Mcr-1 Gene Research Articles

Convergence of mcr-1 and broad-spectrum β-lactamase genes in Escherichia coli strains from the environmental sector

The mcr-type gene encodes the main plasmid-mediated mechanism of colistin resistance and has been reported in several bacterial species obtained from different sources. Anthropogenic activities in the environment favor the evolution of antimicrobial resistance. Indeed, mcr-1-positive Escherichia coli strains were susceptible to non-polymyxins antimicrobials, but now emerging as multidrug-resistant (MDR) lineages. In this regard, hundreds of surface water and agricultural soil samples were screened for the presence of E. coli carrying the mcr-type genes and mcr-1-positive strains were subjected to in-depth genomic analysis. Almost all colistin-resistant strains were classified as MDR, highlighting those obtained from soils that showed resistance to extended-spectrum cephalosporins and carbapenems. International and high-risk clones of E. coli were identified, with ST10 and ST1720 shared between water and soil samples. Resistome analysis showed a broad resistome (AMR, metal tolerance, and biocide resistance). The mcr-1.1 and mcr-1.26 allelic variants were detected on IncX4 and IncI2 plasmids. Curiously, mcr-1-positive E. coli strains from agricultural soils harbored plasmid-mediated blaCTX-M-1, blaCTX-M-8, or blaKPC-2 genes. Virulome analysis demonstrated traits of a high putative virulence potential, with the presence of extraintestinal pathogenic E. coli. Comparative analysis revealed the persistence and dissemination of plasmid-mediated antimicrobial resistance genes in genetically diversity E. coli strains at the human-animal-environmental interface. These findings demonstrate a possible emerging AMR trend with the convergence of resistance to colistin and broad-spectrum β-lactams in environmental-derived E. coli strains.

Rapid detection of enterobacteria in wastewater treated by microalgal consortia using loop-mediated isothermal amplification (LAMP)

In the present study, nine Enterobacteriaceae species present in wastewater were isolated and identified, and loop-mediated isothermal amplification (LAMP) was developed for the detection of Enterobacteriaceae by designing primers based on the mcr-1, KPC, OXA-23, and VIM genes, which are recognized markers of antimicrobial resistance (AMR) transmission during microalgal bioremediation treatment. The developed assays successfully detected four strains positive for mcr-1 gene-asociated resistance (Acinetobacter baylyi, Klebsiella pneumoniae, Morganella morganii, and Serratia liquefaciens), three strains for KPC gene-associated resistance (Acinetobacter sp., Escherichia coli 15499, and Morganella morganii), seven strains for OXA-23 gene-associated resistance (Acinetobacter baylyi, Enterobacter hormaechi, Enterobacter cloacae, Escherichia coli 15922, Escherichia coli 51446, Morganella morganii, and Serratia liquefaciens), and three strains for resistance to the VIM gene-associated resistance (Acinetobacter baylyi, Acinetobacter sp., and Enterobacter hormaechi) from a single colony. A reduction in microbiological load of 93.6% was achieved at 15 colony-forming units (CFU) mL−1, utilizing EMB agar and LAMP values of 0.142 ± 0.011 for the mcr-1 gene, 0.212 ± 0.02 for the KPC gene, 0.233 ± 0.006 for the OXA-23 gene, and 0.219 ± 0.035 for the VIM gene. Furthermore, bioremediation efficiency values of 71.6% and 75% for total nitrogen and phosphorus, respectively, were observed at 72 h of treatment in open pond microalgal remediation systems (MRS). This study demonstrated that the LAMP technique is faster and more sensitive than traditional detection methods, such as CFU, for Enterobacteriaceae. Consequently, this method may be considered for the detection of microbiological quality indicators within the water treatment industry.

Prevalence, transmission and genomic epidemiology of mcr-1-positive colistin-resistant Escherichia coli strains isolated from international airplane waste, local resident fecal and wastewater treatment plants.

