Co-production of Tet(X) and MCR-1, two resistance enzymes by a single plasmid

Tigecycline and colistin are few of ‘last-resort’ antibiotic defences used in anti-infection therapies against carbapenem-resistant bacterial pathogens. The successive emergence of plasmid-borne tet(X) tigecycline resistance mechanism and mobile colistin resistance (mcr) determinant, renders them clinically useless. Here, we report that co-carriage of tet(X6) and mcr-1 gives co-resistance to both classes of antibiotics by a single plasmid in Escherichia coli. Tet(X6), the new tigecycline resistance enzyme is functionally defined. Both Tet(X6) and MCR-1 robustly interfere accumulation of antibiotic-induced reactive oxygen species (ROS). Unlike that mcr-1 exerts fitness cost in E. coli, tet(X6) does not. In the tet(X6)-positive strain that co-harbors mcr-1, tigecycline resistance is independently of colistin resistance caused by MCR-1-mediated lipid A remodelling, and vice versa. In general consistency with that of MCR-1, Tet(X6) leads to the failure of tigecycline treatment in the infection model of G. mellonella. Taken together, the co-production of Tet(X) and MCR-1 appears as a major clinic/public health concern.     

Peptide MSI-1 inhibited MCR-1 and regulated outer membrane vesicles to combat immune evasion of Escherichia coli

Polymyxin resistance is conferred by MCR-1 (mobile colistin resistance 1)-induced lipopolysaccharide (LPS) modification of G⁻ bacteria. However, the peptide MSI-1 exerts potent antimicrobial activity against mcr-1-carrying bacteria. To further investigate the potential role of MCR-1 in improving bacterial virulence and facilitating immune evasion, and the immunomodulatory effect of peptide MSI-1, we first explored outer membrane vesicle (OMV) alterations of mcr-1-carrying bacteria in the presence and absence of sub-MIC MSI-1, and host immune activation during bacterial infection and OMV stimulation. Our results demonstrated that LPS remodelling induced by MCR-1 negatively affected OMV formation and protein cargo by E. coli. In addition, MCR-1 diminished LPS-stimulated pyroptosis but facilitated mitochondrial dysfunction, further aggravating apoptosis in macrophages induced by OMVs of E. coli. Similarly, TLR4-mediated NF-κB activation was markedly alleviated once LPS was modified by MCR-1. However, peptide MSI-1 at the sub-MIC level inhibited the expression of MCR-1, further partly rescuing OMV alteration and attenuation of immune responses in the presence of MCR-1 during both infection and OMV stimulation, which can be exploited for anti-infective therapy.     

A ProQ/FinO family protein involved in plasmid copy number control favours fitness of bacteria carrying mcr-1-bearing IncI2 plasmids

The plasmid-encoded colistin resistance gene mcr-1 challenges the use of polymyxins and poses a threat to public health. Although IncI2-type plasmids are the most common vector for spreading the mcr-1 gene, the mechanisms by which these plasmids adapt to host bacteria and maintain resistance genes remain unclear. Herein, we investigated the regulatory mechanism for controlling the fitness cost of an IncI2 plasmid carrying mcr-1. A putative ProQ/FinO family protein encoded by the IncI2 plasmid, designated as PcnR (plasmid copy number repressor), balances the mcr-1 expression and bacteria fitness by repressing the plasmid copy number. It binds to the first stem-loop structure of the repR mRNA to repress RepA expression, which differs from any other previously reported plasmid replication control mechanism. Plasmid invasion experiments revealed that pcnR is essential for the persistence of the mcr-1-bearing IncI2 plasmid in the bacterial populations. Additionally, single-copy mcr-1 gene still exerted a fitness cost to host bacteria, and negatively affected the persistence of the IncI2 plasmid in competitive co-cultures. These findings demonstrate that maintaining mcr-1 plasmid at a single copy is essential for its persistence, and explain the significantly reduced prevalence of mcr-1 following the ban of colistin as a growth promoter in China.     

