Colistin-resistant Strains Research Articles

Colistin Interaction and Surface Changes Associated with mcr-1 Conferred Plasmid Mediated Resistance in E. coli and A. veronii Strains.

Colistin, a polycationic antimicrobial peptide, is one of the last-resort antibiotics for treating infections caused by carbapenem-resistant Gram-negative bacteria. The antibacterial activity of colistin occurs through electrostatic interaction between the polycationic peptide group of colistin and the negatively charged phosphate groups of lipid A membrane. This study investigated the interaction of colistin with the outer membrane and surface constituents of resistant and susceptible strains of Escherichia coli and Aeromonas veronii harboring mcr-1 resistance gene. Bacterial membrane and lipopolysaccharide used in this study were isolated from susceptible as well as colistin-resistant strains of E. coli and A. veronii. Interaction of colistin with the bacterial surface was studied by deoxycholate and lysozyme sensitivity test, N-phenyl-1-naphthylamine (NPN) uptake assay, Atomic force microscopy (AFM), Zeta potential measurements and 1H NMR. The binding affinity of colistin was found to be lower with outer membrane from resistant strains in comparison with the susceptible strains. Colistin exposure enhances the outer membrane permeability of the susceptible strains to deoxycholate and lysozyme. However, on the other hand, colistin dose of 256 µg/mL did not permeabilize the outer membrane of resistant bacteria. The NPN permeability in resistant strains was greater in comparison with susceptible strains. Atomic force microscopy images depicted smooth, featherless and deformed membranes in treated susceptible cells. Contrary to the above, resistant treated cells displayed surface roughness topography even at 256 µg/mL colistin concentration. Surface charge alterations were confirmed by Zeta potential measurements as a function of the growth phase. Mid-logarithmic phase susceptible strains showed a greater negative charge than resistant strains upon exposure to colistin. However, there was no statistical variation in the Zeta potential measurements between resistant and susceptible strains at the stationary phase. NMR analysis revealed line broadening in susceptible strains with increasing colistin: LPS aggregates mass ratio. Moreover, resistant strains did not show line broadening for the outer membrane, even at the highest mass ratio. The findings of this study suggest that the resistant strains of E. coli and A. veronii can block the electrostatic contact between the cationic peptide and anionic lipid A component that drives the first phase of colistin action, thereby preventing hydrophobically driven second-tier action of colistin on the outer lipopolysaccharide layer.

Molecular and Genomic Insights of mcr-1-Producing Escherichia coli Isolates from Piglets.

The use of colistin in food-producing animals favors the emergence and spread of colistin-resistant strains. Here, we investigated the occurrence and molecular mechanisms of colistin resistance among E. coli isolates from a Mexican piglet farm. A collection of 175 cephalosporin-resistant colonies from swine fecal samples were recovered. The colistin resistance phenotype was identified by rapid polymyxin test and the mcr-type genes were screened by PCR. We assessed the colistin-resistant strains by antimicrobial susceptibility test, pulse-field gel electrophoresis, plasmid profile, and mating experiments. Whole-Genome Sequencing data was used to explore the resistome, virulome, and mobilome of colistin-resistant strains. A total of four colistin-resistant E. coli were identified from the cefotaxime-resistant colonies. All harbored the plasmid-borne mcr-1 gene, which was located on conjugative 170-kb IncHI-2 plasmid co-carrying ESBLs genes. Thus, high antimicrobial resistance rates were observed for several antibiotic families. In the RC2-007 strain, the mcr-1 gene was located as part of a prophage carried on non-conjugative 100-kb-plasmid, which upon being transformed into K. variicola strain increased the polymyxin resistance 2-fold. The genomic analysis showed a broad resistome and virulome. Our findings suggest that colistin resistance followed independent acquisition pathways as clonal and non-genetically related mcr-1-harboring strains were identified. These E. coli isolates represent a reservoir of antibiotic resistance and virulence genes in animals for human consumption which could be potentially propagated into other interfaces.

Colistin Resistance Mechanisms in Human Salmonella enterica Strains Isolated by the National Surveillance Enter-Net Italia (2016-2018).

