PEtN-Modified O-Antigen Enhances Shigella Pathogenesis by Promoting Epithelial Cell Invasion and Inhibiting Complement Binding.

Shigellosis poses an ongoing global public health threat. The presence and length of the O-antigen in lipopolysaccharide play critical roles in Shigella pathogenesis. The plasmid-mediated opt gene encodes a phosphoethanolamine (PEtN) transferase that catalyzes the addition of PEtN to the O-antigen of Shigella flexneri serotype X and Y strains, converting them into serotype Xv and Yv strains, respectively. Since 2002, these modified strains have become prevalent in China. Here we demonstrate that PEtN-mediated O-antigen modification in S. flexneri increase the severity of corneal infection in guinea pigs without any adaptive cost. This heightened virulence is associated with epithelial cell adhesion and invasion, as well as an enhanced inflammatory response of macrophage. Notably, PEtN addition allow S. flexneri to attenuate the binding of complement C3 and better resist phagocytosis, potentially contributing to the retention of S. flexneri in the host environment.

MCR Expression Conferring Varied Fitness Costs on Host Bacteria and Affecting Bacteria Virulence.

Since the first report of the plasmid-mediated, colistin-resistant gene, mcr-1, nine mcr genes and their subvariants have been identified. The spreading scope of mcr-1~10 varies greatly, suggesting that mcr-1~10 may have different evolutionary advantages. Depending on MCR family phylogeny, mcr-6 is highly similar to mcr-1 and -2, and mcr-7~10 are highly similar to mcr-3 and -4. We compared the expression effects of MCR-1~5 on bacteria of common physiological background. The MCR-1-expressing strain showed better growth than did MCR-2~5-expressing strains in the presence of colistin. LIVE/DEAD staining analysis revealed that MCR-3~5 expression exerted more severe fitness burdens on bacteria than did MCR-1 and -2. Bacteria expressing MCRs except MCR-2 showed enhanced virulence with increased epithelial penetration ability determined by trans-well model (p < 0.05). Enhanced virulence was also observed in the Galleria mellonella model, which may have resulted from bacterial membrane damage and different levels of lipopolysaccharide (LPS) release due to MCR expression. Collectively, MCR-1-expressing strain showed the best survival advantage of MCR-1~5-expressing strains, which may partly explain the worldwide distribution of mcr-1. Our results suggested that MCR expression may cause increased bacterial virulence, which is alarming, and further attention will be needed to focus on the control of infectious diseases caused by mcr-carrying pathogens.

Mobile Colistin Resistance Enzyme MCR-3 Facilitates Bacterial Evasion of Host Phagocytosis.

Mobile colistin resistance enzyme MCR-3 is a phosphoethanolamine transferase modifying lipid A in Gram-negative bacteria. MCR-3 generally mediates low-level (≤8 mg L-1 ) colistin resistance among Enterobacteriaceae, but occasionally confers high-level (>128 mg L-1 ) resistance in aeromonads. Herein, it is determined that MCR-3, together with another lipid A modification mediated by the arnBCADTEF operon, may be responsible for high-level colistin resistance in aeromonads. Lipid A is the critical site of pathogens for Toll-like receptor 4 recognizing. However, it is unknown whether or how MCR-3-mediated lipid A modification affects the host immune response. Compared with the wild-type strains, increased mortality is observed in mice intraperitoneally-infected with mcr-3-positive Aeromonas salmonicida and Escherichia coli strains, along with sepsis symptoms. Further, mcr-3-positive strains show decreased clearance rates than wild-type strains, leading to bacterial accumulation in organs. The increased mortality is tightly associated with the increased tissue hypoxia, injury, and post-inflammation. MCR-3 expression also impairs phagocytosis efficiency both in vivo and in vitro, contributing to the increased persistence of mcr-3-positive bacteria in tissues compared with parental strains. This study, for the first time, reveals a dual function of MCR-3 in bacterial resistance and pathogenicity, which calls for caution in treating the infections caused by mcr-positive pathogens.

Molecular genetic characteristics of mcr-9-harbouring Salmonella enterica serotype Typhimurium isolated from raw milk.