Association of the colistin resistance gene mcr-1 with faecal pollution in water environments in Hanoi,Vietnam

Colistin is one of the antibiotics of last resort for human health. However, the dissemination of the plasmid-mediated colistin resistance gene mcr-1 is of great concern globally. In the One Health framework, the environment is an important component for managing antimicrobial resistance. However, little information is available concerning the prevalence of mcr-1 in water environments. We aimed to reveal the prevalence of mcr-1 in different water environments in Hanoi, Vietnam. Quantitative PCR was applied to detect mcr-1 in four urban drainages receiving untreated domestic wastewater, three rivers, five lakes and two groundwater samples. Urban drainages contained higher concentrations of mcr-1, suggesting that urban residents carry the gene. The class 1 integron-integrase gene was identified as a good surrogate of antibiotic resistance genes including mcr-1. A significant correlation was found between the levels of mcr-1 and the human-specific cross-assembly phage, which is an indicator of human faecal pollution. These results indicated that the primary source of mcr-1 in urban water environments is human faeces, which is consistent with the fact that most domestic wastewater is untreated in Hanoi. The control of untreated wastewater is critical for alleviating the spread of mcr-1 in water environments in Vietnam.     

Screening of mcr-1 Among Gram-Negative Bacteria from Different Clinical Samples from ICU Patients in Alexandria,Egypt: One-Year Study

Antimicrobial resistance represents a global dilemma. Our present study aimed to investigate the presence of mcr-1 among different Gram-negative bacteria including Enterobacteriaceae (except intrinsically resistant to colistin) and Pseudomonas aeruginosa. Gram-negative bacterial isolates were collected from different ICUs in several Alexandria hospitals from June 2019 to June 2020. The identification of these Gram-negative isolates was made using the VITEK-2^® system (BioMérieux, France). SYBR Green-based PCR was used to screen for the presence of mcr-1 using a positive control that we amplified and sequenced earlier in our pilot study. All isolates were screened for the presence of mcr-1 regardless of their colistin susceptibility. Isolates that harbored mcr-1 were tested for colistin susceptibility and for the presence of some beta-lactamase genes. Klebsiella pneumoniae isolates harboring mcr-1 were capsule typed using the wzi sequence analysis. Four hundred eighty isolates were included in this study. Only six isolates harbored mcr-1.1. Of these, four were resistant to colistin, while two (K. pneumoniae and P. aeruginosa) were susceptible to colistin. Five of the six isolates were resistant to carbapenems. They harbored bla_OXA-48, and three of them co-harbored bla_NDM-1. K-58 was the most often found among our K. pneumoniae harboring mcr-1.1. To our knowledge, this is the first time to report colistin susceptible P. aeruginosa and K. pneumoniae harboring the mcr-1.1 gene in Egypt. Further studies are needed to investigate the presence of the mcr genes among colistin susceptible isolates to shed more light on its significance as a potential threat. Open in a separate window     

A rapid MALDI-TOF mass spectrometry-based method for colistin susceptibility testing in Escherichia coli

Colistin is recognized as a last-resort treatment option against multi-drug resistant bacteria including carbapenem-resistant Enterobacteriaceae (CRE). However, the plasmid-mediated colistin-resistance gene mcr-1 has been reported globally resulting in an increase of colistin-resistant bacteria. A quick and accurate method for determining the pathogen resistance of colistin is therefore crucial in the clinic. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a potential tool forto be applied for antimicrobial susceptibility testing. We compared the growth of Escherichia coli strains in the presence or absence of colistin. Automated analyses of the spectra were performed with a prototype software tool written with package R. Three mcr-1-positive and six mcr-1-negative E. coli were used for establishing the model to obtain the optimal incubation time, the breakpoint concentration of colistin and cut-off of the relative growth (RG) value. The distinction between susceptible and resistant strains was already noticeable after 2 h of incubation. The best separation between the susceptible and resistant strains was achieved at a concentration of 4 µg ml^-1 and a relative growth cut-off value of 0.6. Application of the model for the analysis of 128 E. coli isolates, a sensitivity of 97.4% and a specificity of 88.2% were achieved compared with colistin MIC results. The rapid MALDI-TOF MS-based method approach is simple to set-up, uses a short incubation time, and had excellent outcomes with respect to sensitivity and specificity for colistin sensitivity testing in Escherichia coli.     

