Mechanisms Of Colistin Resistance Research Articles

Identification of combinatorial mutations associated with colistin resistance in Shewanella algae

Colistin is a last-resort antibiotic used to treat infections caused by drug-resistant gram-negative bacteria. However, the genetic mechanisms underlying colistin resistance in Shewanella algae are not well understood. In this study, we sequenced and compared the genomes of 23 mcr-negative colistin-resistant and sensitive S. algae samples from various sources. We applied a computational approach to identify combinatorial mutations associated with colistin resistance. Our analysis revealed a combination of three mutations (PmrB 451, PmrE168, PmrH292) that were strongly associated with colistin resistance in S. algae. This study provides insights into the genetic mechanisms of colistin resistance in S. algae and demonstrates the utility of a computational approach for identifying epistatic interactions among mutations. Identifying the genetic mutations responsible for colistin resistance in S. algae can inform the development of new treatments or strategies to combat infections caused by this emerging pathogen.

EptA of Riemerella anatipestifer mediates phenotypes involved in colistin resistance and virulence.

Colistin (polymyxin E) is a group of cationic antimicrobial cyclic peptides and is recognized as a last-resort defense against lethal infections with carbapenem-resistant pathogens. In addition to the plasmid-borne mobilized phosphoethanolamine (PEA) transferases, the functional expression of lipid A-modifying enzymes encoded on chromosomes has been attributed to intrinsic bacterial colistin resistance. However, the mechanisms of colistin resistance in Riemerella anatipestifer remain unknown. Herein, the GE296_RS09715 gene-encoded Lipid A PEA transferases (RaEptA) was identified in R. anatipestifer. Genetic and structural analyses revealed that the amino acid sequence of RaEptA shared 26.6%-33.1% similarities with the family of Lipid A PEA transferases (EptA) and MCR-like proteins and have defined 12 residues that contribute to the formation of phosphatidylethanolamine (PE)-recognizable cavities. Comparative analyses of colistin resistance in RA-LZ01 and RA-LZ01ΔRaEptA showed the level of colistin has fallen from 96 μg mL-1 down to 24 ~ 32 μg mL-1 . Site-directed mutagenesis assay of the PE-binding cavity and expression of the mutants reveals that K309-rRaEptA can remodel the surface of Escherichia coli and rendering it resistant to colistin, suggesting this point-mutation of P309K is necessary for EptA-mediated lipid A modification. Moreover, the virulence of RA-LZ01ΔRaEptA was attenuated compared with RA-LZ01 both in vivo and vitro. Taken together, the results represent the RaEptA involved in the colistin resistance and pathogenicity, and the P309K mutation might alter bacterial adaptation and increase the spread of colistin resistance from R. anatipestifer to other gram-negative bacteria. The findings of this study suggest another scenario for the spread of colistin resistance genes and should be considered by a wide audience.

Antimicrobial resistance and plasmid-mediated colistin resistance mechanism of diarrheagenic Escherichia coli recovered from foods in parts of China in 2020

Objective: To investigate the antimicrobial resistance of food-borne diarrheagenic Escherichia coli (DEC) and the prevalence of mcr genes that mediates mobile colistin resistance in parts of China, 2020. Methods: For 91 DEC isolates recovered from food sources collected from Fujian province, Hebei province, Inner Mongolia Autonomous Region and Shanghai city in 2020, Vitek2 Compact biochemical identification and antimicrobial susceptibility testing platform was used for the detection of antimicrobial susceptibility testing (AST) against to 18 kinds of antimicrobial compounds belonging to 9 categories, and multi-polymerase chain reaction (mPCR) was used to detect the mcr-1-mcr-9 genes, then a further AST, whole genome sequencing (WGS) and bioinformatics analysis were platformed for these DEC isolates which were PCR positive for mcr genes. Results: Seventy in 91 isolates showed different antimicrobial resistance levels to the drugs tested with a resistance rate of 76.92%. The isolates showed the highest antimicrobial resistance rates to ampicillin (69.23%, 63/91) and trimethoprim-sulfamethoxazole (59.34%, 54/91), respectively. The multiple drug-resistant rate was 47.25% (43/91). Two mcr-1 gene and ESBL (extended-spectrum beta-lactamase) positive EAEC (enteroaggregative Escherichia coli) strains were detected. One of them was identified as serotype of O11:H6, which showed a resistance profile to 25 tested drugs referring to 10 classes, and 38 drug resistance genes were predicted by genome analysis. The other one was O16:H48 serotype, which was resistant to 21 tested drugs belonging to 7 classes and carried a new variant of mcr-1 gene (mcr-1.35). Conclusion: An overall high-level antimicrobial resistance was found among foodborne DEC isolates recovered from parts of China in 2020, and so was the MDR (multi-drug resistance) condition. MDR strains carrying multiple resistance genes such as mcr-1 gene were detected, and a new variant of mcr-1 gene was also found. It is necessary to continue with a dynamic monitoring on DEC contamination and an ongoing research into antimicrobial resistance mechanisms.

