The Occurrence of Colistin Resistance in Potential Lactic Acid Bacteria of Food-Producing Animals in India.

The overuse of colistin, the last-resort antibiotic, has led to the emergence of colistin-resistant bacteria, which is a major concern. Lactic acid bacteria which are generally regarded as safe are known to be reservoirs of antibiotic resistance that possibly pose a threat to human and animal health. Therefore, this study assessed the prevalence of colistin antimicrobial resistance in livestock in India, that is lactic acid bacteria in healthy chickens, sheep, beef, and swine of Mysore. Diverse phenotypic and genotypic colistin resistance were examined among the lactic acid bacterial species (n = 84) isolated from chicken (n = 44), sheep (n = 16), beef (n = 14), and swine (n = 10). Hi-comb, double-disk diffusion tests, Minimum Inhibitory Concentration (MIC), and biofilm formation were assessed for phenotypic colistin resistance. Specific primers for colistin-resistant genes were used for the determination of genotypic colistin resistance. Around 20%, 18%, and 1% were colistin-resistant Lactobacillus, Enterococcus, and Pediococcus species, respectively. Among these, 66.67% exhibited MDR phenotypes, including colistin antibiotic. The identified resistant isolates are Levilactobacillus brevis LBA and LBB (2), Limosilactobacillus fermentum LBF (1), and Pediococcus acidilactici CHBI (1). The mcr-1 and mcr-3 genes were detected in Levilactobacillus brevis LBA, LBB, and Pediococcus acidilactici CHBI isolated from chicken and sheep intestines respectively. The study identified colistin resistance determinants in lactobacilli from food animals, emphasizing the need for enhanced surveillance and monitoring of resistance spread. These findings underscore colistin resistance as a significant medical concern and should be integrated into India's ongoing antimicrobial resistance monitoring programs.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of polymixin B1, B2, ile-B1, E1, and E2 in human plasma and its clinical pharmacokinetic application.

Polymyxin B (PB) and Polymyxin E (PE, also called colistin) are used as the last treatment resort for multidrug-resistant Gram-negative bacterial infections. The nephrotoxicity and neurotoxicity of polymyxins limit their clinical use, and guidelines recommend therapeutic drug monitoring (TDM) to optimize efficacy and reduce toxicity. However, there are limited analytical methods available for the determination of PB and PE. This study aimed to develop a simple and robust liquid chromatography with tandem mass spectrometry (LC-MS/MS) analytical method for determining the main compounds of PB and PE, namely PB1, PB2, ile-PB1, PE1, and PE2, in human plasma and to investigate of their pharmacokinetics in critically ill patients with the use of PB and PE, respectively. Plasma PB1, PB2, ile-PB1, PE1, and PE2 were chromatographically separated on a Welch LP-C18 column and detected using electrospray ionization mode coupled with multiple reaction monitoring. The calibration curve showed acceptable linearity over 20-10,000 ng/mL for PB1, PE1, and PE2 and 10-5000 ng/mL for PB2 and ile-PB1 in the plasma, respectively. After validation following approved guidelines, this method was successfully applied for PB and PE pharmacokinetic analysis and TDM in critically ill patients. Additionally, the composition of PB1, PB2, ile-PB1, PE1, and PE2 remains unchanged from 0 to 12 h after entering the patient's body.

Molecular characterization of NDM and OXA-48-like-producing Klebsiella pneumoniae ST16 and hypervirulent ST337 clone among two patients; a case report.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are a major public health problem, requiring the use of last-resort antibiotics such as colistin. However, there is concern regarding the emergence of isolates resistant to this agent. The report describes two patients with urinary tract infection (UTI) and ventilator-associated pneumonia (VAP) infection caused by CRKP strains. The first case was a 23-year-old male with UTI caused by a strain of ST16 co-harboring blaCTX-M, blaTEM, blaSHV, blaNDM, blaOXA-48-like genes. The second case was a 39-year-old woman with VAP due to hypervirulent ST337-K2 co-harboring blaSHV, blaNDM, blaOXA-48-like, iucA, rmpA2 and rmpA. The patients' general condition improved after combination therapy with colistin (plus meropenem and rifampin, respectively) and both of them recovered and were discharged from the hospital. This study highlights the necessary prevention and control steps to prevent the further spread of CRKP strains should be a priority in our hospital.