The emergence and dissemination of mcr-1-positive Escherichia coli (MCRPEC) represent a critical public health threat. Here, we conducted a prospective analysis of MCRPEC isolates from wastewater treatment plants (WWTPs), local residents' fecal (LRF), and international airplane waste (IAW) to investigate their genetic characteristics and transmission patterns circulating in human-environment domains. The MCRPEC prevalence was 2.43 % in WWTPs, 1.37 % in IAW and 0.69 % in LRF. MCRPEC showed substantial genetic diversity, encompassing 61 sequence types (primarily ST1011, ST101, and ST2705), 7 plasmid types (primarily IncI2), 8 phylogroups (primarily A and B1), 9 mcr-1-flanked lineages (primarily L5), 6 clusters (primarily C2 and C4), diverse serotypes, and 61.95 % transposon-containing strains. The mcr-1 gene co-existed with 46 antibiotic resistance genes (ARGs) and 19 virulence factor genes (VFGs). Notably, 6 IncI2 plasmids carried the blaCTX-M, IS1380, and mcr-1 genes. MCRPEC from WWTPs harbored a greater number of ARGs (56.95 ± 5.99) but fewer VFGs (15.03 ± 6.40) compared to those from human-associated sources (LRF and IAW). ST1011, ST2705, IncHI2, and L7 were prevalent in WWTP-derived MCRPEC, whereas IncX4 and L3 were more common in human-derived MCRPEC. Genetic features such as ST101, ST48, IncI2, L4, L5, C2, and C4 were simultaneously present in strains from LRF, IAW, and WWTPs. Core genetic analyses also showed genetically similar MCRPEC strains across various geographic locations. The findings underscore the extensive dissemination, strong environmental adaptation, and clonal transmission of MCRPEC across diverse reservoirs, reinforcing the urgent need for coordinated multisectoral surveillance of human and environment interfaces to effectively mitigate further transmission.

Culture-Independent Quantitative PCR Detected Mobilized Colistin Resistance Genes (mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5) in Chicken Gut Contents in Bangladesh

Inappropriate antimicrobial use in food animal farming propels antimicrobial resistance (AMR) that affects all health domains. Colistin is a ‘Reserve’ antibiotic for human treatment to be conserved for multidrug-resistant pathogens; however, it is being used as an animal growth promoter in many developing countries. The evolution of mobilized colistin resistance (mcr) gene-mediated colistin resistance has been reported to be associated with rampant colistin use. This study investigated the current variants of the mcr gene in chicken gut contents in Bangladesh. A cross-sectional study was designed to assess the mcr-1 to mcr-5 genes in 80 fresh poultry droppings from commercial poultry farms and 40 poultry droppings from household farms. DNA was extracted from each poultry dropping using commercial kits (Qiagen GmbH, Hilden, Germany). Real-time quantitative polymerase chain reaction (RT-qPCR) was employed using the qTOWER3 thermal cycler (Analytik Jena GmbH, Jena, Germany) to analyze the mcr gene variants in the extracted DNA. This study observed that 47.5% (57/120) of the samples exhibited the presence of at least one mcr gene out of the five variants investigated. The individual detection rates of the mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 genes were 42.5% (51/120), 2.5% (3/120), 1.7% (2/120), 5% (6/120), and 9.2% (11/120), respectively. The co-carriage of two or more genes was found in over 10% (10/57) of the samples. The triple occurrence of mcr genes was identified in three samples with the combination of mcr-1+mcr-2+mcr-4, mcr-1+mcr-3+mcr-5, and mcr-1+mcr-4+mcr-5. Overall, a significantly higher number of mcr genes were identified in the commercial farm chicken droppings compared to the household chicken droppings (p = 0.007). The existence of mcr genes in poultry feces in Bangladesh emphasizes the importance of proper poultry waste disposal and good hygiene practices in poultry livestock and its value chain. The potential impact of environmental ARGs should be considered in national and global policy documents. An integrated and combined approach to the One Health concept should be applied in all domains to understand and control the environment’s role in the evolution and transmission of AMR.

Antibiotic resistance genes, colistin-resistant Escherichia coli, and physicochemicals in health care wastewater in Vinh Long General Hospital, Vietnam.