CRISPR-Cas9技术在细菌耐药性防控研究进展

近年来,多种新型耐药基因的出现和全球性流行,严重威胁了全球公众健康。CRISPR-Cas9系统(clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 system)是细菌的一种适应性免疫系统,可切割耐药基因、抵御外来核酸入侵,现已作为一种新型基因编辑工具应用于防控细菌耐药性研究。本团队已建立了一种单质粒介导靶向mcr-1基因的CRISPR-Cas9系统,能有效并特异性消除黏菌素耐药大肠杆菌中的mcr-1,恢复其对黏菌素的敏感性。同时也发现在临床中应用还需要优化其递送方式。本文对近几年该技术在细菌耐药性防控方面的研究进展进行了综述,包括CRISPR-Cas9系统的发现过程、作用机制、递送方式、在体外检测实验结果的进展以及当前存在的问题等方面,以期为防控细菌耐药性提供新思路。     

From farm to fork: Colistin voluntary withdrawal in Portuguese farms reflected in decreasing occurrence of mcr-1-carrying Enterobacteriaceae from chicken meat

Expansion of mcr-carrying Enterobacteriaceae (MCR-E) is a well-recognized problem affecting animals, humans and the environment. Ongoing global control actions involve colistin restrictions among food-animal production, but their impact on poultry-derived products is largely unknown, justifying comprehensive farm-to-fork studies. Occurrence of MCR-E among 53 chicken-meat batches supplied from 29 Portuguese farms shortly after colistin withdrawal was evaluated. Strains (FT-IR/MLST/WGS), mcr plasmids and their adaptive features were characterized by cultural, molecular and genomic approaches. We found high rates of chicken-meat batches (80%–100% – 4 months; 12% – the last month) with multiple MDR + mcr-1-carrying Escherichia coli (Ec-including ST117 and ST648-Cplx) and Klebsiella pneumoniae (Kp-ST147-O5:K35) clones, some of them persisting over time. The mcr-1 was located in the chromosome (Ec-ST297/16-farms) or dispersed IncX4 (Ec-ST602/ST6469/5-farms), IncHI2-ST2/ST4 (Ec-ST533/ST6469/5 farms and Kp-ST147/6-farms) or IncI2 (Ec-ST117/1-farm) plasmids. WGS revealed high load and diversity in virulence, antibiotic resistance and metal tolerance genes. This study supports colistin withdrawal potential efficacy in poultry production and highlights both poultry-production chain as a source of mcr-1 and the risk of foodborne transmission to poultry-meat consumers. Finally, in the antibiotic reduction/replacement context, other potential co-selective pressures (e.g., metals-Cu as feed additives) need to be further understood to guide concerted, effective and durable actions under 'One Health' perspective.     

Performance of different methods for testing polymyxin B: comparison of broth microdilution,agar dilution and MIC test strip in mcr-1 positive and negative Escherichia coli

Antimicrobial susceptibility testing with the last-resort antibiotics polymyxins (polymyxin B and colistin) is associated with several methodological issues. Currently, broth microdilution (BMD) is recommended for colistin and polymyxin B. BMD is laborious and the utility of alternative methods needs to be evaluated for polymyxin B susceptibility testing. In this study, using BMD as a reference method, the performance of agar dilution (AD) and MIC test strips (MTS) were evaluated in polymyxin B susceptibility testing. BMD, AD and MTS were used to determine MICs of 193 clinical isolates of Escherichia coli. Seventy-nine were positive for the polymyxin resistance gene mcr-1. Method performances were evaluated based on pair-wise agreements with the reference method (BMD) and statistical testing. AD and MTS showed an unacceptable number of very major errors (VMEs) compared with BMD, 9·3 and 10·7%, respectively. The essential agreement (EA) was low for AD (49·7%), but high for MTS (97·8%). However, statistical testing showed that MTS tended to yield a one-step lower MIC (P < 0·01) compared with BMD. The discordances observed with MTS and AD in comparison with BMD for polymyxin B susceptibility testing for E. coli suggest their inapplicability in routine testing. A large number of isolates clustered around the susceptibility breakpoint (2–4 mg l⁻¹) and several mcr-1 positive isolates (17%) were determined as susceptible with BMD. A screening breakpoint for mcr-1 of 2 mg l⁻¹ should also be considered.     

