Detection of Colistin Resistance in Salmonella enterica Using MALDIxin Test on the Routine MALDI Biotyper Sirius Mass Spectrometer. - Related Documents




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504401.0000Detection of Colistin Resistance in Salmonella enterica Using MALDIxin Test on the Routine MALDI Biotyper Sirius Mass Spectrometer. Resistance to polymyxins in most Gram-negative bacteria arises from chemical modifications to the lipid A portion of their lipopolysaccharide (LPS) mediated by chromosomally encoded mutations or the recently discovered plasmid-encoded mcr genes that have further complicated the landscape of colistin resistance. Currently, minimal inhibitory concentration (MIC) determination by broth microdilution, the gold standard for the detection of polymyxin resistance, is time consuming (24 h) and challenging to perform in clinical and veterinary laboratories. Here we present the use of the MALDIxin to detect colistin resistant Salmonella enterica using the MALDxin test on the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system.202032582090
504310.9997Detection of Colistin Resistance in Escherichia coli by Use of the MALDI Biotyper Sirius Mass Spectrometry System. Polymyxin antibiotics are a last-line treatment for multidrug-resistant Gram-negative bacteria. However, the emergence of colistin resistance, including the spread of mobile mcr genes, necessitates the development of improved diagnostics for the detection of colistin-resistant organisms in hospital settings. The recently developed MALDIxin test enables detection of colistin resistance by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in less than 15 min but is not optimized for the mass spectrometers commonly found in clinical microbiology laboratories. In this study, we adapted the MALDIxin test for the MALDI Biotyper Sirius MALDI-TOF MS system (Bruker Daltonics). We optimized the sample preparation protocol by using a set of 6 mobile colistin resistance (MCR) protein-expressing Escherichia coli clones and validated the assay with a collection of 40 E. coli clinical isolates, including 19 confirmed MCR protein producers, 12 colistin-resistant isolates that tested negative for commonly encountered mcr genes (i.e., likely chromosomally resistant isolates), and 9 polymyxin-susceptible isolates. We calculated polymyxin resistance ratio (PRR) values from the acquired spectra; PRR values of 0, indicating polymyxin susceptibility, were obtained for all colistin-susceptible E. coli isolates, whereas positive PRR values, indicating resistance to polymyxins, were obtained for all resistant strains, independent of the genetic basis of resistance. Thus, we report a preliminary feasibility study showing that an optimized version of the MALDIxin test adapted for the routine MALDI Biotyper Sirius system provides an unbiased, fast, reliable, cost-effective, and high-throughput way of detecting colistin resistance in clinical E. coli isolates.201931597744
250320.9996Rapid detection and discrimination of chromosome- and MCR-plasmid-mediated resistance to polymyxins by MALDI-TOF MS in Escherichia coli: the MALDIxin test. BACKGROUND: Polymyxins are currently considered a last-resort treatment for infections caused by MDR Gram-negative bacteria. Recently, the emergence of carbapenemase-producing Enterobacteriaceae has accelerated the use of polymyxins in the clinic, resulting in an increase in polymyxin-resistant bacteria. Polymyxin resistance arises through modification of lipid A, such as the addition of phosphoethanolamine (pETN). The underlying mechanisms involve numerous chromosome-encoded genes or, more worryingly, a plasmid-encoded pETN transferase named MCR. Currently, detection of polymyxin resistance is difficult and time consuming. OBJECTIVES: To develop a rapid diagnostic test that can identify polymyxin resistance and at the same time differentiate between chromosome- and plasmid-encoded resistances. METHODS: We developed a MALDI-TOF MS-based method, named the MALDIxin test, which allows the detection of polymyxin resistance-related modifications to lipid A (i.e. pETN addition), on intact bacteria, in <15 min. RESULTS: Using a characterized collection of polymyxin-susceptible and -resistant Escherichia coli, we demonstrated that our method is able to identify polymyxin-resistant isolates in 15 min whilst simultaneously discriminating between chromosome- and plasmid-encoded resistance. We validated the MALDIxin test on different media, using fresh and aged colonies and show that it successfully detects all MCR-1 producers in a blindly analysed set of carbapenemase-producing E. coli strains. CONCLUSIONS: The MALDIxin test is an accurate, rapid, cost-effective and scalable method that represents a major advance in the diagnosis of polymyxin resistance by directly assessing lipid A modifications in intact bacteria.201830184212