Complete Genome Sequence of the First Colistin-Resistant Raoultella electrica Strain

We present the full genome sequence of a colistin-resistant Raoultella electrica strain (MIC, >4 μg/mL) that was isolated from the stool of a healthy person living in India. The sequence consists of a chromosome and three plasmids (5,455,992-bp and 98,913-bp, 4,232-bp, and 3,961-bp, respectively). No previously described colistin resistance mechanisms were detected.

Farm to table: colistin resistance hitchhiking through food.

Colistin is a high priority, last-resort antibiotic recklessly used in livestock and poultry farms. It is used as an antibiotic for treating multi-drug resistant Gram-negative bacterial infections as well as a growth promoter in poultry and animal farms. The sub-therapeutic doses of colistin exert a selection pressure on bacteria leading to the emergence of colistin resistance in the environment. Colistin resistance gene, mcr are mostly plasmid-mediated, amplifying the horizontal gene transfer. Food products such as chicken, meat, pork etc. disseminate colistin resistance to humans through zoonotic transfer. The antimicrobial residues used in livestock and poultry often leaches to soil and water through faeces. This review highlights the recent status of colistin use in food-producing animals, its association with colistin resistance adversely affecting public health. The underlying mechanism of colistin resistance has been explored. The prohibition of over-the-counter colistin sales and as growth promoters for animals and broilers has exhibited effective stewardship of colistin resistance in several countries.

Colistin Resistance in Acinetobacter baumannii: Molecular Mechanisms and Epidemiology.

Acinetobacter baumannii is recognized as a clinically significant pathogen causing a wide spectrum of nosocomial infections. Colistin was considered a last-resort antibiotic for the treatment of infections caused by multidrug-resistant A. baumannii. Since the reintroduction of colistin, a number of mechanisms of colistin resistance in A. baumannii have been reported, including complete loss of LPS by inactivation of the biosynthetic pathway, modifications of target LPS driven by the addition of phosphoethanolamine (PEtN) moieties to lipid A mediated by the chromosomal pmrCAB operon and eptA gene-encoded enzymes or plasmid-encoded mcr genes and efflux of colistin from the cell. In addition to resistance to colistin, widespread heteroresistance is another feature of A. baumannii that leads to colistin treatment failure. This review aims to present a critical assessment of relevant published (>50 experimental papers) up-to-date knowledge on the molecular mechanisms of colistin resistance in A. baumannii with a detailed review of implicated mutations and the global distribution of colistin-resistant strains.

Colistin resistance mechanisms in Gram-negative bacteria: a Focus on Escherichia coli.

Multidrug-resistant (MDR) Escherichia coli strains have rapidly increased worldwide, and effective antibiotic therapeutic options are becoming more restricted. As a polymyxin antibiotic, colistin has a long history of usage, and it is used as a final line of treatment for severe infections by Gram-negative bacteria (GNB) with high-level resistance. However, its application has been challenged by the emergence of E. coli colistin resistance. Hence, determining the mechanism that confers colistin resistance is crucial for monitoring and controlling the dissemination of colistin-resistant E. coli strains. This comprehensive review summarizes colistin resistance mechanisms in E. coli strains and concentrates on the history, mode of action, and therapeutic implications of colistin. We have mainly focused on the fundamental mechanisms of colistin resistance that are mediated by chromosomal or plasmid elements and discussed major mutations in the two-component systems (TCSs) genes and plasmids that transmit the mobilized colistin resistance resistant genes in E. coli strains.