This study collected ten treated wastewater samples from Vinh Long General Hospital to determine their physicochemical characteristics and antibiotic properties. All treated wastewater samples collected during the monitoring periods complied with national regulations. In addition, these samples did not contain bacteria such as Salmonella, Shigella, and Vibrio cholerae. The investigation yielded a total of 25 Escherichia coli isolates. The E. coli isolates exhibied highest antibiotic resistance rate to ampicillin (100%), followed by ciprofloxacin, amoxicillin/clavulanic acid, and cefazolin (96%, 92%, and 92%, respectively). The resistance rate to fosfomycin was 88%, whereas 80% of the isolates were resistant to sulfamethoxazole-trimethoprim. The resistance rate to gentamicin was 72%, whereas that to imipenem and tetracycline was 52%. In addition, 44% isolates were resistant to chloramphenicol, and 32% of isolates were colistin-resistant. Among analyzed isolates, three were resistant to 10 of 11 tested antibiotics but only displayed intermediate resistance to imipenems (carbapenems). Surprisingly, 23 out of 25 isolates showed a positive ESBL phenotype. Eleven of them had both the blaTEM and blaCTX-M-1 group structural genes, while twelve only had the blaCTX-M-1 group gene. Furthermore, none of the isolated E. coli isolates exhibited the blaSHV gene. The minimum inhibitory concentration (MIC) of colistin exceeded 4μg/mL in 8 out of 25 (32%) isolates. Seven of eight isolates (87.5%) carried the mcr-1 gene, while one (12.5%) carried the mcr-8 gene. None of the other mcr (mcr-2 to mcr-9) genes were found.

Bacteria carrying mobile colistin resistance genes and their control measures, an updated review.

The plasmid encoded mobile colistin resistance (MCRs) enzyme poses a significant challenge to the clinical efficacy of colistin, which is frequently employed as a last resort antibiotic for treating infections caused by multidrug resistant bacteria. This transferase catalyzes the addition of positively charged phosphoethanolamine to lipid A of the outer membrane of gram-negative bacteria, thereby facilitating the acquired colistin resistance. This review aims to summarize and critically discuss recent advancements in the distribution and pathogenesis of mcr-positive bacteria, as well as the various control measures available for treating these infections. In addition, the ecology of mcr genes, colistin-resistance mechanism, co-existence with other antibiotic resistant genes, and their impact on clinical treatment are also analyzed to address the colistin resistance crisis. These insights provide a comprehensive perspective on MCRs and serve as a valuable reference for future therapeutic approaches to effectively combat mcr-positive bacterial infections.

Molecular characterization of colistin-resistant Klebsiella pneumoniae isolates and their conjugative mcr-carrying plasmids

BackgroundThis study aimed to characterize colistin-resistant K. pneumoniae (CoRKp) strains isolated from patients with urinary tract infections and bacteremia between 1999 and 2022 at a tertiary teaching hospital in Taiwan. MethodsA total of 1,966K. pneumoniae isolates were collected, among which 21 strains were identified as CoRKp. The antimicrobial susceptibility of these CoRKp strains to 19 antibiotics was assessed. The genome characteristics of 21 CoRKp strains were determined by Nanopore-Illumina hybrid whole genome sequencing. Additionally, conjugation assays were conducted to determine the transferability of plasmids carrying mcr genes to K. pneumoniae ATCC BAA-1706 and E. coli C600. The larvae infection model was used to analyze the differences in virulence between transconjugants and recipient strains. ResultsAmong the 21 CoRKp, 12 were multidrug-resistant, and four were extensively drug-resistant. The distribution of sequence types (STs) and K types among the CoRKp strains was quite diverse, and ST307 (5 strains) and K64 (3 strains) dominated in CoRKp. The insertion elements IS903B and ISVsa5, were found to inactivate mgrB of 1 and 2 CoRKp isolates, respectively. Moreover, 1, 4, 6, and 1 missense mutations of PhoQ, PmrA, PmrB, and MgrB, were identified in 21 CoRKp. Only two isolates SC-KP169 and SC-KP585 carried mcr-1 and mcr-8, respectively. The plasmid pSC-KP169-1 could be transferred inter- and intra-genus and contributed to the virulence of K. pneumoniae to larvae. In contrast, the plasmid pSC-KP585-1 could be transferred to E. coli but could not affect its virulence to larvae. ConclusionsWe identified 21 CoRKp from 1,966 isolates and found a conjugative plasmid carrying mcr-1 gene that contributed to the virulence of K. pneumoniae to larvae.

