Colistin Concentrations Research Articles

Antibacterial activity and mechanism of colistin-loaded polymeric nanoparticles for combating multidrug-resistant Pseudomonas aeruginosa biofilms: A synergistic approach

Multidrug-resistant P. aeruginosa (MDR-P. aeruginosa), associated with elevated morbidity, mortality, and readmission rates, presents a formidable challenge to eradication due to its robust resistance to antimicrobial agents and biofilm formation. Herein, self-assembling nanoparticles (NO-PE/PLL NPs) comprised of NO donor-conjugated γ-polyglutamic acid (GSNO-PGA), epsilon-poly-l-lysine (PLL) and colistin were fabricated. The negatively charged NO-PE/PLL NPs exhibited effective penetration through airway mucus, reaching the infection site where GSNO-PGA released NO in response to glutathione within biofilm. PLL worked synergistically with colistin (fractional inhibitory concentration index: 0.281), reducing the minimum inhibitory concentration (MIC) of colistin from 2 to 0.5 μg/mL. Benefiting from this synergistic antibacterial action and NO-mediated biofilm disruption, NO-PE/PLL NPs achieved a 99.99 % eradication rate against MDR-P. aeruginosa biofilms. Additionally, NO-PE/PLL NPs efficiently inhibited endotoxins-stimulated inflammation response. In a chronic pulmonary infection model, NO-PE/PLL NPs displayed the highest eradication efficiency (99.78 %) to infected mice, while having no adverse effects on their major organs or pulmonary functions. These results highlight NO-PE/PLL NPs as a promising therapeutic strategy for treating recalcitrant infections caused by MDR-P. aeruginosa biofilms.

Assessment of inhaled cationic antibiotics in an in vitro model of Pseudomonas aeruginosa lung biofilm

ObjectivesThis study aimed to evaluate the efficacy of inhaled cationic antibiotics, including tobramycin (TOB) and colistin (CST), using an in vitro alginate bead model that simulates Pseudomonas aeruginosa lung biofilms. MethodsBioluminescent P. aeruginosa were encapsulated within alginate beads and dispersed in either Mueller-Hinton broth (MHB) or artificial sputum medium (ASM). The impact of bead size and culture medium on TOB and CST efficacy was assessed by monitoring bioluminescence kinetics, followed by colony-forming unit (CFU/mL) measurements. Antibiotic efficacy was quantified using a Hill inhibitory model to analyze variations in CFU/mL in response to TOB and CST concentrations. ResultsThe TOB EC50 was found to be 8-fold higher when P. aeruginosa was encapsulated in larger beads (1200 μm) compared to smaller beads (60 μm). TOB efficacy further decreased twofold when larger beads were dispersed in ASM. In contrast, CST demonstrated superior efficacy, being four times more potent than TOB, with corresponding EC50 values of 20.5 ± 2.8 times MIC and 78.4 ± 10.2 times MIC, respectively. No change in MICs was observed for either antibiotic, even after exposing bacteria to 200 times the MIC. ConclusionsThis P. aeruginosa biofilm model highlights how alginate and mucus modulated the efficacy of TOB and CST, and suggested the superior efficacy of CST in eradicating pulmonary biofilms.

Emerging Colistin Resistance: Experience from an Intensive Care Unit of a Tertiary Care Center in India.

