. The isolates were screened for mcr genes using polymerase chain reaction. Whole-genome sequencing (WGS) and bioinformatics analysis were performed to characterize the genomes of PDR isolates. No plasmid-borne mcr genes were detected, and WGS analysis revealed that PDR isolates belonged to the high-risk clones: ST14 (n = 1), ST147 (n = 2), and ST383 (n = 2). They carried genes encoding extended-spectrum β-lactamases and carbapenemases, blaCTX-M-15 and blaNDM-5, on conjugative IncHI1B/IncFIB plasmids, illustrating the convergence of virulence and resistance genes. The most common mechanism of colistin resistance involved alterations in mgrB. Furthermore, deleterious amino acid substitutions were also detected within PhoQ, PmrC, CrrB, ArnB, and ArnT. Seven colistin-containing combinations were compared using the checkerboard experiment. Synergy was observed when combining colistin with tigecycline, doxycycline, levofloxacin, ciprofloxacin, sulfamethoxazole/trimethoprim, imipenem, or meropenem. The efficacy of colistin combined with either doxycycline or levofloxacin was assessed in vitro using a resistance modulation assay, and in vivo, using a murine infection model. In vitro, doxycycline and levofloxacin reversed colistin resistance in 80% and 73.3% of the population, respectively. In vivo, the colistin + doxycycline combination demonstrated superiority over colistin + levofloxacin, rescuing 80% of infected animals, and reducing bacterial bioburden in the liver and kidneys while preserving nearly intact lung histology.

This study represents the first comparative in vitro and in vivo investigation of the efficacy of colistin + doxycycline and colistin + levofloxacin combinations in clinical PDR ColRKp isolates characterized at a genomic level.

The emergence of the mobile colistin resistance (mcr) gene is a demonstrable threat contributing to the worldwide antibiotic resistance crisis. The gene is encoded on plasmids and can easily spread between different bacterial strains. mcr encodes a phosphoethanolamine (pEtN) transferase, which catalyses the transfer of the pEtN moiety from phosphatidylethanolamine to lipid A, the head group of lipopolysaccharides (LPS). This neutralises the overall negative charge of the LPS and prevents the binding of polymyxins to bacterial membranes. We believe that the development of polymyxin adjuvants could be a promising approach to prolong the use of this important class of last-resort antibiotics. This review discusses recent progress in the identification, design and development of adjuvants to restore polymyxin sensitivity in these resistant bacteria, and focuses on both MCR inhibitors as well as alternative approaches that modulate polymyxin resistance.

Major efforts have been made to reduce the use of colistin in livestock since the discovery of the plasmid-borne mobile colistin resistance (mcr) gene in E. coli a decade ago, to curb the burden of its potential transmission to other bacterial species, spread between animals, humans and the environment. This study explored the longitudinal prevalence and characteristics of colistin-resistant and extended-spectrum beta-lactamase-producing (ESBL) E. coli via in vivo fecal and ex vivo carcass swabs from two batches of conventional indoor and organic outdoor Wallon meat sheep from birth to slaughter in 2020 and 2021. Antimicrobial susceptibility testing via broth microdilution revealed that n = 16/109 (15%) E. coli isolates from conventional meat lamb fecal samples had a reduced colistin sensitivity (MIC = 0.5 μg/mL) and thereof, n = 9/109 (8%) were multi-drug-resistant E. coli, while no resistant isolates were recovered from their carcasses. Sequencing revealed causative pmrB genes, indicating that the reduced sensitivity to colistin was not plasmid-borne. While the sample size was small (n = 32), no colistin-resistant and ESBL-producing E. coli were isolated from the organic meat sheep and their carcasses, potentially due to the different husbandry conditions. Prudent and judicious antimicrobial use and strict slaughter hygiene remain imperative for effective risk management to protect consumers in a sustainable One Health approach.