Biochemical characterization of metabolism‐based atrazine resistance in Amaranthus tuberculatus and identification of an expressed GST associated with resistance

Rapid detoxification of atrazine in naturally tolerant crops such as maize (Zea mays) and grain sorghum (Sorghum bicolor) results from glutathione S‐transferase (GST) activity. In previous research, two atrazine‐resistant waterhemp (Amaranthus tuberculatus) populations from Illinois, U.S.A. (designated ACR and MCR), displayed rapid formation of atrazine‐glutathione (GSH) conjugates, implicating elevated rates of metabolism as the resistance mechanism. Our main objective was to utilize protein purification combined with qualitative proteomics to investigate the hypothesis that enhanced atrazine detoxification, catalysed by distinct GSTs, confers resistance in ACR and MCR. Additionally, candidate AtuGST expression was analysed in an F₂ population segregating for atrazine resistance. ACR and MCR showed higher specific activities towards atrazine in partially purified ammonium sulphate and GSH affinity‐purified fractions compared to an atrazine‐sensitive population (WCS). One‐dimensional electrophoresis of these fractions displayed an approximate 26‐kDa band, typical of GST subunits. Several phi‐ and tau‐class GSTs were identified by LC‐MS/MS from each population, based on peptide similarity with GSTs from Arabidopsis. Elevated constitutive expression of one phi‐class GST, named AtuGSTF2, correlated strongly with atrazine resistance in ACR and MCR and segregating F₂ population. These results indicate that AtuGSTF2 may be linked to a metabolic mechanism that confers atrazine resistance in ACR and MCR.     

Antimicrobial resistance and biotechnological potential of plastic-associated bacteria isolated from an urban estuary

Plastics have quickly become one of the major pollutants in aquatic environments worldwide and solving the plastic pollution crisis is considered a central goal of modern society. In this study, 10 different plastic samples, including high- and low-density polyethylene and polypropylene, were collected from a deeply polluted urban estuary in Brazil. By employing different isolation and analysis approaches to investigate plastic-associated bacteria, a predominance of potentially pathogenic bacteria such as Acinetobacter, Aeromonas, and Vibrio was observed throughout all plastic samples. Bacteria typically found in the aquatic environment harboured clinically relevant genes encoding resistance to carbapenems (bla_KPC) and colistin (such as mcr-3 and mcr-4), along with genetic determinants associated with potentially active gene mobilization. Whole genome sequencing and annotation of three plastic-associated Vibrio strains further demonstrated the carriage of mobile genetic elements and antimicrobial resistance and virulence genes. On the other hand, bacteria isolated from the same samples were also able to produce esterases, lipases, and bioemulsifiers, thus highlighting that the plastisphere could also be of special interest from a biotechnological perspective.     

Tryptamine derivatives disarm colistin resistance in polymyxin-resistant gram-negative bacteria

The last three decades have seen a dwindling number of novel antibiotic classes approved for clinical use and a concurrent increase in levels of antibiotic resistance, necessitating alternative methods to combat the rise of multi-drug resistant bacteria. A promising strategy employs antibiotic adjuvants, non-toxic molecules that disarm antibiotic resistance. When co-dosed with antibiotics, these compounds restore antibiotic efficacy in drug-resistant strains. Herein we identify derivatives of tryptamine, a ubiquitous biochemical scaffold containing an indole ring system, capable of disarming colistin resistance in the Gram-negative bacterial pathogens Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli while having no inherent bacterial toxicity. Resistance was overcome in strains carrying endogenous chromosomally-encoded colistin resistance machinery, as well as resistance conferred by the mobile colistin resistance-1 (mcr-1) plasmid-borne gene. These compounds restore a colistin minimum inhibitory concentration (MIC) below the Clinical & Laboratory Sciences Institute (CLSI) breakpoint in all resistant strains.     

