Colistin and tigecycline resistance in carbapenemase-producing Gram-negative bacteria: emerging resistance mechanisms and detection methods. - Related Documents




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503501.0000Colistin and tigecycline resistance in carbapenemase-producing Gram-negative bacteria: emerging resistance mechanisms and detection methods. A literature review was undertaken to ascertain the molecular basis for tigecycline and colistin resistance mechanisms and the experimental basis for the detection and delineation of this resistance particularly in carbapenemase-producing Gram-negative bacteria. Pubmed, Google Scholar and Science Direct were searched with the keywords colistin, tigecycline, resistance mechanisms and detection methods. Trans-complementation and comparative MIC studies, mass spectrometry, chromatography, spectrofluorometry, PCR, qRT-PCR and whole genome sequencing (WGS) were commonly used to determine tigecycline and colistin resistance mechanisms, specifically modifications in the structural and regulatory efflux (acrAB, OqxAB, kpgABC adeABC-FGH-IJK, mexAB-XY-oprJM and soxS, rarA robA, ramRAB marRABC, adeLRS, mexRZ and nfxb) and lipid A (pmrHFIJFKLM, lpxA, lpxC lpxD and mgrB, pmrAB, phoPQ,) genes respectively. Mutations in the ribosomal 16S rRNA operon rrnBC, also yielded resistance to tigecycline through target site modifications. The mcr-1 gene conferring resistance to colistin was identified via WGS, trans-complementation and a murine thigh infection model studies. Common detection methods are mainly antibiotic sensitivity testing with broth microdilution while molecular identification tools are mostly PCR and WGS. Spectrofluorometry, MALDI-TOF MS, micro-array and real-time multiplex PCR hold much promise for the future as new detection tools.201627153928
250210.9990Rapid 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
250320.9989Rapid 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
245630.9988MgrB Alterations Mediate Colistin Resistance in Klebsiella pneumoniae Isolates from Iran. Colistin is one of the last-resort therapeutic agents to combat multidrug-resistant Gram-negative bacteria (GNB) including Klebsiella pneumoniae. Although it happens rarely, resistance to colistin has been reported for several GNB. A total of 20 colistin resistant (col-R) and three colistin susceptible (col-S) clinical isolates of K. pneumoniae were studied to explore the underlying mechanisms of colistin resistance. The presence of plasmid encoded resistance genes, mcr-1, mcr-2, mcr-3, and mcr-4 genes were examined by PCR. The nucleotide sequences of pmrA, pmrB, phoP, phoQ, and mgrB genes were determined. To evaluate the association between colistin resistance and upregulation of pmrHFIJKLM and pmrCAB operons, transcriptional level of the pmrK and pmrC genes encoding for lipopolysaccharide target modifying enzymes was quantified by RT-qPCR analysis. None of the plasmid encoded resistance genes were detected in the studied isolates. Inactivation of MgrB due to nonsense mutations and insertion of IS elements was observed in 15 col-R isolates (75%). IS elements (IS5-like and IS1-like families) most commonly targeted the coding region and in one case the promoter region of the mgrB. Complementation with wild-type MgrB restored colistin susceptibility in isolates with altered mgrB. All col-R isolates lacked any genetic alterations in the pmrA, phoP, and phoQ genes and substitutions identified in the pmrB were not found to be involved in resistance conferring determined by complementation assay. Colistin resistance linked with upregulation of pmrHFIJKLM and pmrCAB operons with the pmrK and pmrC being overexpressed in 20 and 11 col-R isolates, respectively. Our results demonstrated that MgrB alterations are the major mechanisms contributing to colistin resistance in the tested K. pneumoniae isolates from Iran.201729326662
504040.9988Rapid 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
245550.9988Molecular 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
245760.9987Prevalence 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
504670.9987Molecular 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
504480.9987Detection 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
227890.9987First detected OXA-50 carbapenem-resistant clinical isolates Pseudomonas aeruginosa from Bulgaria and interplay between the expression of main efflux pumps, OprD and intrinsic AmpC. Introduction. Carbapenems are often described as the most effective weapon against infections caused by multidrug-resistant bacteria especially those belonging to the group of non-fermenting bacteria such as Pseudomonas. The main mechanisms leading to resistance are the hyperexpression of certain efflux pumps belonging to the resisto-nodular division and the lower expression of the transmembrane porin OprD, sometimes in combination with excessive production of the intrinsic AmpC. Carbapenemases are assumed to play a secondary role.Aim. The aim of this study was to determine the exact mechanisms of carbapenem resistance in Pseudomonas aeruginosa isolates from the largest Bulgarian University hospital 'St. George'- Plovdiv.Methodology. A total of 32 clinical isolates collected from different patients' samples resistant to imipenem and/or meropenem were examined via phenotypic and molecular-genetic tests.Results. No metallo-enzyme production was detected. Three isolates were positive for OXA-50-encoding genes in two of them in combination with other oxacillinases or the bla (VEB-1) gene. For the first time, OXA-50-producing P. aeruginosa have been reported in Bulgaria. The increased expression or hyperexpression of MexXY-OprM efflux pump was observed as the main mechanism of resistance. In most cases, it was combined with lower expression or lack of OprD with or without MexAB-OprM hyperexpression. No excessive production of AmpC was detected in comparison to the reference ATCC 27853 P. aeruginosa strain.Conclusion. The increased expression or overexpression of MexXY-OprM efflux pumps is the leading cause of carbapenem resistance in our isolates Pseudomonas, detected in 94 % of the bacteria investigated.201931746726