The rapid increase of mcr-positive Klebsiella pneumoniae (K. pneumoniae) has received considerable attention and poses a major public health concern. Here, we systematically analyzed the global distribution of mcr-positive K. pneumoniae isolates based on published articles as well as publicly available genomes. Combining strain information from 78 articles and 673 K. pneumoniae genomes, a total of 1000 mcr-positive K. pneumoniae isolates were identified. We found that mcr-positive K. pneumoniae has disseminated widely worldwide, especially in Asia, with a higher diversity of sequence types (STs). These isolates were disseminated in 57 countries and were associated with 12 different hosts. Most of the isolates were found in China and were isolated from human sources. Moreover, MLST analysis showed that ST15 and ST11 accounted for the majority of mcr-positive K. pneumoniae, which deserve sustained attention in further surveillance programs. mcr-1 and mcr-9 were the dominant mcr variants in mcr-positive K. pneumoniae. Furthermore, a Genome-wide association study (GWAS) demonstrated that mcr-1- and mcr-9-producing genomes exhibited different antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), thereby indicating a distinct evolutionary path. Notably, the phylogenetic analysis suggested that certain mcr-positive K. pneumoniae genomes from various geographical areas and hosts harbored a high degree of genetic similarities (<20 SNPs), suggesting frequent cross-region and cross-host clonal transmission. Overall, our results emphasize the significance of monitoring and exploring the transmission and evolution of mcr-positive K. pneumoniae in the context of "One health".

Colistin resistance in Escherichia coli is of public health significance for its use to treat multidrug-resistant Gram-negative infections. Amino acid variations in PmrB have been implicated in colistin resistance in E. coli. In this cross-sectional study, 288 generic E. coli isolates from surveillance of broiler chicken and feedlot cattle feces, retail meat, wastewater, and well water were whole-genome sequenced. Phylogroup designation and screening for two amino acid substitutions in PmrB putatively linked to colistin resistance (Y358N, E123D) were performed in silico. Three additional data sets of publicly available E. coli assemblies were similarly scrutinized: (i) E. coli isolates from studies identifying the Y358N or E123D substitutions, (ii) colistin-susceptible E. coli isolates reported in the literature, and (iii) a random sampling of 14,700 E. coli assemblies available in the National Center for Biotechnology Information public database. Within all data sets, ≥95% of phylogroup B1 and C isolates have the PmrB Y358N variation. The PmrB E123D amino acid substitution was only identified in phylogroup B2 isolates, of which 94%-100% demonstrate the substitution. Both PmrB amino acid variations were infrequent in other phylogroups. Among published colistin susceptible isolates, colistin minimum inhibitory concentrations (MICs) were not higher in isolates bearing the E123D and Y358N amino acid variations than in isolates without these PmrB substitutions. The E123D and Y358N PmrB amino acid substitutions in E. coli appear strongly associated with phylogroup. The previously observed associations between Y358N and E123D amino acid substitutions in PmrB and colistin resistance in E. coli may be spurious.

Colistin is a critical last-resort treatment for extensively drug-resistant Gram-negative infections in humans. Therefore, accurate identification of the genetic mechanisms of resistance to this antimicrobial is crucial to effectively monitor and mitigate the spread of resistance. Examining over 16,000 whole-genome sequenced Escherichia coli isolates, this study identifies that PmrB E123D and Y358N amino acid substitutions previously associated with colistin resistance in E. coli are strongly associated with phylogroup and are alone not sufficient to confer a colistin-resistant phenotype. This is a critical clarification, as both substitutions are identified as putative mechanisms of colistin resistance in many publications and a common bioinformatic tool. Given the potential spurious nature of initial associations of these substitutions with colistin resistance, this study's findings emphasize the importance of appropriate experimental design and consideration of relevant biological factors such as phylogroup when ascribing causal mechanisms of resistance to chromosomal variations.

Colistin (COL) was once considered to be the last line of defence against multidrug-resistant bacteria belonging to the family Enterobacteriaceae. Due to the misuse of COL, COL-resistant (COL-R) Enterobacteriaceae have emerged. To address this clinical issue and combat COL resistance, novel approaches are urgently needed.

In this study, the in vitro and in vivo antimicrobial and antibiofilm effects of the immunomodulator AS101 were investigated in combination with COL against COL-R Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae).