Carbonyl Cyanide 3-Chloro Phenyl Hydrazone (CCCP) Restores the Colistin Sensitivity in Brucella intermedia.

Brucella intermedia (formerly Ochrobactrum intermedium), a non-fermentative bacterium, has been isolated from animals and human clinical specimens. It is naturally resistant to polymyxins, including colistin (CO), and may cause opportunistic infections in humans. We isolated six Brucella intermedia strains from Senegalese monkey stool. In order to determine whether an efflux pump mechanism was involved in CO resistance in B. intermedia, we evaluated the effects of verapamil (VRP), reserpine (RSP), phe-arg β-naphthylamide dihydrochloride (PAβN) and carbonyl cyanide 3-chloro phenyl hydrazone (CCCP), four efflux pump inhibitors, on these colistin-resistant strains. Using the broth microdilution method, a CO and CCCP combination of 2 µg/mL and 10 µg/mL, respectively, significantly reduced the CO minimal inhibitory concentration (MIC) of B. intermedia, supporting an efflux pump mechanism. In contrast, VRP, PAβN and RSP did not restore CO susceptibility. A time kill assay showed a bactericidal effect of the CO-CCCP combination. Genomic analysis revealed a potential implication in the CO resistance mechanism of some conserved efflux pumps, such as YejABEF, NorM and EmrAB, as previously reported in other bacteria. An inhibitory effect of the CO-CCCP combination was observed on biofilm formation using the crystal violet method. These results suggest that the intrinsic CO resistance in Brucella intermedia is linked to an efflux pump mechanism and that the synergistic effect of CO-CCCP may open a new field to identify new treatments to restore antibiotic efficacy in humans.

Regulatory Landscape of the Pseudomonas aeruginosa Phosphoethanolamine Transferase Gene eptA in the Context of Colistin Resistance

Pseudomonas aeruginosa has the genetic potential to acquire colistin resistance through the modification of lipopolysaccharide by the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) or phosphoethanolamine (PEtN), mediated by the arn operon or the eptA gene, respectively. However, in vitro evolution experiments and genetic analysis of clinical isolates indicate that lipopolysaccharide modification with L-Ara4N is invariably preferred over PEtN addition as the colistin resistance mechanism in this bacterium. Since little is known about eptA regulation in P. aeruginosa, we generated luminescent derivatives of the reference strain P. aeruginosa PAO1 to monitor arn and eptA promoter activity. We performed transposon mutagenesis assays to compare the likelihood of acquiring mutations leading to arn or eptA induction and to identify eptA regulators. The analysis revealed that eptA was slightly induced under certain stress conditions, such as arginine or biotin depletion and accumulation of the signal molecule diadenosine tetraphosphate, but the induction did not confer colistin resistance. Moreover, we demonstrated that spontaneous mutations leading to colistin resistance invariably triggered arn rather than eptA expression, and that eptA was not induced in resistant mutants upon colistin exposure. Overall, these results suggest that the contribution of eptA to colistin resistance in P. aeruginosa may be limited by regulatory restraints.

Molecular epidemiology and genomic characterization of a plasmid-mediated mcr-10 and blaNDM-1 co-harboring multidrug-resistant Enterobacter asburiae