Carbapenems and colistin resistance genes isolated in Musca domestica from a garbage dump near a hospital in Lima. / Genes resistentes a carbapenémicos y colistina aislados en Musca domestica proveniente de un basural cercano a un hospital de Lima.

Motivation for the study. The presence of antibiotic resistance genes in bacteria isolated from common flies is a potential public health hazard because it facilitates the presence and spread of antibiotic resistance genes in the environment. Main findings. Thirty-eight bacterial strains identified in 14 species were isolated from within the fly bodies, of which 31 strains showed resistance to carbapenems and 26 strains showed resistance to colistin. Seven bacterial strains showed carbapenem resistance genes and one Escherichia coli strain had resistance to KPC, OXA-48 and mcr-1. Implications. This is the first report of antibiotic resistance genes in bacteria carried by common flies in Peru. The objective was to determine the presence of carbapenem resistance genes and plasmid resistance to colistin (mcr-1) in bacteria isolated from Musca domestica in a garbage dump near a hospital in Lima, Peru. Bacteria with phenotypic resistance to carbapenemics were isolated on CHROMagar mSuperCARBATM medium and colistin resistance profiling was performed using the colistin disk elution method. Detection of blaKPC, blaNDM, blaIMP, blaOXA-48, blaVIM and mcr-1 genes was performed by conventional PCR. The antimicrobial susceptibility profile was determined using the automated MicroScan system. We found that 31/38 strains had phenotypic resistance to carbapenemics and 26/38 strains had phenotypic resistance to colistin with a minimum inhibitory concentration ≥ 4 µg/ml. Finally, we identified seven bacterial strains with carbapenem resistance genes (OXA-48 and KPC) and one bacterial strain with plasmid resistance to colistin (mcr-1). One Escherichia coli strain had three resistance genes: KPC, OXA-48 and mcr-1.

El objetivo fue determinar la presencia de genes de resistencia a carbapenémicos y resistencia plasmídica a colistina (mcr-1) en bacterias aisladas de Musca domestica en un basural cercano a un hospital de Lima, Perú. Las bacterias con resistencia fenotípica a los carbapénemicos se aislaron en medio CHROMagar mSuperCARBATM y el perfil de resistencia a colistina se realizó mediante el método de elución de discos de colistina. La detección de genes blaKPC, blaNDM, blaIMP, blaOXA-48, blaVIM y mcr-1 se realizó mediante PCR convencional. El perfil de susceptibilidad antimicrobiana se determinó mediante el sistema automatizado MicroScan. Las bacterias con resistencia fenotípica a carbapenémicos fueron 31/38 cepas y a colistina fueron 26/38 cepas con una concentración inhibitoria mínima ≥ 4 µg/ml. Finalmente, se identificaron siete cepas bacterianas con genes de resistencia a carbapenémicos (OXA-48 Y KPC) y una cepa bacteriana con resistencia plasmídica a colistina (mcr-1). Una cepa de Escherichia coli presentó tres genes de resistencia: KPC, OXA-48 y mcr-1. Motivación para realizar el estudio. La presencia de genes de resistencia a antibióticos en bacterias aisladas de moscas comunes es un peligro potencial para la salud pública debido a que facilita la presencia y dispersión de genes de resistencia a antibióticos en el medio ambiente. Principales hallazgos. Se aislaron 38 cepas bacterianas identificadas en 14 especies dentro del cuerpo de las moscas, de las cuales 31 cepas mostraron resistencia a los carbapenémicos y 26 cepas mostraron resistencia a colistina. Siete cepas bacterianas presentaron genes de resistencia a carbapenémicos y una cepa de Escherichia coli con resistencia a KPC, OXA-48 y mcr-1. Implicancias. Se realiza el primer reporte en el Perú de genes de resistencia a antibióticos en bacterias movilizadas por moscas comunes.

Predicting early appropriate therapy for patients infected by carbapenem-resistant Gram-negative pathogens in intensive care units in Italy.