Resistance to the currently available classes of antimicrobials has emerged as a major health concern, with a growing number of isolated organisms demonstrating resistance to available antimicrobials. With the escalation of carbapenem resistance, the use of polymyxin B/E (colistin) has increased over the years, which has, in turn, contributed to the worldwide emergence of colistin resistance. The available data on colistin resistance from Southeast Asian nations, including India, is limited, especially in intensive care unit (ICU) settings. The primary objective of our study was to analyze the clinical profile of patients with polymyxin B/E (colistin) resistant positive cultures and to study their outcome in terms of length of ICU stay and outcome at discharge. This observational, single-center, prospective study was conducted in a 20-bed adult ICU serving both medical and surgical patients at a 1,600-bed tertiary care institute in northern India between Jan 2020 and Dec 2021. In this study, all adult patients, defined as individuals older than 18 years of age, admitted to our ICU with cultures detecting polymyxin B/E (colistin) resistant organisms were included as cases, and all adult ICU patients (age >18 years) with polymyxin B/E (colistin) sensitive cultures were taken as controls. Clinical, laboratory, and demographic parameters, along with ICU variables like severity of illness, length of ICU stay, mechanical ventilation, days of shock, renal replacement therapy (RRT), etc., and outcome at discharge were collected. Identification of resistance to colistin and minimal inhibitory concentration (MIC) of colistin is based on Clinical and Laboratory Standards Institute (CLSI) guidelines. For statistical analysis, Mann-Whitney U test, Fisher's exact test, and binary logistic regression were used. Twenty-eight cases and 55 controls (n = 83) were included for analysis. Abdominal [gastrointestinal (GI)] sepsis was the most common diagnosis (41.8%) at admission to the ICU, and Klebsiella pneumoniae was the most common species isolated in both the cases (96.4%) and the controls (50.9%). The most common site of isolation of Gram-negative bacteria in both cases and controls was blood (71.4 vs 74.5%), followed by deep-seated pus (21.4 vs 23.64%). The most common class of drugs to which the cases were sensitive was tetracyclines in 60.7%, followed by ceftazidime-avibactam. Factors such as prior exposure to colistin, exposure to monobactams in the ICU, and ICU stay in days were identified as independent predictors for colistin resistance. Overall mortality was not statistically different between cases and controls (p -value 0.38). Factors like prolonged ICU stay, exposure to monobactams in the ICU, and a history of prior exposure to colistin in the previous 90 days are independent predictors of colistin resistance. The most common organism to develop colistin resistance identified was K. pneumoniae, and the most common site of isolation was blood. There was no difference in mortality between colistin-resistant and colistin-sensitive patients.

Изменение бактериального фитнеса в ходе экспериментальной адаптации Pseudomonas aeruginosa к колистину

Pseudomonas aeruginosa, the opportunistic pathogen, occupies one of the leading places in the structure of pathogens causing nosocomial infections, which is due to high adaptive potential and the ability to quickly develop antimicrobial resistance. The study aimed to assess the influence of the P. aeruginosa adaptation to colistin on bacterial fitness. A total of nine isolates obtained during the experimental evolution of the P. aeruginosa strain (laboratory number 1202) under conditions of increasing colistin concentrations, the growth kinetics of which was compared to that of wild type strain, were included in the study; the whole genome sequencing of all isolates was performed, and the minimum inhibitory concentration of colistin was determined. Growth rate was estimated using the Varioskan LUX multimodal reader (Thermo Scientific, USA) throughout 18 h at 37 °С; optical density (OD) at λ = 600 nm was measured every 15 min. The maximum growth rate (GRmax, i.e. the maximum change in OD within 1h) and the time to reach 50% of the maximum OD reported when growing the wild type Ра_1202_0 strain (T_OD50%) were considered. Isolates of the clone carrying mutations of the genes phoQ, lptA, and prs showed low fitness values compared to wild type strains. For example, GRmax of the isolate Ра_1202_43 was 0.029 OD/h vs. 0.182 OD/h reported for the original isolate Ра_1202_0, and it reached OD50% 4.6 h later. The growth characteristics of the clone carrying mutations of lpxL and lptB, as well as the clone carrying mutant pmrB were generally comparable with the characteristics of the wild type strain. Thus, the genome modifications observed during the P. aeruginosa adaptation to colistin have an ambiguous effect on bacterial fitness.

