Connecting Gut Microbial Diversity with Plasma Metabolome and Fecal Bile Acid Changes Induced by the Antibiotics Tobramycin and Colistin Sulfate.

The diversity of microbial species in the gut has a strong influence on health and development of the host. Further, there are indications that the variation in expression of gut bacterial metabolic enzymes is less diverse than the taxonomic profile, underlying the importance of microbiome functionality, particularly from a toxicological perspective. To address these relationships, the gut bacterial composition of Wistar rats was altered by a 28 day oral treatment with the antibiotics tobramycin or colistin sulfate. On the basis of 16S marker gene sequencing data, tobramycin was found to cause a strong reduction in the diversity and relative abundance of the microbiome, whereas colistin sulfate had only a marginal impact. Associated plasma and fecal metabolomes were characterized by targeted mass spectrometry-based profiling. The fecal metabolome of tobramycin-treated animals had a high number of significant alterations in metabolite levels compared to controls, particularly in amino acids, lipids, bile acids (BAs), carbohydrates, and energy metabolites. The accumulation of primary BAs and significant reduction of secondary BAs in the feces indicated that the microbial alterations induced by tobramycin inhibit bacterial deconjugation reactions. The plasma metabolome showed less, but still many alterations in the same metabolite groups, including reductions in indole derivatives and hippuric acid, and furthermore, despite marginal effects of colistin sulfate treatment, there were nonetheless systemic alterations also in BAs. Aside from these treatment-based differences, we also uncovered interindividual differences particularly centering on the loss of Verrucomicrobiaceae in the microbiome, but with no apparent associated metabolite alterations. Finally, by comparing the data set from this study with metabolome alterations in the MetaMapTox database, key metabolite alterations were identified as plasma biomarkers indicative of altered gut microbiomes resulting from a wide activity spectrum of antibiotics.

Whole-genome epidemiology and characterisation of mcr-1-encoding Escherichia coli in aquatic bird farms from the Pearl River Delta, China, 2019-2020.

Due to their unique breeding pattern, aquatic bird farms are increasingly considered as hotspots in the development and spread of antimicrobial resistance. However, comprehensive studies addressing the whole-genomic features of colistin-resistant bacteria in aquatic bird farms are scarce. Over a 2-year period, we conducted surveillance to determine the whole-genome epidemiology and characterisation of mcr-1-positive Escherichia coli in aquatic bird farms in southeastern coastal China. A total of 100 mcr-1-producing isolates among 654 E. coli strains were recovered from 781 samples collected in 11 aquatic bird farms and 1 veterinary clinic in the Pearl River Delta area. Higher resistance phenotypes to 17 antibiotics were found in mcr-1-positive isolates compared with other isolates. Subsequently, 20 mcr-1-carrying isolates were sequenced to analyse the whole-genomic features. Molecular typing as well as antimicrobial resistance gene and virulence factor profiles of the isolates showed considerable diversity. Three types of genetic backbones of mcr-1 in the isolates were assembled and were identified in diverse broad-host-range plasmids and bacterial species. Pangenome analyses revealed a large genetic pool composed of the isolates. Furthermore, phylogenetic trees both of the isolates in this study and a global data set were built, indicating the spread of the three mcr-1 backbones and the mcr-1-positive isolates among different habitats, farms and even countries. This study highlights that aquatic bird farms may act as an important reservoir for mcr-1-producing E. coli, from which colistin resistance may be spread to diverse habitats, different geographical locations and even across bacterial species.

The surveillance of colistin resistance and mobilized colistin resistance genes in multidrug-resistant Enterobacteriaceae isolated in Japan.