250230.9995Rapid detection of colistin resistance in Acinetobacter baumannii using MALDI-TOF-based lipidomics on intact bacteria. With the dissemination of extremely drug resistant bacteria, colistin is now considered as the last-resort therapy for the treatment of infection caused by Gram-negative bacilli (including carbapenemase producers). Unfortunately, the increase use of colistin has resulted in the emergence of resistance as well. In A. baumannii, colistin resistance is mostly caused by the addition of phosphoethanolamine to the lipid A through the action of a phosphoethanolamine transferase chromosomally-encoded by the pmrC gene, which is regulated by the two-component system PmrA/PmrB. In A. baumannii clinical isolate the main resistance mechanism to colistin involves mutations in pmrA, pmrB or pmrC genes leading to the overexpression of pmrC. Although, rapid detection of resistance is one of the key issues to improve the treatment of infected patient, detection of colistin resistance in A. baumannii still relies on MIC determination through microdilution, which is time-consuming (16-24 h). Here, we evaluated the performance of a recently described MALDI-TOF-based assay, the MALDIxin test, which allows the rapid detection of colistin resistance-related modifications to lipid A (i.e phosphoethanolamine addition). This test accurately detected all colistin-resistant A. baumannii isolates in less than 15 minutes, directly on intact bacteria with a very limited sample preparation prior MALDI-TOF analysis.201830442963
245540.9994Molecular Mechanisms of Colistin Resistance Among Klebsiella Pneumoniae Strains. BACKGROUND: The increasing rate of infections caused by multiple drug resistant gram-negative bacteria has led to resuscitation of colistin. As a result, colistin resistance, mainly among Klebsiella pneumoniae strains has also been increased. The aim of this study was to investigate molecular mechanisms behind colistin resistance. METHODS: Twenty colistin-resistant K. pneumoniae strains isolated from clinical samples of different patients were involved in this study. VITEK2 automated ID/AST system (Biomeriux, France) was used for the identification and also the susceptibility testing for antibiotics other than colistin. Colistin susceptibility was determined by broth microdilution method. To identify the mechanisms of resistance, mutations on mgrB genes, expression levels of pmrA, pmrB, pmrC, pmrD, pmrE, pmrK, phoQ, and phoP genes, and the presence of plasmid mediated colistin resistance genes, mcr-1 and mcr-2 were investigated. RESULTS: As a result of the study, increased expression levels of the pmrA, pmrB, pmrD, pmrK, phoP, and phoQ genes were observed. All colistin resistant strains were found wild type for the mgrB gene which is thought to be esponsible for colistin resistance. Also, no mcr-1 or mcr-2 genes which are the causes of plasmid mediated colistin resistance have been detected in any of the strains. CONCLUSIONS: Among the colistin resistant K. pneumoniae strains included in our study, increased expression Levels of the genes responsible for cell membrane modifications related with colistin resistance were the most common mechanisms.201931307167
502450.9993Colistin Resistance in Enterobacterales Strains - A Current View. Colistin is a member of cationic polypeptide antibiotics known as polymyxins. It is widely used in animal husbandry, plant cultivation, animal and human medicine and is increasingly used as one of the last available treatment options for patients with severe infections with carbapenem-resistant Gram-negative bacilli. Due to the increased use of colistin in treating infections caused by multidrug-resistant (MDR) bacteria, the resistance to this antibiotic ought to be monitored. Bacterial resistance to colistin may be encoded on transposable genetic elements (e.g. plasmids with the mcr genes). Thus far, nine variants of the mcr gene, mcr-1 - mcr-9, have been identified. Chromosomal resistance to colistin is associated with the modification of lipopolysaccharide (LPS). Various methods, from classical microbiology to molecular biology methods, are used to detect the colistin-resistant bacterial strains and to identify resistance mechanisms. The broth dilution method is recommended for susceptibility testing of bacteria to colistin. Colistin is a member of cationic polypeptide antibiotics known as polymyxins. It