High-dose colistin pharmacokinetics in critically ill patients receiving continuous renal replacement therapy

BackgroundColistin, administered as intravenous colistimethate (CMS), is still used in the critical care setting and current guidelines recommend high dosage CMS in patients undergoing continuous renal replacement therapy (CRRT). Due to the paucity of real-life data, we aimed to describe colistin pharmacokinetic/pharmacodynamic (PK/PD) profile in a cohort of critically ill patients with infections due to carbapenem-resistant (CR) bacteria undergoing CRRT.ResultsAll consecutive patients admitted to three Intensive Care Units (ICUs) of a large metropolitan University Hospital, treated with colistin for at least 48 h at the dosage of 6.75 MUI q12, after 9 MIU loading dose, and undergoing CRRT were included. After the seventh dose, patients underwent blood serial sampling during a time frame of 24 h. We included 20 patients, who had CR-Acinetobacter baumannii ventilator-associated pneumonia and were characterized by a median SAPS II and SOFA score of 41 [34.5–59.3] and 9 [6.7–11], respectively. Fifteen patients died during ICU stay and six recovered renal function. Median peak and trough colistin concentrations were 16.6 mcg/mL [14.8–20.6] and 3.9 mcg/mL [3.3–4.4], respectively. Median area under the time–concentration curve (AUC0 − 24) and average steady-state concentration (Css, avg) were 193.9 mcg h/mL [170.6–208.6] and 8.07 mcg/mL [7.1–8.7]. Probability of target attainment of colistin pharmacodynamics according to the fAUC0 − 24/MIC target ≥ 12 was 100% for MIC ≤ 2 mcg/mL and 85% for MIC = 4 mcg/ML, although exceeding the toxicity limit of Css, avg 3–4 mcg/mL.ConclusionsIn critically ill patients with CR infections undergoing CRRT, recommended CMS dosage resulted in colistin plasmatic levels above bacterial MIC90, but exceeding the safety Css, avg. limit.Trial registrationThis trial was registered in ClinicalTrials.gov on 23/07/2021 with the ID NCT04995133 (https//clinicaltrials.gov/study/NCT04995133).

The effect of combinations of a glyphosate-based herbicide with various clinically used antibiotics on phenotypic traits of Gram-negative species from the ESKAPEE group

The emission of glyphosate and antibiotic residues from human activities threatens the diversity and functioning of the microbial community. This study examines the impact of a glyphosate-based herbicide (GBH) and common antibiotics on Gram-negative bacteria within the ESKAPEE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli). Ten strains, including type and multidrug-resistant strains for each species were analysed and eight antibiotics (cefotaxime, meropenem, aztreonam, ciprofloxacin, gentamicin, tigecycline, sulfamethoxazole-trimethoprim, and colistin) were combined with the GBH. While most combinations yielded additive or indifferent effects in 70 associations, antagonistic effects were observed with ciprofloxacin and gentamicin in five strains. GBH notably decreased the minimum inhibitory concentration of colistin in eight strains and displayed synergistic activity with meropenem against metallo-β-lactamase (MBL)-producing strains. Investigation into the effect of GBH properties on outer membrane permeability involved exposing strains to a combination of this GBH and vancomycin. Results indicated that GBH rendered strains sensitive to vancomycin, which is typically ineffective against Gram-negative bacteria. Furthermore, we examined the impact of GBH in combination with three carbapenem agents on 14 strains exhibiting varying carbapenem-resistance mechanisms to assess its effect on carbapenemase activity. The GBH efficiently inhibited MBL activity, demonstrating similar effects to EDTA (ethylenediaminetetraacetic acid). Chelating effect of GBH may have multifaceted impacts on bacterial cells, potentially by increasing outer membrane permeability and inactivating metalloenzyme activity.