Among the 10 reported mcr genes, mcr-9 was first identified in Salmonella enterica serotype Typhimurium, which is a leading cause of foodborne illness worldwide. However, information about the prevalence and genetic features of mcr-9 is still lacking, especially among food samples. This study reports the presence of mcr-9 in raw milk samples from China; the prevalence rate was low (0.83%, 1/120). mcr-9 was located on a transferable plasmid, and was stable in wild-type S. enterica. However, it had a biological fitness cost when transferred to an Escherichia coli recipient. Whole-genome sequencing revealed that mcr-9 was located on the IncHI2A-type plasmid, and was surrounded by IS903B and IS26 in its flanking regions. The mcr-9-carrying S. enterica 19SE belonged to ST26 and had a multi-drug-resistant phenotype. It was confirmed that mcr-9 did not mediate colistin resistance in this study, indicating that its transfer may not facilitate the dissemination of colistin resistance.

A method for screening tigecycline-resistant gene tet(X) from human gut.

OBJECTIVES: To develop an effective enrichment method for tet(X) detection, we performed PCR and Sanger sequencing to screen and confirm the presence of tet(X) gene. METHODS: Species were identified by MALDI-TOF MS analysis. The minimum inhibitory concentrations (MICs) of common antibiotics were determined by broth microdilution and interpreted according to the CLSI guidelines and EUCAST breakpoints. RESULTS: We obtained 29 (2.26%, 29/1284) tet(X4)-positive Escherichia coli, and 96.6% of those (28 isolates) exhibited resistance to tigecycline. CONCLUSION: This specific screening strategy for functional tet(X) mediating tigecycline resistance will be useful to facilitate development and advancement of our knowledge of tet(X).

Heterogeneity and Diversity of mcr-8 Genetic Context in Chicken-Associated Klebsiella pneumoniae.

Increasing mobile colistin resistance, mediated by the mcr gene family, in Enterobacteriaceae has become a global concern. Among the 10 reported mcr genes, mcr-8 was first identified in Klebsiella pneumoniae, which could cause severe infections with high mortality. Information about the prevalence and genetic context of mcr-8 is still lacking. In this study, we found that mcr-8 was present in 9.83% of K. pneumoniae isolates of chicken origin. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting showed that the mcr-8 gene was located on a plasmid in all of the isolates. The genetic context of the plasmids exhibited considerable diversity from the whole-genome sequence through Illumina and MinION long-read sequencing. Mutations in two-component systems may function synergistically with mcr-8, resulting in extremely high resistance to colistin. In addition to colistin resistance, these plasmids also contained genes conferring resistance to beta-lactams, tetracycline, aminoglycosides, sulfonamides, macrolides, chloramphenicol, and florfenicol. Therefore, these findings indicate that the genetic context of mcr-8 is heterogeneous and diverse and that mcr-8 and certain chromosomal mechanisms jointly contribute to high-level colistin resistance in K. pneumoniae strains, which provides new insights into the resistance mechanisms of K. pneumoniae.

One-step icELISA developed with novel antibody for rapid and specific detection of diclazuril residue in animal-origin foods.

Diclazuril, a broad-spectrum anticoccidial drug, may be accumulated in edible tissues of animals through illegal use, which poses potential threats to human health through the food chain. In this study, an innovative hapten was designed and an immunogen of diclazuril was successfully synthesised with keyhole limpet haemocyanin as carrier protein; then a monoclonal antibody with high specificity was obtained. Furthermore, based on the novel antibody, a one-step indirect competitive enzyme-linked immunosorbent assay (icELISA) was established for rapid and specific detection of diclazuril residues. Compared with the traditional icELISA method, this method saves at least 0.5 hours and one washing step. Under the optimal conditions, the one-step icELISA for diclazuril exhibited good performance with a 50% inhibition concentration (IC50) value of 0.952 µg/kg. The average recoveries of the icELISA ranged from 73.1% to 115.5% with the coefficient of variation lower than 12.7%, which was evaluated by detecting spiked animal-origin food samples. Finally, the one-step icELISA shows a good correlation with an ultra-high liquid chromatography-tandem mass spectrometry method. Those results demonstrate that the one-step icELISA developed for diclazuril detection is time-saving, low-cost, specific, sensitive, and reliable. It shows good potential for social, environmental, and economic benefits in future use.

Epidemiology of mobile colistin resistance genes mcr-1 to mcr-9.