is widely used in animal husbandry, plant cultivation, animal and human medicine and is increasingly used as one of the last available treatment options for patients with severe infections with carbapenem-resistant Gram-negative bacilli. Due to the increased use of colistin in treating infections caused by multidrug-resistant (MDR) bacteria, the resistance to this antibiotic ought to be monitored. Bacterial resistance to colistin may be encoded on transposable genetic elements (e.g. plasmids with the mcr genes). Thus far, nine variants of the mcr gene, mcr-1 – mcr-9, have been identified. Chromosomal resistance to colistin is associated with the modification of lipopolysaccharide (LPS). Various methods, from classical microbiology to molecular biology methods, are used to detect the colistin-resistant bacterial strains and to identify resistance mechanisms. The broth dilution method is recommended for susceptibility testing of bacteria to colistin.201931880886
504660.9993Molecular mechanisms of colistin- and multidrug-resistance in bacteria among patients with hospital-acquired infections. AIM: The increasing burden of resistance in Gram-negative bacteria (GNB) is becoming a major issue for hospital-acquired infections. Therefore, understanding the molecular mechanisms is important. METHODOLOGY: Resistance genes of phenotypically colistin-resistant GNB (n = 60) were determined using whole genome sequencing. Antimicrobial susceptibility patterns were detected by Vitek®2 & broth microdilution. RESULTS: Of these phenotypically colistin-resistant isolates, 78% were also genetically resistant to colistin. Activation of efflux pumps, and point-mutations in pmrB, and MgrB genes conferred colistin resistance among GNB. Eight different strains of K. pneumoniae were identified and ST43 was the most prominent strain with capsular type-specific (cps) gene KL30. DISCUSSION: These results, in combination with rapid diagnostic methods, will help us better advice appropriate antimicrobial regimens.202337753358
166070.9993Emergence of Plasmid-Mediated Fosfomycin-Resistance Genes among Escherichia coli Isolates, France. FosA, a glutathione S-transferase that inactivates fosfomycin, has been reported as the cause of enzymatic resistance to fosfomycin. We show that multiple lineages of FosA-producing extended spectrum β-lactamase Escherichia coli have circulated in France since 2012, potentially reducing the efficacy of fosfomycin in treating infections with antimicrobial drug-resistant gram-negative bacilli.201728820368
504080.9992Rapid detection and differentiation of mobile colistin resistance (mcr-1 to mcr-10) genes by real-time PCR and melt-curve analysis. BACKGROUND: The emergence of multi-drug-resistant (MDR) micro-organisms prompted new interest in older antibiotics, such as colistin, that had been abandoned previously due to limited efficacy or high toxicity. Over the years, several chromosomal-encoded colistin resistance mechanisms have been described; more recently, 10 plasmid-mediated mobile colistin resistance (mcr) genes have been identified. Spread of these genes among MDR Gram-negative bacteria is a matter of serious concern; therefore, reliable and timely mcr detection is paramount. AIM: To design and validate a multiplex real-time polymerase chain reaction (PCR) assay for detection and differentiation of mcr genes. METHODS: All available mcr alleles were downloaded from the National Center for Biotechnology Information Reference Gene Catalogue, aligned with Clustal Omega and primers designed using Primer-BLAST. Real-time PCR monoplexes were optimized and validated using a panel of 120 characterized Gram-negative strains carrying a wide range of resistance genes, often in combination. Melt-curve analysis was used to confirm positive results. FINDINGS: In-silico analysis enabled the design of a 'screening' assay for detection of mcr-1/2/6, mcr-3, mcr-4, mcr-5, mcr-7, mcr-8 and mcr-9/10, paired with an internal control assay to discount inhibition. A 'supplementary' assay was subsequently designed to differentiate mcr-1, mcr-2, mcr-6, mcr-9 and mcr-10. Expected results were obtained for all strains (100% sensitivity and specificity). Melt-curve analysis showed consistent melting temperature results. Inhibition was not observed. CONCLUSIONS: The assay is rapid and easy to perform, enabling unequivocal mcr detection and differentiation even when more than one variant is present. Adoption by clinical and veterinary microbiology laboratories would aid the surveillance of mcr genes amongst Gram-negative bacteria.202133485969