First detection of multidrug-resistant and toxigenic Pasteurella aerogenes in sow vaginal discharge: a novel threat to swine health in Thailand

Pasteurella aerogenes has been implicated in reproductive disorders in sows, yet its prevalence and characteristics in vaginal discharge are not well understood. This study aimed to detect P. aerogenes in sow vaginal discharge samples and investigate its antibiotic resistance profile, toxin genes, and toxicity. P. aerogenes was isolated from 40% (8/20) of samples. Antimicrobial susceptibility testing revealed universal resistance to amoxicillin-clavulanate (4:1), with 87.5% of isolates also resistant to oxytetracycline, amoxicillin, ceftriaxone, and enrofloxacin. The colistin resistance gene mcr-2 was detected in 75% of isolates, while class 1 integron (int1) was found in 12.5%. The pax toxin gene cluster was present in 75% of isolates. Toxicity assays using Panagrellus redivivus demonstrated dose-dependent effects of P. aerogenes supernatant containing pax toxins. This study represents the first report of P. aerogenes isolation from sow vaginal discharge in Thailand. The high prevalence of antibiotic resistance, presence of the mcr-2 gene, and toxicity of pax toxin-positive isolates suggest that P. aerogenes may be an underestimated factor in swine reproductive health. These findings highlight the need for further investigation into the role of P. aerogenes in sow reproductive disorders and its potential impact on swine production.

Bla SED-1 beta-lactamase-producing Citrobacter sedlakii isolated from horses and genomic comparison with human-derived isolates.

We aim to detect beta-lactamase-producing Citrobacter sedlakii from horses and compare the genomic characteristics with isolates from humans.Methods and Result: We characterized phenotypically and genotypically nine C. sedlakii isolates from the feces of horses and then compared them to human-derived isolates using whole genome sequencing and phylogenomic methods. Seven isolates (7/9) were ampicillin-resistant, while at least one isolate was resistant to ceftriaxone, gentamicin, meropenem, and streptomycin. All nine isolates were carriers of the chromosomal-mediated blaSED-1 beta-lactamase gene which confer resistance to ampicillin. One isolate was positive for the mcr-9 gene that confers resistance to colistin, and another isolate had the aac(6')-lid gene that confers resistance to aminoglycosides. Seven isolates (7/9) were carriers of genes that confer metal resistance to copper, silver, and arsenic. Phylogenetically, two horse-derived isolates clustered together with two human-derived isolates from the NDARO database. The results from our study provide insight into the antimicrobial susceptibility of C. sedlakii in horses which was previously lacking, and the specific beta-lactamase gene mediating resistance.

Aeromonas and mcr–3: A Critical Juncture for Transferable Polymyxin Resistance in Gram-Negative Bacteria

Polymyxin antibiotics B and colistin are considered drugs of last resort for the treatment of multi-drug and carbapenem-resistant Gram-negative bacteria. With the emergence and dissemination of multi-drug resistance, monitoring the use and resistance to polymyxins imparted by mobilised colistin resistance genes (mcr) is becoming increasingly important. The Aeromonas genus is widely disseminated throughout the environment and serves as a reservoir of mcr–3, posing a significant risk for the spread of resistance to polymyxins. Recent phylogenetic studies and the identification of insertion elements associated with mcr–3 support the notion that Aeromonas spp. may be the evolutionary origin of the resistance gene. Furthermore, mcr–3-related genes have been shown to impart resistance in naïve E. coli and can increase the polymyxin MIC by up to 64-fold (with an MIC of 64 mg/L) in members of Aeromonas spp. This review will describe the genetic background of the mcr gene, the epidemiology of mcr-positive isolates, and the relationship between intrinsic and transferable mcr resistance genes, focusing on mcr–3 and mcr–3-related genes.