Background: A collection of human-epidemiologically unrelated S. enterica strains collected over a 3-year period (2016 to 2018) in Italy by the national surveillance Enter-Net Italia was analysed. Methods: Antimicrobial susceptibility tests, including the determination of minimal inhibitory concentrations (MICs) for colistin, were performed. Colistin resistant strains were analysed by PCR to detect mobile colistin resistance (mcr) genes. In mcr-negative S. enterica serovar Enteritidis strains, chromosomal mutations potentially involved in colistin resistance were identified by a genomic approach. Results: The prevalence of colistin-resistant S. enterica strains was 7.7%, the majority (87.5%) were S. Enteritidis. mcr genes were identified only in one strain, a S. Typhimurium monophasic variant, positive for both mcr-1.1 and mcr-5.1 genes in an IncHI2 ST4 plasmid. Several chromosomal mutations were identified in the colistin-resistant mcr-negative S. Enteritidis strains in proteins involved in lipopolysaccharide and outer membrane synthesis and modification (RfbN, LolB, ZraR) and in a component of a multidrug efflux pump (MdsC). These mutated proteins were defined as possible candidates for colistin resistance in mcr-negative S. Enteritidis of our collection. Conclusions: The colistin national surveillance in Salmonella spp. in humans, implemented with genomic-based surveillance, permitted to monitor colistin resistance, determining the prevalence of mcr determinants and the study of new candidate mechanisms for colistin resistance.

Prevalence, Risk Factors, Antimicrobial Resistance and Molecular Characterization of Salmonella in Northeast Tunisia Broiler Flocks.

This study was conducted in northeastern Tunisia to estimate both the prevalence and the risk factors of Salmonella in broiler flocks as well as to characterize the isolated multidrug-resistant (MDR) Salmonella strains. In the present study, a total number of 124 farms were sampled; Salmonella isolates were identified by the alternative technique VIDAS Easy Salmonella. The susceptibility of Salmonella isolates was assessed against 21 antimicrobials using the disk diffusion method on Mueller–Hinton agar using antimicrobial discs. Some antimicrobial resistance genes were identified using PCR. The prevalence rate of Salmonella infection, in the sampled farms, was estimated at 19.9% (64/322). Moreover, a total number of 13 different serotypes were identified. High rate of resistance was identified against nalidixic acid (82.85%), amoxicillin (81.25%), streptomycin (75%), and ciprofloxacin (75%). Alarming level of resistance to ertapenem (12.5%) was noticed. A total of 87.5% (56/64) of isolated strains were recognized as MDR. Three MDR strains were extended-spectrum β-lactamases (ESBL)-producers and three MDR strains were cephalosporinase-producers. The blaCTX-M gene was amplified in all the three ESBL strains. The qnrB gene was not amplified in fluoroquinolones-resistant strains. The tetA and tetB genes were amplified in 5% (2/40) and 2.5% (1/40) of tetracycline-resistant strains, respectively. The dfrA1 gene was amplified in five of the 20 trimethoprim-resistant strains. The mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 genes were not amplified in any of the phenotypically colistin-resistant strains. In terms of integrase genes int1 and int2, only gene class 2 was amplified in 11% (7/64) of analyzed strains. Risk factors, such as the poor level of cleaning and disinfection, the lack of antimicrobial treatment at the start of the breeding, and a crawl space duration lower than 15 days, were associated with high Salmonella infection in birds. These data should be considered when preparing salmonellosis control programs in Tunisian broiler flocks.

Lacticaseicin 30 and Colistin as a Promising Antibiotic Formulation against Gram-Negative β-Lactamase-Producing Strains and Colistin-Resistant Strains.

Antimicrobial resistance is a global health concern across the world and it is foreseen to swell if no actions are taken now. To help curbing this well announced crisis different strategies are announced, and these include the use of antimicrobial peptides (AMP), which are remarkable molecules known for their killing activities towards pathogenic bacteria. Bacteriocins are ribosomally synthesized AMP produced by almost all prokaryotic lineages. Bacteriocins, unlike antibiotics, offer a set of advantages in terms of cytotoxicity towards eukaryotic cells, their mode of action, cross-resistance and impact of microbiota content. Most known bacteriocins are produced by Gram-positive bacteria, and specifically by lactic acid bacteria (LAB). LAB-bacteriocins were steadily reported and characterized for their activity against genetically related Gram-positive bacteria, and seldom against Gram-negative bacteria. The aim of this study is to show that lacticaseicin 30, which is one of the bacteriocins produced by Lacticaseibacillus paracasei CNCM I-5369, is active against Gram-negative clinical strains (Salmonella enterica Enteritidis H10, S. enterica Typhimurium H97, Enterobacter cloacae H51, Escherichia coli H45, E. coli H51, E. coli H66, Klebsiella oxytoca H40, K. pneumoniae H71, K. variicola H77, K. pneumoniae H79, K. pneumoniae H79), whereas antibiotics failed. In addition, lacticaseicin 30 and colistin enabled synergistic interactions towards the aforementioned target Gram-negative clinical strains. Further, the combinations of lacticaseicin 30 and colistin prompted a drastic downregulation of mcr-1 and mcr-9 genes, which are associated with the colistin resistance phenotypes of these clinical strains. This report shows that lacticaseicin 30 is active against Gram-negative clinical strains carrying a rainbow of mcr genes, and the combination of these antimicrobials constitutes a promising therapeutic option that needs to be further exploited.

