Plasmid-mediated Colistin Resistance Gene Research Articles

Co-existence of two plasmids harboring transferable resistance-nodulation-division pump gene cluster, tmexCD1-toprJ1, and colistin resistance gene mcr-8 in Klebsiella pneumoniae

BackgroundThe emergence of plasmid-mediated mobile colistin resistance (mcr) gene poses a great challenge to the clinical application of polymyxins. To date, mcr-1 to mcr-10 have been found in animals, humans, and the environment. Among them, mcr-8 was first identified in Klebsiella pneumoniae (K. pneumoniae) of swine origin, and then mcr-8.1 to mcr-8.5 were successively identified. Notably, K. pneumoniae is the major host of the mcr-8 gene in both animals and humans. This study aims to explore the characteristics of K. pneumoniae strains carrying the mcr-8 gene and tmexCD1-toprJ1 gene cluster and investigate the correlation between these two antibiotic resistance genes.MethodsThe isolates from the poultry farms and the surrounding villages were identified by mass spectrometer, and the strains positive for mcr-1 to mcr-10 were screened by polymerase chain reaction (PCR). The size of the plasmid and the antimicrobial resistance genes carried were confirmed by S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern hybridization, and the transferability of the plasmid was verified by conjugation experiments. Antimicrobial susceptibility testing (AST) and whole genome sequencing (WGS) were used to characterize the strains.ResultsTwo K. pneumoniae isolates (KP26 and KP29) displaying polymyxin resistance were identified as mcr-8 gene carriers. Besides that, tigecycline-resistant gene cluster tmexCD1-toprJ1 was also found on the other plasmid which conferred strain resistance to tigecycline. Through epidemiological analysis, we found that the mcr-8 gene has dispersed globally, circulating in the human, animals, and the environment. Furthermore, our analysis suggests that the coexistence of mcr-8 and tmexCD1-toprJ1 on a single plasmid might evolved through plasmid recombination.ConclusionsAlthough the mcr-8 and tmexCD1-toprJ1 gene clusters in the two strains of K. pneumoniae in this study were on two different plasmids, they still pose a potential threat to public health, requiring close monitoring and further study.

Novel functions of o-cymen-5-ol nanoemulsion in reversing colistin resistance in multidrug-resistant Klebsiella pneumoniae infections

Multidrug resistance (MDR) Klebsiella pneumoniae (K. pneumoniae) is a major emerging threat to human health, and leads to very high mortality rate. The effectiveness of colistin, the last resort against MDR Gram-negative bacteria, is significantly compromised due to the widespread presence of plasmid- or chromosome-mediated resistance genes. In this study, o-cymen-5-ol has been found to greatly restore colistin sensitivity in MDR K. pneumoniae. Importantly, this compound does not impact bacterial viability, induce resistance, or cause any noticeable cell toxicity. Various routes disclosed the potential mechanism of o-cymen-5-ol potentiating colistin activity against MDR K. pneumoniae. These include inhibiting the activity of plasmid-mediated mobile colistin resistance gene (mcr-1), accelerating lipopolysaccharide (LPS) − mediated membrane damage, and promoting the ATP-binding cassette (ABC) transporter pathway. To enhance the administration and bioavailability of o-cymen-5-ol, a nanoemulsion has been designed, which significantly improves the loading efficiency and solubility of o-cymen-5-ol, resulting in antimicrobial potentiation of colistin against K. pneumoniae infection. This study has revealed a new understanding of the o-cymen-5-ol nanoemulsion as a means to enhance the effectiveness of colistin against resistant factors. The finding also suggests that o-cymen-5-ol nanoemulsion could be a promising approach in the development of potential treatments for multidrug-resistant Gram-negative bacterial infections.

Mobile Colistin-Resistant Genes mcr-1, mcr-2, and mcr-3 Identified in Diarrheal Pathogens among Infants, Children, and Adults in Bangladesh: Implications for the Future.

Colistin is a last-resort antimicrobial for treating multidrug-resistant Gram-negative bacteria. Phenotypic colistin resistance is highly associated with plasmid-mediated mobile colistin resistance (mcr) genes. mcr-bearing Enterobacteriaceae have been detected in many countries, with the emergence of colistin-resistant pathogens a global concern. This study assessed the distribution of mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 genes with phenotypic colistin resistance in isolates from diarrheal infants and children in Bangladesh. Bacteria were identified using the API-20E biochemical panel and 16s rDNA gene sequencing. Polymerase chain reactions detected mcr gene variants in the isolates. Their susceptibilities to colistin were determined by agar dilution and E-test by minimal inhibitory concentration (MIC) measurements. Over 31.6% (71/225) of isolates showed colistin resistance according to agar dilution assessment (MIC > 2 μg/mL). Overall, 15.5% of isolates carried mcr genes (7, mcr-1; 17, mcr-2; 13, and mcr-3, with co-occurrence occurring in two isolates). Clinical breakout MIC values (≥4 μg/mL) were associated with 91.3% of mcr-positive isolates. The mcr-positive pathogens included twenty Escherichia spp., five Shigella flexneri, five Citrobacter spp., two Klebsiella pneumoniae, and three Pseudomonas parafulva. The mcr-genes appeared to be significantly associated with phenotypic colistin resistance phenomena (p = 0.000), with 100% colistin-resistant isolates showing MDR phenomena. The age and sex of patients showed no significant association with detected mcr variants. Overall, mcr-associated colistin-resistant bacteria have emerged in Bangladesh, which warrants further research to determine their spread and instigate activities to reduce resistance.