5024100.9986Colistin 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
1823110.9986Finding the Missing IMP Gene: Overcoming the Imipenemase IMP Gene Drop-Out in Automated Molecular Testing for Carbapenem-Resistant Bacteria Circulating in Latin America. Carbapenem resistance is considered one of the greatest current threats to public health, particularly in the management of infections in clinical settings. Carbapenem resistance in bacteria is mainly due to mechanisms such as the production of carbapenemases (such as the imipenemase IMP, or other enzymes like VIM, NDM, and KPC), that can be detected by several laboratory tests, including immunochromatography and automated real-time PCR (qPCR). Methods: As part of local studies to monitor carbapenem-resistant bacteria in Costa Rica, two cases were initially identified with inconsistent IMP detection results. A possible gene drop-out in the automated qPCR test was suggested based on the negative result, contrasting with the positive result by immunochromatography and whole-genome sequencing. We hypothesized that molecular testing could be optimized through the development of tailored assays to improve the detection of IMP genes. Thus, using IMP gene sequences from the local isolates and regional sequences in databases, primers were redesigned to extend the detection of IMP alleles of regional relevance. Results: The tailored qPCR was applied to a local collection of 119 carbapenem-resistant isolates. The genomes of all 14 positive cases were sequenced, verifying the results of the custom qPCR, despite the negative results of the automated testing. Conclusions: Guided by whole-genome sequencing, it was possible to extend the molecular detection of IMP alleles circulating in Latin America using a tailored qPCR to overcome IMP gene drop-out and false-negative results in an automated qPCR.202540867967
5839120.9986Computer Program for Detection and Analyzing the Porin-Mediated Antibiotic Resistance of Bacteria. The aim of this work was to develop a new software tool for identifying gene mutations that determine the porin-mediated resistance to antibiotics in gram-negative bacteria and to demonstrate the functionality of this program by detecting porin-mediated resistance to carbapenems in clinical isolates of Pseudomonas aeruginosa. MATERIALS AND METHODS: The proposed algorithm is based on searching for a correspondence between the reference and the studied genes. When the sought nucleotide sequence is found in the analyzed genome, it is compared with the reference one and analyzed. The genomic analysis is then verified by comparing between the amino acid sequences encoded by the reference and studied genes. The genes of the susceptible P. aeruginosa ATCC 27853 strain were used as the reference nucleotide sequences encoding for porins (OprD, OpdD, and OpdP) involved in the transport of carbapenems into the bacterial cell. The complete genomes of clinical P. aeruginosa isolates from the PATRIC database 3.6.9 and our own collection were used to test the functionality of the proposed program. The analyzed isolates were phenotypically characterized according to the CLSI standard. The search for carbapenemase genes in the studied genomes of P. aeruginosa was carried out using the ResFinder 4.1. RESULTS: The developed program for detecting the genetic determinants of non-plasmid antibiotic resistance made it possible to identify mutations of various types and significance in the porin genes of P. aeruginosa clinical isolates. These mutations led to modifications of the peptide structure of porin proteins. Single amino acid substitutions prevailed in the OpdD and OpdP porins of carbapenem-susceptible and carbapenem-resistant isolates. In the carbapenem-resistant strains, the gene encoding for OprD porin was found heavily modified, including insertions and/or deletions, which led to premature termination of porin synthesis. In several isolates resistant to meropenem, no mutations were detected in the gene encoding for OprD, which might be associated with alternative mechanisms of resistance to carbapenems. CONCLUSION: The proposed software product can become an effective tool for deciphering the molecular genetic mechanisms of bacterial chromosomal resistance to antibiotics. Testing the program revealed differences between the occurrences of mutations significant for carbapenem resistance in the oprD, opdD, and opdP genes.202135265355
5042130.9986Multiplex 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