Predation of colistin- and carbapenem-resistant bacterial pathogenic populations and their antibiotic resistance genes in simulated microgravity

As mankind evaluates moving toward permanently inhabiting outer space and other planetary bodies, alternatives to antibiotic that can effectively control drug-resistant pathogens are needed. The activity of one such alternative, Bdellovibrio bacteriovorus HD100, was explored here, and was found to be as active or better in simulated microgravity (SMG) conditions as in flask and normal gravity (NG) cultures, with the prey viabilities decreasing by 3- to 7-log CFU/mL in 24 h. The activity of B. bacteriovorus HD100 under SMG was also appraised with three different carbapenem- and colistin-resistant pathogenic bacterial strains. In addition to being more efficient at killing two of these pathogens under SMG conditions (with losses of 5- to 6-log CFU/mL), we also explored the ability of B. bacteriovorus HD100 to hydrolyze the carbapenem- and colistin-resistant gene pools, i.e., mcr-1, blaKPC-2 and blaOXA-51, present in these clinical isolates. We found removal efficiencies of 97.4 ± 0.9 %, 97.8 ± 0.4 % and 99.3 ± 0.1 %, respectively, in SMG cultures, while similar reductions were also seen in the flask and NG cultures. These results illustrate the potential applicability of B. bacteriovorus HD100 as an antibiotic to combat the ever-growing threat of multidrug-resistant (MDR) pathogens during spaceflight, such as in the International Space Station (ISS).

Virulence Determinants and Plasmid-Mediated Colistin Resistance mcr Genes in Gram-Negative Bacteria Isolated From Bovine Milk.

A major increase of bacterial resistance to colistin, a last-resort treatment for severe infections, was observed globally. Using colistin in livestock rearing is believed to be the ground of mobilized colistin resistance (mcr) gene circulation and is of crucial concern to public health. This study aimed to determine the frequency and virulence characteristics of colistin-resistant Gram-negative bacteria from the milk of mastitic cows and raw unpasteurized milk in Egypt. One hundred and seventeen strains belonging to Enterobacteriaceae (n = 90), Pseudomonas aeruginosa (n = 10), and Aeromonas hydrophila (n = 17) were screened for colistin resistance by antimicrobial susceptibility testing. The genetic characteristics of colistin-resistant strains were investigated for mcr-1–9 genes, phylogenetic groups, and virulence genes. Moreover, we evaluated four commonly used biocides in dairy farms for teat disinfection toward colistin-resistant strains. Multidrug-resistant (MDR) and extensive drug-resistant (XDR) phenotypes were detected in 82.91% (97/117) and 3.42% (4/117) of the isolates, respectively. Of the 117 tested isolates, 61 (52.14%) were colistin resistant (MIC >2 mg/L), distributed as 24/70 (34.29%) from clinical mastitis, 10/11 (90.91%) from subclinical mastitis, and 27/36 (75%) from raw milk. Of these 61 colistin-resistant isolates, 47 (19 from clinical mastitis, 8 from subclinical mastitis, and 20 from raw milk) harbored plasmid-borne mcr genes. The mcr-1 gene was identified in 31.91%, mcr-2 in 29.79%, mcr-3 in 34.04%, and each of mcr-4 and mcr-7 in 2.13% of the colistin-resistant isolates. Among these isolates, 42.55% (20/47) were E. coli, 21.28% (10/47) A. hydrophila, 19.12% (9/47) K. pneumoniae, and 17.02% (8/47) P. aeruginosa. This is the first report of mcr-3 and mcr-7 in P. aeruginosa. Conjugation experiments using the broth-mating technique showed successful transfer of colistin resistance to E. coli J53-recipient strain. Different combinations of virulence genes were observed among colistin-resistant isolates with almost all isolates harboring genes. Hydrogen peroxide has the best efficiency against all bacterial isolates even at a low concentration (10%). In conclusion, the dissemination of mobile colistin resistance mcr gene and its variants between MDR- and XDR-virulent Gram-negative isolates from dairy cattle confirms the spread of mcr genes at all levels; animals, humans, and environmental, and heralds the penetration of the last-resort antimicrobial against MDR bacteria. Consequently, a decision to ban colistin in food animals is urgently required to fight XDR and MDR bacteria.

Elastic Nanofibrous Membranes for Medical and Personal Protection Applications: Manufacturing, Anti-COVID-19, and Anti-Colistin Resistant Bacteria Evaluation.