Detection of PhoP-mediated colistin resistance in Gram-negative bacteria without mcr genes in human population in the Ho Municipality, Ghana

IntroductionAntimicrobial resistance (AMR) has become a global public health threat, with colistin emerging as a last-resort treatment option for multidrug-resistant Gram-negative infections. However, the emergence of colistin resistance, mediated by mechanisms like mutations in the PhoP gene, raises concerns about the future utility of this antibiotic. This study aimed to determine the prevalence of PhoP-mediated colistin resistance in Gram-negative bacteria isolated from the stool of residents in the Ho Municipality, Ghana. MethodsIn this cross-sectional study, 110 stool samples were collected from June 2021 to December 2022. Gram-negative bacteria were isolated, and colistin susceptibility was determined by broth microdilution. Genomic DNA from resistant isolates was extracted and sequenced using the Nanopore platform to detect the presence of the PhoP gene. ResultsOf the 107 Gram-negative isolates, 57 % were resistant to colistin. The PhoP gene was detected in 61.4 % of the colistin-resistant isolates, with the highest prevalence observed in Proteus mirabilis, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. ConclusionThe study reveals a high prevalence of PhoP-mediated colistin resistance among Gram-negative bacteria colonizing residents in the Ho Municipality, highlighting the role of the gut microbiota as a reservoir for antibiotic resistance. Continued surveillance and a collaborative One Health approach are crucial to address this growing threat.

Conventionally Reared Wallon Meat Lambs Carry Transiently Multi-Drug-Resistant Escherichia coli with Reduced Sensitivity to Colistin Before Slaughter.

Major efforts have been made to reduce the use of colistin in livestock since the discovery of the plasmid-borne mobile colistin resistance (mcr) gene in E. coli a decade ago, to curb the burden of its potential transmission to other bacterial species, spread between animals, humans and the environment. This study explored the longitudinal prevalence and characteristics of colistin-resistant and extended-spectrum beta-lactamase-producing (ESBL) E. coli via in vivo fecal and ex vivo carcass swabs from two batches of conventional indoor and organic outdoor Wallon meat sheep from birth to slaughter in 2020 and 2021. Antimicrobial susceptibility testing via broth microdilution revealed that n = 16/109 (15%) E. coli isolates from conventional meat lamb fecal samples had a reduced colistin sensitivity (MIC = 0.5 μg/mL) and thereof, n = 9/109 (8%) were multi-drug-resistant E. coli, while no resistant isolates were recovered from their carcasses. Sequencing revealed causative pmrB genes, indicating that the reduced sensitivity to colistin was not plasmid-borne. While the sample size was small (n = 32), no colistin-resistant and ESBL-producing E. coli were isolated from the organic meat sheep and their carcasses, potentially due to the different husbandry conditions. Prudent and judicious antimicrobial use and strict slaughter hygiene remain imperative for effective risk management to protect consumers in a sustainable One Health approach.

Validation of Recombinase Polymerase Amplification with In-House Lateral Flow Assay for mcr-1 Gene Detection of Colistin Resistant Escherichia coli Isolates.

The emergence of the mobilized colistin resistance 1 (mcr-1) gene, which causes colistin resistance, is a serious concern in animal husbandry, particularly in pigs. Although antibiotic regulations in many countries have prohibited the use of colistin in livestock, the persistence and dissemination of this plasmid-mediated gene require effective and rapid monitoring. Therefore, a rapid, sensitive, and specific method combining recombinase polymerase amplification (RPA) with an in-house lateral flow assay (LFA) for the mcr-1 gene detection was developed. The colistin agar test and broth microdilution were employed to screen 152 E. coli isolates from pig fecal samples of five antibiotic-used farms. The established RPA-in-house LFA was validated with PCR for mcr-1 gene detection. The RPA-in-house LFA was completed within 35 min (20 min of amplification and 5-15 min on LFA detection) at 37 °C. The sensitivity, specificity, and accuracy were entirely 100% in concordance with PCR results. No cross-reactivity was detected with seven common pathogenic bacteria or other mcr gene variants. Therefore, the in-house RPA-LFA serves as a point-of-care testing tool that is rapid, simple, and portable, facilitating effective surveillance of colistin resistance in both veterinary and clinical settings, thereby enhancing health outcomes.