Checkerboard assay, time-kill assay, and scanning electron microscopy confirmed the in vitro antimicrobial phenotype, whereas, crystal violet staining and multidimensional confocal laser scanning microscopy with live/dead staining confirmed the antibiofilm capability of the combination therapy. Moreover, the Galleria mellonella infection model and the mouse infection model indicated the high in vivo efficacy of the combination therapy. Additionally, cytotoxicity experiments performed using human kidney-derived HK-2 cells and haemolysis assays performed using human erythrocytes collectively demonstrated safety at effective combination concentrations. Furthermore, quantification of the expression of inflammatory cytokines via enzyme-linked immunosorbent assay confirmed the anti-inflammatory advantage of combination therapy. At the mechanistic level, changes in outer and inner membrane permeability and accumulation of ROS levels, which might be potential mechanisms for synergistic antimicrobial effects.

This study found that AS101 can restore COL susceptibility in clinical COL-R E. coli and K. pneumoniae and also has synergistic antibiofilm and anti-inflammatory capabilities. This study provided a novel strategy to combat clinical infections caused by COL-R E. coli and K. pneumoniae.

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.

The upsurge of antimicrobial resistance demands innovative strategies to fight bacterial infections. With traditional antibiotics becoming less effective, anti-virulence agents or pathoblockers, arise as an alternative approach that seeks to disarm pathogens without affecting their viability, thereby reducing selective pressure for the emergence of resistance mechanisms.

To elucidate the mechanism of action of compound N'-(thiophen-2-ylmethylene)benzohydrazide (A16B1), a potent synthetic hydrazone inhibitor against the Salmonella PhoP/PhoQ system, essential for virulence.

The measurement of the activity of PhoP/PhoQ-dependent and -independent reporter genes was used to evaluate the specificity of A16B1 to the PhoP regulon. Autokinase activity assays with either the native or truncated versions of PhoQ were used to dissect the A16B1 mechanism of action. The effect of A16B1 on Salmonella intramacrophage replication was assessed using the gentamicin protection assay. The checkerboard assay approach was used to analyse potentiation effects of colistin with the hydrazone. The Galleria mellonella infection model was chosen to evaluate A16B1 as an in vivo therapy against Salmonella.

A16B1 repressed the Salmonella PhoP/PhoQ system activity, specifically targeting PhoQ within the second transmembrane region. A16B1 demonstrates synergy with the antimicrobial peptide colistin, reduces the intramacrophage proliferation of Salmonella without being cytotoxic and enhances the survival of G. mellonella larvae systemically infected with Salmonella.

A16B1 selectively inhibits the activity of the Salmonella PhoP/PhoQ system through a novel inhibitory mechanism, representing a promising synthetic hydrazone compound with the potential to function as a Salmonella pathoblocker. This offers innovative prospects for combating Salmonella infections while mitigating the risk of antimicrobial resistance emergence.

Colistin is renowned as a last-resort antibiotic due to the emergence of multidrug-resistant pathogens. However, its potential toxicity significantly hampers its clinical utilization. Melatonin, chemically known as N-acetyl-5-hydroxytryptamine, is an endogenous hormone produced by the pineal gland and possesses diverse biological functions. However, the protective role of melatonin in alleviating antibiotic-induced intestinal inflammation remains unknown. Herein, we reveal that colistin stimulation markedly elevates intestinal inflammatory levels and compromises the gut barrier. In contrast, pretreatment with melatonin safeguards mice against intestinal inflammation and mucosal damage. Microbial diversity analysis indicates that melatonin supplementation prevents a reduction in the abundance of Erysipelotrichales and Bifidobacteriales, as well as an increase in Desulfovibrionales abundance, following colistin exposure. Remarkably, short-chain fatty acids (SCFAs) analysis shows that propanoic acid contributes to the protective effect of melatonin on colistin-induced intestinal inflammation. Furthermore, the protection effects of melatonin and propanoic acid on LPS-induced cellular inflammation in RAW 264.7 cells are confirmed. Mechanistic investigations suggest that intervention with melatonin and propanoic acid can repress the activation of the TLR4 signal and its downstream NF-κB and MAPK signaling pathways, thereby mitigating the toxic effects of colistin. Our work highlights the unappreciated role of melatonin in preventing the potential detrimental effects of colistin on intestinal health and suggests a combined therapeutic strategy to effectively manage intestinal infectious diseases.