Investigation of Plasmid-Mediated Colistin Resistance Genes (mcr-1-8) in Enterobacterales Isolates.

Background The escalating global rise in multidrug-resistant gram-negative bacteria presents an increasingly substantial threat to patient safety. Over the past decade, carbapenem-resistant Enterobacterales (CRE) have emerged as one of the most critical pathogens in hospital-acquired infections, notably within intensive care units. Colistin has become one of the last-resort antimicrobial agents utilized to combat infections caused by CRE. However, the use of colistin has been accompanied by a notable increase in the prevalence of colistin-resistant bacteria. This study aimed to investigate plasmid-mediated colistin resistance genes ranging from mcr-1 to mcr-8 among members of the Enterobacterales order. Materials and methods This prospective study was conducted in the microbiology laboratory of Afyonkarahisar Health Sciences UniversityHealth Research and Practice Center between May 1, 2021 and July 31, 2022. A total of 2,646 Enterobacterales isolates were obtained from all culture-positive clinical samples sent from various clinics.Of these, 79 isolates exhibiting resistance to carbapenem antibiotics were included in the study. Among the 79 isolates, the presence of mcr-1 to mcr-8 genes was investigated in 27 isolates that were shown to be resistant to colistin. The identification of bacteria at the species level and antibiotic susceptibility tests were conducted using the VITEK 2 automated system (bioMérieux, USA). Colistin resistance among Enterobacterales strains exhibiting carbapenem resistance was evaluated using the broth microdilution technique (ComASP™ Colistin, Liofilchem, Italy), in accordance with the manufacturer's instructions. Results In our in vitro investigations, the minimum inhibitory concentration (MIC) values for meropenem were determined to be >8 µg/ml, whereas for colistin, the MIC50 value was >16 µg/ml and the MIC90 value was 8 µg/ml. A total of 27 colistin-resistant strains were identified among the 79 carbapenem-resistant Enterobacterales strains analyzed. The most prevalent agent among colistin-resistant strains was Klebsiella pneumoniae (K. pneumoniae), representing 66.7% of the isolates. This was followed by Proteus mirabilis (P. mirabilis) with 29.6%and Escherichia coli (E. coli) with 3.7%. The colistin resistance rate among carbapenem-resistant strains was found to be 34.2%, with colistin MIC values in strains tested by the broth microdilution method ranging from 4 to >16 µg/ml concentrations. In polymerase chain reaction (PCR) studies, the mcr-1 gene region was successfully detected by real-time PCR in the positive control isolate. Nevertheless, none of the gene regions from mcr-1 to mcr-8 were identified in our study investigating the presence of plasmid-mediated genes using a multiplex PCR kit. Conclusion Although our study demonstrated the presence of increased colistin resistance rates in carbapenem-resistant Enterobacterales isolates, it resulted in the failure to detect genes from mcr-1 to mcr-8 by the multiplex PCR method. Therefore, it is concluded that the colistin resistance observed in Enterobacteriaceae isolates in our region is not due to the mcr genes screened, but to different resistance development mechanisms.

River waters in Greece: A reservoir for clinically relevant extended-spectrum-β-lactamases-producing Escherichia coli