Synergism of colistin and globular endolysins against multidrug-resistant gram-negative bacteria

Endolysins (lysins), a novel class of antibacterial agents derived from bacteriophages, efficiently lyse bacteria by degrading the peptidoglycan layer within the bacterial wall. Colistin, a classic peptide antibiotic with the ability to permeabilize the outer membrane, has recently shown great promise in synergizing with lysins against gram-negative bacteria. However, the exact mechanisms responsible for their synergy remain unclear. Here, we first demonstrated the synergistic bacterial killing of various lysin and colistin combinations. With a model lysin, LysAB2, we then confirmed that there is a threshold concentration of colistin causing sufficient permeabilization of the outer membrane for lysin to access the peptidoglycan layer and subsequently exert its lytic ability. The threshold colistin concentrations were found to range 0.2−0.8 μM for the tested bacteria, with the exact value largely depending on the density of lipopolysaccharides on the outer membrane. Beyond the threshold colistin level, LysAB2 could synergize with colistin at a concentration as low as 0.31 μM. Next, we proved for the first time that lysin-induced degradation of the peptidoglycan layer facilitated the disruption of cytoplasmic membrane by colistin, elevated the level of reactive oxygen species in bacterial cells, and boosted the killing effect of colistin. Additionally, the colistin-lysin combination could effectively eliminate established biofilms due to the biofilm dispersal ability of lysin. The in-vivo efficacy was preliminary confirmed in a Galleria mellonella infection model for combination with colistin doses (≥ 1.8 μg/larvae), which could reach beyond the threshold concentration, and a fixed LysAB2 dose (10 μg/larvae). In summary, our study provided the first experimental evidence unravelling the mechanisms behind the synergy of colistin and lysins. All these findings provided important insights in guiding the dosing strategy for applying this combination in future development.

Alteration in the Morphological and Transcriptomic Profiles of Acinetobacter baumannii after Exposure to Colistin.

Acinetobacter baumannii is often highly resistant to multiple antimicrobials, posing a risk of treatment failure, and colistin is a "last resort" for treatment of the bacterial infection. However, colistin resistance is easily developed when the bacteria are exposed to the drug, and a comprehensive analysis of colistin-mediated changes in colistin-susceptible and -resistant A. baumannii is needed. In this study, using an isogenic pair of colistin-susceptible and -resistant A. baumannii isolates, alterations in morphologic and transcriptomic characteristics associated with colistin resistance were revealed. Whole-genome sequencing showed that the resistant isolate harbored a PmrBL208F mutation conferring colistin resistance, and all other single-nucleotide alterations were located in intergenic regions. Using scanning electron microscopy, it was determined that the colistin-resistant mutant had a shorter cell length than the parental isolate, and filamented cells were found when both isolates were exposed to the inhibitory concentration of colistin. When the isolates were treated with inhibitory concentrations of colistin, more than 80% of the genes were upregulated, including genes associated with antioxidative stress response pathways. The results elucidate the morphological difference between the colistin-susceptible and -resistant isolates and different colistin-mediated responses in A. baumannii isolates depending on their susceptibility to this drug.

Genetic Alterations Associated with Colistin Resistance Development in Escherichia coli.

Background: The increased incidence of infections due to multidrug-resistant Gram-negative bacteria has led to the renewed interest in the use of 'forgotten' antibiotics such as colistin. In this work, we studied the chromosomal colistin resistance mechanisms among laboratory-induced colistin-resistant Escherichia coli isolates. Methods: Three colistin-susceptible (ColS) clinical isolates of E. coli assigning to ST131, ST405, and ST361 were exposed to successively increasing concentrations of colistin. The nucleotide sequences of pmrA, pmrB, pmrD, phoP, phoQ, and mgrB genes were determined. The fitness burden associated with colistin resistance acquisition was determined by measuring the in vitro growth rate. Results: Colistin resistance induction resulted in 16-64 times increase in colistin MICs in mutants (n = 8) compared with parental isolates. Analysis of chromosomal genes in colistin-resistant mutants compared with those of ColS ancestors revealed genetic alterations confined to PmrAB two-component system and included PmrA G53R/R81S/L105P and PmrB E121K/E121A/A159P/A159V/G302E changes. The PmrB E121 was found as a critical position for colistin resistance development being altered in three mutants with different ancestors. The acquired colistin-resistance phenotype was stable following 10 consecutive passages in the absence of selective pressure of colistin and it did not alter the susceptibility of mutants to other antimicrobial agents. All mutants exhibited growth rates similar to their respective ColS ancestors, except for one isolate, which revealed a significant growth defect. Conclusion: Our results revealed that colistin resistance in E. coli was more related to PmrAB alterations, which did not impose a fitness cost in most cases.