BACKGROUND: Antibiotic resistance among Gram-negative bacteria in intensive care units (ICUs) is linked with high morbidity and mortality in patients. In this study, we estimated the therapeutic coverage of various antibiotics, focusing on cefiderocol and comparators, administered empirically against an infection of unknown origin in the ICU. METHODS: In the ARTEMIS surveillance study, susceptibilities of 624 Italian Gram-negative isolates to amikacin, aztreonam-avibactam, cefiderocol, ceftazidime-avibactam, ceftolozane-tazobactam, colistin, imipenem-relebactam, meropenem, and meropenem-vaborbactam were tested by broth microdilution, and results were interpreted by European Committee on Antimicrobial Susceptibility Testing breakpoints. The susceptibility rates from the ARTEMIS study were extrapolated to Gram-negative isolates obtained from 5,774 patients in Italian ICUs in 2021. The sum of the predicted susceptibilities of individual pathogens represented the overall likelihood of in vitro activity of each antibiotic as early targeted therapy for ICU patients. RESULTS: A total of 624 Italian Gram-negative isolates included 206 Pseudomonas aeruginosa, 138 Acinetobacter baumannii, 187 Klebsiella pneumoniae, and 93 Escherichia coli. Against A. baumannii, K. pneumoniae, P. aeruginosa, and E. coli, the overall susceptibility rates for cefiderocol were 87.7%, 96.8%, 99%, and 100%, respectively; and for comparator agents, 8.7-96.4%, 25.7-100%, 73.3-100%, and 89.2-100%, respectively. Among the subset of meropenem-resistant isolates, susceptibility rates of A. baumannii, K. pneumoniae, and P. aeruginosa to cefiderocol were 86.4%, 96.2% and 100%, respectively. Corresponding susceptibility rates to comparator agents were 0-96.8%, 0-100%, and 6.4-100%, respectively. There were no meropenem-resistant isolates of E. coli. The extrapolation of data to isolates from Italian ICUs showed that the highest likelihood of therapeutic coverage, both overall and among meropenem-resistant isolates, was reported for colistin (96.8% and 72.2%, respectively) and cefiderocol (95.7% and 71.4%, respectively). All other antibiotics were associated with a likelihood below 73% overall and between 0% and 41.4% for meropenem-resistant isolates. CONCLUSIONS: Based on confirmed susceptibility rates and reported ICU prevalence of multiple Gram-negative species, cefiderocol showed a higher predicted therapeutic coverage and utility in ICUs compared with comparator beta-lactam-beta-lactamase inhibitor antibiotics. Cefiderocol may be a promising early treatment option for patients at high risk of carbapenem-resistant Gram-negative bacterial infections in the ICU.

Rapid evolution of colistin resistance in a bioreactor model of infection of Klebsiella pneumoniae.

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.

Potent synergy and sustained bactericidal activity of polymyxins combined with Gram-positive only class of antibiotics versus four Gram-negative bacteria.

BACKGROUND: Gram-negative bacteria (GNB) are becoming increasingly resistant to a wide variety of antibiotics. There are currently limited treatments for GNB, and the combination of antibiotics with complementary mechanisms has been reported to be a feasible strategy for treating GNB infection. The inability to cross the GNB outer membrane (OM) is an important reason that a broad spectrum of Gram-positive only class of antibiotics (GPOAs) is lacking. Polymyxins may help GPOAs to permeate by disrupting OM of GNB. OBJECTIVE: To identify what kind of GPOAs can be aided to broaden their anti-GNB spectrum by polymyxins, we systematically investigated the synergy of eight GPOAs in combination with colistin (COL) and polymyxin B (PMB) against GNB in vitro. METHODS: The synergistic effect of COL or PMB and GPOAs combinations against GNB reference strains and clinical isolates were determined by checkerboard tests. The killing kinetics of the combinations were assessed using time-kill assays. RESULTS: In the checkerboard tests, polymyxins-GPOAs combinations exert synergistic effects characterized by species and strain specificity. The synergistic interactions on P. aeruginosa strains are significantly lower than those on strains of A. baumannii, K. pneumoniae and E. coli. Among all the combinations, COL has shown the best synergistic effect in combination with dalbavancin (DAL) or oritavancin (ORI) versus almost all of the strains tested, with FICIs from 0.16 to 0.50 and 0.13 to < 0.28, respectively. In addition, the time-kill assays demonstrated that COL/DAL and COL/ORI had sustained bactericidal activity. CONCLUSIONS: Our results indicated that polymyxins could help GPOAs to permeate the OM of specific GNB, thus showed synergistic effects and bactericidal effects in the in vitro assays. In vivo combination studies should be further conducted to validate the results of this study.

