Mcr-1 Gene Research Articles

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.

Molecular Characterization of Carbapenem and Colistin Resistance in Klebsiella pneumoniae Isolates Obtained from Clinical Samples at a University Hospital Center in Algeria.

The current study aimed to determine the molecular mechanisms of carbapenem and colistin resistance among the clinical isolates of Klebsiella pneumoniae from hospitalized patients admitted to a university hospital in Eastern Algeria. In total, 124 non-duplicate isolates of K. pneumoniae were collected from September 2018 to April 2019. Bacterial identification was performed using MALDI-TOF MS. The presence of extended spectrum β-lactamase (ESBL) genes, carbapenemase genes, chromosomal mutation and mcr genes in colistin-resistant K. pneumoniae were evaluated by PCR. ESBLs represented a rate of 49.1% and harbored blaCTX-M, blaTEM and blaSHV genes. Concerning carbapenems, 12 strains (9.6%) were resistant to ertapenem (MIC: 1-32 μg/mL), of which one strain (0.8%) was also resistant to imipenem (MIC: 32 μg/mL). Among these strains, nine (75%) harbored blaOXA-48 gene. Seven strains (5.6%) expressed resistance to colistin (MIC: 2-32 μg/mL), of which two harbored mcr-8 and mgrB genes simultaneously. The existence of a double resistance to colistin in the same strain is new in Algeria, and this could raise concerns about the increase in levels of resistance to this antibiotic (MIC: 32 μg/mL). The mgrB gene alone was observed in five isolates (71.4%), including two strains harboring blaOXA-48. This is the first report revealing the presence of K. pneumoniae strains carrying the blaOXA-48 gene as well as a mutation in the mgrB gene. Large-scale surveillance and effective infection control measures are also urgently needed to prevent the outbreak of various carbapenem- and colistin-resistant isolates.

Thymus algeriensis essential oil: Phytochemical investigation, bactericidal activity, synergistic effect with colistin, molecular docking, and dynamics analysis against Gram-negative bacteria resistant to colistin

Due to the increasing resistance prevalence to the last line of antibiotics, such as colistin, and the rising threat of multi-drug resistant bacteria, it is crucial to find alternative therapeutic options. The current study focuses on evaluating antibacterial activities alone and in combination with colistin of Thymus algeriensis essential oil (TA-EO) against colistin-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli co-harboring mcr-1 gene. GC/MS was used to determine the chemical composition of TA-EO. Disc diffusion and microdilution techniques were used to evaluate the antimicrobial activities of TA-EO. Synergism between colistin and TA-EO was evaluated by checkerboard assay. The major compounds of TA-EO were docked with known enzymes involved in resistance to colistin, as well as the biosynthesis of peptidoglycan and amino acids. GC/MS revealed that TA-EO was of carvacrol chemotype (67.94 %). The TA-EO showed remarkable antibacterial activities against all Gram-negative bacterial strains, with the diameter of inhibition zones varied between 30 and 50 mm and a ratio MBC/MIC equal to 1 for the vast majority of bacterial isolates. Interestingly, the checkerboard showed synergism between TA-EO and colistin against colistin-resistant Escherichia coli co-harboring mcr-1 gene (FICI˂1) and reduced the MIC of colistin by 16- to 512-fold and those of TA-EO by 4- to 16-fold. The docking study demonstrated that carvacrol had high binding free energies against MCR-1, a phosphoethanolamine transferase extracellular domain, and its catalytic domain implicated in resistance to colistin, and undecaprenyl pyrophosphate synthase in complex with magnesium which is involved in bacterial peptidoglycan biosynthesis. The molecular dynamics study for 100-ns also revealed the stability of the MCR-1/carvacrol complex with a constant surface area over the simulation. These results support using carvacrol or TA-EO as a bactericidal agent, either alone or in combination with colistin, to treat infections caused by colistin-resistant Gram-negative bacteria.