Mutations in the pmrB gene constitute the major mechanism underlying chromosomally encoded colistin resistance in clinical Escherichia coli

ObjectivesThe mechanisms underlying chromosomally encoded colistin resistance in Escherichia coli remain insufficiently investigated. In this study, we investigated the contribution of various pmrB mutations from E. coli clinical isolates to colistin resistance. MethodsThe resistance mechanisms in eight mcr-negative colistin-resistant E. coli isolates obtained from a nationwide surveillance program in Taiwan using recombinant DNA techniques and complementary experiments were investigated. The minimal inhibitory concentrations (MICs) of colistin in the recombinant strains were compared with those in the parental strains. The expression levels of pmrA and pmrK (which are part of the pmrCAB and pmrHFIJKLM operons associated with colistin resistance) were measured using reverse transcription-quantitative real-time polymerase chain reaction. ResultsIn the complementation experiments, various mutated pmrB alleles from the eight mcr-negative colistin-resistant E. coli strains were introduced into an ATCC25922 mutant with a PmrB deletion, which resulted in colistin resistance. The MIC levels of colistin in the most complemented strains were comparable to those of the parental colistin-resistant strains. Increased expression levels of pmrA and pmrK were consistently detected in most complemented strains. The impact for colistin resistance was confirmed for various novel amino acid substitutions, P94L, G19E, L194P, L98R and R27L in PmrB from the parental clinical strains. The detected amino acid substitutions are distributed in the different functional domains of PmrB. ConclusionsColistin resistance mediated by amino acid substitutions in PmrB is a major chromosomally encoded mechanism in E. coli of clinical origin.

Colistin Resistance Mediated by Mcr-3-Related Phosphoethanolamine Transferase Genes in Aeromonas Species Isolated from Aquatic Environments in Avaga and Pakro Communities in the Eastern Region of Ghana.

Colistin is classified by the World Health Organization (WHO) as a critically important and last-resort antibiotic for the treatment of infections caused by carbapenem-resistant bacteria. However, colistin resistance mediated by chromosomal mutations or plasmid-linked mobilized colistin resistance (mcr) genes has emerged. Thirteen mcr-positive Aeromonas species isolated from water samples collected in Eastern Ghana were analyzed using whole-genome sequencing (WGS). Antimicrobial susceptibility was tested using the broth microdilution method. Resistome analysis was performed in silico using a web-based platform. The minimum inhibitory concentration (MIC) of colistin for all except three isolates was >4 µg/mL. Nine new sequence types were identified and whole-genome analysis revealed that the isolates harbored genes (mcr-3-related genes) that code for Lipid A phosphoethanolamine transferases on their chromosomes. BLAST analysis indicated that the amino acid sequences of the mcr-3-related genes detected varied from those previously reported and shared 79.04-99.86% nucleotide sequence identity with publicly available mcr-3 variants and mcr-3-related phosphoethanolamine transferases. Analysis of the genetic context of mcr-3-related genes revealed that the genetic environment surrounding mcr-3-related genes was diverse among the different species of Aeromonas but conserved among isolates of the same species. Mcr-3-related-gene-IS-mcr-3-related-gene segment was identified in three Aeromonas caviae strains. The presence of mcr-3-related genes close to insertion elements is important for continuous monitoring to better understand how to control the mobilization and dissemination of antibiotic resistance genes.