In the current study, the genotypic characteristics such as antimicrobial resistance and virulence genes, and plasmid replicons and phenotypic characteristics such as biofilm formation and antimicrobial resistance of 87 extended-spectrum beta-lactamase (ESBL)-producing E. coli (ESBL-Ec) isolated from 7 water bodies in northern Greece were investigated. Our data show a high prevalence (60.0 %) of ESBL-Ec in surface waters that exhibit high genetic diversity, suggesting multiple sources of their transmission into the aquatic environment. When evaluating the antimicrobial resistance of isolates, wide variation in their resistance profiles has been detected, with all isolates being multi-drug resistant (MDR). Regarding biofilm formation capacity and phylogenetic groups, the majority (54.0 %, 47/87) of ESBL-Ec were classified as no biofilm producers mainly assigned to phylogroup A (35.6 %; 31/87), followed by B2 (26.5 %; 23/87). PCR screening showed that a high proportion of the isolates tested positive for the blaCTX-M-1 group genes (69 %, 60/87), followed by blaTEM (55.2 %, 48/87), blaOXA (25.3 %, 22/87) and blaCTX-M-9 (17.2 %, 15/87). A subset of 28 ESBL-Ec strains was further investigated by applying whole genome sequencing (WGS), and among them, certain clinically significant sequence types were identified, such as ST131 and ST10. The corresponding in silico analysis predicted all these isolates as human pathogens, while a significant proportion of WGS-ESBL-Ec were assigned to extraintestinal pathogenic E. coli (ExPEC; 32.1 %), and urinary pathogenic E. coli (UPEC; 28.6 %) pathotypes. Comparative phylogenetic analysis, showed that the genomes of the ST131-O25:H4-H30 isolates are genetically linked to the human clinical strains. Here, we report for the first time the detection of a plasmid-mediated mobile colistin resistance gene in ESBL-Ec in Greece isolated from an environmental source. Overall, this study underlines the role of surface waters as a reservoir for antibiotic resistance genes and for presumptive pathogenic ESBL-Ec.

Development and evaluation of rapid and accurate one-tube RPA-CRISPR-Cas12b-based detection of mcr-1 and tet(X4).

The emergence and quick spread of the plasmid-mediated tigecycline resistance gene tet(X4) and colistin resistance gene mcr-1 have posed a great threat to public health and raised global concerns. It is imperative to develop rapid and accurate detection systems for the onsite surveillance of mcr-1 and tet(X4). In this study, we developed one-tube recombinase polymerase amplification (RPA) and CRISPR-Cas12b integrated mcr-1 and tet(X4) detection systems. We identified mcr-1- and tet(X4)-conserved and -specific protospacers through a comprehensive BLAST search based on the NCBI nt database and used them for assembling the detection systems. Our developed one-tube RPA-CRISPR-Cas12b-based detection systems enabled the specific detection of mcr-1 and tet(X4) with a sensitivity of 6.25 and 9 copies within a detection time of ~ 55 and ~ 40min, respectively. The detection results using pork and associated environmental samples collected from retail markets demonstrated that our developed mcr-1 and tet(X4) detection systems could successfully monitor mcr-1 and tet(X4), respectively. Notably, mcr-1- and tet(X4)-positive strains were isolated from the positive samples, as revealed using the developed detection systems. Whole-genome sequencing of representative strains identified an mcr-1-carrying IncI2 plasmid and a tet(X4)-carrying IncFII plasmid, which are known as important vectors for mcr-1 and tet(X4) transmission, respectively. Taken together, our developed one-tube RPA-CRISPR-Cas12b-based mcr-1 and tet(X4) detection systems show promising potential for the onsite detection of mcr-1 and tet(X4). KEY POINTS: • One-tube RPA-CRISPR-Cas12b-based mcr-1 and tet(X4) detection systems were developed based on identified novel protospacers. • Both detection systems exhibited high sensitivity and specification with a sample-to-answer time of less than 1 h. • The detection systems show promising potential for onsite detection of mcr-1 and tet(X4).

The prevalent dynamic and genetic characterization of mcr-1 encoding multidrug resistant Escherichia coli strains recovered from poultry in Hebei, China

ObjectivesColistin is known as the last resort antibiotic to treat the infections caused by multidrug resistant foodborne pathogens. The emergence and widespread dissemination of plasmid-mediated colistin resistance gene mcr-1 in the Escherichia coli (E. coli) incurs potential threat to public health. Here, we investigated the epidemiology, transmission dynamics, and genetic characterization of mcr-1 harbouring E. coli isolates from poultry originated in Hebei Province, China. MethodsA total of 297 faecal samples were collected from the two large poultry farms in Hebei Province, China. The samples were processed for E. coli identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry and 16S rDNA sequencing. Then, the mcr-1 gene harbouring E. coli strains were identified by polymerase chain reaction and subjected to antimicrobial susceptibility testing by broth microdilution assay. The genomic characterization of the isolates was done by whole genome sequencing using the various bioinformatics tools, and multi-locus sequence typing was done by sequence analysis of the seven housekeeping genes. The conjugation experiment was done to check the transferability of mcr-1 along with the plasmid stability testing. ResultsA total of six mcr-1 E. coli isolates with minimum inhibitory concentration of 4 μg/mL were identified from 297 samples (2.02%). The mcr-1 harbouring E. coli were identified as multidrug resistant and belonged to ST101 (n = 4) and ST410 (n = 2). The genetic environment of mcr-1 presented its position on IncHI2 plasmid in 4 isolates and p0111 in 2 isolates, which is a rarely reported plasmid type for mcr-1. Moreover, both type of plasmids was transferable to recipient J53, and mcr-1 was flanked by 3 mobile elements ISApl1, Tn3, and IS26 forming a novel backbone Tn3–IS26-mcr-1– pap2-ISApl1 on the p0111 plasmid. The phylogenetic analysis shared a common lineage with mcr-1 harbouring isolates from the environment, humans, and animals, which indicate its horizontal spread among the diverse sources, species, and hosts. ConclusionThis study recommends the one health approach for future surveillance across multiple sources and bacterial species to adopt relevant measures and reduce global resistance crises.