A Systematic Review and Comprehensive Analysis of mcr Gene Prevalence in Bacterial Isolates in Arab Countries.

The resurgence of colistin has become critical in combating multidrug-resistant Gram-negative bacteria. However, the emergence of mobilized colistin resistance (mcr) genes presents a crucial global challenge, particularly in the Arab world, which includes regions with unique conditions and ongoing conflicts in some parts. To address this issue, a systematic review was conducted using multiple databases, including Cochrane, PubMed, Scopus, Web of Science, and Arab World Research Source. A total of 153 studies were included, revealing substantial heterogeneity in the prevalence of mcr genes across 15 Arab countries, with notable findings indicating that Egypt and Lebanon reported the highest number of cases. The analysis indicated that the most prevalent sequence types were ST10, ST101, and ST1011, all of which are Escherichia coli strains linked to significant levels of colistin resistance and multiple antimicrobial resistance profiles. By analyzing the diverse findings from different Arab countries, this review lays a critical foundation for future research and highlights the necessity for enhanced surveillance and targeted interventions to address the looming threat of colistin resistance in the region. PROSPERO CRD42024584379.

Evidence for high-risk pollutants and emerging microbial contaminants at two major bathing ghats of the river Ganga using high-resolution mass spectrometry and metagenomics

An efficient wastewater treatment plant is imperative to limit the entry of emerging pollutants (EPs) and emerging microbial contaminants (EMCs) in the river ecosystem. The detection of emerging EPs in aquatic environments is challenging due to complex sample preparation methods, and the need for sophisticated accurate analytical tools. In Varanasi (India), the river Ganga holds immense significance as a holy river but is consistently polluted with municipal (MWW) and hospital wastewater (HWW). We developed an efficient method for untargeted detection of EPs in the water samples using High-resolution mass spectrometry (HRMS), and identified 577 and 670 chemicals (or chemical components) in the water samples from two major bathing ghats, Assi Ghat (AG) and Dashashwamedh Ghat (DG), respectively. The presence of EPs of different categories viz chemicals from research labs, diagnostic labs, lifestyle and industrial chemicals, toxins, flavor and food additives indicated the unsafe disposal of MWW and HWW or inefficient wastewater treatment plants (WWTPs). Besides, shotgun metagenomic analysis depicted the presence of bacteria associated with MWW viz Cloacibacterium normanse, Sphaerotilus natans (sewage fungi), E. coli, and Prevotella. Also, the presence of human pathogens Arcobacter, Polynucleobacter, Pseudomonas, Klebsiella, Aeromonas, Acinetobacter, Vibrio, and Campylobacter suggests the discharge of HWW. EPs are linked to the development, and transmission of antimicrobial resistance (AMR). Occurrence of antibiotic resistance genes (ARGs), plasmid-borne β-lactamases, aminoglycoside transferases, and ARGs associated with integrons, transposons and plasmids viz mcr-3 gene that confer resistance to colistin, the last resort of antibiotics confirmed the presence of emerging microbial contaminants. Subsequent genome reconstruction studies showed the presence of uncultivable ARB and transmission of ARGs through horizontal gene transfer. This study can be used to monitor the health of aquatic bodies as well as the efficiency of WWTPs and raise an urgent need for efficient WWTPs to safeguard the river, Ganga.

Whole genome analysis of multidrug-resistant Escherichia coli isolate collected from drinking water in Armenia revealed the plasmid-borne mcr-1.1-mediated colistin resistance.