In vitro interactions of combinations of colistin with meropenem, rifampicin and tigecycline in colistin-resistant, biofilm-forming Klebsiella pneumoniae

In this study, it was aimed to reveal the in vitro interactions of combinations of colistin with meropenem, rifampicin and tigecycline in colistin-resistant, biofilm-forming Klebsiella pneumonia. A total of 30 isolates, 15 of which formed biofilms and 15 did not form biofilms, were randomly selected from K. pneumoniae isolates growing in blood samples. The synergy rates of colistin-meropenem, colistin-tigecycline, colistin-rifampicin combinations in planktonic/sessile bacteria are; It was determined as 83,3%/73,3%, 66,6%/33,3%, 100%/60% respectively. Biofilm inhibitory concentration (BIC) of colistin, meropenem, tigecycline, and rifampicin significantly increased after biofilm formation. The synergistic activity seen in the sessile form was independent of the planktonic form. Although a high synergistic effect was observed in the meropenem-colistin combination on sessile bacteria, colistin had very high BIC ​​in all combinations. Large-scale studies are needed in which the number of isolates studied is large, bacterial resistance profiles are evaluated genomically, and various antimicrobial groups are included.

Implementation of Modern Therapeutic Drug Monitoring and Lipidomics Approaches in Clinical Practice: A Case Study with Colistin Treatment.

Nowadays, lipidomics plays a crucial role in the investigation of novel biomarkers of various diseases. Its implementation into the field of clinical analysis led to the identification of specific lipids and/or significant changes in their plasma levels in patients suffering from cancer, Alzheimer's disease, sepsis, and many other diseases and pathological conditions. Profiling of lipids and determination of their plasma concentrations could also be helpful in the case of drug therapy management, especially in combination with therapeutic drug monitoring (TDM). Here, for the first time, a combined approach based on the TDM of colistin, a last-resort antibiotic, and lipidomic profiling is presented in a case study of a critically ill male patient suffering from Pseudomonas aeruginosa-induced pneumonia. Implementation of innovative analytical approaches for TDM (online combination of capillary electrophoresis with tandem mass spectrometry, CZE-MS/MS) and lipidomics (liquid chromatography-tandem mass spectrometry, LC-MS/MS) was demonstrated. The CZE-MS/MS strategy confirmed the chosen colistin drug dosing regimen, leading to stable colistin concentrations in plasma samples. The determined colistin concentrations in plasma samples reached the required minimal inhibitory concentration of 1 μg/mL. The complex lipidomics approach led to monitoring 545 lipids in collected patient plasma samples during and after the therapy. Some changes in specific individual lipids were in good agreement with previous lipidomics studies dealing with sepsis. The presented case study represents a good starting point for identifying particular individual lipids that could correlate with antimicrobial and inflammation therapeutic management.

Investigation of the Rapid Emergence of Colistin Resistance in a Newborn Infected with KPC-2–Producing Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae

ObjectivesHypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKp) poses a significant threat to public health. This study reports an infection related to hv-CRKp in a premature infant and reveals its colistin resistance and evolutionary mechanisms within the host. MethodsThree KPC-producing CRKp strains were isolated from a patient with sepsis and CRKp osteoarthritis who had been receiving colistin antimicrobial therapy. The minimum inhibitory concentrations (MICs) of ceftazidime, ceftazidime-avibactam (CAZ-AVI), meropenem, imipenem, tigecycline, amikacin, minocycline, sulfamethoxazole/trimethoprim, ciprofloxacin, levofloxacin, aztreonam, cefepime, cefoperazone/sulbactam, piperacillin/tazobactam, and colistin were determined using the microbroth dilution method. The whole-genome sequencing analysis was conducted to determine the sequence types (STs), virulence genes, and antibiotic resistance genes of the three CRKp strains. ResultsWhole-genome sequencing revealed that all three CRKp strains belonged to the ST11 clone and carried a plasmid encoding blaKPC-2. The three strains all possessed the iucABCDiutA virulence cluster, peg-344 gene, and rmpA/rmpA2 genes, defining them as hv-CRKp. Further experiments and whole-genome analysis revealed that a strain of K. pneuomniae had developed resistance to colistin. The mechanism found to be responsible for colistin resistance was a deletion mutation of approximately 9000 bp including the mgrB gene. ConclusionThis study characterizes colistin resistance of the ST11 clone hv-CRKp during colistin treatment and its rapid evolution within the host.