Investigation of Escherichia coli isolates from pigs and humans for colistin resistance in Lao PDR- a cross-sectional study

BackgroundIn Laos, colistin is not currently registered for use in humans. This One Health study aimed to estimate the prevalence of meat-producing pigs carrying colistin-resistant Escherichia coli, and investigate if E. coli causing invasive human infections were colistin-resistant. MethodsBetween September 2022 and March 2023, rectal swabs were collected from 895 pigs from abattoirs in 9/17 Lao provinces. Pig rectal swabs and stored E. coli isolates from human blood cultures, submitted to Mahosot Hospital Microbiology laboratory between 2005 and 2022, were screened for colistin resistance on selective chromogenic agar with organism identification confirmed using MALDI-TOF MS. Suspected colistin-resistant isolates underwent colistin susceptibility testing by broth microdilution following European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Isolates with MIC values of ≥2 μg/ml were tested for plasmid-mediated colistin resistance genes (mcr-1, mcr-2, and mcr-3) by multiplex SYBR Green PCR. ResultsA total of 15/620 (2.41%) invasive human E. coli isolates were phenotypically colistin-resistant by broth microdilution (MIC values 4 to 8 μg/ml). The earliest isolate was from 2015 in a patient from Phongsaly province in Northern Laos. A total of 582/895 (65.02%) pig rectal swab samples contained colistin-resistant E. coli. The detected colistin resistance genes were predominantly mcr-1 (57.8%, 346/598), followed by mcr-3 (20.23%,121/598), and 22.24% (133/598) were found to co-harbour mcr-1 and mcr-3. Among the 15 human isolates with colistin MIC values of ≥4 μg/ml, 12/15 were mcr-1. ConclusionsWe found that colistin resistant E. coli is causing invasive infection in humans in Laos despite the fact it is not available for human use. Use in animals seems to be widespread, confirmed by high carriage rates of colistin-resistant E. coli in pigs. It is probable that food-producing animals are the source of colistin-resistant E. coli bloodstream infection in Laos, although these have been infrequent to date. This is a serious public health concern in the region that needs to be addressed by appropriate enforceable legislation.

Repeated Occurrence of Mobile Colistin Resistance Gene-Carrying Plasmids in Pathogenic Escherichia coli from German Pig Farms.

The global spread of plasmid-mediated mobile colistin resistance (mcr) genes threatens the vital role of colistin as a drug of last resort. We investigated whether the recurrent occurrence of specific E. coli pathotypes and plasmids in individual pig farms resulted from the continued presence or repeated reintroduction of distinct E. coli strains. E. coli isolates (n = 154) obtained from three pig farms with at least four consecutive years of mcr detection positive for virulence-associated genes (VAGs) predicting an intestinal pathogenic pathotype via polymerase chain reaction were analyzed. Detailed investigation of VAGs, antimicrobial resistance genes and plasmid Inc types was conducted using whole genome sequencing for 87 selected isolates. Sixty-one E. coli isolates harbored mcr-1, and one isolate carried mcr-4. On Farm 1, mcr-positive isolates were either edema disease E. coli (EDEC; 77.3%) or enterotoxigenic E. coli (ETEC; 22.7%). On Farm 2, all mcr-positive strains were ETEC, while mcr-positive isolates from Farm 3 showed a wider range of pathotypes. The mcr-1.1 gene was located on IncHI2 (Farm 1), IncX4 (Farm 2) or IncX4 and IncI2 plasmids (Farm 3). These findings suggest that various pathogenic E. coli strains play an important role in maintaining plasmid-encoded colistin resistance genes in the pig environment over time.

A multiplex TaqMan real-time PCR assays for the rapid detection of mobile colistin resistance (mcr-1 to mcr-10) genes.