The increase in antimicrobial resistance leads to complications in treatments, prolonged hospitalization, and increased mortality. Glabridin (GLA) is a hydroxyisoflavan from Glycyrrhiza glabra L. that exhibits multiple pharmacological activities. Colistin (COL), a last-resort antibiotic, is increasingly being used in clinic against Gram-negative bacteria. Previous reports have shown that GLA is able to sensitize first line antibiotics such as norfloxacin and vancomycin on Staphylococcus aureus, implying that the use of GLA as an antibiotic adjuvant is a promising strategy for addressing the issue of drug resistance. However, the adjuvant effect on other antibiotics, especially COL, on Gram-negative bacteria such as Escherichia coli has not been studied.

The objective of our study was to investigate the targets of GLA and the synergistic effect of GLA and COL in E. coli, and to provide further evidence for the use of GLA as an antibiotic adjuvant to alleviate the problem of drug resistance.

We first investigated the interaction between GLA and enoyl-acyl carrier protein reductase, also called "FabI", through enzyme inhibition assay, differential scanning fluorimetry, isothermal titration calorimetry and molecular docking assay. We tested the transmembrane capacity of GLA on its own and combined it with several antibiotics. The antimicrobial activities of GLA and COL were evaluated against six different susceptible and resistant E. coli in vitro. Their interactions were analyzed using checkerboard assay, time-kill curve and CompuSyn software. A series of sensitivity tests was conducted in E. coli overexpressing the fabI gene. The development of COL resistance in the presence of GLA was tested. The antimicrobial efficacy of GLA and COL in a mouse model of urinary tract infection was assessed. The anti-biofilm effects of GLA and COL were investigated.

In this study, enzyme kinetic analysis and thermal analysis provided evidence for the interaction between GLA and FabI in E. coli. GLA enhanced the antimicrobial effect of COL and synergistically suppressed six different susceptible and resistant E. coli with COL. Overexpression experiments showed that targeted inhibition of FabI was a key mechanism by which GLA synergistically enhanced COL activity. The combination of GLA and COL slowed the development of COL resistance in E. coli. Combined GLA and COL treatment significantly reduced bacterial load and mitigated urinary tract injury in a mouse model of E. coli urinary tract infection. Additionally, GLA + COL inhibited the formation and eradication of biofilms and the synthesis of curli.

Our findings indicate that GLA synergistically enhances antimicrobial activities of COL by targeting inhibition of FabI in E. coli. GLA is expected to continue to be developed as an antibiotic adjuvant to address drug resistance issues.

The surge in mobile colistin-resistant genes (mcr) has become an increasing public health concern, especially in carbapenem-resistant Enterobacterales (CRE). Prospective surveillance was conducted to explore the genomic characteristics of clinical CRE isolates harbouring mcr in 2015-2020. In this study, we aimed to examine the genomic characteristics and phonotypes of mcr-8 and mcr-9 harbouring carbapenem-resistant K. pneumoniae complex (CRKpnC). Polymerase chain reaction test and genome analysis identified CRKpnC strain AMR20201034 as K. pneumoniae (CRKP) ST147 and strain AMR20200784 as K. quasipneumoniae (CRKQ) ST476, harbouring mcr-8 and mcr-9, respectively. CRKQ exhibited substitutions in chromosomal-mediated colistin resistance genes (pmrB, pmrC, ramA, and lpxM), while CRKP showed two substitutions in crrB, pmrB, pmrC, lpxM and lapB. Both species showed resistance to colistin, with minimal inhibitory concentrations of 8 µg/ml for mcr-8-carrying CRKP isolate and 32 µg/ml for mcr-9-carrying CRKQ isolate. In addition, CRKP harbouring mcr-8 carried blaNDM, while CRKQ harbouring mcr-9 carried blaIMP, conferring carbapenem resistance. Analysis of plasmid replicon types carrying mcr-8 and mcr-9 showed FIA-FII (96,575 bp) and FIB-HI1B (287,118 bp), respectively. In contrast with the plasmid carrying the carbapenemase genes, the CRKQ carried blaIMP-14 on an IncC plasmid, while the CRKP harboured blaNDM-1 on an FIB plasmid. This finding provides a comprehensive insight into another mcr-carrying CRE from patients in Thailand. The other antimicrobial-resistant genes in the CRKP were blaCTX-M-15, blaSHV-11, blaOXA-1, aac(6')-Ib-cr, aph(3')-VI, ARR-3, qnrS1, oqxA, oqxB, sul1, catB3, fosA, and qacE, while those detected in CRKQ were blaOKP-B-15, qnrA1, oqxA, oqxB, sul1, fosA, and qacE. This observation highlights the importance of strengthening official active surveillance efforts to detect, control, and prevent mcr-harbouring CRE and the need for rational drug use in all sectors.