Colistin, the last resort antibiotic: challenges in the implementation of its routine susceptibility testing

Background: colistin has become a critical antibiotic for lifethreatening multidrug resistance Gram-negative infections, particularly carbapenemase-producing bacteria. Detecting colistin resistance in routine microbiology laboratories is crucial for combating these fatal infections poses a challenge. Especially in developing countries, there is a need for a cost-effective, rapid, and user-friendly diagnostic method. Objective: implementing the various available methods for colistin testing is a significant challenge in resource-limited settings due to logistic difficulties and the need for technical expertise. Materials and Methods: this study shares experiences and insights gained while implementing in-vitro colistin susceptibility testing in a high-load bacteriology laboratory of a tertiary care center in Delhi, India. The following test methods for colistin susceptibility testing were incorporated in the routine antimicrobial susceptibility testing of our laboratory: Colistin Agar Test, Colistin Broth Disk Elution Test, Broth Microdilution susceptibility testing. Results: inconsistent growth patterns were observed in the colistin agar dilution Minimum Inhibitory Concentration (MIC) method, which could be resolved only after the preparation of fresh plates containing that specific concentration of colistin. The contamination issue of plates on use over a few days was addressed by pouring agar containing various concentrations of colistin in cottonplugged glass tubes. In the colistin broth disk elution test, due to the non-availability of screw-capped 10 mL glass tubes, MacCornety bottles (30 mL) were used. Subcultures were performed from the turbid wells to rule out the growth of contaminants when encountering discordant MIC values or skipped wells on the colistin broth microdilution test. Conclusions: despite several technical issues in in-vitro colistin susceptibility testing, we have successfully implemented it in our laboratory. Our experiences can offer guidance to laboratories that are still in the process of implementing it.

Genetic Alternatives for Experimental Adaptation to Colistin in Three Pseudomonas aeruginosa Lineages

Pseudomonas aeruginosa is characterized by a high adaptive potential, developing resistance in response to antimicrobial pressure. We employed a spatiotemporal evolution model to disclose the pathways of adaptation to colistin, a last-resort polymyxin antimicrobial, among three unrelated P. aeruginosa lineages. The P. aeruginosa ATCC-27833 reference strain (Pa_ATCC), an environmental P. aeruginosa isolate (Pa_Environment), and a clinical isolate with multiple drug resistance (Pa_MDR) were grown over an increasing 5-step colistin concentration gradient from 0 to 400 mg/L. Pa_Environment demonstrated the highest growth pace, achieving the 400 mg/L band in 15 days, whereas it took 37 and 60 days for Pa_MDR and Pa_ATCC, respectively. To identify the genome changes that occurred during adaptation to colistin, the isolates selected during the growth of the bacteria (n = 185) were subjected to whole genome sequencing. In total, 17 mutation variants in eight lipopolysaccharide-synthesis-associated genes were detected. phoQ and lpxL/PA0011 were affected in all three lineages, whereas changes in pmrB were found in Pa_Environment and Pa_MDR but not in Pa_ATCC. In addition, mutations were detected in 34 general metabolism genes, and each lineage developed mutations in a unique set of such genes. Thus, the three examined distinct P. aeruginosa strains demonstrated different capabilities and genetic pathways of colistin adaptation.

Comparative analysis of three methods for screening for colistin resistant <i>Escherichia coli</i>