BACKGROUND: The plasmid-mediated bacterial colistin-resistant gene, mcr, is of global concern in clinical healthcare. However, there are few reports of surveillance for mcr in Japan. The aim of this study was to assess the prevalence of colistin resistance by identifying nine mcr genes in extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae and carbapenem-resistant Enterobacteriaceae (CRE) isolates in Japan. METHODS: A total of 273 ESBL and CRE clinical isolates were collected from patients in five tertiary hospitals from August 2016 to March 2017. Minimum inhibitory concentration (MIC) of colistin was measured using the microdilution method. Polymerase chain reaction (PCR) was performed to detect mcr-1 to mcr-9 genes in all strains. Whole-genome sequencing (WGS) analysis was conducted for any mcr-genes identified that had not been previously reported in patients from Japan. RESULTS: The rate of colistin resistance was 7.7% in all strains, with a higher rate in the CRE strains than in the ESBL-producing strains (20.4% versus 1.1%). The mcr-5 and mcr-9 gene were detected in one ESBL-producing Escherichia coli strain (1/273, 0.37%) and three CRE strains (3/273, 1.1%), respectively. As the ESBL-producing E. coli strain was the first clinical strain with mcr-5 in Japan, WGS analysis was performed for the strain. The sequence type of the mcr-5-positive strain was ST1642 and it carried two distinct plasmids, ESBL gene-carrying pN-ES-6-1, and mcr-5.1-carrying pN-ES-6-2. CONCLUSIONS: The results of this study showed that the frequency of colistin resistance and mcr-positive strains is not high in Japan. As the MIC for colistin was low in the mcr-5.1 and mcr-9 gene-positive strain, continuous monitoring of mcr genes is necessary.

Development of a liquid chromatography-tandem mass spectrometry method to determine colistin, bacitracin and virginiamycin M1 at cross-contamination levels in animal feed.

Cross-contamination of animal feed with antibiotics may occur during manufacturing in feed mills, because shared production lines can be used for medicated and non-medicated feed, but may also occur during transport, storage and at the farm level. This is a major issue in the current context where antimicrobial usage must be controlled in order to maintain their effectiveness. A LC-MS/MS method was developed for the determination of colistin, bacitracin A and virginiamycin M1 in feed for pigs, poultry and rabbits at concentrations similar to those encountered in cross-contamination. After investigating various issues related to colistin behaviour and matrix effects, we successfully validated this method according to the requirements of European regulations in terms of linearity, trueness, precision, limit of quantification and limit of decision. Trueness ranged 88.6-107.8% and precision ranged 12.6-21.2%. We then applied this method to the analysis of medicated pig feed to check the performance of the method on "real" samples of medicated feed. We subsequently analysed non-medicated pig, and rabbit feed samples, collected directly on farms, to check the rate of cross-contamination. No samples were contaminated by colistin, bacitracin, or virginiamycin.

A portable fluorescent microsphere-based lateral flow immunosensor for the simultaneous detection of colistin and bacitracin in milk.

The polypeptide antibiotics colistin (COL) and bacitracin (Baci) are extensively used as veterinary drugs and feedstock additives in the livestock industry, which inevitably causes residues in animal-origin food, which can accelerate human tolerance to antibiotics. In this study, a portable lateral flow immunoassay (LFIA) for the simultaneous determination of COL and Baci residues in milk was developed. The replacement of gold nanoparticles used in the traditional LFIA with fluorescent microspheres (FMs) to label monoclonal antibodies (mAbs) allowed qualitative and quantitative analyses within a few minutes. Based on the principle of competitive binding to FM-labelled mAbs between analytes in samples and fixed antigens on the membrane, the assay provided qualitative cut-off values of 100 and 50 ng mL-1 for Baci and COL in milk samples. Furthermore, a strip reader-based semi-quantitative detection system could detect lower limits of 7.85 and 1.89 ng mL-1 for Baci and COL, respectively. In conclusion, the proposed multiplex LFIA immunosensor provides an auxiliary analytical tool for the rapid and simultaneous screening of COL and Baci in large cohorts of samples.

Evaluation of three ELISA kits for the screening of colistin residue in porcine and poultry muscle according to the European guideline for the validation of screening methods.