577190.9992Peptide nucleic acid-mediated re-sensitization of colistin resistance Escherichia coli KP81 harboring mcr-1 plasmid. Escherichia coli is a gram-negative bacterium and it causes a variety of diseases in humans. It causes a wide range of clinical infections in humans; urinary tract infections is the most prevalent infection caused by uropathogenic Escherichia coli. In recent years, the observation of antibiotic-resistant genes such as resistance to colistin, makes the Escherichia coli resistant to antibiotics like colistin (polymyxin E), because of that the use of new therapies like peptide nucleic acid (PNA) has attracted the consideration of scientists. The aim of this study is the assessment of the inhibitory role of PNA against mcr-1 gene and reduction of mcr-1 gene expression and MIC in colistin resistant E. coli by PNA. NCBI database was used to design PNA. Our study was carried out on E. coli KP81 bacteria containing the mcr-1 gene. Microbroth dilution (MIC) method was used to survey phenotypic sensitivity and determine the sensitivity of the bacteria to the colistin antibiotic. E. coli KP81 isolates were further investigated by polymerase chain reaction to assess the presence of mcr-1 genes and target genes were quantified by real-time PCR assay using specific primers. The MIC result after treatment with specific PNA showed that the resistance to colistin reduced about three fold and the resistance level dropped from 32 μg/ml to 4 μg/ml. The expression analysis of mcr-1 gene in E. coli KP81 isolate indicates the PNA, 95% reduced the expression of the mcr-1 gene. Our observations showed that by inhibiting the expression of mcr-1, sensitivity to colistin can be defeated. Using higher concentrations of PNA and an in vivo study can reveal more clinical application of this method.201931344478
5049100.9992Colistin Resistance Mechanisms in Human and Veterinary Klebsiella pneumoniae Isolates. Colistin (polymyxin E) is increasingly used as a last-resort antibiotic for the treatment of severe infections with multidrug-resistant Gram-negative bacteria. In contrast to human medicine, colistin is also used in veterinary medicine for metaphylaxis. Our objective was to decipher common colistin resistance mechanisms in Klebsiella pneumoniae isolates from animals. In total, 276 veterinary K. pneumoniae isolates, derived from companion animals or livestock, and 12 isolates from human patients were included for comparison. Six out of 276 veterinary isolates were colistin resistant (2.2%). Human isolates belonging to high-risk clonal lineages (e.g., ST15, ST101, ST258), displayed multidrug-resistant phenotypes and harboured many resistance genes compared to the veterinary isolates. However, the common colistin resistance mechanism in both human and animal K. pneumoniae isolates were diverse alterations of MgrB, a critical regulator of lipid A modification. Additionally, deleterious variations of lipopolysaccharide (LPS)-associated proteins (e.g., PmrB P95L, PmrE P89L, LpxB A152T) were identified. Phylogenetic analysis and mutation patterns in genes encoding LPS-associated proteins indicated that colistin resistance mechanisms developed independently in human and animal isolates. Since only very few antibiotics remain to treat infections with MDR bacteria, it is important to further analyse resistance mechanisms and the dissemination within different isolates and sources.202236421315
1849110.9992Carbapenemase-Producing Elizabethkingia Meningoseptica from Healthy Pigs Associated with Colistin Use in Spain. Carbapenems are considered last-resort antimicrobials, especially for treating infections involving multidrug-resistant Gram-negative bacteria. In recent years, extended-spectrum β-lactamase (ESBL) and carbapenemase-producing Gram-negative bacteria have become widespread in hospitals, community settings, and the environment, reducing the range of effective therapeutic alternatives. The use of colistin to treat infection caused by these multi-drug bacteria may favour the selection and persistence of carbapenem-resistant bacteria. In this study, it is described, for the first time to our knowledge, a carbapenemase-producing isolate of Elizabethkingia meningoseptica from healthy pigs in Spain. The isolate we report was recovered during a study to detect colistin-resistant bacteria from faecal samples of healthy food-production animals using a chromogenic selective medium. Unexpectedly, we found an isolate of Elizabethkingia meningoseptica with high Minimum Inhibitory Concentration (MIC) values for several antibiotics tested. Molecular analysis did not show any mcr family genes related with colistin resistance, but two carbapenemase genes, bla(B-12_1) and bla(GOB-17_1), were detected. This finding in healthy animals could suggest that colistin may favour the selection and persistence of carbapenem-resistant bacteria.201931514353