Genomic and phenotypic inconsistencies in Pseudomonas aeruginosa resistome among intensive care patients.

Objective: Pseudomonas aeruginosa, a difficult-to-manage nosocomial pathogen, poses a serious threat to clinical outcomes in intensive care (ICU) patients due to its high antimicrobial resistance (AMR). To promote effective management, it is essential to investigate the genomic and phenotypic differences in AMR expression of the isolates. Methods: A prospective observational study was conducted from July 2022 to April 2023 at Liepaja Regional Hospital in Latvia. The study included all adult patients who were admitted to the ICU and had a documented infection with P. aeruginosa, as confirmed by standard laboratory microbiological testing and short-read sequencing. Since ResFinder is the only sequencing-based database offering antibacterial susceptibility testing (AST) data for each antibiotic, we conducted a comparison of the resistance profile with the results of phenotypic testing, evaluating if ResFinder met the US Food and Drug Administration (FDA) requirements for approval as a new AMR diagnostic test. Next, to improve precision, AST data from ResFinder was compared with two other databases - AMRFinderPlus and RGI. Additionally, data was gathered from environmental samples to inform the implementation of appropriate infection control measures in real time. Results: Our cohort consisted of 33 samples from 29 ICU patients and 34 environmental samples. The presence of P. aeruginosa infection was found to be associated with unfavourable clinical outcomes. A third of the patient samples were identified as multi-drug resistant isolates. Apart from resistance against colistin, significant discrepancies were observed when phenotypic data were compared to genotypic data. For example, the aminoglycoside resistance prediction of ResFinder yielded a major errors value of 3.03% for amikacin, which was marginally above the FDA threshold. Among the three positive environmental samples, one sample exhibited multiple AMR genes similar to the patient samples in its cluster. Conclusion: Our findings underscore the importance of utilizing a combination of diagnostic methods for the identification of resistance mechanisms, clusters, and environmental reservoirs in ICUs.

Integration of individual preclinical and clinical anti-infective PKPD data to predict clinical study outcomes.

The AIDA randomized clinical trial found no significant difference in clinical failure or survival between colistin monotherapy and colistin-meropenem combination therapy in carbapenem-resistant Gram-negative infections. The aim of this reverse translational study was to integrate all individual preclinical and clinical pharmacokinetic-pharmacodynamic (PKPD) data from the AIDA trial in a pharmacometric framework to explore whether individualized predictions of bacterial burden were associated with the trial outcomes. The compiled dataset included for each of the 207 patients was (i) information on the infecting Acinetobacter baumannii isolate (minimum inhibitory concentration, checkerboard assay data, and fitness in a murine model), (ii) colistin plasma concentrations and colistin and meropenem dosing history, and (iii) disease scores and demographics. The individual information was integrated into PKPD models, and the predicted change in bacterial count at 24 h for each patient, as well as patient characteristics, was correlated with clinical outcomes using logistic regression. The in vivo fitness was the most important factor for change in bacterial count. A model-predicted growth at 24 h of ≥2-log10 (164/207) correlated positively with clinical failure (adjusted odds ratio, aOR = 2.01). The aOR for one unit increase of other significant predictors were 1.24 for SOFA score, 1.19 for Charlson comorbidity index, and 1.01 for age. This study exemplifies how preclinical and clinical anti-infective PKPD data can be integrated through pharmacodynamic modeling and identify patient- and pathogen-specific factors related to clinical outcomes - an approach that may improve understanding of study outcomes.

Colistin-, cefepime-, and levofloxacin-resistant Salmonella enterica serovars isolated from Egyptian chicken carcasses.