Herein, in the present work two series of thermoplastic polyurethane (TPU) nanofibers were manufactured using the electrospinning techniques with ZnO and CuO nanoparticles for a potential use as an elastic functional layer in antimicrobial applications. Percentages of 0%, 2 wt%, and 4 wt% of the nanoparticles were used. The morphological characterization of the electrospun TPU and TPU/NPs composites nanofibers were observed by using scanning electron microscopy to show the average fiber diameter and it was in the range of 90–150 nm with a significant impact of the nanoparticle type. Mechanical characterization showed that TPU nanofiber membranes exhibit excellent mechanical properties with ultra-high elastic properties. Elongation at break reached up to 92.5%. The assessment of the developed nanofiber membranes for medical and personal protection applications was done against various colistin resistant bacterial strains and the results showed an increment activity by increasing the metal oxide concentration up to 83% reduction rate by using TPU/ZnO 4% nanofibers against K. pneumoniae strain 10. The bacterial growth was completely eradicated after 8 and 16 h incubation with TPU/ZnO and TPU/CuO nanofibers, respectively. The nanofibers SEM study reveals the adsorption of the bacterial cells on the metal oxides nanofibers surface which led to cell lysis and releasing of their content. Finally, in vitro study against Spike S-protein from SARS-CoV-2 was also evaluated to investigate the potent effectiveness of the proposed nanofibers in the virus deactivation. The results showed that the metal oxide concentration is an effective factor in the antiviral activity due to the observed pattern of increasing the antibacterial and antiviral activity by increasing the metal oxide concentration; however, TPU/ZnO nanofibers showed a potent antiviral activity in relation to TPU/CuO.

Combining Colistin with Furanone C-30 Rescues Colistin Resistance of Gram-Negative Bacteria in Vitro and in Vivo.

ABSTRACTThe spread of multidrug-resistant (MDR) Gram-negative bacteria (GNB) has led to serious public health problems worldwide. Colistin, as a “last resort” for the treatment of MDR bacterial infections, has been used significantly in recent years and has led to the continuous emergence of colistin-resistant strains. In this study, we aimed to investigate the synergistic effect on the antimicrobial and antibiofilm activities of a colistin/furanone C-30 combination against colistin-resistant GNB in vitro and in vivo. According to antimicrobial resistance profiles, most of the colistin-resistant strains we collected showed MDR phenotypes. The checkerboard method and time-kill curve showed that the combination with furanone C-30 increases the antibacterial activity of colistin significantly. In addition, the furanone C-30/colistin combination can not only inhibit the formation of bacterial biofilm but also has a better eradication effect on preformed mature biofilms. The result of scanning electron microscopy (SEM) demonstrated that the furanone C-30/colistin combination led to a significant reduction in the number of cells in biofilms. Furthermore, furanone C-30 at 50 μg/ml did not cause any additional toxicity to RAW264.7 cells according to a cytotoxicity assay. In in vivo infection experiments, the furanone C-30/colistin combination increased the survival rate of infected Galleria mellonella larvae as well as decreased the microbial load in a mouse thigh infection model. The synergistic effect of the furanone C-30/colistin combination against colistin-resistant GNB is encouraging, and this work may shed light on a new therapeutic approach to combat colistin-resistant pathogens.IMPORTANCE Colistin is among the few antibiotics effective against multidrug-resistant Gram-negative bacteria (GNB) clinical isolates. However, colistin-resistant GNB strains have emerged in recent years. Therefore, the combination of colistin and nonantibacterial drugs has attracted much attention. In this study, the furanone C-30/colistin combination showed good antibacterial and antibiofilm activity in vitro and in vivo. In addition, increased membrane permeability leads to the synergistic effect of the furanone C-30/colistin combination. Because of the low cytotoxicity of furanone C-30, this combination has good application prospects in clinical anti-infective therapy. This finding might shed light on the discovery of combination therapy for infections caused by colistin-resistant GNB pathogens.

Activity of the combination of colistin and fosfomycin against NDM-1-producing Escherichia coli with variable levels of susceptibility to colistin and fosfomycin in a murine model of peritonitis.