Evolutionary patterns of Escherichia coli show that they usually develop into highly pathogenic forms by acquiring fitness advantages such as antimicrobial resistance (AMR) and various virulence factors through horizontal gene transfer mediated by mobile elements. This has led to high prevalence of multidrug-resistant (MDR) strains, which highlights the relevancy of enhanced surveillance to monitor and prevent transmission of the MDR bacteria to human and animal populations. However, the limited number of reports regarding the whole genome sequencing (WGS) investigation of MDR E. coli strains isolated from drinking water and harboring mcr genes hampers the adoption of a comprehensive approach to address the relationship between environmental E. coli populations and human and veterinary infections. Our results highlight the relevance of analyzing the environment, especially water, as a part of the surveillance programs to understand the origins and dissemination of antimicrobial resistance within the One Health concept.

Coexistence and genomics characterization of mcr-1 and extended-spectrum-β-lactamase-producing Escherichia coli, an emerging extensively drug-resistant bacteria from sheep in China

The emergence of pathogens harboring multiple resistance genes poses a great threat to global public health. However, the coexistence of mobile resistance genes that provide resistance to both third-generation cephalosporins and colistin in sheep-origin Escherichia coli has not been previously investigated in China. This study is the first to characterize five E. coli isolates from sheep in Shaanxi province that harbor both Extended-Spectrum β-Lactamase (ESBL) and mcr-1 resistance genes. The isolates were identified and characterized by Illumina sequencing, nanopore sequencing, bioinformatic analysis, conjugation experiments, and antimicrobial susceptibility testing. Genetic analysis revealed that blaCTX-M-55 gene, mediated by the IS26, was located on the IncFIB-IncFIC plasmid, while the mcr-1 gene was located on the IncI2(Delta) plasmid. Notably, two copies of blaCTX-M-55 gene were also identified on the chromosome of one isolate (SX45), facilitated by the ISEcp1 insertion sequence. Additionally, the plasmid pSX23–2 was identified as a complex plasmid derived through homologous recombination of pMG337 from E. coli (MK878890) and pZY-1 from Citrobacter freundii (CP055248). Data mining of publicly available databases revealed that isolates carrying both blaCTX-M-55 and mcr-1 genes have been found in humans, animals, and the environment, indicating the widespread presence of these critical resistance genes across different niches. Antimicrobial susceptibility testing showed that the five isolates were resistant to a nearly all tested antibiotics, except meropenem. Conjugative transfer experiments demonstrated that the IncFIB-IncFIC and IncI2(Delta) plasmids carrying mcr-1 and blaCTX-M-55 were capable of transferring between different sequence types (STs) of sheep-origin E. coli, including ST10, ST162, and ST457. This finding suggests the potential for wide dissemination of these resistance markers among diverse E. coli strains. Overall, the characterization of these ESBL and mcr-1 co-harboring isolates enhances our understanding of the spread of these resistance genes in sheep-origin E. coli. Global surveillance of these isolates, particularly within the One Health framework, is essential to monitor and mitigate the risks posed by the dissemination of these resistance genes across various settings.

Investigation of Colistin resistance and method comparison in Klebsiella pneumoniae strains

PurposeThis study aimed to assess the prevalence of colistin resistance in the study group and compare alternative methods with the gold standard. It sought to evaluate the prevalence of plasmid-mediated colistin resistance genes. Material and methodsThe colistin susceptibility of 151 K. pneumoniae strains was determined using Sensititre™, CBDE, ETEST®, and VITEK®2. Results were compared with BMD. The presence of the mcr gene was assessed using polymerase chain reaction. ResultsThe colistin resistance rate was 16,6 %. The categorical agreement of Sensititre™, CBDE, and ETEST® was 100 %. VITEK®2 had a CA of 98 %, a major error of 0.79 %, and a very major error of 8 %. Essential agreement for Sensititre™, ETEST®, and VITEK®2 was 92.7 %, 52.3 %, and 78.1 %, respectively. There were no mcr genes in any strains. ConclusionsDue to the difficulty of applying BMD, colistin resistance data are insufficient globally. Continuous epidemiological studies and validation of alternative methods are needed.