OBJECTIVES: The emergence of multidrug-resistant (MDR) Salmonella strains, especially resistant ones toward critically important antimicrobial classes such as fluoroquinolones and third- and fourth-generation cephalosporins, is a growing public health concern. The current study, therefore, aimed to determine the prevalence, and existence of virulence genes (invA, stn, and spvC genes), antimicrobial resistance profiles, and the presence of ß-lactamase resistance genes (blaOXA, blaCTX-M1, blaSHV, and blaTEM) in Salmonella strains isolated from native chicken carcasses in Egypt marketed in Mansoura, Egypt, as well as spotlight the risk of isolated MDR, colistin-, cefepime-, and levofloxacin-resistant Salmonella enterica serovars to public health. METHODS: One hundred fifty freshly dressed native chicken carcasses were collected from different poultry shops in Mansoura City, Egypt between July 2022 and November 2022. Salmonella isolation was performed using standard bacteriological techniques, including pre-enrichment in buffered peptone water (BPW), selective enrichment in Rappaport Vassiliadis broth (RVS), and cultivating on the surface of xylose-lysine-desoxycholate (XLD) agar. All suspected Salmonella colonies were subjected to biochemical tests, serological identification using slide agglutination test, and Polymerase Chain Reaction (PCR) targeting the invasion A gene (invA; Salmonella marker gene). Afterward, all molecularly verified isolates were screened for the presence of virulence genes (stn and spvC). The antimicrobial susceptibility testing for isolated Salmonella strains towards the 16 antimicrobial agents tested was analyzed by Kirby-Bauer disc diffusion method, except for colistin, in which the minimum inhibition concentration (MIC) was determined by broth microdilution technique. Furthermore, 82 cefotaxime-resistant Salmonella isolates were tested using multiplex PCR targeting the ß-lactamase resistance genes, including blaOXA, blaCTX-M1, blaSHV, and blaTEM genes. RESULTS: Salmonella enterica species were molecularly confirmed via the invA Salmonella marker gene in 18% (27/150) of the freshly dressed native chicken carcasses. Twelve Salmonella serotypes were identified among 129 confirmed Salmonella isolates with the most predominant serotypes were S. Kentucky, S. Enteritidis, S. Typhimurium, and S. Molade with an incidence of 19.4% (25/129), 17.1% (22/129), 17.1% (22/129), and 10.9% (14/129), respectively. All the identified Salmonella isolates (n = 129) were positive for both invA and stn genes, while only 31.8% (41/129) of isolates were positive for the spvC gene. One hundred twenty-one (93.8%) of the 129 Salmonella-verified isolates were resistant to at least three antibiotics. Interestingly, 3.9%, 14.7%, and 75.2% of isolates were categorized into pan-drug-resistant, extensively drug-resistant, and multidrug-resistant, respectively. The average MAR index for the 129 isolates tested was 0.505. Exactly, 82.2%, 82.2%, 63.6%, 51.9%, 50.4%, 48.8%, 11.6%, and 10.1% of isolated Salmonella strains were resistant to cefepime, colistin, cefotaxime, ceftazidime/clavulanic acid, levofloxacin, ciprofloxacin, azithromycin, and meropenem, respectively. Thirty-one out (37.8%) of the 82 cefotaxime-resistant Salmonella isolates were ß-lactamase producers with the blaTEM as the most predominant ß-lactamase resistance gene, followed by blaCTX-M1 and blaOXA genes, which were detected in 21, 16, and 14 isolates respectively). CONCLUSION: The high prevalence of MDR-, colistin-, cefepime-, and levofloxacin-resistant Salmonella serovars among Salmonella isolates from native chicken is alarming as these antimicrobials are critically important in treating severe salmonellosis cases and boost the urgent need for controlling antibiotic usage in veterinary and human medicine to protect public health.

Antimicrobial resistance genes harbored in invasive Acinetobacter calcoaceticus-baumannii complex isolated from Korean children during the pre-COVID-19 pandemic periods, 2015-2020.