Alternative treatments are needed against NDM-1-producing Escherichia coli. Colistin (COL) and fosfomycin (FOS) often remain active in vitro but selection of resistant mutants is frequent if used separately. We determined whether the combination of colistin and fosfomycin may be useful to treat infections with NDM-1-producing E. coli with varying levels of resistance. Isogenic derivatives of E. coli CFT073 with blaNDM-1 and variable levels of resistance to colistin and fosfomycin (CFT073-NDM1, CFT073-NDM1-COL and CFT073-NDM1-FOS, respectively) were used. The combination (colistin + fosfomycin) was tested in vitro and in a fatal peritonitis murine model. Mortality and bacterial loads were determined and resistant mutants detected. Colistin MICs were 0.5, 16 and 0.5 mg/L and fosfomycin MICs were 1, 1 and 32 mg/L against CFT073-NDM1, CFT073-NDM1-COL and CFT073-NDM1-FOS, respectively. In time-kill curves, combining colistin with fosfomycin was synergistic and bactericidal against CFT073-NDM1 and CFT073-NDM1-FOS, with concentrations of 4× MIC (for both drugs), but not against CFT073-NDM1-COL (concentrations of colistin = 0.5× MIC), due to regrowth with fosfomycin-resistant mutants. Mice died less and bacterial counts were lower in spleen with the combination compared with monotherapy against all strains; the combination prevented selection of resistant mutants except for CFT073-NDM1-COL where fosfomycin-resistant mutants were found in all mice. Combining colistin and fosfomycin was beneficial in vitro and in vivo against NDM-1-producing E. coli, even with strains less susceptible to colistin and fosfomycin. However, the combination failed to prevent the emergence of fosfomycin-resistant mutants against colistin-resistant strains. Combining colistin and fosfomycin constitutes an alternative for treatment of NDM-1 E. coli, except against colistin-resistant strains.

Rapid Identification of Escherichia coli Colistin-Resistant Strains by MALDI-TOF Mass Spectrometry.

Colistin resistance is one of the major threats for global public health, requiring reliable and rapid susceptibility testing methods. The aim of this study was the evaluation of a MALDI-TOF mass spectrometry (MS) peak-based assay to distinguish colistin resistant (colR) from susceptible (colS) Escherichia coli strains. To this end, a classifying algorithm model (CAM) was developed, testing three different algorithms: Genetic Algorithm (GA), Supervised Neural Network (SNN) and Quick Classifier (QC). Among them, the SNN- and GA-based CAMs showed the best performances: recognition capability (RC) of 100% each one, and cross validation (CV) of 97.62% and 100%, respectively. Even if both algorithms shared similar RC and CV values, the SNN-based CAM was the best performing one, correctly identifying 67/71 (94.4%) of the E. coli strains collected: in point of fact, it correctly identified the greatest number of colS strains (42/43; 97.7%), despite its lower ability in identifying the colR strains (15/18; 83.3%). In conclusion, although broth microdilution remains the gold standard method for testing colistin susceptibility, the CAM represents a useful tool to rapidly screen colR and colS strains in clinical practice.

Equisetin Restores Colistin Sensitivity against Multi-Drug Resistant Gram-Negative Bacteria.

The overuse of antibiotics and the scarcity of new drugs have led to a serious antimicrobial resistance crisis, especially for multi-drug resistant (MDR) Gram-negative bacteria. In the present study, we investigated the antimicrobial activity of a marine antibiotic equisetin in combination with colistin against Gram-negative bacteria and explored the mechanisms of synergistic activity. We tested the synergistic effect of equisetin in combination with colistin on 23 clinical mcr-1 positive isolates and found that 4 µg/mL equisetin combined with 1 µg/mL colistin showed 100% inhibition. Consistently, equisetin restored the sensitivity of 10 species of mcr-1 positive Gram-negative bacteria to colistin. The combination of equisetin and colistin quickly killed 99.9% bacteria in one hour in time-kill assays. We found that colistin promoted intracellular accumulation of equisetin in colistin-resistant E. coli based on LC-MS/MS analysis. Interestingly, equisetin boosted ROS accumulation in E. coli in the presence of colistin. Moreover, we found that equisetin and colistin lost the synergistic effect in two LPS-deficient A. baumannii strains. These findings suggest that colistin destroys the hydrophobic barrier of Gram-negative bacteria, facilitating equisetin to enter the cell and exert its antibacterial effect. Lastly, equisetin restored the activity of colistin in a G. mellonella larvae infection model. Collectively, these results reveal that equisetin can potentiate colistin activity against MDR Gram-negative bacteria including colistin-resistant strains, providing an alternative approach to address Gram-negative pathogens associated with infections in clinics.