Polymyxins [colistin and polymyxin B (PMB)] comprise an important class of natural product lipopeptide antibiotics used to treat multidrug-resistant Gram-negative bacterial infections. These positively charged lipopeptides interact with lipopolysaccharide (LPS) located in the outer membrane and disrupt the permeability barrier, leading to increased uptake and bacterial cell death. Many bacteria counter polymyxins by upregulating genes involved in the biosynthesis and transfer of amine-containing moieties to increase positively charged residues on LPS. Although 4-deoxy-l-aminoarabinose (Ara4N) and phosphoethanolamine (PEtN) are highly conserved LPS modifications in Escherichia coli, different lineages exhibit variable PMB susceptibilities and frequencies of resistance for reasons that are poorly understood. Herein, we describe a mechanism prevalent in E. coli B strains that depends on specific insertion sequence 1 (IS1) elements that flank genes involved in the biosynthesis and transfer of Ara4N to LPS. Spontaneous and transient chromosomal amplifications mediated by IS1 raise the frequency of PMB resistance by 10- to 100-fold in comparison to strains where a single IS1 element located 90 kb away from the end of the arn operon has been deleted. Amplification involving IS1 becomes the dominant resistance mechanism in the absence of PEtN modification. Isolates with amplified arn operons gradually lose their PMB-resistant phenotype with passaging, consistent with classical PMB heteroresistance behavior. Analysis of the whole genome transcriptome profile showed altered expression of genes residing both within and outside of the duplicated chromosomal segment, suggesting complex phenotypes including PMB resistance can result from tandem amplification events.IMPORTANCEPhenotypic variation in susceptibility and the emergence of resistant subpopulations are major challenges to the clinical use of polymyxins. While a large database of genes and alleles that can confer polymyxin resistance has been compiled, this report demonstrates that the chromosomal insertion sequence (IS) content and distribution warrant consideration as well. Amplification of large chromosomal segments containing the arn operon by IS1 increases the Ara4N content of the lipopolysaccharide layer in Escherichia coli B lineages using a mechanism that is orthogonal to transcriptional upregulation through two-component regulatory systems. Altogether, our work highlights the importance of IS elements in modulating gene expression and generating diverse subpopulations that can contribute to phenotypic polymyxin B heteroresistance.

Colistin remains an important antibiotic for the therapeutic management of drug-resistant Klebsiella pneumoniae. Despite the numerous reports of colistin resistance in clinical strains, it remains unclear exactly when and how different mutational events arise resulting in reduced colistin susceptibility. Using a bioreactor model of infection, we modelled the emergence of colistin resistance in a susceptible isolate of K. pneumoniae. Genotypic, phenotypic and mathematical analyses of the antibiotic-challenged and un-challenged population indicates that after an initial decline, the population recovers within 24 h due to a small number of "founder cells" which have single point mutations mainly in the regulatory genes encoding crrB and pmrB that when mutated results in up to 100-fold reduction in colistin susceptibility. Our work underlines the rapid development of colistin resistance during treatment or exposure of susceptible K. pneumoniae infections having implications for the use of cationic antimicrobial peptides as a monotherapy.