Background: Acinetobacter baumannii (AB) has emerged as one of the most challenging pathogens worldwide, causing invasive infections in the critically ill patients due to their ability to rapidly acquire resistance to antibiotics. This study aimed to analyze antibiotic resistance genes harbored in AB and non-baumannii Acinetobacter calcoaceticus-baumannii (NB-ACB) complex causing invasive diseases in Korean children. Methods: ACB complexes isolated from sterile body fluid of children in three referral hospitals were prospectively collected. Colistin susceptibility was additionally tested via broth microdilution. Whole genome sequencing was performed and antibiotic resistance genes were analyzed. Results: During January 2015 to December 2020, a total of 67 ACB complexes were isolated from sterile body fluid of children in three referral hospitals. The median age of the patients was 0.6 (interquartile range, 0.1-7.2) years old. Among all the isolates, 73.1% (n=49) were confirmed as AB and others as NB-ACB complex by whole genome sequencing. Among the AB isolates, only 22.4% susceptible to carbapenem. In particular, all clonal complex (CC) 92 AB (n=33) showed multi-drug resistance, whereas 31.3% in non-CC92 AB (n=16) (P<0.001). NB-ACB showed 100% susceptibility to all classes of antibiotics except 3rd generation cephalosporin (72.2%). The main mechanism of carbapenem resistance in AB was the bla oxa23 gene with ISAba1 insertion sequence upstream. Presence of pmr gene and/or mutation of lpxA/C gene were not correlated with the phenotype of colistin resistance of ACB. All AB and NB-ACB isolates carried the abe and ade multidrug efflux pumps. Conclusions: In conclusion, monitoring and research for resistome in ACB complex is needed to identify and manage drug-resistant AB, particularly CC92 AB carrying the bla oxa23 gene.

Real-world data on the effects of colistin sulfate and polymyxin B sulfate in the treatment of pneumonia induced by CR-GNB.

INTRODUCTION: The aim of this study was to compare the efficacy and safety of colistin sulfate (CS) with polymyxin B sulfate (PMB) in the treatment of pneumonia induced by carbapenem-resistant Gram-negative bacteria (CR-GNB). METHODOLOGY: Patients diagnosed with pneumonia caused by CR-GNB and admitted to the intensive care unit (ICU) from January 2020 to September 2022 were enrolled in this study. The patients were divided into the CS group and the PMB group according to their medication regimens. Group-wise demographic data, clinical efficacy, prognosis, and adverse events were analyzed and compared. RESULTS: A total of 120 patients (68 in the CS group and 52 in the PMB group) with pneumonia were included in the study. The majority of the pathogens were CR-Acinetobacter baumannii, followed by CR-Klebsiella pneumoniae, and CR-Pseudomonas aeruginosa. The clinical response rates in the CS and PMB groups after treatment were 62.0% and 65.4%, bacterial clearances were 44.0% and 36.5%, 28-day mortality rates were 16.0% and 13.5%, respectively; no significant differences between the two treatments were found. Nevertheless, the adverse effects were significantly less common in the CS group than in the PMB group, especially when treatments were administered intravenously. CONCLUSIONS: CS, a novel polymyxin E formulation, is as effective as PMB in treating pneumonia induced by CR-GNB while causing less side effects.

High Prevalence of Colistin-Resistant Encoding Genes Carriage among Patients and Healthy Residents in Vietnam.

Background and Objectives: We aimed to investigate the carriage of colistin-resistant genes among both patients with a history of antibiotic exposure and apparently healthy adults with no recent healthcare contact. Materials and Methods: Stool swabs were collected from healthy people, and specimens were collected at the infection foci from the patients. Eleven primer/probe sets were used to perform the Multiplex Real-Time PCR assay with the QuantiNova Multiplex Probe PCR kit for screening the carriage of colistin-resistant genes (mcr-1 to mcr-10) and 16S rRNA gene as internal control. Results: In total, 86 patients and 96 healthy residents were included. Twenty two patients (25.9%) were positive with at least one colistin-resistance encoding gene. The mcr-1 gene was the most frequent (16.5%), followed by mcr-9, mcr-6, and mcr-4 genes, where the prevalence was 11.8%, 10.6%, and 9.4%, respectively. No patient was positive with mcr-3, mcr-7, and mcr-8 genes. Eight patients (9.4%) were positive with multiple colistin-encoding genes. Twenty-three healthy people (24.0%) were positive with at least one colistin-resistance encoding gene, and the mcr-10 gene was the most frequent (27.0%), followed by the mcr-1, mcr-8, and mcr-9 genes, where the prevalence was 24.3%, 21.6%, and 13.5%, respectively. No person was positive with the mcr-2 and mcr-5 genes. Conclusions: Our findings underscore the urgent need for enhanced surveillance, infection control measures, and stewardship interventions to mitigate the spread of colistin resistance in Vietnam.