Colistin is a polypeptide antibiotic mainly used in porcine and poultry to treat gastrointestinal infections. It has been included by the World Health Organisation (WHO) in the list of critically important human antibiotics of high priority for antimicrobial resistance since 2017. Therefore, it is necessary to develop specific and sensitive screening methods for this molecule. Screening for colistin with immunoassays is an interesting alternative to LC-MS/MS screening methods. The performance of three commercially available ELISA kits was evaluated in poultry and porcine muscles for the detection of colistin in regards to its European maximum residue limit (MRL) (150 µg/kg). The applicability of the three ELISA kits to the detection of colistin at or below the MRL in porcine and poultry muscles was demonstrated. The detection capabilities (CCß) of two kits were or lower than or equal to the MRL (150 µg/kg). The lowest detection capability (30 µg/kg) was achieved with the third ELISA kit. The specificity of the three kits was very satisfactory (false positive rates 0%). The three kits are very specific for the detection of colistin (colistin A and B) and polymyxin B.

A fluorescent artificial receptor with specific imprinted cavities to selectively detect colistin.

A novel and facile fluorescent artificial receptor on the basis of the molecularly imprinted polymer-coated graphene quantum dots was engineered successfully to detect colistin. The colistin imprinted graphene quantum dots (CMIP-GQDs) was synthesized by vinyl-based radical polymerization between functional monomers and crosslinker at around the template molecule on the surface of graphene quantum dots. The size of bare, CNIP-GQDs, and CMIP-GQDs was about 4.8 ± 0.6 nm, 18.4 ± 1.7 nm, and 19.7 ± 1.3 nm, respectively. The CMIP-GQDs, which showed the strong fluorescence emission at 440 nm with the excitation wavelength fixed at 380 nm, had excellent selectivity and specificity to rapidly recognize and detect colistin. The linear range of fluorescence quenching of this fluorescent artificial receptor for detection colistin was 0.016-2.0 µg mL-1 with a correlation coefficient (R2) of 0.99919, and the detection limit was 7.3 ng mL-1 in human serum samples. The designed receptor was successfully applied to detect colistin in human serum samples and it achieved excellent recoveries shifted from 93.8 to 105%. Graphical abstract.

Quantitative determination of colistin A/B and colistin methanesulfonate in biological samples using hydrophilic interaction chromatography tandem mass spectrometry.

Colistin (polymyxin E) is a polycation antibiotic which is increasingly used (administered as colistin methanesulfonate, CMS) as a salvage therapy in critically ill patients with multidrug resistant Gram-negative infections. Even though colistin has been used for more than 50 years, its metabolic fate is poorly understood. One of the current challenges for studying the pharmacokinetics (PK) is the precise and accurate determination of colistin in in vitro and in vivo studies. In the present study, we developed and validated a series of sensitive and robust liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for analysing biological samples obtained from in vitro and in vivo disposition assays. After a zinc acetate-mediated precipitation, hydrophilic-lipophilic-balanced solid phase extraction (HLB-SPE) was used for the extraction of colistin. The compounds were retained on a hydrophilic interaction liquid chromatography (HILIC) column and were detected by MS/MS. CMS was quantified by determining the produced amount of colistin during acidic hydrolysis. The developed methods are sensitive with lower limits of quantification varying between 0.009 µg/mL and 0.071 µg/mL for colistin A, and 0.002 µg/mL to 0.013 µg/mL for colistin B. The intra- and inter-day precision and accuracy were within ±15%. Calibration curves of colistin were linear (0.063 µg/mL to 8.00 µg/mL) within clinically relevant concentration ranges. Zinc acetate-mediated precipitation and the use of a HILIC column were found to be essential. The developed methods are sensitive, accurate, precise, highly efficient and allow monitoring colistin and CMS in biological samples without the need for an internal standard.

Emergence of mcr-1 conferred colistin resistance among bacterial isolates from urban sewage water in India.