Antimicrobial resistance (AMR) poses a serious global health concern, resulting in a significant number of deaths annually due to infections that are resistant to treatment. Amidst this crisis, antimicrobial peptides (AMPs) have emerged as promising alternatives to conventional antibiotics (ATBs). These cationic peptides, naturally produced by all kingdoms of life, play a crucial role in the innate immune system of multicellular organisms and in bacterial interspecies competition by exhibiting broad-spectrum activity against bacteria, fungi, viruses, and parasites. AMPs target bacterial pathogens through multiple mechanisms, most importantly by disrupting their membranes, leading to cell lysis. However, bacterial resistance to host AMPs has emerged due to a slow co-evolutionary process between microorganisms and their hosts. Alarmingly, the development of resistance to last-resort AMPs in the treatment of MDR infections, such as colistin, is attributed to the misuse of this peptide and the high rate of horizontal genetic transfer of the corresponding resistance genes. AMP-resistant bacteria employ diverse mechanisms, including but not limited to proteolytic degradation, extracellular trapping and inactivation, active efflux, as well as complex modifications in bacterial cell wall and membrane structures. This review comprehensively examines all constitutive and inducible molecular resistance mechanisms to AMPs supported by experimental evidence described to date in bacterial pathogens. We also explore the specificity of these mechanisms toward structurally diverse AMPs to broaden and enhance their potential in developing and applying them as therapeutics for MDR bacteria. Additionally, we provide insights into the significance of AMP resistance within the context of host-pathogen interactions.

Transplant recipients commonly harbor multidrug-resistant organisms (MDROs), as a result of frequent hospital admissions and increased exposure to antimicrobials and invasive procedures.

To investigate the impact of patient demographic and clinical characteristics on MDRO acquisition, as well as the impact of MDRO acquisition on intensive care unit (ICU) and hospital length of stay, and on ICU mortality and 1-year mortality post heart transplantation.

This retrospective cohort study analyzed 98 consecutive heart transplant patients over a ten-year period (2013-2022) in a single transplantation center. Data was collected regarding MDROs commonly encountered in critical care.

Among the 98 transplanted patients (70% male), about a third (32%) acquired or already harbored MDROs upon transplantation (MDRO group), while two thirds did not (MDRO-free group). The prevalent MDROs were Acinetobacter baumannii (14%), Pseudomonas aeruginosa (12%) and Klebsiella pneumoniae (11%). Compared to MDRO-free patients, the MDRO group was characterized by higher body mass index (P = 0.002), higher rates of renal failure (P = 0.017), primary graft dysfunction (10% vs 4.5%, P = 0.001), surgical re-exploration (34% vs 14%, P = 0.017), mechanical circulatory support (47% vs 26% P = 0.037) and renal replacement therapy (28% vs 9%, P = 0.014), as well as longer extracorporeal circulation time (median 210 vs 161 min, P = 0.003). The median length of stay was longer in the MDRO group, namely ICU stay was 16 vs 9 d in the MDRO-free group (P = 0.001), and hospital stay was 38 vs 28 d (P = 0.006), while 1-year mortality was higher (28% vs 7.6%, log-rank-χ 2: 7.34).

Following heart transplantation, a predominance of Gram-negative MDROs was noted. MDRO acquisition was associated with higher complication rates, prolonged ICU and total hospital stay, and higher post-transplantation mortality.

A concern with the ESKAPE pathogen, Enterobacter bugandensis, and other species of the Enterobacter cloacae complex, is the frequent appearance of multidrug resistance against last-resort antibiotics, such as polymyxins.

Here, we investigated the responses to polymyxin B (PMB) in two PMB-resistant E. bugandensis clinical isolates by global transcriptomics and deletion mutagenesis.