Genome analysis of an OXA-48-producing carbapenem- and colistin-resistant Klebsiella pneumoniae sequence type 11 clone isolated from an inpatient

BackgroundColistin is one of the last-resort therapeutic antibiotics used to control carbapenem-resistant Klebsiella pneumoniae. However, the emergence of colistin-resistant strains with high mortality and morbidity has become a new challenge. Therefore, it is crucial to characterize the genotypic features of carbapenem- and colistin-resistant K. pneumoniae (CCKP) isolates to implement public health intervention strategies. MethodsIn this study, we analyzed the whole genome of an OXA-48-producing CCKP strain for molecular characterization of antimicrobial resistance (AMR) as well as comparative genome and plasmid analysis. ResultsThe whole-genome analysis showed that IRAN-43 genome possessed 140 contigs with 5,585,691 bp length, GC content of 56.9% and N50 score of 200,728. Multi-locus sequence typing (MLST) analysis showed that this K. pneumoniae strain belonged to sequence type (ST) 11. Single amino acid substitutions associated with colistin resistance were found in PhoP (D150G), PmrB (R256G) and RamA (I25T). In addition, two plasmids harboring qnrS1, tetR, tetB (plasmid-1) and blaOXA-48 (plasmid-2) were discovered. No plasmid-mediated colistin resistance (mcr) genes were detected. Integrative conjugative element of K. pneumoniae (ICEKp)4 variant was detected as well as cps locus length of 22,156 bp encoding 17 open reading frames (ORFs) which belonged to K. pneumoniae capsular locus (KL)15-1. ConclusionCCKP has been increasingly reporting from Iran. High frequency of antibiotic resistance genes among ST11 suggests that stricter control measures are needed to prevent the spread of this clone in hospital settings.

МОЛЕКУЛЯРНО-ГЕНЕТИЧЕСКИЕ МЕХАНИЗМЫ УСТОЙЧИВОСТИ НОЗОКОМИАЛЬНЫХ ШТАММОВ KLEBSIELLA PNEUMONIAE К ПОЛИМИКСИНАМ И АНТИБИОТИКАМ ДРУГИХ ГРУПП ПО ДАННЫМ ПОЛНОГЕНОМНОГО СЕКВЕНИРОВАНИЯ

Identification of numerous mechanisms of resistance to colistin and other antibiotics is possible using whole genome sequencing. Objectives. To assess the molecular-genetic mechanisms of resistance to polymyxins and antibiotics of other groups in nosocomial Klebsiella pneumoniae strains. Material and methods. For 13 multidrug- and extensively drug-resistant K.pneumoniae strains semiconductor sequencing was performed in the Ion PGM System genomic sequencer (Thermo Fisher Scientific, USA). The assembly of genomic sequences and their annotation were carried out. The PROVEAN software tool was used to predict the influence of nucleotide replacements on the structure of amino acid sequences and functional activity of proteins. The identification of antibiotic resistance genes and the search for efflux mechanisms were performed by the ResFinder v.4.1 and CARD web resources. Results. Several types of β-lactamase genes were detected simultaneously in all strains, as well as genes of resistance to fosfomycin. Genes of resistance to aminoglycosides were identified in 11 strains, to chloramphenicol - in 10, to rifampicin - in 5, to macrolides - in 4. The mcr phosphoethanolamine transferase genes were absent in all strains. Functionally significant substitutions were revealed in the pmrB gene (D150Y, T157P, G207S) comparing the studied samples with the reference K. pneumoniae strain ATCC 700603. Changes in the mgrB gene were also found in colistin-resistant strains (W20R replacement, insertional inactivation of the gene by transposons of the IS1, IS4 and IS5 families). Conclusions. The results of whole genome sequencing represent the significant resistance of nosocomial Klebsiella pneumoniae strains to the majority of antibiotics including β-lactams, aminoglycosides, fluoroquinolones, fosfomycin, chloramphenicol, polymyxins. Genetic determinants of colistin resistance were revealed (insertional inactivation and deletion of the mgrB gene; D150Y, T157P and G207S substitutions in the pmrB gene) in strains with colistin MIC 64-128 mg/l and their absence in colistin-susceptible strains.

Synergistic Activity of Capsaicin and Colistin Against Colistin-Resistant Acinetobacter baumannii: In Vitro/Vivo Efficacy and Mode of Action.

Acinetobacter baumannii is an opportunistic pathogen predominantly associated with nosocomial infections. With emerging resistance against polymyxins, synergistic combinations of drugs are being investigated as a new therapeutic approach. Capsaicin is a common constituent of the human diet and is widely used in traditional alternative medicines. The present study evaluated the antibacterial activities of capsaicin in combination with colistin against three unrelated colistin-resistant Acinetobacter baumannii strains in vitro and in vivo, and then further studied their synergistic mechanisms. Using the checkerboard technique and time-kill assays, capsaicin and colistin showed a synergistic effect on colistin-resistant A. baumannii. A mouse bacteremia model confirmed the in vivo effects of capsaicin and colistin. Mechanistic studies shown that capsaicin can inhibit the biofilm formation of both colistin-resistant and non-resistant A. baumannii. In addition, capsaicin decreased the production of intracellular ATP and disrupted the outer membrane of A. baumannii. In summary, the synergy between these drugs may enable a lower concentration of colistin to be used to treat A. baumannii infection, thereby reducing the dose-dependent side effects. Hence, capsaicin–colistin combination therapy may offer a new treatment option for the control of A. baumannii infection.