Detection of PhoP-mediated Colistin Resistance in Gram-negative bacteria without mcr genes in Water in the Ho Municipality, Ghana

Detection of PhoP-mediated Colistin Resistance in Gram-negative bacteria without mcr genes in Water in the Ho Municipality, Ghana

Decoding the origins, spread, and global risks of mcr-9 gene

The global spread of the plasmid-mediated mcr (mobilized colistin resistance) gene family presents a significant threat to the efficacy of colistin, a last-line defense against numerous Gram-negative pathogens. The mcr-9 is the second most prevalent variant after mcr-1. A dataset of 698 mcr-9-positive isolates from 44 countries is compiled. The historical trajectory of the mcr-9 gene is reconstructed using Bayesian analysis. The effective reproduction number is used innovatively to study the transmission dynamics of this mobile-drug-resistant gene. Our investigation traces the origins of mcr-9 back to the 1960s, revealing a subsequent expansion from Western Europe to the America and East Asia in the late 20th century. Currently, its transmissibility remains high in Western Europe. Intriguingly, mcr-9 likely emerged from human-associated Salmonella and exhibits a unique propensity for transmission within the Enterobacter. Our research provides a new perspective that this host preference may be driven by codon usage biases in plasmids. Specifically, mcr-9-carrying plasmids prefer the nucleotide C over T compared to mcr-1-carrying plasmids among synonymous codons. The same bias is seen in Enterobacter compared to Escherichia (respectively as their most dominant genus). Furthermore, we uncovered fascinating patterns of coexistence between different mcr-9 subtypes and other resistance genes. Characterized by its low colistin resistance, mcr-9 has used this seemingly benign feature to silently circumnavigate the globe, evading conventional detection methods. However, colistin-resistant Enterobacter strains with high mcr-9 expression have emerged clinically, implying a strong risk of mcr-9 evolving into a global "true-resistance-gene". This study explores the mcr-9 gene, emphasizing its origin, adaptability, and dissemination potential. Given the high mcr-9 expression colistin-resistant strains was observed in clinically the prevalence of mcr-9 poses a significant challenge to drug resistance prevention and control within the One Health framework. This work was partially supported by the National Natural Science Foundation of China (Grant No. 32141001 and 81991533).

Emergence of multidrug resistant mobile colistin resistance (mcr) carrying Aeromonas spp. isolated from retail foods in Mymensingh district, Bangladesh

The emergence of plasmid-mediated mobile colistin resistance (mar) in bacteria under Enterobacteriaceae, and aeromonads isolated from foods has been reported in many parts of the world. So far, we know there is no report of mcr carrying Aeromonas spp. Isolated from retail foods in Bangladesh. Therefore, this study was carried out to determine the prevalence of mcr carrying Aeromonas spp. isolated from retail foods (meat, milk, and fish) in Mymensingh district and their characterization for antimicrobial resistance patterns. The prevalence of mcr-1 gene carrying Aeromonas spp. was 30 % (15/50) in chickens, 23 % (8/35) in fish, and 10 % (2/20) in milk. No other tested mcr gene variants (mcr-2 to 4) were detected. The minimum inhibitory concentration (MIC) for colistin was determined to be 80% (12/15) and 75% (6/8) of the mcr-1 gene carrying Aeromonas spp. isolated from chicken and fish, respectively, had an MIC of 8-16 µg/ml whereas 100% (2/2) of the isolate from milk showed a MIC of 4–8 µg/ml. Most of the mcr-1 gene-carrying isolates from chicken meats, fish, and milk were resistant to oxytetracycline, and ampicillin (80%–100%). Apart from multidrug resistant (MDR), extensive MDR was detected in mcr-1 gene-carrying Aeromonas spp., irrespective of the sample’s origin. Overall, these data suggested that the mcr-1 gene-carrying Aeromonas spp. in retail foods could be a potential reservoir for antimicrobial resistance determinants. To the best of our knowledge, this is the first report of the mcr gene carrying Aeromonas spp. isolated from retail foods such as chicken meat, fish, and milk in Bangladesh. Res. Agric. Livest. Fish. Vol. 11, No. 2, August 2024: 115-124

High-throughput screening identification of apigenin that reverses the colistin resistance of mcr-1-positive pathogens.