Colistin is considered as one of the last-resort antimicrobial agents for treating multidrug-resistant bacterial infections. Multidrug-resistant E. asburiae has been increasingly isolated from clinical patients, which posed a great challenge for antibacterial treatment. This study aimed to report a mcr-10 and blaNDM-1 co-carrying E. asburiae clinical isolate 5549 conferred a high-level resistance against colistin. Antibiotic susceptibility testing was performed using the microdilution broth method. Transferability of mcr-10 and blaNDM-1-carrying plasmids were investigated by conjugation experiments. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to identify modifications in lipid A. Whole genome sequencing and phylogenetic analysis between strain 5549 and a total of 301 E. asburiae genomes retrieved from NCBI database were performed. The genetic characteristics of mcr-10 and blaNDM-1-bearing plasmids were also analyzed. Our study indicated that strain 5549 showed extensively antibiotic-resistant trait, including colistin and carbapenem resistance. The mcr-10 and blaNDM-1 were carried by IncFIB/IncFII type p5549_mcr-10 (159417 bp) and IncN type p5549_NDM-1 (63489 bp), respectively. Conjugation assays identified that only the blaNDM-1-carrying plasmid could be successfully transferred to E. coli J53. Interestingly, mcr-10 did not mediate colistin resistance when it was cloned into E. coli DH5α. Mass spectrometry analysis showed the lipid A palmitoylation of the C-lacyl-oxo-acyl chain to the chemical structure of lipid A at m/z 2063 in strain 5549. In summary, this study is the first to report a mcr-10 and blaNDM-1 co-occurrence E. asburiae recovered from China. Our investigation revealed the distribution of different clonal lineage of E. asburiae with epidemiology perspective and the underlying mechanisms of colistin resistance. Active surveillance is necessary to control the further dissemination of multidrug-resistant E. asburiae.

Comparative genomics and molecular epidemiology of colistin-resistant Acinetobacter baumannii

This study aimed to investigate the prevalence and resistance mechanisms of colistin-resistant Acinetobacter baumannii (ColRAB) isolates in Serbia, assess their genetic relatedness to other circulating A. baumannii isolates in the neighbouring European countries, and analyse the global genomic epidemiology of ColRAB isolates. A total of 784 isolates of A. baumannii were recovered from hospitalised patients in Serbia between 2018 and 2021. The antimicrobial susceptibility testing was performed using disk diffusion and broth microdilution. All ColRAB isolates were subjected to DNA isolation and whole-genome sequencing (WGS). Overall, 3.94 % (n = 30) isolates were confirmed as ColRAB. Results of mutational and transcriptional analysis of genes associated with colistin resistance indicate the central role of the two-component regulating system, PmrAB, and increased expression of the pmrC gene in ColRAB. Most of the isolates (n = 29, 96.6 %) belonged to international clone II, with the most common sequence type being STPas2 (n = 23, 76.6 %). Based on the WGS analysis, ColRAB isolates belonging to the same ST isolated in various countries were grouped into the same clusters, indicating the global dissemination of several high-risk clonal lineages. Phylogenomic analysis of ColRAB isolates, together with all previously published A. baumannii genomes from South-Eastern European countries, showed that colistin resistance arose independently in several clonal lineages. Comparative genomic analysis revealed multiple genes with various roles (transcriptional regulation, transmembrane transport, outer membrane assembly, etc.), which might be associated with colistin resistance in A. baumannii. The obtained findings serve as the basis for further studies, contributing to a better understanding of colistin resistance mechanisms in A. baumannii.

147. Identification of Novel Colistin Resistance Genes in an Extremely Colistin Resistant Pseudomonas aeruginosa Clinical Isolate