Detection of Colistin Resistance in Pseudomonas aeruginosa Using the MALDIxin Test on the Routine MALDI Biotyper Sirius Mass Spectrometer.

Colistin is frequently a last resort treatment for Pseudomonas aeruginosa infections caused by multidrug-resistant (MDR) and extensively drug resistant (XDR) strains, and detection of colistin resistance is essential for the management of infected patients. Therefore, we evaluated the recently developed MALDIxin test for the detection of colistin resistance in P. aeruginosa clinical strains using the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system. The test is based on the detection by mass spectrometry of modified lipid A by the addition of 4-amino-l-arabinose (l-ara4N) molecules on one or two phosphate groups, in strains resistant to colistin. Overproduction of l-Ara4N molecules is mainly due to the constitutive activation of the histidine kinase (PmrB) or the response regulator (PmrA) following an amino-acid substitution in clinical strains. The performance of the test was determined on a panel of 14 colistin-susceptible and 14 colistin-resistant P. aeruginosa clinical strains, the reference strain PAO1 and positive control mutants PmrB (V28G), PmrB (D172), PhoQ (D240–247), and ParR (M59I). In comparison with the broth microdilution (BMD) method, all the susceptible strains (n=14) and 8/14 colistin-resistant strains were detected in less than 1h, directly on whole bacteria. The remaining resistant strains (n=6) were all detected after a short pre-exposure (4h) to colistin before sample preparation. Validation of the method on a larger panel of strains will be the next step before its use in diagnostics laboratories. Our data showed that the MALDIxin test offers rapid and efficient detection of colistin resistant P. aeruginosa and is thus a valuable diagnostics tool to control the spread of these emerging resistant strains.

Slaughterhouse wastewater as a reservoir for extended-spectrum β-lactamase (ESBL)-producing, and colistin-resistant Klebsiella spp. and their impact in a “One Health” perspective

Klebsiella spp. are ubiquitous bacteria capable of colonizing humans and animals, and sometimes leading to severe infections in both. Due to their high adaptability against environmental/synthetic conditions as well as their potential in aquiring antimicrobial/metal/biocide resistance determinants, Klebsiella spp. are recognized as an emerging threat to public health, worldwide. Currently, scarce information on the impact of livestock for the spread of pathogenic Klebsiella spp. is available. Thus, the phenotypic and genotypic properties of extended-spectrum β-lactamase-producing, and colistin-resistant Klebsiella strains (n = 185) from process- and wastewater of two poultry and pig slaughterhouses as well as their receiving municipal wastewater treatment plants (mWWTPs) were studied to determine the diversity of isolates that might be introduced into the food-production chain or that are released into the environment by surviving the wastewater treatment.Selectively-isolated Klebsiella spp. from slaughterhouses including effluents and receiving waterbodies of mWWTPs were assigned to various lineages, including high-risk clones involved in human outbreaks, and exhibited highly heterogeneous antibiotic-resistance patterns. While isolates originating from poultry slaughterhouses showed the highest rate of colistin resistance (32.4%, 23/71), carbapenem-resistant Klebsiella spp. were only detected in mWWTP samples (n = 76). The highest diversity of resistance genes (n = 77) was detected in Klebsiella spp. from mWWTPs, followed by isolates from pig (n = 56) and poultry slaughterhouses (n = 52). Interestingly, no carbapenemase-encoding genes were detected and mobile colistin resistance genes were only obeserved in isolates from poultry and pig slaughterhouses.Our study provides in-depth information into the clonality of livestock-associated Klebsiella spp. and their determinants involved in antimicrobial resistance and virulence development. On the basis of their pathogenic potential and clinical importance there is a potential risk of colonization and/or infection of wildlife, livestock and humans exposed to contaminated food and/or surface waters.