Acetylshikonin reduces the spread of antibiotic resistance via plasmid conjugation

The plasmid-mediated conjugative transfer of antibiotic resistance genes (ARGs) stands out as the primary driver behind the dissemination of antimicrobial resistance (AMR). Developing effective inhibitors that target conjugative transfer represents an efficient strategy for addressing the issue of AMR. Here, we studied the effect of acetylshikonin (ASK), a botanical derivative, on plasmid conjugation. The conjugative transfer of RP4-7 plasmid inter and intra species was notably reduced by ASK. The conjugation process of IncI2 and IncX4 plasmids harboring the mobile colistin resistance gene (mcr-1), IncX4 and IncX3 plasmids containing the carbapenem resistance gene (blaNDM-5), and IncFI and IncFII plasmids possessing the tetracycline resistance gene [tet(X4)] were also reduced by ASK. Importantly, the conjugative transfer frequency of mcr-1 positive IncI2 plasmid in mouse peritoneal conjugation model and gut conjugation model was reduced by ASK. The mechanism investigation showed that ASK disrupt the functionality of the bacterial cell membrane. Furthermore, the proton motive force (PMF) was dissipated. In addition, ASK blocked the electron transmission in bacteria's electron transport chain (ETC) through disturbing the quinone interaction, resulting in an insufficient energy supply for conjugation. Collectively, ASK is a potential conjugative transfer inhibitor, providing novel strategies to prevent the spread of AMR.

In vitro activity of zidebactam/cefepime (WCK 5222), a β-lactam enhancer/ β-lactam combination against carbapenem- and colistin-resistant Klebsiella pneumoniae isolates

ObjectivesIn vitro activity of β-lactam enhancer/β-lactam combination zidebactam/cefepime was evaluated against carbapenem- and colistin-resistant Klebsiella pneumoniae isolates. MethodsNon duplicate K. pneumoniae (n=185), resistant to colistin as well as non-susceptible to carbapenems were collected (2018-2019) at two large tertiary care hospitals in India. Colistin resistance-conferring genes mcr1 and mcr3 were screened among 123 of 185 randomly-selected isolates. These isolates were also subjected to multi-locus sequence typing (MLST). Additionally, alterations in mgrB were screened in 109 of these 123 isolates. All the study isolates were screened for presence of carbapenemases genes. MICs of zidebactam/cefepime, colistin, carbapenems, ceftazidime/avibactam, imipenem/relebactam, amikacin and piperacillin/tazobactam were determined by reference CLSI broth dilution method. ResultsAmong the isolates, 65.4% (121/185) carried blaOXA-48-like gene and 27.6% isolates (51/185) carried dual carbapenemase genes; blaOXA-48-like and blaNDM. Of the remainder, 8 isolates carried blaNDM and 5 isolates lacked carbapenemases gene despite being carbapenem-resistant. None of the isolates showed presence of mcr1 and mcr3. Out of 109 isolates analysed for mgrB, 36 showed mutational changes. The MLST profile revealed at least 14 unique sequence types with ST231 being the dominant clone. All the isolates showed colistin MICs >2 mg/L and were non-susceptible to carbapenems. Zidebactam/cefepime demonstrated potent activity with MIC50 and MIC90 of 1 and 2 mg/L, respectively. MIC90s of amikacin, ceftazidime/avibactam and imipenem/relebactam were >32 mg/L. ConclusionZidebactam/cefepime combination was highly active against multi-clonal, carbapenem-non-susceptible and colistin-resistant K. pneumoniae isolates producing OXA-48-like (Ambler class D) or/and NDM (Ambler class B) carbapenemases, thus potentially offering a valuable treatment options for infections caused by such pan-drug resistant resistotypes. Though, zidebactam is not an inhibitor of class B and D β-lactamases, potent activity of zidebactam/cefepime combination is attributable to β-lactam enhancer mechanism.