Abstract Background Pseudomonas aeruginosa (PA) readily acquires genomic mutations and exogenous genetic elements that confer antimicrobial resistance (AMR). With the rise in AMR, there are limited antibiotics available to treat multidrug-resistant (MDR) PA. As such, clinicians have returned to previously used antibiotics. Colistin, sidelined for neurotoxicity and nephrotoxicity, has returned to clinical practice as a viable but suboptimal option for MDR-PA treatment. The most common mechanism of resistance to colistin involves modifications of the lipid A moiety within the bacterial lipopolysaccharide (LPS). Following the identification of a MDR PA isolate, BWH047, we experimentally determined its colistin MIC to be > 1,280 µg/mL and used genomic approaches to identify novel genetic mechanisms of extreme colistin resistance. Methods We created a random, saturated transposon (Tn) insertion library in PA BWH047 using the Himar1 mariner system. After exposure of the library to 640 µg/mL colistin for 10 hours, genomic DNA was harvested, and the Tn insertion sites were sequenced. Insertion sequencing (INSeq) analysis was performed. We identified 27 genes conditionally important for BWH047 growth in the presence of colistin. We selected five initial targets arnC, dedA, wapH, speE2, and bchE and tested their impact on colistin resistance using standard microbroth dilution methods. Results Of our deletion mutants, three showed loss of resistance to colistin. ArnC was chosen as a positive control as its role in colistin resistance in PA is well described. Colistin MICs of BWH047 ΔarnC, ΔdedA, ΔwapH, ΔspeE2, and ΔbchE were determined to be 0.5, 0.5, 1, > 1,280 and > 1280 μg/mL, respectively. Conclusion Here, we used INSeq to identify novel genes involved in extreme colistin resistance. Thus far, we have identified two new candidate genes dedA and wapH, critical for colistin resistance in PA BWH047. Neither gene has been associated with colistin resistance in PA; However, dedA orthologs in Burkholderia thailandenesis and Klebsiella pneumoniae have been shown to be important for colistin resistance. The gene wapH is part of the LPS core oligosaccharide biosynthetic pathway and its discovery hints that additional alterations in the bacterial outer membrane may impact colistin resistance. Disclosures All Authors: No reported disclosures.

Molecular mechanisms of colistin resistance among multi-drug resistant (MDR) Klebsiella pneumoniae and Escherichia coli isolated from ICU patients and their susceptibility towards eravacycline

Molecular mechanisms of colistin resistance among multi-drug resistant (MDR) Klebsiella pneumoniae and Escherichia coli isolated from ICU patients and their susceptibility towards eravacycline

Molecular mechanisms and clonal lineages of colistin-resistant bacteria across the African continent: a scoping review.

Colistin (also known as polymyxin E), a polymyxin antibiotic discovered in the late 1940s, has recently reemerged as a last-line treatment option for multidrug-resistant infections. However, in recent years, colistin-resistant pathogenic bacteria have been increasingly reported worldwide. Accordingly, the presented review was undertaken to identify, integrate and synthesize current information regarding the detection and transmission of colistin-resistant bacteria across the African continent, in addition to elucidating their molecular mechanisms of resistance. PubMed, Google Scholar and Science Direct were employed for study identification, screening and extraction. Overall, based on the developed literature review protocol and associated inclusion/exclusion criteria, 80 studies published between 2000 and 2021 were included comprising varying bacterial species and hosts. Numerous mechanisms of colistin resistance were reported, including chromosomal mutation(s) and transferable plasmid-mediated colistin resistance (encoded by mcr genes). Perhaps unexpectedly, mcr-variants have exhibited rapid emergence and spread across most African regions. The genetic variant mcr-1 is predominant in humans, animals and the natural environment, and is primarily carried by IncHI2- type plasmid. The highest number of studies reporting the dissemination of colistin-resistant Gram-negative bacteria were conducted in the North African region.

Colistin 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.

Preferential Selection of Low-Frequency, Lipopolysaccharide-Modified, Colistin-Resistant Mutants with a Combination of Antimicrobials in Acinetobacter baumannii.