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

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

Diclofenac Sodium Restores the Sensitivity of Colistin-Resistant Gram-Negative Bacteria to Colistin.

The continuous rise of multidrug-resistant (MDR) Gram-negative bacteria poses a severe threat to public health worldwide. Colistin(COL), employed as the last-line antibiotic against MDR pathogens, is now at risk due to the emergence of colistin-resistant (COL-R) bacteria, potentially leading to adverse patient outcomes. In this study, synergistic activity was observed when colistin and diclofenac sodium (DS) were combined and used against clinical COL-R strains of Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumannii (A. baumannii), and Pseudomonas aeruginosa (P. aeruginosa) both in vitro and in vivo. The checkerboard method and time-killing assay showed that DS, when combined with COL, exhibited enhanced antibacterial activity compared to DS and COL monotherapies. Crystal violet staining and scanning electron microscopy showed that COL-DS inhibited biofilm formation compared with monotherapy. The in vivo experiment showed that the combination of DS and COL reduced bacterial loads in infected mouse thighs. Synergistic activity was observed when COL and DS were use in combination against clinical COL-R strains of E. coli, K. pneumoniae, A. baumannii and P. aeruginosa both in vitro and in vivo. The synergistic antibacterial effect of the COL-DS combination has been confirmed by performing various in vitro and in vivo experiments, which provides a new treatment strategy for infections caused by MDR bacteria.

Melatonin Protects Against Colistin-Induced Intestinal Inflammation and Microbiota Dysbiosis.

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.

Colistin: Lights and Shadows of an Older Antibiotic.

The emergence of antimicrobial resistance represents a serious threat to public health and for infections due to multidrug-resistant (MDR) microorganisms, representing one of the most important causes of death worldwide. The renewal of old antimicrobials, such as colistin, has been proposed as a valuable therapeutic alternative to the emergence of the MDR microorganisms. Although colistin is well known to present several adverse toxic effects, its usage in clinical practice has been reconsidered due to its broad spectrum of activity against Gram-negative (GN) bacteria and its important role of "last resort" agent against MDR-GN. Despite the revolutionary perspective of treatment with this old antimicrobial molecule, many questions remain open regarding the emergence of novel phenotypic traits of resistance and the optimal usage of the colistin in clinical practice. In last years, several forward steps have been made in the understanding of the resistance determinants, clinical usage, and pharmacological dosage of this molecule; however, different points regarding the role of colistin in clinical practice and the optimal pharmacokinetic/pharmacodynamic targets are not yet well defined. In this review, we summarize the mode of action, the emerging resistance determinants, and its optimal administration in the treatment of infections that are difficult to treat due to MDR Gram-negative bacteria.

A pilot clinical risk model to predict polymyxin-induced nephrotoxicity: a real-world, retrospective cohort study.

OBJECTIVES: Polymyxin-induced nephrotoxicity (PIN) is a major safety concern and challenge in clinical practice, which limits the clinical use of polymyxins. This study aims to investigate the risk factors and to develop a scoring tool for the early prediction of PIN. METHODS: Data on critically ill patients who received intravenous polymyxin B or colistin sulfate for over 24 h were collected. Logistic regression with the least absolute shrinkage and selection operator (LASSO) was used to identify variables that are associated with outcomes. The eXtreme Gradient Boosting (XGB) classifier algorithm was used to further visualize factors with significant differences. A prediction model for PIN was developed through binary logistic regression analysis and the model was assessed by temporal validation and external validation. Finally, a risk-scoring system was developed based on the prediction model. RESULTS: Of 508 patients, 161 (31.6%) patients developed PIN. Polymyxin type, loading dose, septic shock, concomitant vasopressors and baseline blood urea nitrogen (BUN) level were identified as significant predictors of PIN. All validation exhibited great discrimination, with the AUC of 0.742 (95% CI: 0.696-0.787) for internal validation, of 0.708 (95% CI: 0.605-0.810) for temporal validation and of 0.874 (95% CI: 0.759-0.989) for external validation, respectively. A simple risk-scoring tool was developed with a total risk score ranging from -3 to 4, corresponding to a risk of PIN from 0.79% to 81.24%. CONCLUSIONS: This study established a prediction model for PIN. Before using polymyxins, the simple risk-scoring tool can effectively identify patients at risk of developing PIN within a range of 7% to 65%.