Recently, 10 plasmid-mediated mobile colistin resistance genes, mcr-1 to mcr-10, and their variants have been identified, posing a new threat to the treatment of clinical infections caused by Gram-negative bacteria. Our objective was to develop a rapid, sensitive, and accurate molecular assay for detecting mcr genes in clinical isolates. The primers and corresponding TaqMan-MGB probes were designed based on the sequence characteristics of all reported MCR family genes, multiplex Taqman-MGB probe-based qPCR assays were developed and optimized, and the sensitivity, specificity and reproducibility of the method were evaluated. The assay contained 8 sets of primers and probes in 4 reaction tubes, each containing 2 sets of primers and probes. The standard curves for both the single and multiplex systems showed good linearity (R2 > 0.99) between the starting template amount and the Ct value, with a lower limit of detection of 102 copies/μL. The specificity test showed positive amplification results only for strains containing the mcr genes, whereas the other strains were negative. The results of intra-and inter-group repeatability experiments demonstrated the stability and reliability of the newly developed method. It was used to detect mcr genes in 467 clinically-obtained Gram-negative isolates, which were multidrug-resistant. Twelve strains containing the mcr genes were detected (seven isolates carrying mcr-1, four isolates carrying mcr-10, and one isolate carrying mcr-9). The products amplified by the full-length PCR primer were identified by sequencing, and the results were consistent with those of the multiplex qPCR method. The assay developed in this study has the advantages of high specificity, sensitivity, and reproducibility. It can be used to specifically detect drug-resistant clinical isolates carrying the mcr genes (mcr-1 to mcr-10), thus providing a better basis for clinical drug treatment and drug resistance research.

Molecular Detection of Plasmid-mediated colistin-resistance genes in Multi-drug resistant Escherichia coli isolated from human and poultry in Duhok city, Iraq

Molecular Detection of Plasmid-mediated colistin-resistance genes in Multi-drug resistant Escherichia coli isolated from human and poultry in Duhok city, Iraq

Hidden threats in the plastisphere: Carbapenemase-producing Enterobacterales colonizing microplastics in river water

Carbapenem resistance poses a significant burden on healthcare systems worldwide. Microplastics (MPs) have emerged as potential contributors to antibiotic resistance spread in the environment. However, the link between MPs and carbapenem resistance remains unexplored. We investigated the prevalence of carbapenem-resistant bacteria colonizing MPs placed in a river. Three replicates of a mixture of polypropylene (PP), polyethylene (PE) and polyethylene terephthalate (PET) and of PET alone were placed both upstream and downstream a wastewater treatment plant (WWTP) discharge. Carbapenem-resistant Enterobacterales (CRE) were further characterized by phenotypic tests and whole-genome sequencing. The abundance of carbapenem-resistant bacteria on MPs increased significantly downstream the WWTP. Their prevalence was higher in the MPs mixture compared to PET alone. CRE strains colonizing MPs included Klebsiella pneumoniae (n = 3), Klebsiella quasipneumoniae (n = 3), Raoultella ornithinolytica (n = 2), Enterobacter kobei (n = 1) and Citrobacter freundii (n = 1), most (n = 8) recovered after the WWTP discharge. All strains exhibited at least one of the tested virulence traits (biofilm formation at 37 °C, haemolytic activity and siderophore production), were multi-drug resistant and carried carbapenemase-encoding genes [blaKPC-3 (n = 5), blaGES-5 (n = 2) or blaKPC-3 + blaGES-5 (n = 3)]. Uncommon phenotypes of resistance to imipenem/relebactam (n = 3) and ceftazidime/avibactam (n = 2) were observed. Two blaKPC−3-positive K. pneumoniae successfully transfer this gene trough conjugation. Genome analysis predicted all strains as human pathogens. The blaKPC−3 was associated with the Tn4401d transposon on a pBK30683-like plasmid in most of the isolates (n = 7). The blaGES-5 was mostly linked to class 3 integrons. A K. pneumoniae strain belonging to the outbreak-causing high-risk clone ST15 carried both blaKPC-3 and blaCTX-M-15. Two K. quasipneumoniae isolates carried the plasmid-mediated colistin resistance gene mcr-9. Our results underscore the role of MPs as vectors for CRE dissemination, particularly following WWTPs discharges. MPs may act as carriers, facilitating the dissemination of carbapenemase-encoding genes and potentially contributing to increased CRE incidence in the environment.

A new variant of the colistin resistance gene MCR-1 with co-resistance to β-lactam antibiotics reveals a potential novel antimicrobial peptide.