1664120.9992Emergence of colistin resistance in Enterobacter aerogenes from Croatia. A colistin-resistant Enterobacter aerogenes [study code 12264] was isolated from the tracheal aspirate of a 71-year-old male patient in the General Hospital [GH] in Pula, Croatia. The patient was previously treated in University Hospital Centre in Rijeka with colistin in order to eradicate Acinetobacter baumannii isolate, susceptible only to colistin and tigecycline. Genes encoding ESBLs [bla(TEM), bla(SHV), bla(CTX-M), bla(PER-1)] were screened by PCR. The strain was shown to possess bla(CTX-M-15) and bla(TEM-1) genes. To asses genes possibly involved in resistance to colistin the chromosomal enconding mgrB gene and the plasmid-mediated mcr-1 and mcr-2 genes were screened as described previously. Mcr-1 and mcr-2 genes were not detected and mgrB gene presented a wild-type sequence. PCR-based Replicon typing method [PBRT] conducted on an E. aerogenes isolate, showed that the strain carried an IncN plasmid. Adaptive mechanisms such as changes of the bacterial cell outer membrane that cause porin decrease or presence of an efflux pump, due to selection pressure exerted by the therapeutic administration of colistin, could be responsible for the development of colistin resistance in our strain, as recently reported in E. aerogenes from France. Due to effective infection control measures, the colistin-resistant strain did not spread to other patients or hospital wards. This is the first report of an ESBL-producing, colistin-resistant E. aerogenes in clinically relevant samples such as endotracheal aspirate and blood culture, showing the presence of this rare resistance profile among Gram-negative bacteria.201829063811
5773130.9992LBJMR medium: a new polyvalent culture medium for isolating and selecting vancomycin and colistin-resistant bacteria. BACKGROUND: Multi-drug resistant bacteria are a phenomenon which is on the increase around the world, particularly with the emergence of colistin-resistant Enterobacteriaceae and vancomycin-resistant enterococci strains. The recent discovery of a plasmid-mediated colistin resistance with the description of the transferable mcr-1 gene raised concerns about the need for an efficient detection method for these pathogens, to isolate infected patients as early as possible. The LBJMR medium was developed to screen for all polymyxin-resistant Gram-negative bacteria, including mcr-1 positive isolates, and vancomycin-resistant Gram-positive bacteria. RESULTS: The LBJMR medium was developed by adding colistin sulfate salt at a low concentration (4 μg/mL) and vancomycin (50 μg/mL), with glucose (7.5 g/L) as a fermentative substrate, to a Purple Agar Base (31 g/L). A total of 143 bacterial strains were used to evaluate this universal culture medium, and the sensitivity and specificity of detection were 100% for the growth of resistant strains. 68 stool samples were cultured on LBJMR, and both colistin-resistant Gram-negative and vancomycin-resistant Gram-positive strains were specifically detected. CONCLUSIONS: The LBJMR medium is a multipurpose selective medium which makes it possible to identify bacteria of interest from clinical samples and to isolate contaminated patients in hospital settings. This is a simple medium that could be easily used for screening in clinical microbiology laboratories.201729169321
5041140.9992Development and Validation of a Clinical Laboratory Improvement Amendments-Compliant Multiplex Real-Time PCR Assay for Detection of mcr Genes. Increased use of colistin in both human and veterinary medicine has led to the emergence of plasmid-mediated colistin resistance (mcr genes). In this study, we report the development of a real-time PCR assay using TaqMan probe-based chemistry for detection of mcr genes from bacterial isolates. Positive control isolates harboring mcr-1 and mcr-2 yielded exponential amplification curves with the assay, and the amplification efficiency was 98% and 96% for mcr-1 and mcr-2, respectively. Each target gene could be reproducibly detected from a sample containing 10(3) cfu/mL of mcr-harboring bacteria, and there was no cross-reactivity with DNA extracted from several multidrug-resistant bacteria harboring other resistance genes, but lacking mcr genes. Both sensitivity and specificity of the mcr real-time PCR assay were 100% in a method validation performed with a set of 25 previously well-characterized bacterial isolates containing mcr-positive and -negative bacteria. This newly developed assay is a rapid and sensitive tool for detecting emerging mcr genes in cultured bacterial isolates. The assay was successfully validated according to quality standards of the Clinical Laboratory Improvement Amendments (CLIA).201930942652