Analysis of mcr family of colistin resistance genes in Gram-negative isolates from a tertiary care hospital in India.

AIM: Colistin serves as the drug of last resort for combating numerous multidrug-resistant (MDR) Gram-negative infections. Its efficacy is hampered by the prevalent issue of colistin resistance, which severely limits treatment options for critically ill patients. Identifying resistance genes is crucial for controlling resistance spread, with horizontal gene transfer being the primary mechanism among bacteria. This study aimed to assess the prevalence of plasmid-mediated mcr genes associated with colistin resistance in Gram-negative bacteria, utilizing both genotypic and phenotypic tests. METHODS AND RESULTS: The clinical isolates (n = 913) were obtained from a tertiary care center in Chennai, India. Colistin resistance was seen among Gram-negative isolates. These strains underwent screening for mcr-1, mcr-3, mcr-4, and mcr-5 genes via conventional PCR. Additionally, mcr-positive isolates were confirmed through Sanger sequencing and phenotypic testing. The bacterial isolates predominantly comprised Klebsiella pneumoniae (62.43%), Escherichia coli (19.71%), Pseudomonas aeruginosa (10.73%), and Acinetobacter baumannii (4.81%), along with other species. All isolates exhibited multidrug resistance to three or more antibiotic classes. Colistin resistance, determined via broth microdilution (BMD) using CLSI guidelines, was observed in 13.08% of the isolates studied. Notably, mcr-5 was detected in K. pneumoniae in PCR, despite its absence in Sanger sequencing and phenotypic tests (including the combined-disk test, colistin MIC in the presence of EDTA, and Zeta potential assays). This finding underscores the importance of employing multiple diagnostic approaches to accurately identify colistin resistance mechanisms.

L-carnitine co-administration prevents colistin-induced mitochondrial permeability transition and reduces the risk of acute kidney injury in mice.

Colistin is a polymyxin antibiotic currently experiencing renewed clinical interest due to its efficacy in the treatment of multidrug resistant (MDR) bacterial infections. The frequent onset of acute dose-dependent kidney injury, with the potential of leading to long-term renal damage, has limited its use and hampered adequate dosing regimens, increasing the risk of suboptimal plasma concentrations during treatment. The mechanism of colistin-induced renal toxicity has been postulated to stem from mitochondrial damage, yet there is no direct evidence of colistin acting as a mitochondrial toxin. The aim of this study was to evaluate whether colistin can directly induce mitochondrial toxicity and, if so, uncover the underlying molecular mechanism. We found that colistin leads to a rapid permeability transition of mitochondria isolated from mouse kidney that was fully prevented by co-incubation of the mitochondria with desensitizers of the mitochondrial transition pore cyclosporin A or L-carnitine. The protective effect of L-carnitine was confirmed in experiments in primary cultured mouse tubular cells. Consistently, the relative risk of colistin-induced kidney damage, calculated based on histological analysis as well as by the early marker of tubular kidney injury, Kim-1, was halved under co-administration with L-carnitine in vivo. Notably, L-carnitine neither affected the pharmacokinetics of colistin nor its antimicrobial activity against relevant bacterial strains. In conclusion, colistin targets the mitochondria and induces permeability transition thereof. L-carnitine prevents colistin-induced permeability transition in vitro. Moreover, L-carnitine co-administration confers partial nephroprotection in mice treated with colistin, without interfering with its pharmacokinetics and antibacterial activity.