Increased use of colistin, a last resort drug due to failure of carbapenems, has possibly contributed in development and spread of resistance to colistin among Enterobacteriaceae. The colistin belongs to the family of polymyxins, cationic polypeptides, with broad-spectrum activity against Gram-negative bacteria. In this study, we obtained 253 non-duplicate bacterial isolates from sewage water in Delhi and phenotypically screened for colistin resistance. Of the 47 positive isolates, the colistin resistance gene mcr-1 was detected among 5 isolates. Based on 16S ribosomal RNA-based identification, bacterial isolates were found to be Escherichia coli, Aeromonas veronii, and Aeromonas dhakensis. Extended spectrum ß-lactamases (ESBL)-resistant determinants CTX-M and TEM were detected in all five mcr-1 positive isolates. On the basis of literature survey, this is the first report of mcr-1 gene from Aeromonas veronii and Aeromonas dhakensis worldwide. Furthermore, mcr-1 gene has not been reported earlier from sewage water in India. Antibiotic susceptibility test of all five isolates against 9 different classes of drugs revealed multidrug-resistant phenotype with high minimum inhibitory concentration values. In vitro transconjugation studies showed successful transfer of mcr-1 and other ESBL-resistant determinants. The occurrence of colistin resistance phenotype conferred by plasmid-based mcr-1 gene in the environment and an ever-increasing list of bacterial isolates is a cause of concern. A comprehensive survey of different water bodies and epidemiological studies are required to assess the risk of dissemination of resistance determinants.

Association of colistin residues and manure treatment with the abundance of mcr-1 gene in swine feedlots.

BACKGROUND: The extensive use of colistin in swine production may have contributed to the recent emergence of corresponding mobile resistance gene mcr-1. The use of colistin as a feed additive was banned in China in April 2017. OBJECTIVES: To examine the occurrence of colistin and dissemination of mcr-1 in swine feedlots before and after the colistin ban and effects of different manure treatments. METHODS: Environmental samples were collected from swine feedlots before (December 2016) and after (December 2017) the colistin ban. Colistin concentrations were determined by ultra-high performance liquid chromatography coupled to tandem mass spectrometry. The prevalence of mcr-1 were determined by quantitative PCR analysis, while bacterial community composition was investigated by 16S rRNA sequencing. RESULTS: In 2016, colistin was detected in feed and fresh manure samples at 67 mg/kg and 17 mg/kg, respectively, but was absent from all samples in 2017. In 2016, the relative abundance of mcr-1 in fresh manure was lower than that in solid samples after natural drying, while a higher relative abundance was detected in fresh manure samples compared with biogas slurry samples. A strong correlation between colistin concentration and relative abundance of mcr-1 was observed in fresh manure. The samples collected in 2017 showed a lower relative abundance of mcr-1 compared with those collected in 2016. Bacterial community analysis showed that the abundance of Enterobacteriaceae, which act as a vehicle and reservoir of mcr-1, increased with natural dying but decreased with anaerobic digestion. CONCLUSIONS: The presence of colistin exerts direct selection pressure for the accumulation of mcr-1 in manure, while the ban on colistin likely halted the dissemination of mcr-1 on pig farms. Anaerobic digestion is an effective waste treatment process for removing mcr-1, which might be mainly driven by the shift in bacterial community structure.

Analytical methodologies used for the determination of colistin in biological fluids. Is it still a challenge?

Colistin is a multicomponent polypeptide antibiotic consisting mainly of colistin A and colistin B, produced by selected strains of Bacillus polymyxa var. Colistinus. Only recently, the prodrug of colistin, colistimethate sodium, is widely used as last resort antibiotic for infections caused by resistant gram-negative bacteria. Colistin having been discovered several years ago, has not subjected to the drug development and regulatory approval processes that are applied today. However, pharmacological and pharmacokinetic information are necessary for its optimal use thus, during the last decades several studies are carried out in order to shed light on this issue. In the current review, the analytical methodologies of colistin assessment in biological material are summarized and the analytical challenges are critically discussed and critical aspects of the determinations such as the method of detection, the sample pretreatment methodology etc. are compared. Furthermore, critical quality aspects of the assessment methodologies such as the sensitivity of the currently developed methodologies are presented. Lastly, some future trends that should be incorporated in the determination pipeline of modern drugs are suggested.