The emerging and global spread of a novel plasmid-mediated colistin resistance gene, mcr-1, threatens human health. Expression of the MCR-1 protein affects bacterial fitness and this cost correlates with lipid A perturbation. However, the exact molecular mechanism remains unclear. Here, we identified the MCR-1 M6 variant carrying two-point mutations that conferred co-resistance to β-lactam antibiotics. Compared to wild-type (WT) MCR-1, this variant caused severe disturbance in lipid A, resulting in up-regulation of L, D-transpeptidases (LDTs) pathway, which explains co-resistance to β-lactams. Moreover, we show that a lipid A loading pocket is localized at the linker domain of MCR-1 where these 2 mutations are located. This pocket governs colistin resistance and bacterial membrane permeability, and the mutated pocket in M6 enhances the binding affinity towards lipid A. Based on this new information, we also designed synthetic peptides derived from M6 that exhibit broad-spectrum antimicrobial activity, exposing a potential vulnerability that could be exploited for future antimicrobial drug design.

A new function of thymol nanoemulsion for reversing colistin resistance in Salmonella enterica serovar Typhimurium infection.

Adjuvant addition of approved drugs or foodborne additives to colistin might be a cost-effective strategy to overcome the challenge of plasmid-mediated mobile colistin resistance gene emergence, which poses a threat in the clinic and in livestock caused by infections with Gram-negative bacteria, especially carbapenem-resistant Enterobacteriaceae. Chequerboard assay was applied to screen the colistin adjuvants from natural compounds. The killing-time curve, combined disc test and membrane permeation assay were conducted to identify the synergy efficacy of thymol and colistin in vitro. Thin-layer chromatography (TLC), LC-MS and fluorescence spectra were used to indicate the interaction of thymol and MCR-1. The potential binding sites were then investigated by molecular simulation dynamics. Finally, a thymol nanoemulsion was prepared with high-pressure homogenization as the clinical dosage form. Thymol presented an excellent synergistic effect in vitro with colistin against Salmonella enterica serovar Typhimurium and Escherichia coli bacteria. Thymol addition, forming a complex with MCR-1, might interfere with the efficacy of MCR-1. Moreover, thymol strengthened colistin activity associated with potentiating membrane damage, destroying the biofilm and enhancing reactive oxygen species-mediated oxidative damage. Thymol nanoemulsion combined with colistin remarkably prevented the intestinal damage caused by S. Typhimurium infection, resulting in a survival rate higher than 60%. This study achieved a promising thymol oral formulation as colistin adjuvant to combat S. Typhimurium infection, which could be used to extend the lifespan of colistin in clinical veterinary medicine.

First Report and Characterization of the mcr-1 Positive Multidrug-Resistant Escherichia coli Strain Isolated from Pigs in Croatia.

The emergence and rapid spread of the plasmid-mediated colistin-resistant mcr-1 gene introduced a serious threat to public health. In 2021, a multi-drug resistant, mcr-1 positive Escherichia coli EC1945 strain, was isolated from pig caecal content in Croatia. Antimicrobial susceptibility testing and whole genome sequencing were performed. Bioinformatics tools were used to determine the presence of resistance genes, plasmid Inc groups, serotype, sequence type, virulence factors, and plasmid reconstruction. The isolated strain showed phenotypic and genotypic resistance to nine antimicrobial classes. It was resistant to colistin, gentamicin, ampicillin, cefepime, cefotaxime, ceftazidime, sulfamethoxazole, chloramphenicol, nalidixic acid, and ciprofloxacin. Antimicrobial resistance genes included mcr-1, blaTEM-1B, blaCTX-M-1, aac(3)-IId, aph(3')-Ia, aadA5, sul2, catA1, gyrA (S83L, D87N), and parC (A56T, S80I). The mcr-1 gene was located within the conjugative IncX4 plasmid. IncI1, IncFIB, and IncFII plasmids were also detected. The isolate also harbored 14 virulence genes and was classified as ST744 and O101:H10. ST744 is a member of the ST10 group which includes commensal, extraintestinal pathogenic E. coli isolates that play a crucial role as a reservoir of genes. Further efforts are needed to identify mcr-1-carrying E. coli isolates in Croatia, especially in food-producing animals to identify such gene reservoirs.

Characteristics and genomic epidemiology of colistin-resistant Enterobacterales from farmers, swine, and hospitalized patients in Thailand, 2014–2017