1556150.9992Resistance to Colistin in Klebsiella Pneumoniae: A 4.0 Strain? The global rise of multidrug-resistant gram-negative bacteria represents an increasing threat to patient safety. From the first observation of a carbapenem-resistant gram-negative bacteria a global spread of extended-spectrum beta-lactamases and carbapenemases producing Klebsiella pneumoniae has been observed. Treatment options for multidrug-resistant K. pneumoniae are actually limited to combination therapy with some aminoglycosides, tigecycline and to older antimicrobial agents. Unfortunately, the prevalence of colistin-resistant and tigecycline-resistant K. pneumoniae is increasing globally. Infection due to colistin-resistant K. pneumoniae represents an independent risk factor for mortality. Resistance to colistin in K. pneumoniae may be multifactorial, as it is mediated by chromosomal genes or plasmids. The emergence of transmissible, plasmid-mediated colistin resistance is an alarming finding. The absence of new agents effective against resistant Gram-negative pathogens means that enhanced surveillance, compliance with infection prevention procedures, and antimicrobial stewardship programs will be required to limit the spread of colistin-resistant K. pneumoniae.201728626539
2457160.9992Prevalence and molecular mechanisms of colistin resistance in Acinetobacter baumannii clinical isolates in Tehran, Iran. Colistin is one of the last remaining active antibiotics against multidrug resistant Gram-negative bacteria. However, several recent studies reported colistin-resistant (ColR) Acinetobacter baumannii from different countries. In the current study, we investigated molecular mechanisms involved in colistin resistance in A. baumannii isolates from different clinical samples. A total of 110 clinical A. baumannii isolates were collected from two hospitals in Tehran. Minimum inhibitory concentrations (MICs) were determined by broth microdilution according to the Clinical and Laboratory Standards Institute. For the ColR isolates, mutation was detected in pmrA, pmrB, lpxA, lpxC, and lpxD genes using the polymerase chain reaction (PCR) and sequencing. Moreover, the relative expression of the pmrC gene was calculated using quantitative reverse transcription PCR. Three colistin resistant isolates were identified with MIC between 8 and 16 μg/mL and were resistant to all the tested antimicrobial agents. All the three isolates had a mutation in the pmrB, pmrA, lpxA, lpxD, and lpxC genes. Moreover, the overexpression of pmrC gene was observed in all isolates. Our results showed that the upregulation of the PmrAB two component system was the primary mechanism linked to colistin resistance among the studied colistin resistant A. baumannii isolates.202134370684
5047170.9992Phenotypic and Genotypic Characterization of Pan-Drug-Resistant Klebsiella pneumoniae Isolated in Qatar. In secondary healthcare, carbapenem-resistant Enterobacterales (CREs), such as those observed in Klebsiella pneumoniae, are a global public health priority with significant clinical outcomes. In this study, we described the clinical, phenotypic, and genotypic characteristics of three pan-drug-resistant (PDR) isolates that demonstrated extended resistance to conventional and novel antimicrobials. All patients had risk factors for the acquisition of multidrug-resistant organisms, while microbiological susceptibility testing showed resistance to all conventional antimicrobials. Advanced susceptibility testing demonstrated resistance to broad agents, such as ceftazidime-avibactam, ceftolozane-tazobactam, and meropenem-vaborbactam. Nevertheless, all isolates were susceptible to cefiderocol, suggested as one of the novel antimicrobials that demonstrated potent in vitro activity against resistant Gram-negative bacteria, including CREs, pointing toward its potential therapeutic role for PDR pathogens. Expanded genomic studies revealed multiple antimicrobial-resistant genes (ARGs), including bla(NMD-5) and bla(OXA) derivative types, as well as a mutated outer membrane porin protein (OmpK37).202438534710