Simultaneous determination of aminoglycosides and colistins in food.

A novel method for the simultaneous identification and quantification of twelve aminoglycosides (AGs) and two colistins in meat and bovine milk has been developed. The analysis was carried out using liquid chromatography coupled to quadrupole-Orbitrap mass spectrometry (LC-Q-Orbitrap). Among the HILIC (Hydrophilic Interaction Liquid Chromatography) stationary phases tested, the bare silica Poroshell 120 provided the best results. The samples were extracted with an aqueous solution followed by an SPE clean up based on the weak cation exchange mechanism. The validation study was performed carrying out 72 experiments per matrix at six different concentrations in a range encompassing the Maximum Residue Limits. The recoveries were from 72 to 87% in meat (except colistins) and from 82 to 96% in milk. Repeatabilities and intra-lab reproducibilities were lower than 10 and 15%, respectively. Limits of detection were lower than or equal to 33 µg kg-1. Finally, test materials containing AGs prepared for interlaboratory studies were successfully analysed.

Determination of colistin in animal tissues, egg, milk, and feed by ultra-high performance liquid chromatography-tandem mass spectrometry.

A confirmatory method for the determination of colistin in animal tissues, egg, milk, and feed was developed and validated. Colistin A and colistin B were extracted from samples with the mixture of 10% trichloroacetic acid-acetonitrile and isolated with mixed-mode weak cation exchange cartridge. Analytes were separated from matrix components using ultra-high performance liquid chromatography, and detected with electrospray ionization on a triple quadrupole mass spectrometer. Mean recoveries ranged from 78.0% to 115.6% with intra-day and inter-day relative standard deviation lower than 8.4% and 12.4%, respectively. The quantitation limits for different matrices were between 5 and 30 µg/kg, which was satisfactory for surveillance monitoring. The developed method was applied to the analysis of real samples collected from different provinces of China, and 19 out of 348 samples were found to be contaminated, with the highest concentration of approximately 12,000 µg/kg colistin A and 10,000 µg/kg colistin B in feed.

Analysis of Major Components of Bacitracin, Colistin and Virginiamycin in Feed Using Matrix Solid-phase Dispersion Extraction by Liquid Chromatography-electrospray Ionization Tandem Mass Spectrometry.

A quantitative LC-MS/MS method has been developed for simultaneous determination of bacitracin A, bacitracin B, colistin A, colistin B and virginiamycin M1 in feed. This rapid simple and effective extraction method was based on matrix solid-phase dispersion. Qualitative and quantitative analyses were performed by LC-ESI-MS/MS. CCß of polypeptide antibiotics upon the method ranged from 9.6 to 15.8 µg kg-1 and 19.4 to 27.5 µg kg-1, respectively. The limit of quantification of polypeptide antibiotics was 25 µg kg-1 in feed samples. The recoveries of polypeptide antibiotics spiked in feed samples at a concentration range of 25-100 µg kg-1 were found above 75.9-87.9% with relative standard deviations within days less than 15.7% and between days less than 20.6%. This rapid and reliable method can be used to efficiently separate, characterize and quantify the residues of polypeptide antibiotics in feed with advantages of simple pretreatment and environmental friendly.

Resistencia antimicrobiana en bacilos gramnegativos: una amenaza actual y global / Antimicrobial resistance in Gram-negative bacilli: a current and global threat