5083140.9986Multiplex Microarrays in 96-Well Plates Photoactivated with 4-Azidotetrafluorobenzaldehyde for the Identification and Quantification of β-Lactamase Genes and Their RNA Transcripts. Antibiotic-resistant bacteria represent a global issue that calls for novel approaches to diagnosis and treatment. Given the variety of genetic factors that determine resistance, multiplex methods hold promise in this area. We developed a novel method to covalently attach oligonucleotide probes to the wells of polystyrene plates using photoactivation with 4-azidotetrafluorobenzaldehyde. Then, it was used to develop the technique of microarrays in the wells. It consists of the following steps: activating polystyrene, hybridizing the probes with biotinylated target DNA, and developing the result using a streptavidin-peroxidase conjugate with colorimetric detection. The first microarray was designed to identify 11 different gene types and 16 single-nucleotide polymorphisms (SNPs) of clinically relevant ESBLs and carbapenemases, which confer Gram-negative bacteria resistance to β-lactam antibiotics. The detection of bla genes in 65 clinical isolates of Enterobacteriaceae demonstrated the high sensitivity and reproducibility of the technique. The highly reproducible spot staining of colorimetric microarrays allowed us to design a second microarray that was intended to quantify four different types of bla mRNAs in order to ascertain their expressions. The combination of reliable performance, high throughput in standard 96-well plates, and inexpensive colorimetric detection makes the microarrays suitable for routine clinical application and for the study of multi-drug resistant bacteria.202338275665
5754150.9986Efflux pump inhibitor CCCP to rescue colistin susceptibility in mcr-1 plasmid-mediated colistin-resistant strains and Gram-negative bacteria. OBJECTIVES: Efflux in bacteria is a ubiquitous mechanism associated with resistance to antimicrobials agents. Efflux pump inhibitors (EPIs) have been developed to inhibit efflux mechanisms and could be a good alternative to reverse colistin resistance, but only CCCP has shown good activity. The aim of our study was to identify CCCP activity in a collection of 93 Gram-negative bacteria with known and unknown colistin resistance mechanisms including isolates with mcr-1 plasmid-mediated colistin resistance. METHODS: Colistin MIC was evaluated with and without CCCP and the fold decrease of colistin MIC was calculated for each strain. In order to evaluate the effect of this combination, a time-kill study was performed on five strains carrying different colistin resistance mechanisms. RESULTS: Overall, CCCP was able to reverse colistin resistance for all strains tested. The effect of CCCP was significantly greater on intrinsically colistin-resistant bacteria (i.e. Proteus spp., Serratia marcescens, Morganella morganii and Providencia spp.) than on other Enterobacteriaceae (P < 0.0001). The same was true for bacteria with a heteroresistance mechanism compared to bacteria with other colistin resistance mechanisms (P < 0.0001). A time-kill study showed the combination was bacteriostatic on strains tested. CONCLUSIONS: These results suggest an efflux mechanism, especially on intrinsically resistant bacteria and Enterobacter spp., but further analysis is needed to identify the molecular support of this mechanism. EPIs could be an alternative for restoring colistin activity in Gram-negative bacteria. Further work is necessary to identify new EPIs that could be used in humans.201829718423
5047160.9986Phenotypic 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
5827170.9986Duplex dPCR System for Rapid Identification of Gram-Negative Pathogens in the Blood of Patients with Bloodstream Infection: A Culture-Independent Approach. Early and accurate detection of pathogens is important to improve clinical outcomes of bloodstream infections (BSI), especially in the case of drug-resistant pathogens. In this study, we aimed to develop a culture-independent digital PCR (dPCR) system for multiplex detection of major sepsiscausing gram-negative pathogens and antimicrobial resistance genes using plasma DNA from BSI patients. Our duplex dPCR system successfully detected nine targets (five bacteria-specific targets and four antimicrobial resistance genes) through five reactions within 3 hours. The minimum detection limit was 50 ag of bacterial DNA, suggesting that 1 CFU/ml of bacteria in the blood can be detected. To validate the clinical applicability, cell-free DNA samples from febrile patients were tested with our system and confirmed high consistency with conventional blood culture. This system can support early identification of some drug-resistant gram-negative pathogens, which can help improving treatment outcomes of BSI.202134528911
5060180.9986Nonclonal 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
5693190.9986Evaluation of an expanded microarray for detecting antibiotic resistance genes in a broad range of gram-negative bacterial pathogens. A microarray capable of detecting genes for resistance to 75 clinically relevant antibiotics encompassing 19 different antimicrobial classes was tested on 132 Gram-negative bacteria. Microarray-positive results correlated >91% with antimicrobial resistance phenotypes, assessed using British Society for Antimicrobial Chemotherapy clinical breakpoints; the overall test specificity was >83%. Microarray-positive results without a corresponding resistance phenotype matched 94% with PCR results, indicating accurate detection of genes present in the respective bacteria by microarray when expression was low or absent and, hence, undetectable by susceptibility testing. The low sensitivity and negative predictive values of the microarray results for identifying resistance to some antimicrobial resistance classes are likely due to the limited number of resistance genes present on the current microarray for those antimicrobial agents or to mutation-based resistance mechanisms. With regular updates, this microarray can be used for clinical diagnostics to help accurate therapeutic options to be taken following infection with multiple-antibiotic-resistant Gram-negative bacteria and prevent treatment failure.201323129055