5060180.9992Nonclonal Emergence of Colistin Resistance Associated with Mutations in the BasRS Two-Component System in Escherichia coli Bloodstream Isolates. Infections by multidrug-resistant Gram-negative bacteria are increasingly common, prompting the renewed interest in the use of colistin. Colistin specifically targets Gram-negative bacteria by interacting with the anionic lipid A moieties of lipopolysaccharides, leading to membrane destabilization and cell death. Here, we aimed to uncover the mechanisms of colistin resistance in nine colistin-resistant Escherichia coli strains and one Escherichia albertii strain. These were the only colistin-resistant strains of 1,140 bloodstream Escherichia isolates collected in a tertiary hospital over a 10-year period (2006 to 2015). Core-genome phylogenetic analysis showed that each patient was colonized by a unique strain, suggesting that colistin resistance was acquired independently in each strain. All colistin-resistant strains had lipid A that was modified with phosphoethanolamine. In addition, two E. coli strains had hepta-acylated lipid A species, containing an additional palmitate compared to the canonical hexa-acylated E. coli lipid A. One E. coli strain carried the mobile colistin resistance (mcr) gene mcr-1.1 on an IncX4-type plasmid. Through construction of chromosomal transgene integration mutants, we experimentally determined that mutations in basRS, encoding a two-component signal transduction system, contributed to colistin resistance in four strains. We confirmed these observations by reversing the mutations in basRS to the sequences found in reference strains, resulting in loss of colistin resistance. While the mcr genes have become a widely studied mechanism of colistin resistance in E. coli, sequence variation in basRS is another, potentially more prevalent but relatively underexplored, cause of colistin resistance in this important nosocomial pathogen.IMPORTANCE Multidrug resistance among Gram-negative bacteria has led to the use of colistin as a last-resort drug. The cationic colistin kills Gram-negative bacteria through electrostatic interaction with the anionic lipid A moiety of lipopolysaccharides. Due to increased use in clinical and agricultural settings, colistin resistance has recently started to emerge. In this study, we used a combination of whole-genome sequence analysis and experimental validation to characterize the mechanisms through which Escherichia coli strains from bloodstream infections can develop colistin resistance. We found no evidence of direct transfer of colistin-resistant isolates between patients. The lipid A of all isolates was modified by the addition of phosphoethanolamine. In four isolates, colistin resistance was experimentally verified to be caused by mutations in the basRS genes, encoding a two-component regulatory system. Our data show that chromosomal mutations are an important cause of colistin resistance among clinical E. coli isolates.202032161146
5042190.9992Multiplex loop-mediated isothermal amplification (multi-LAMP) assay for rapid detection of mcr-1 to mcr-5 in colistin-resistant bacteria. Purpose: The discovery of the plasmid-mediated colistin resistance genes, mcr, revealed a mechanism of transmission of colistin resistance, which is a major, global public health concern especially among individuals infected with carbapenem-resistant Gram-negative bacteria. To monitor the spread and epidemiology of mcr genes, a convenient and reliable method to detect mcr genes in clinical isolates is needed, especially in the primary care institutions. This study aimed to establish a restriction endonuclease-based multiplex loop-mediated isothermal amplification (multi-LAMP) assay to detect mcr genes (mcr-1 to mcr-5) harbored by colistin-resistant bacteria. Methods: A triple-LAMP assay for mcr-1, mcr-3, and mcr-4 and a double-LAMP assay for mcr-2 and mcr-5 were established. The sensitivity and specificity of the LAMP reactions were determined via electrophoresis and visual detection. Results: The sensitivity of the LAMP assay was 10-fold greater than that of PCR, with high specificity among the screened primers. Specific mcr genes were distinguished in accordance with band numbers and the fragment length of the digested LAMP amplification products. Furthermore, the LAMP assay was confirmed as a rapid and reliable diagnostic technique upon application for clinical samples, and the results were consistent with those of conventional PCR assay. Conclusion: The multi-LAMP assay is a potentially promising method to detect mcr genes and will, if implemented, help prevent infections by drug-resistant bacteria in primary-care hospitals due to rapid and reliable surveillance. To our knowledge, this is the first study to report the application of LAMP to detect mcr-2 to mcr-5 genes and the first time that multi-LAMP has been applied to detect mcr genes.201931308708