La resistencia a los antimicrobianos se ha incrementado de manera preocupante en los bacilos gramnegativos. La dispersión de clones de alto riesgo multirresistentes con determinantes genéticos responsables de la producción de betalactamasas de espectro extendido y, más recientemente, de carbapenemas ha reducido notablemente las alternativas terapéuticas. En España es preocupante la dispersión de las enterobacterias productoras de carbapenemasas de tipo OXA-48 y KPC. Se ha notificado el hallazgo de aislados de Klebsiella pneumoniae con KPC de clones similares a los descritos en Italia, con resistencia a la colistina por mutaciones en los sistemas que afectan a la estructura del lipopolisacárido. En Pseudomonas aeruginosa y Acinetobacter baumannii, las tasas de aislados multirresistentes son similares a las de los países de nuestro entorno y se han detectado clones de alto riesgo con carbapenemasas. Recientemente se ha descrito el gen mcr-1 en Escherichia coli y K. pneumoniae asociado a plásmidos que presentan genes de betalactamasas de espectro extendido y de carbapenemasas. También preocupa el posible incremento de la resistencia a la fosfomicina en E. coli, que se está produciendo asociado a genes tipo fosA ligados a plásmidos y que podrían limitar el uso futuro de este antimicrobiano. El llamamiento de la Organización de las Naciones Unidas a la lucha unificada de los distintos estados miembros frente a la resistencia debe impulsar el desarrollo de nuevos antimicrobianos y la puesta en marcha a nivel local de los planes de contención de la resistencia (AU)

Antimicrobial resistance has dramatically increased in gram-negative bacilli. The dispersion of multiresistant high-risk clones with genetic determinants responsible for the production of extended spectrum beta-lactamases (ESBL) and, more recently, carbapenemases has markedly reduced current therapeutic alternatives. In Spain, the dispersion of OXA-48 and KPC carbapenemase-producing enterobacteria (EPC) is worrisome. KPC producing Klebsiella pneumoniae clones with resistance to colistin similar to those described in Italy has been reported in our country. Resistance mechanisms include mutations affecting the lipopolysaccharide structure. In Pseudomonas aeruginosa and Acinetobacter baumannii the rates of multiresistant isolates are similar to those in surrounding countries and high-risk clones with carbapenemases within these organisms have been detected. Recently, the mcr-1 gene has been described in Escherichia coli and K. pneumoniae associated with plasmids presenting ESBL and carbapenemase genes. Moreover, there is also concern about the potential increase of fosfomycin resistance in E. coli. This resistance has been associated with plasmid-mediated fosA-like gene and might limit future use of this antimicrobial. The high-level meeting of the United Nations celebrated with the aim that all member states fight against antimicrobial resistance must include promotion of the development of new antimicrobials and the implementation of resistance containment plans at the local level (AU)

A simple method for assaying colistimethate sodium in pharmaceutical aerosol samples using high performance liquid chromatography.

A rapid and simple reversed-phase high performance liquid chromatography (HPLC) method for the quantitation of colistimethate sodium in pharmaceutical formulations has been developed. The chromatographic separation was performed using a Phenomenex Kinetex XB-C18 column with gradient elution using a mobile phase containing acetonitrile and 32mM sodium sulphate. Quantitation is based on the sum of the areas of two prominent peaks in the chromatogram, which produces a total peak area that is stable for 120 sample injections. The HPLC method was validated over the range 0.05-7mg/mL, and was shown to be suitable for the analysis of aerosolised pharmaceuticals in terms of aerosol output onto filter and for the analysis of samples from a cascade impactor, which is used for the determination of aerosol particle size.

Fecal microbiota transplantation: is there a role in the eradication of carbapenem-resistant Klebsiella pneumoniae intestinal carriage?

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How Much Surface Coating of Hydrophobic Azithromycin Is Sufficient to Prevent Moisture-Induced Decrease in Aerosolisation of Hygroscopic Amorphous Colistin Powder?