ABSTRACTColistin, which targets lipopolysaccharide (LPS), is used as a last-resort drug against severe infections caused by drug-resistant Acinetobacter baumannii. However, A. baumannii possesses two colistin-resistance mechanisms. LPS modification caused by mutations in pmrAB genes is often observed in clinical isolates of multidrug-resistant Gram-negative pathogens. In addition to LPS modification, A. baumannii has a unique colistin resistance mechanism, a complete loss of LPS due to mutations in the lpxACD genes, which are involved in LPS biosynthesis. This study aimed to elucidate the detailed mechanism of the emergence of colistin-resistant A. baumannii using strains with the same genetic background. Various colistin-resistant strains were generated experimentally using colistin alone and in combination with other antimicrobials, such as meropenem and ciprofloxacin, and the mutation spectrum was analyzed. In vitro selection of A. baumannii in the presence of colistin led to the emergence of strains harboring mutations in lpxACD genes, resulting in LPS-deficient colistin-resistant strains. However, combination of colistin with other antimicrobials led to the selection of pmrAB mutant strains, resulting in strains with modified LPS (LPS-modified strains). Further, the LPS-deficient strains showed decreased fitness and increased susceptibility to many antibiotics and disinfectants. As LPS-deficient strains have a higher biological cost than LPS-modified strains, our findings suggested that pmrAB mutants are more likely to be isolated in clinical settings. We provide novel insights into the mechanisms of resistance to colistin and provide substantial solutions along with precautions for facilitating current research and treatment of colistin-resistant A. baumannii infections.IMPORTANCE Acinetobacter baumannii has developed resistance to various antimicrobial drugs, and its drug-resistant strains cause nosocomial infections. Controlling these infections has become a global clinical challenge. Carbapenem antibiotics are the frontline treatment drugs for infectious diseases caused by A. baumannii. For patients with infections caused by carbapenem-resistant A. baumannii, colistin-based therapy is often the only treatment option. However, A. baumannii readily acquires resistance to colistin. Many patients infected with colistin-resistant A. baumannii undergo colistin treatment before isolation of the colistin-resistant strain, and it is hypothesized that colistin resistance predominantly emerges under selective pressure during colistin therapy. Although the concomitant use of colistin and carbapenems has been reported to have a synergistic effect in vitro against carbapenem-resistant A. baumannii strains, our observations strongly suggest the need for attention to the emergence of strains with a modified lipopolysaccharide during treatment.

Dissemination of Carbapenemases and MCR-1 Producing Gram-Negative Bacteria in Aquatic Environments in Batna, Algeria.

Antibiotic-resistant-bacteria are being considered as emerging environmental contaminants where the importance of the surrounding environment in their emergence and dissemination has been emphasized. The aim of this study was to screen for the presence and diversity of carbapenem- and colistin-resistant Gram-negative bacteria (GNBs) in different aquatic environments. Water samples were collected in Batna, Algeria. Carbapenem- and colistin-resistant GNBs were selectively isolated and then identified using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. After phenotypic antibiotic susceptibility testing, the molecular mechanisms of β-lactams and colistin-resistance were investigated by PCR and sequencing. The clonality of mcr-1 positive Escherichia coli was determined by multi-locus sequence typing. We noticed a high level of resistance in both tap water and wastewater. The most commonly found carbapenem-resistance mechanism was the OXA-48 enzyme, but other carbapenemases were also detected. In addition, the mcr-1 gene was detected in 18 E. coli of different sequence types. Our findings highlight the role of aquatic environments in the dissemination of resistant-bacteria, especially considering that water is a connecting medium between different ecological systems and can easily transmit resistant-bacteria and promote horizontal gene transfer. Thus, the development of effective treatment strategies for eliminating antibiotic-resistance is seriously needed.

Detection of different colistin resistance mechanisms among multidrug resistant Klebsiella pneumoniae isolates in Bulgaria.

The more frequent usage of colistin resulted in an increase of colistin resistance due to lipopolysaccharide modifications. The aim of this study was to reveal the prevalence and mechanisms of colistin resistance among multidrug-resistant Klebsiella pneumoniae isolates collected in Bulgaria. One hundred multidrug resistant K. pneumoniae isolates were collected in a period between 2017 and 2018. Among them, 29 colistin resistant and 8 heteroresistant isolates were observed and further investigated. Clonal relatedness was detected by RAPD and MLST. Сarbapenemases, two component system phoQ/phoP, pmrA/B, and mgrB were investigated by PCR amplification and Sanger sequencing. Among 37 colistin nonsusceptible isolates, we detected 25 NDM-1 producers. The isolates belonged mainly to ST11 (80%), and also to ST147, ST35, ST340, ST219 (1-2 members per clone). Nine colistin resistant isolates showed changes in mgrB. IS903B-like elements truncated mgrB in five isolates. In two isolates, premature stopcodon (Q30stopcodon) was observed and another two isolates did not amplify mgrB, possibly due to bigger deletion or insertion. No isolates showed phoQ/phoP and pmrA/B mutations except for pmrB (four isolates had R256G). All isolates with IS903B insertions belonged to ST11 clone. The mgrB alterations play major role in colistin resistance in K. pneumoniae isolates studied in the current work. We report truncation of mgrB by IS903 like element in colistin resistant NDM-1 producing K. pneumoniae ST11 clone in Bulgaria.