Enterobacteriaceae genome-wide analysis reveals roles for P1-like phage-plasmids in transmission of mcr-1, tetX4 and other antibiotic resistance genes

P1 -like phage-plasmids (PPs) are important gene vehicles in isolated pathogens. In this study, we conducted genome-wide and cross-species analysis of antimicrobial resistance genes (ARGs) from 35 ARG-positive P1-like PPs. LS-BSR analysis reveal that P1-like PPs had in common 7 highly variable regions and carried 48 different ARG subtypes. The most prevalent gene groups were the colistin resistance gene mcr-1 and a class 1 integron. Analysis of the flanking sequences of mcr-1 indicated an “IS30-mcr-1-ORF-IS30” as the core cluster. In particular, we found an mcr-1- and bla_CTX-M-55-coharboring large fusion P1-like PP. Also, tet(X4) was detected and flanking sequences indicated tet(X4)-bearing cluster can formed a larger size fusion plasmid mediated a wider spread via IS26 hotspots. Overall, this study demonstrated that P1-like PPs can not only mobilize a large number of ARGs in variable regions but also form larger hybrid P1-like PPs that would increase their ability to spread antimicrobial resistance.     

Cell surface hydrophobicity of colistin-susceptible vs resistant Acinetobacter baumannii determined by contact angles: methodological considerations and implications

Contact angle analysis of cell surface hydrophobicity (CSH) describes the tendency of a water droplet to spread across a lawn of filtered bacterial cells. Colistin‐induced disruption of the Gram‐negative outer membrane necessitates hydrophobic contacts with lipopolysaccharide (LPS). We aimed to characterize the CSH of Acinetobacter baumannii using contact angles, to provide insight into the mechanism of colistin resistance. Contact angles were analysed for five paired colistin‐susceptible and resistant Ac. baumannii strains. Drainage of the water droplet through bacterial layers was demonstrated to influence results. Consequently, measurements were performed 0·66 s after droplet deposition. Colistin‐resistant cells exhibited lower contact angles (38·8±2·8–46·8±1·3°) compared with their paired colistin‐susceptible strains (40·7±3·0–48·0±1·4°; anova ; P < 0·05). Contact angles increased at stationary phase (50·3±2·9–61·5±2·5° and 47·4±2·0–50·8±3·2°, susceptible and resistant, respectively, anova ; P < 0·05) and in response to colistin 32 mg l^?1 exposure (44·5±1·5–50·6±2·8° and 43·5±2·2–48·0±2·2°, susceptible and resistant, respectively; anova ; P < 0·05). Analysis of complemented strains constructed with an intact lpxA gene, or empty vector, highlighted the contribution of LPS to CSH. Compositional outer‐membrane variations likely account for CSH differences between Ac. baumannii phenotypes, which influence the hydrophobic colistin–bacterium interaction. Important insight into the mechanism of colistin resistance has been provided. Greater consideration of contact angle methodology is necessary to ensure accurate analyses are performed.     

两株分离自鸡粪便费格森埃希菌的耐药性

【背景】费格森埃希菌(Escherichia fergusonii)是与大肠杆菌同属、近源的病原菌,目前耐药性鲜有报道。【目的】对在浙江省鸡粪便中分离到的2株费格森埃希菌EFCF053和EFCF056进行耐药性检测和分析。【方法】通过微量肉汤稀释法进行MIC测定,二代高通量测序获得全基因组序列,并通过ResFinder数据库预测获得性耐药基因。利用S1-PFGE和Southernblotting杂交进行质粒和耐药基因的确认。【结果】两种菌均对氨苄西林、庆大霉素、氟苯尼考、磺胺异噁唑、复方新诺明、四环素耐药,其中菌株EFCF056还对粘菌素、头孢噻呋、大观霉素、恩诺沙星、氧氟沙星耐药。预测到耐药基因β-内酰胺类blaTEM-1A、blaCTX-M-65、blaOXA-1、blaTEM-1B、blaCTX-M-55;氨基糖苷类aac(3)-IId、aph(3')-Ia、aph(3')-Ib、aph(6)-Id、rmtB、aac(6')-Ib-cr、aadA2;粘菌素mcr-1;喹诺酮类qnrS2、aac(6')-Ib-cr、oqxA、oqxB;磷霉素fosA3;大环内酯类mph(A);苯丙醇类catA1、floR、catB3;利福霉素ARR-3;磺胺类sul1、sul2、sul3、dfrA12、dfrA14;四环素类tet(A)。另外,含有mcr-1基因的质粒通过实验证实可发生接合转移。【结论】结果显示费格森埃希菌可能是重要耐药基因存储库,费格森埃希菌与大肠埃希菌要在抗药性流行病学中加以区分,深入研究其MIC频率分布、重要耐药基因mcr-1及ESBL等,保障临床检测的准确性。     