Aerosolisation performance of hygroscopic particles of colistin could be compromised at elevated humidity due to increased capillary forces. Co-spray drying colistin with a hydrophobic drug is known to provide a protective coating on the composite particle surfaces against moisture-induced reduction in aerosolisation performance; however, the effects of component ratio on surface coating quality and powder aerosolisation at elevated relative humidities are unknown. In this study, we have systematically examined the effects of mass ratio of hydrophobic azithromycin on surface coating quality and aerosolisation performance of the co-spray dried composite particles. Four combination formulations with varying drug ratios were prepared by co-spray drying drug solutions. Both of the drugs in each combination formulation had similar in vitro deposition profiles, suggesting that each composite particle comprises two drugs in the designed mass ratio, which is supported by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) data. XPS and ToF-SIMS measurements also revealed that 50% by weight (or 35% by molecular fraction) of azithromycin in the formulation provided a near complete coating of 96.5% (molar fraction) on the composite particle surface, which is sufficient to prevent moisture-induced reduction in fine particle fraction (FPF)recovered and FPFemitted. Higher azithromycin content did not increase coating coverage, while contents of azithromycin lower than 20% w/w did not totally prevent the negative effects of humidity on aerosolisation performance. This study has highlighted that a critical amount of azithromycin is required to sufficiently coat the colistin particles for short-term protection against moisture.

Development and validation of a UHPLC-MS/MS assay for colistin methanesulphonate (CMS) and colistin in human plasma and urine using weak-cation exchange solid-phase extraction.

A rapid ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay method was developed for determination of CMS and formed colistin in human plasma and urine. After extraction on a 96-well SPE Supra-Clean Weak Cation Exchange (WCX) plate, the eluents were mixed and injected into the UHPLC-MS/MS system directly. A Phonomenex Kinetex XB-C18 analytical column was employed with a mobile phase consisting of solution "A" (acetonitrile:methanol, 1:1, v/v) and solution "B" (0.1% formic acid in water, v/v). The flow rate was 0.4 mL/min with gradient elution over 3.5 min. Ions were detected in ESI positive ion mode and the precursor-product ion pairs were m/z 390.7/101.3 for colistin A, m/z 386.0/101.2 for colistin B, and m/z 402.3/101.2 for polymyxin B1 (IS), respectively. The lower limit of quantification (LLOQ) was 0.0130 and 0.0251 mg/L for colistin A and colistin B in both plasma and urine with accuracy (relative error, %) <± 12.6% and precision (relative standard deviation, %) <± 10.8%. Stability of CMS was demonstrated in biological samples before and during sample treatment, and in the extract. This new analytical method provides high-throughput treatment and optimized quantification of CMS and colistin, which offers a highly efficient tool for the analysis of a large number of clinical samples as well as routine therapeutic drug monitoring.

Validation of a colistin plasma concentration breakpoint as a predictor of nephrotoxicity in patients treated with colistin methanesulfonate.

Nephrotoxicity limits the effective use of colistin for the treatment of multidrug-resistant Gram-negative bacteria (MDR-GNB) infections. We previously defined a steady-state colistin plasma concentration (Css) of 2.42 mg/L that predicted nephrotoxicity at end of treatment (EOT). The objective of this study was to validate this breakpoint in a prospective cohort. This was a multicentre, prospective, observational study conducted at three hospitals with a cohort of patients treated for MDR-GNB infection with colistin methanesulfonate from September 2011 until January 2015. Nephrotoxicity was evaluated at Day 7 and at EOT using the RIFLE criteria. Css values were measured and analysed using HPLC. Taking the previously defined breakpoint for colistin concentration as a criterion, patients were divided into two groups (Css, ≤2.42 mg/L vs. >2.42 mg/L). Sixty-four patients were included. Seven patients (10.9%) had a Css > 2.42 mg/L and were compared with the remaining patients. Bivariate analysis showed that patients with a Css > 2.42 mg/L were older and had a significantly higher incidence of nephrotoxicity at Day 7 and EOT. Although not statistically significant, nephrotoxicity occurred earlier in these patients (6.2 days vs. 9.2 days in patients with lower Css; P = 0.091). Multivariate analysis of nephrotoxicity showed that Css > 2.42 mg/L was the only predictive factor. Nephrotoxicity was more frequent and occurred earlier in patients with colistin plasma concentrations higher than the previously defined breakpoint (2.42 mg/L). Colistin therapeutic drug monitoring should be routinely considered to avoid reaching this toxicity threshold and potential clinical consequences.