BackgroundColistin is one of the last resort therapeutic options for treating carbapenemase-producing Enterobacterales, which are resistant to a broad range of beta-lactam antibiotics. However, the increased use of colistin in clinical and livestock farming settings in Thailand and China, has led to the inevitable emergence of colistin resistance. To better understand the rise of colistin-resistant strains in each of these settings, we characterized colistin-resistant Enterobacterales isolated from farmers, swine, and hospitalized patients in Thailand.MethodsEnterobacterales were isolated from 149 stool samples or rectal swabs collected from farmers, pigs, and hospitalized patients in Thailand between November 2014–December 2017. Confirmed colistin-resistant isolates were sequenced. Genomic analyses included species identification, multilocus sequence typing, and detection of antimicrobial resistance determinants and plasmids.ResultsThe overall colistin-resistant Enterobacterales colonization rate was 26.2% (n = 39/149). The plasmid-mediated colistin-resistance gene (mcr) was detected in all 25 Escherichia coli isolates and 9 of 14 (64.3%) Klebsiella spp. isolates. Five novel mcr allelic variants were also identified: mcr-2.3, mcr-3.21, mcr-3.22, mcr-3.23, and mcr-3.24, that were only detected in E. coli and Klebsiella spp. isolates from farmed pigs.ConclusionOur data confirmed the presence of colistin-resistance genes in combination with extended spectrum beta-lactamase genes in bacterial isolates from farmers, swine, and patients in Thailand. Differences between the colistin-resistance mechanisms of Escherichia coli and Klebsiella pneumoniae in hospitalized patients were observed, as expected. Additionally, we identified mobile colistin-resistance mcr-1.1 genes from swine and patient isolates belonging to plasmids of the same incompatibility group. This supported the possibility that horizontal transmission of bacterial strains or plasmid-mediated colistin-resistance genes occurs between humans and swine.

Gigantol restores the sensitivity of mcr carrying multidrug-resistant bacteria to colistin

BackgroundThe emergence and wide spread of plasmid-mediated colistin resistance gene (mcr-1) and its mutants have immensely limited the efficacy of colistin in treating multidrug-resistant (MDR) Gram-negative bacterial infections. The development of synergistic combinations of antibiotics with a natural product that coped with the resistance of MDR bacteria was an economic strategy to restore antibiotics activity. Herein, we investigated gigantol, a bibenzyl phytocompound, for restoring in vitro and in vivo, the sensitivity of mcr-positive bacteria to colistin. MethodsThe synergistic activity of gigantol and colistin against multidrug-resistant Enterobacterales was studied via checkerboard assay and time-killing curve. Subsequently, the transcription and protein expression levels of mcr-1 gene were determined by RT-PCR and Western blots. The interaction of gigantol and MCR-1 was simulated via molecular docking and verified via site-directed mutagenesis of MCR-1. Hemolytic activity and cytotoxicity assay were used to evaluate the safety of gigantol. Finally, the in vivo synergistic effect was evaluated via two animal infection models. ResultsGigantol restored the activity of colistin against mcr-positive bacteria E.coli B2 (MIC from 4 μg/ml to 0.25 μg/ml), Salmonella 15E343 (MIC from 8 μg/ml to 1 μg/ml), K. pneumoniae 19-2-1 (MIC from 32 μg/ml to 2 μg/ml) carrying mcr-1, mcr-3, mcr-8, respectively. Mechanistic studies revealed that gigantol down-regulated the expression of genes involved in LPS-modification, reduced the MCR-1 products and inhibited the activity of MCR-1 by binding to amino acid residues Tyr287 and Pro481 in its D-glucose-binding pocket. Safety evaluation showed that the addition of gigantol relieves the hemolysis caused by colistin. Compared with monotherapy, the combination of gigantol and colistin significantly improved the survival rate of Gallgallella mellonella larvae and mice infected by E.coli B2. Moreover, there was a considerable decrease in the bacterial load present in the viscera of mice. ConclusionOur results confirmed that gigantol was a potential colistin adjuvant, and could be used to tackle multi-drug resistant Gram-negative pathogen infections combined with colistin.

Cajanin stilbene acid: A direct inhibitor of colistin resistance protein MCR-1 that restores the efficacy of polymyxin B against resistant Gram-negative bacteria

BackgroundThe resistance of Gram-negative bacteria to polymyxin B, caused by the plasmid-mediated colistin resistance gene mcr-1, which encodes a phosphoethanolamine transferase (MCR-1), is a serious threat to global public health. Therefore, it is urgent to find new drugs that can effectively alleviate polymyxin B resistance. Through the screening of 78 natural compounds, we found that cajanin stilbene acid (CSA) can significantly restore the susceptibility of polymyxin B to mcr-1 positive Escherichia coli (E. coli). PurposeIn this study, we tried to evaluate the ability of CSA to restore the susceptibility of polymyxin B towards the E. coli, and explore the mechanism of sensitivity recovery. Study design and methodsCheckerboard MICs, time-killing curves, scanning electron microscope, lethal and semi-lethal models of infection in mice were used to assess the ability of CSA to restore the susceptibility of polymyxyn to E. coli. The interaction between CSA and MCR-1 was evaluated using surface plasmon resonance (SPR), and molecular docking experiments. ResultsHere, we find that CSA, a potential direct inhibitor of MCR-1, effectively restores the sensitivity of E. coli to polymyxin B. CSA can restore the sensitivity of polymyxin B to drug-resistant E. coli, and the MIC value can be reduced to 1 μg/ml. The time killing curve and scanning electron microscopy results also showed that CSA can effectively restore polymyxin B sensitivity. In vivo experiments showed that the simultaneous use of CSA and polymyxin B can effectively reduce the infection of drug-resistant E. coli in mice. SPR and molecular docking experiments confirmed that CSA strongly bound to MCR-1. The 17-carbonyl oxygen and 12- and 18‑hydroxyl oxygens of CSA were the key sites binding to MCR-1. ConclusionCSA is able to significantly restore the sensitivity of polymyxin B to E. coli in vivo and in vitro. CSA inhibits the enzymatic activity of the MCR-1 protein by binding to key amino acids at the active center of the MCR-1 protein.