First detection of a colistin-resistant Klebsiella aerogenes isolate from a critically ill patient with septic shock in Bulgaria.

Colistin is considered as the last-line antibiotic for the treatment of infections caused by extensively drug-resistant Gram-negative pathogens belonging to the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) group. The present study aimed to explore the colistin resistance mechanisms of a Klebsiella aerogenes (formerly Enterobacter aerogenes) isolate (Kae1177-1bg) obtained from a Bulgarian critically ill patient with septic shock in 2020. Antimicrobial susceptibility testing and whole-genome sequencing using DNA nanoball technology were performed. The resulting read pairs were used for draft genome assembly, MLST analysis and mutation screening in the pmrA/B, phoP/Q, and mgrB genes. Kae1177-1bg demonstrated high-level resistance to colistin, resistance to 3rd generation cephalosporins and susceptibility to all other antibiotics tested. In our strain a CMY-2-type class C cephalosporinase was the only β-lactamase identified. No mobile colistin resistance (mcr) genes were detected. A total of three missense variants in the genes for the two-component PmrA/PmrB system were identified. Two of them were located in the pmrB (pR57K and pN275K) and one in the pmrA gene (pL162M). The pN275K variant emerged as the most likely cause for colistin resistance because it affected a highly conservative position and was the only nonconservative amino acid substitution. In conclusion, to the best of our knowledge, this is the first documented clinical case of a high-level colistin-resistant K. aerogenes in Bulgaria and the first identification of the nonconservative amino acid substitution pN275K worldwide. Colistin-resistant Gram-negative pathogens of ESKAPE group are serious threat to public health and should be subjected to infection control stewardship practices.

Carbapenemase- and Colistin Resistant Escherichia coli Strains from Children in China: High Genetic Diversity and First Report of bla NDM-5, bla CTX-M-65, bla OXA-10, bla TEM-1, and mcr-1.1 Genes Co-Occurrence in E. coli ST156.

BackgroundThe emergence of carbapenem and colistin co-resistant Escherichia coli poses a huge challenge to infection control. The purpose of this study was to clarify the mechanism of the carbapenems and colistin co-resistance in E. coli strains.MethodsAntimicrobial susceptibility test was carried out by agar dilution methods and colistin resistance was confirmed by broth microdilution methods. Whole genome sequencing was carried out, and resistance genes, sequence types and virulence genes of carbapenems and colistin co-resistance E. coli isolates were analyzed.ResultsThe results showed that among the 176 carbapenem-resistant Enterobacteriaceae strains, 5 multidrug resistant E. coli strains exhibiting coresistance to carbapenem and colistin. The main mechanism of 5 E. coli strains in this study was generating carbapenem. Four E. coli strains were mcr-positive, while one mcr-negative strain had a new MgrB mutation. The blaNDM-5, blaCTX-M-65, blaOXA-10, blaTEM-1 and mcr-1.1 genes were simultaneously detected in E. coli 20IR1127 strain belonging to ST156 lineage. Other antimicrobial resistance genes encoding aminoglycosides-, sulfonamide-, chloramphenicol-, tetracyclines- and macrolides resistance were also detected.ConclusionThe main mechanisms of carbapenem and colistin resistance were encoded by blaNDM and mcr1.1, meanwhile mgrB mutations also contribute to colistin resistance. To our knowledge, this study is the first to report of E. coli ST156 strain in which the blaNDM-5, blaCTX-M-65, blaOXA-10, blaTEM-1 and mcr1.1 genes coexist. In addition, there is also an E. coli ST457 strain, which carries blaTEM-1, blaNDM-9, blaCTX-M-199 and is positive for mcr1.1 gene.