The plasmid-mediated gene mcr-1 that makes bacteria resistant to the antibiotic colistin is spreading quickly, which means that colistin is no longer working well to treat Gram-negative bacterial infections. Herein, we utilized a computer-aided high-throughput screening drugs method to identify the natural product apigenin, a potential mcr-protein inhibitor, which effectively enhanced the antimicrobial activity of colistin. Several assays, including a checkerboard minimum inhibitory concentration assay, a time-kill assay, the combined disk test, molecular simulation dynamics, and animal infection models assay, were conducted to verify whether apigenin enhanced the ability of colistin to fight Gram-negative bacterial infections. The results showed that apigenin improved the antimicrobial activity of colistin against multidrug-resistant Enterobacteriaceae infection. Moreover, apigenin not only did not increase the toxic effect of colistin but also had the ability to effectively inhibit the frequency of bacterial resistance mutations to colistin. Studies clearly elucidated that apigenin could interfere with the thermal stability of the protein by binding to the mcr-1 protein. Additionally, the combination of apigenin and colistin could exert multiple effects, including disrupting bacterial membranes, the generation of bacterial nitric oxide and reactive oxygen species, as well as inhibiting bacterial adenosine triphosphate production. Furthermore, the addition of apigenin was able to significantly inhibit colistin-stimulated high expression levels of the bacterial mcr-1 gene. Finally, apigenin exhibited a characteristic anti-inflammatory effect while enhancing the antimicrobial activity of colistin against mcr-1-positive Escherichia coli (E. coli) infected animals. In conclusion, as a potential lead compound, apigenin is promising in combination with colistin in the future treatment of mcr-1-positive E. coli infections.IMPORTANCEThis study found that apigenin was able to inhibit the activity of the mcr-1 protein using a high-throughput virtual screening method. Apigenin effectively enhanced the antimicrobial activity of colistin against multidrug-resistant Enterobacteriaceae, including mcr-1-positive strains, in vitro and in vivo. This study will provide new options and strategies for the future treatment of multidrug-resistant pathogen infections.

Genomic insights into epidemic plasmids carrying bla CTX-M and mcr-1 genes in Escherichia coli from Lebanese broiler production.

In a previous nationwide survey in the Lebanese broiler production, multidrug-resistant CTX-M-producing E. coli were found to carry the mobile colistin resistance gene mcr-1. To investigate the mobile genetic supports responsible for the spread of these resistance genes among E. coli in healthy broilers in Lebanon. Thirty-three bla CTX-M and mcr-1 positive E. coli of various sequence types from 17 broilers farms were subjected to conjugation assays. Long-read sequencing (Oxford Nanopore Technologies) and hybrid assembly were performed to determine complete plasmid sequences and their phylogenetic diversity. Twenty-nine conjugative IncFII plasmids harboured the extended-spectrum β-lactamase genes bla CTX-M-3 (n = 25) or bla CTX-M-55 (n = 4). Highly related IncF2:A-:B-/bla CTX-M-3 plasmids differing only through IS-mediated genetic rearrangements in antibiotic resistance gene clusters were found in genetically diverse E. coli strains isolated from distant farms. The mobile colistin resistance genes mcr-1.1 and mcr-1.26 were carried by IncX4 and IncI2 plasmids. Worryingly, in one isolate, the ISEcp1-bla CTX-M-55 transposable unit was found integrated in a mcr-1.26-carrying IncX4 plasmid. Beside expanded cephalosporins and colistin resistances, all E. coli isolates were multidrug-resistant with different additional resistances against aminoglycosides, (fluoro)quinolones, fosfomycin, phenicols, sulphonamides, tetracycline and trimethoprim. Closely related blaCTX-M-3/55-borne IncF2:A-:B- plasmids harbouring variable MDR regions and mcr-1 carrying IncX4 plasmids are widely disseminated in the E. coli population of healthy broilers in Lebanon. Further surveillance programmes of antimicrobial resistance and interventions to reduce the abusive use of medically important antibiotics are necessary to limit the spread of resistances in food-producing animals in Lebanon.