质粒介导的黏菌素耐药基因mcr-1研究进展

mcr-1基因是迄今为止国际上首次发现的质粒介导的黏菌素耐药基因,可介导肠杆菌科细菌对多粘菌素类药物产生耐药并可通过质粒进行水平转移。最新研究表明该基因已通过IncI2、IncX4和IncHI2等流行性质粒以及可移动元件,在全球35个不同国家和地区的人、动物和环境源多种肠杆菌中广泛传播。这些研究对于深入了解mcr-1基因介导的黏菌素耐药和传播机制、全球流行分布特征奠定了基础,丰富了耐药性形成理论。文章主要介绍了不同来源肠杆菌科细菌中mcr-1的分布流行情况、耐药和传播机制、基因环境等方面的研究进展,探讨了其临床风险性以及后续应对措施,以期为相关科研人员和临床工作者提供参考,共同应对抗生素耐药日益严峻的局面。     

Colistin and tigecycline resistance in carbapenemase‐producing Gram‐negative bacteria: emerging resistance mechanisms and detection methods

A literature review was undertaken to ascertain the molecular basis for tigecycline and colistin resistance mechanisms and the experimental basis for the detection and delineation of this resistance particularly in carbapenemase‐producing Gram‐negative bacteria. Pubmed, Google Scholar and Science Direct were searched with the keywords colistin, tigecycline, resistance mechanisms and detection methods. Trans‐complementation and comparative MIC studies, mass spectrometry, chromatography, spectrofluorometry, PCR, qRT‐PCR and whole genome sequencing (WGS) were commonly used to determine tigecycline and colistin resistance mechanisms, specifically modifications in the structural and regulatory efflux (acrAB, OqxAB, kpgABC adeABC‐FGH‐IJK, mexAB‐XY‐oprJM and soxS, rarA robA, ramRAB marRABC, adeLRS, mexRZ and nfxb) and lipid A (pmrHFIJFKLM, lpxA, lpxC lpxD and mgrB, pmrAB, phoPQ,) genes respectively. Mutations in the ribosomal 16S rRNA operon rrnBC, also yielded resistance to tigecycline through target site modifications. The mcr‐1 gene conferring resistance to colistin was identified via WGS, trans‐complementation and a murine thigh infection model studies. Common detection methods are mainly antibiotic sensitivity testing with broth microdilution while molecular identification tools are mostly PCR and WGS. Spectrofluorometry, MALDI‐TOF MS, micro‐array and real‐time multiplex PCR hold much promise for the future as new detection tools.     

Colistin heteroresistance in Enterobacter cloacae is regulated by PhoPQ‐dependent 4‐amino‐4‐deoxy‐l‐arabinose addition to lipid A

The Enterobacter cloacae complex (ECC) consists of closely related bacteria commonly associated with the human microbiota. ECC are increasingly isolated from healthcare‐associated infections, demonstrating that these Enterobacteriaceae are emerging nosocomial pathogens. ECC can rapidly acquire multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the highly conserved lipid A component of the Gram‐negative outer membrane. Many Enterobacteriaceae fortify their outer membrane with cationic amine‐containing moieties to prevent CAMP binding, which can lead to cell lysis. The PmrAB two‐component system (TCS) directly activates 4‐amino‐4‐deoxy‐l ‐arabinose (l ‐Ara4N) biosynthesis to result in cationic amine moiety addition to lipid A in many Enterobacteriaceae such as E. coli and Salmonella. In contrast, PmrAB is dispensable for CAMP resistance in E. cloacae. Interestingly, some ECC clusters exhibit colistin heteroresistance, where a subpopulation of cells exhibit clinically significant resistance levels compared to the majority population. We demonstrate that E. cloacae lipid A is modified with l ‐Ara4N to induce CAMP heteroresistance and the regulatory mechanism is independent of the PmrAB_Ecl TCS. Instead, PhoP_Ecl binds to the arnB_Ecl promoter to induce l ‐Ara4N biosynthesis and PmrAB‐independent addition to the lipid A disaccharolipid. Therefore, PhoPQ_Ecl contributes to regulation of CAMP heteroresistance in some ECC clusters.