The usage of colistin regarded as a drug of last resort has increased tremendously in recent years. This increase has been followed by an increase in the development of colistin resistant bacteria. Due the large size of colistin and its ability to adhere to plastic, the broth microdilution method using a special medium is the recommended testing method. Resource limited settings struggle with this method and employ alternate methods. This study therefore set out to determine colistin resistance in a group of Escherichia coli using three different methods comprised of colistin agar spot test (COL-AS), colistin drop test (COL-DT) and a disc diffusion method. A total of 51 Escherichia coli isolated from wound samples were screened for colistin resistance using the COLAS, COL-DT and colistin disc diffusion methods. Results showed a combined resistance rate of 96.1% among test isolates. Actual resistance rates varied between testing methods giving values of ranging from 37.3%, 66.0% and 88.2% for COL- DT, colistin disc diffusion methods and COL-A respectively. An assessment of test performance showed categorical agreement values and very major error values of 57.1%/36.7% for COL-DT and 63.3%/8.2% for COL-A. Results of this study show a high-level occurrence of colistin resistance among clinical Escherichia coli isolates. Furthermore, it demonstrates the superiority of the colistin agar test to the colistin drop test. It also points at a need to use higher concentrations of colistin in the screening tests.

Performance of modified colistin broth disc elution vis-a-vis broth microdilution method for susceptibility testing of Enterobacterales

Objectives: Recently, Clinical and Laboratory Standards Institute (CLSI) has approved colistin broth disc elution (CBDE) to be a supplemental test. This requires multiple discs and tubes to get the desired concentrations of colistin -1, 2, and 4 µg/mL and 10 mL volume of cation-adjusted Mueller–Hinton broth for a single isolate. The present study was aimed to evaluate the performance of CBDE in a microtiter plate format modified (mCBDE) with the reference method broth microdilution (BMD) for detection of colistin resistance in carbapenem-resistant Enterobacterales (CRE) isolates. Materials and Methods: One hundred and sixty non-duplicate clinical CRE isolates (May 2021–April 2022) were simultaneously subjected for BMD and mCBDE. For mCBDE, colistin 10 µg discs and Mueller–Hinton broth no-2 control cations were procured from HiMedia, Mumbai, and drug concentrations were prepared following CLSI-M100Ed31. Results of mCBDE were compared with reference BMD (Minimum inhibitory concentration [MIC] ≤2 µg/mL – intermediate and ≥4 µg/mL – resistant). Statistical Analysis: The performance of mCBDE was compared with BMD and expressed in terms of Categorical, essential agreement (EA), very major error (VME), and major error (ME). The sensitivity and specificity were calculated using Fisher’s contingency Table. Results: Of the 160 CRE isolates, 152 had exactly the same minimal inhibitory concentration (MIC) in both the tests with four isolates having higher and four having lower colistin MIC by mCBDE, giving a major error of 2.1% and VME of 5.5%. Categorical and essential agreement of mCBDE were 97.5% and 98.7%, respectively. Conclusions: mCBDE is an easy, economical, and reliable alternative test for determining colistin susceptibility for CRE isolates. Further, large-scale study is needed to strengthen our observation.

Abundance of Colistin-Resistance Genes in Retail Meats in Vietnam.

The degree of contamination of retail meat with colistin-resistant bacteria and its potential contribution to dissemination within communities remains to be determined. Thus, we aimed to elucidate the contamination status of colistin-resistance genes, indicative of colistin-resistant bacteria, in retail meats in Vietnam. In total, 46 chicken and 49 pork meats from stores in Vietnam and Japan were examined. Multiplex real-time polymerase chain reaction with TaqMan probes was performed for detecting mcr-1, mcr-3, and Escherichia coli 16S rRNA. Colistin-resistant bacteria in meats were isolated using selective media. The minimum inhibitory concentrations of colistin were determined using the broth microdilution method. The results showed that 70.7% of chicken meats in Vietnam were contaminated with both mcr-1 and mcr-3. Meanwhile, mcr-1 and mcr-3 were detected in 15.9% and 40.9% of pork meat, respectively. Only mcr-3 was detected in 40% of chicken in Japan. In addition, mcr-1-harboring E. coli and mcr-3-harboring Aeromonas were isolated from chicken meats in Vietnam. Some of these isolates showed colistin resistance. These results showed that most retail meats were highly contaminated with colistin-resistance genes. Notably, our results suggest that mcr-3 is more prevalent in the contaminated samples compared with mcr-1.

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

BackgroundThe 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.ResultsIn 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.ConclusionWhile 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.