Plasmid-Mediated Colistin Resistance Genes mcr-1 and mcr-4 in Multidrug-Resistant Escherichia coli Strains Isolated from a Healthy Pig in Portugal.

Antimicrobial resistance encoded by mobile colistin resistance (mcr) genes is a global and emergent threat. In this study, we report the occurrence of two different populations of colistin-resistant Escherichia coli harboring mcr-1 and mcr-4 variants in the intestinal microbiome of a healthy pig. Following antimicrobial susceptibility determination, the presence of mcr genes in two E. coli strains, isolated according to different selective microbiological procedures, was screened by PCR. Whole-genome sequencing confirmed that both strains were multidrug-resistant; INIAV_002EC was an AmpC producer carrying blaCMY-2, blaTEM-1B, qnrS1, mcr-1.1 genes, and INIAV_001EC carrying blaTEM-1A, tetB, and mcr-4.1 genes, along with mutations in quinolone resistance-determining regions. In addition, both strains harbored sul3, dfrA, and aadA1 determinants. Further genome analysis revealed different plasmid replicons associated with the mcr genes, IncX4 associated with mcr-1.1, and ColE10 with mcr-4.1. In addition, other replicons, including IncFIA, IncI1-Iγ, IncX1, IncY, in INIAV_002EC, and IncX1, IncI1, and p0111, in INIAV_001EC, were identified. Furthermore, both strains belonged to ST215 serotype O68:H12 and ST156 serotype O25:H28, respectively. This finding highlights the pig gut flora as a potential reservoir of mobile colistin resistance genes and reports the presence of the mcr-4.1 gene found for the first time in Portugal.

Quinolone-resistant Escherichia coli at the interface between humans, poultry and their shared environment- a potential public health risk

BackgroundCommensal Escherichia coli residing in the guts of humans and animals are reservoirs of multidrug resistance (MDR) genes, including quinolone resistance genes, in humans and poultry. This study aimed to characterize quinolones resistance in E. coli recovered from poultry workers, chickens, and poultry farm/market environments in Abuja, Nigeria.MethodsThis was a cross-sectional study conducted between December 2018 and April 2019 comprising poultry workers, chickens and their poultry farm/market environments. This study characterized E. coli isolates from stool, faecal and environmental samples using antimicrobial susceptibility testing and whole-genome sequencing methods. Core-genome multilocus sequences-based phylogeny was used to determine the relatedness between quinolone-resistant E. coli isolates. Data were analyzed using descriptive statistics.ResultsOf 110 E. coli isolates, quinolone-resistant phenotypes were observed in 68.2% (n = 75) isolates. Whole-genome sequencing detected plasmid-mediated quinolone resistance (PMQR) genes in 63.6% (n = 70) isolates. The most prevalent PMQR gene detected in 56 of these 70 E. coli isolates was qnrS1, followed by qnrB19 in 14 isolates and aac(6’)-lb-cr in two isolates. Fifteen ciprofloxacin and 19 nalidixic acid-resistant isolates respectively showed double mutations in the quinolone-resistance determining regions (QRDRs) of gyrA, with single or double mutations in parC, and a single mutation in parE. The most prevalent amino-acid substitutions observed were S83L + D87N in gyrA (46.5%, n = 20), S80I in parC (51.2%, n = 22) and S458A in parE (14%, n = 6). About 2.9% (2/70) of PMQR isolates were extended-spectrum beta-lactamase (ESBL) producers while 2.9% (2/70) had plasmid-mediated colistin resistance (PMCR) genes.ConclusionsPMQR genes were prevalent in E. coli isolates recovered from healthy humans, chickens and poultry farm/market environments. PMCR genes (mcr-1.1) occurred in PMQR-positive isolates recovered from manure and drinking water originating from poultry farm/market environments. It was found that the gene encoding ESBL coexisted with qnrS-positive isolates of human and avian origin. Horizontal transfer of PMQR genes among E. coli isolates in the human-poultry-environment interface has public health implications for the spread of antimicrobial resistance. Relevant government agencies should enforce regulations to restrict the use of critically important antimicrobials in poultry production.