In both isolates, the genes of the CrrAB-regulated operon, including crrC and kexD, displayed the highest levels of upregulation in response to PMB. ∆crrC and ∆kexD mutants became highly susceptible to PMB and lost the heteroresistant phenotype. Conversely, heterologous expression of CrrC and KexD proteins increased PMB resistance in a sensitive Enterobacter ludwigii clinical isolate and in the Escherichia coli K12 strain, W3110. The efflux pump, AcrABTolC, and the two component regulators, PhoPQ and CrrAB, also contributed to PMB resistance and heteroresistance. Additionally, the lipid A modification with 4-L-aminoarabinose (L-Ara4N), mediated by the arnBCADTEF operon, was critical to determine PMB resistance. Biochemical experiments, supported by mass spectrometry and structural modelling, indicated that CrrC is an inner membrane protein that interacts with the membrane domain of the KexD pump. Similar interactions were modeled for AcrB and AcrD efflux pumps.

Our results support a model where drug efflux potentiated by CrrC interaction with membrane domains of major efflux pumps combined with resistance to PMB entry by the L-Ara4N lipid A modification, under the control of PhoPQ and CrrAB, confers the bacterium high-level resistance and heteroresistance to PMB.

Colistin (Polymyxin E) has reemerged in the treatment of MDR Gram-negative infections. Traditional Colistin AST methods have long turnaround times and are cumbersome for routine use. We present a SEM-AST technique enabling rapid detection of Colistin resistance through direct observation of morphological and quantitative changes in bacteria exposed to Colistin.

Forty-four Gram-negative reference organisms were chosen based on their Colistin susceptibility profiles. Bacterial suspensions of ∼107 CFU/mL were exposed to Colistin at EUCAST-ECOFF, with controls not exposed, incubated at 37°C, and then sampled at 0, 15, 30, 60, and 120 minutes. Phosphotungstic Acid (PTA) staining was applied, followed by SEM imaging using Hitachi TM4000PlusII-Tabletop-SEM at ×2000, ×5000 and ×7000 magnifications. Bacterial viability analysis was performed for all conditions by quantifying viable and dead organisms based on PTA-staining and morphologic changes.

We identified a significant drop in the percentage of viable organisms starting 30 minutes after exposure in susceptible strains, as compared to nonsignificant changes in resistant strains across all tested organisms. The killing effect of Colistin was best observed after 120 minutes of incubation with the antibiotic, with significant changes in morphologic features, including bacterial inflation, fusion, and lysis, observed as early as 30 minutes. Our observation matched the results of the gold standard-based broth microdilution method.

We provide an extended application of the proof of concept for the utilization of the SEM-AST assay for Colistin for a number of clinically relevant bacterial species, providing a rapid and reliable susceptibility profile for a critical antibiotic.

Colistin resistance is a global concern warning for a one health approach to combat the challenge. Colistin resistant E. coli and their resistance determinants are widely distributed in the environment, and rats could be a potential source of these isolates and resistant determinants to a diverse environmental setting. This study was aimed to determine the presence of colistin resistant E. coli (CREC) in wild rats, their antimicrobial resistance (AMR) phenotypes, and genotypic analysis of mcr-1 CREC through whole genome sequencing (WGS). A total of 39 rats were examined and CREC was isolated from their fecal pellets onto MacConkey agar containing colistin sulfate (1 μg/ mL). AMR of the CREC was determined by disc diffusion and broth microdilution was employed to determine MIC to colistin sulfate. CREC were screened for mcr genes (mcr-1 to mcr-8) and phylogenetic grouping by PCR. Finally, WGS of one mcr-1 CREC was performed to explore its genetic characteristics especially resistomes and virulence determinants. 43.59% of the rats carried CREC with one (2.56%) of them carrying CREC with mcr-1 gene among the mcr genes examined. Examination of seventeen (17) isolates from the CREC positive rats (n = 17) revealed that majority of them belonging to the pathogenic phylogroup D (52.94%) and B2 (11.76%). 58.82% of the CREC were MDR on disc diffusion test. Shockingly, the mcr-1 CREC showed phenotypic resistance to 16 antimicrobials of 8 different classes and carried the ARGs in its genome. The mcr-1 gene was located on a 60 kb IncI2 plasmid. On the other hand, ARGs related to aminoglycosides, phenicols, sulfonamides, tetracyclines and trimethoprims were located on a 288 kb mega-plasmid separately. The mcr-1 CREC carried 58 virulence genes including genes related to adhesion, colonization, biofilm formation, hemolysis and immune-evasion. The isolate belonged to ST224 and closely related to E. coli from different sources including UPEC clinical isolates from human based on cgMLST analysis. The current research indicates that rats might be a possible source of CREC, and the presence of mcr-1 and other ARGs on plasmid increases the risk of ARGs spreading and endangering human health and other environmental components through this infamous pest.