Detection of Colistin Resistant E. coli in Children at Pediatric Hospital of Assiut University, Using Phenotypic and Genotypic Methods

Background: The emergence of colistin-resistant strains is considered a great threat for the children suffering from diarrhea. This study aimed to screen for the presence of mcr-1 in Escherichia coli (E. coli) isolates collected from children with diarrhea and to compare between genotypic and phenotypic methods for detection of colisitin resistant E.coli carrying mcr-1gene. Methods: Isolation of E.coli was done followed by antimicrobial susceptibility test. Kirby-Baur disc diffusion was used to determine antimicrobial susceptibility, whereas broth microdilution (BMD) and the double disc synergy test (DDST) were used to determine colistin resistance. The screening for mcr-1 was used to investigate one probable mechanism of colistin resistance by PCR. Results: All mcr-1 E.coli isolates were resistant to ampicillin, while resistance to ampicillin/sulbactam, cefazolin, cefoxitin, ceftazidime and trimethoprim-sulphamethoxazol was 94.1% (32/34), 94.1% (32/34), 94.1% (32/34), 85.3% (29/34) and 70.6% (24/34) respectively. All mcr-1carrying E. coli strains were sensitive to tobramycin, amikacin and imipenem. Moderate resistance was noticed to piperacillin/ tazobactam(23/34) 67.6%, gentamycin 47.1% (16/34), and ciprofloxacin 44.1% (15/34). Thirty-one (91.2 %) mcr-1 positiveE. coli strains were multidrug resistant (MDR). Forty five out of 95 (47.4%) of E.coli isolates were positive for mcr-1 by DDST and 34 /95 (35.78%) of E. coli isolates were positive for mcr-1 by PCR. Conclusions: This study reported a high prevalence of colistin resistant E. coli harboring mcr-1 gene in young children in Pediatric Hospital of Assiut University. Broth microdilution is more accurate than DDST in detection of colistin resistance.

Emergence and Clonal Spread of CTX-M-65-Producing Escherichia coli From Retail Meat in Portugal.

The emergence and dissemination of resistance to third- and fourth-generation cephalosporins among Enterobacteriaceae from different sources impose a global public health threat. Here, we characterized by whole-genome sequencing four Escherichia coli strains harboring the blaCTX–M–65 gene identified among 49 isolates from beef and pork collected at retail. The genomic content was determined using the Center for Genomic Epidemiology web tools. Additionally, the prediction and reconstruction of plasmids were conducted, the genetic platform of the blaCTX–M–65 genes was investigated, and phylogenetic analysis was carried out using 17 other genomes with the same sequence type and harboring the blaCTX–M–65 gene. All strains harbored blaCTX–M–65, blaOXA–1, and blaTEM–1B, and one also carried the blaSHV–12 gene. Other resistance genes, namely, qnrS2, aac(6′)-Ib-c, dfrA14, sul2, tetA, and mphA, were present in all the genomes; the mcr-1.1 gene was identified in the colistin-resistant strains. They belong to sequence type 2179, phylogenetic group B1, and serotype O9:H9 and carried plasmids IncI, IncFIC(FII), and IncFIB. All strains share an identical genetic environment with IS903 and ISEcp1 flanking the blaCTX–M–65 gene. It seems likely that the blaCTX–M–65 gene is located in the chromosome in all isolates based on deep in silico analysis. Our findings showed that the strains are clonally related and belong to two sub-lineages. This study reports the emergence of CTX-M-65-producing E. coli in Portugal in food products of animal origin. The chromosomal location of the blaCTX–M–65 gene may ensure a stable spread of resistance in the absence of selective pressure.

Antibiotic Potentiation in Multidrug-Resistant Gram-Negative Pathogenic Bacteria by a Synthetic Peptidomimetic.

The peptidomimetic H-[NLys-tBuAla]6-NH2 (CEP-136), which exhibits low inherent antimicrobial activity against Gram-negative bacteria (MIC = 16-64 μM), was shown to significantly potentiate the antibacterial activity of several clinically important antibiotics against the human pathogens Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Thus, the antibacterial spectrum of rifampicin, clarithromycin, and azithromycin could be extended to include also these Gram-negative bacteria. Additionally, the potentiation effect was demonstrated in a panel of clinically relevant multidrug-resistant isolates including extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing as well as colistin-resistant strains. For some peptidomimetic-antibiotic combinations, the strong synergy corresponded to a more than 50-fold reduction of the minimal inhibitory concentration of the antibiotic. Mechanistic studies indicate that the potentiation arises from a permeabilization effect exerted on the outer membrane lipopolysaccharide layer of the Gram-negative bacteria without significant disruption of the inner membrane. Furthermore, the peptidomimetic enhancer exhibited only a marginal effect on the viability of mammalian HepG2 cells even at concentrations 100-fold higher than that enabling the antibiotic enhancement. Also, a low hemolytic activity combined with limited in vivo acute toxicity of CEP-136 in healthy mice allowed in vivo validation of the potentiation effect on both rifampicin and azithromycin treatment in a murine peritonitis model. Thus, CEP-136 is an interesting hit compound for further development of effective adjuvants for repurposing antibiotics for use against infections by multidrug-resistant Gram-negative bacteria.