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.

The current understanding of acquired chromosomal colistin resistance mechanisms in Enterobacterales primarily involves the disruption of the upstream PmrAB and PhoPQ two-component system (TCS) control caused by mutations in the regulatory genes. Interestingly, previous studies have yielded conflicting results regarding the interaction of regulatory genes related to colistin resistance in Escherichia coli, specifically those surrounding PhoPQ and PmrAB TCS.

In our study, we focused on two clinical non-mcr colistin-resistant strains of E. coli, TSAREC02 and TSAREC03, to gain a better understanding of their resistance mechanisms. Upon analysis, we discovered that TSAREC02 had a deletion (Δ27-45) in MgrB, as well as substitutions (G206R, Y222H) in PmrB. On the other hand, TSAREC03 exhibited a long deletion (Δ84-224) in PhoP, along with substitutions (M1I, L14P, P178S, T235N) in PmrB. We employed recombinant DNA techniques to explore the interaction between the PhoPQ and PmrAB two-component systems (TCSs) and examine the impact of the mutated phoPQ and pmrB genes on the minimum inhibitory concentrations (MICs) of colistin. We observed significant changes in the expression of the pmrD gene, which encodes a connector protein regulated by the PhoPQ TCS, in the TSAREC02 wild-type (WT)-mgrB replacement mutant and the TSAREC03 WT-phoP replacement mutant, compared to their respective parental strains. However, the expressions of pmrB/pmrA, which reflect PmrAB TCS activity, and the colistin MICs remained unchanged. In contrast, the colistin MICs and pmrB/pmrA expression levels were significantly reduced in the pmrB deletion mutants from both TSAREC02 and TSAREC03, compared to their parental strains. Moreover, we were able to restore colistin resistance and the expressions of pmrB/pmrA by transforming a plasmid containing the parental mutated pmrB back into the TSAREC02 and TSAREC03 mutants, respectively.

While additional data from clinical E. coli isolates are necessary to validate whether our findings could be broadly applied to the E. coli population, our study illuminates distinct regulatory pathway interactions involving colistin resistance in E. coli compared to other species of Enterobacterales. The added information provided by our study contribute to a deeper understanding of the complex pathway interactions within Enterobacterales.

Antibiotic resistance is a major challenge to public health, but human-caused environmental changes have not been widely recognized as its drivers. Here, we provide a comprehensive overview of the relationships between environmental degradation and antibiotic resistance, demonstrating that the former can potentially fuel the latter with significant public health outcomes. We describe that (i) global warming favors horizontal gene transfer, bacterial infections, the spread of drug-resistant pathogens due to water scarcity, and the release of resistance genes with wastewater; (ii) pesticide and metal pollution act as co-selectors of antibiotic resistance mechanisms; (iii) microplastics create conditions promoting and spreading antibiotic resistance and resistant bacteria; (iv) changes in land use, deforestation, and environmental pollution reduce microbial diversity, a natural barrier to antibiotic resistance spread. We argue that management of antibiotic resistance must integrate environmental goals, including mitigation of further increases in the Earth's surface temperature, better qualitative and quantitative protection of water resources, strengthening of sewage infrastructure and improving wastewater treatment, counteracting the microbial diversity loss, reduction of pesticide and metal emissions, and plastic use, and improving waste recycling. These actions should be accompanied by restricting antibiotic use only to clinically justified situations, developing novel treatments, and promoting prophylaxis. It is pivotal for health authorities and the medical community to adopt the protection of environmental quality as a part of public health measures, also in the context of antibiotic resistance management.