The identification of the first mobile colistin resistance (MCR) gene, mcr-1, in 2015 triggered a rash of mcr screening reports. Subsequently, nine MCR-family genes and their variants have been described. However, a comprehensive overview concerning the epidemiology of the whole MCR family, which is essential for facilitating rational interventions against mcr dissemination, is lacking. Here, based on the National Database of Antibiotic Resistant Organisms and published studies, we have summarized the latest epidemiological characteristics of the mcr genes.

Peptide nucleic acid restores colistin susceptibility through modulation of MCR-1 expression in Escherichia coli.

BACKGROUND: Plasmid-mediated mechanisms of drug resistance accelerate the spread of polymyxin resistance, leaving clinicians with few or no antibacterial options for the treatment of infections caused by MDR bacteria, especially carbapenemase-producing strains. OBJECTIVES: To evaluate the associations among promoter sequence variation, mcr-1 expression, host factors and levels of colistin resistance and to propose antisense agents such as peptide nucleic acids (PNAs) targeting mcr-1 as a tool to restore colistin susceptibility through modulation of MCR-1 expression in Escherichia coli. METHODS: A ß-galactosidase assay was performed to study mcr-1 promoter activity. Quantitative real-time PCR and western blot assays were used to identify the expression level of MCR-1 in WT strains and transformants. Three PNAs targeting different regions of mcr-1 were designed and synthesized to determine whether they can effectively inhibit MCR-1 expression. MIC was measured to test colistin susceptibility in the presence or absence of PNA-1 in mcr-1-carrying E. coli. RESULTS: Variation in the mcr-1 promoter sequence and host species affect promoter activity, MCR-1 expression levels and colistin MICs. One PNA targeting the ribosome-binding site fully inhibited the expression of mcr-1 at a concentration of 4 µM, resulting in significantly increased susceptibility to colistin. The MIC90 of colistin decreased from 8 to 2 mg/L (P < 0.05) in the presence of 4 µM PNA. CONCLUSIONS: These findings suggest that the antisense approach is a possible strategy to combat mcr-1-mediated resistance as well as other causes of emerging global resistance.

Chromosome-Mediated mcr-3 Variants in Aeromonas veronii from Chicken Meat.

Two adjacent colistin resistance gene variants, termed mcr-3.3 and mcr-3-like, were identified in the chromosome of an Aeromonas veronii isolate obtained from retail chicken meat. The variants showed 95.20% and 84.19% nucleotide sequence identity, respectively, to mcr-3 from porcine Escherichia coli Functional cloning indicated that only mcr-3.3 conferred polymyxin resistance in both E. coli and Aeromonas salmonicida The mcr-3.3-mcr-3-like segment was also observed in other Aeromonas species, including A. media, A. caviae, and A. hydrophila.

An extracellular yellow laccase with potent dye decolorizing ability from the fungus Leucoagaricus naucinus LAC-04.

A novel laccase was isolated from fermentation broth of the mycorrhizal fungus Leucoagaricus naucinus LAC-04 by using a protocol that comprising ion-exchange chromatography steps on DEAE-cellulose, SP-Sepharose, and Q-Sepharose, and finally gel filtration by fast protein liquid chromatography on Superdex 75. The laccase (LNL) was purified with a purification fold of 21.19 and a recovery rate of 19.8%. It is a monomeric protein with a molecular mass of 56kDa. LNL lacks absorption around 600nm, which indicates that the purified laccase is a yellow laccases. LNL demonstrates an optimal pH of 2.2 and an optimal temperature range of 30-60°C using ABTS as the substrate. It is inhibited in the presence of EDTA and metal ions including Cd2+, Co2+, Cu2+. The Km of the laccase towards ABTS is estimated to 50.12µM at pH 2.2 and 30°C. Moreover, the purified laccase manifests effective decolorizing activity towards azo, heterocyclic, and aromatic dyes including Bromothymol Blue, Eriochrome Black T, Evans Bue, Fuchsin Basic, and Remazol Brilliant Blue R.

An extracellular laccase with potent dye decolorizing ability from white rot fungus Trametes sp. LAC-01.

A novel laccase was purified from fermentation broth of white rot fungus Trametes sp. LAC-01 using an isolation procedure involving three ion-exchange chromatography steps on DEAE-cellulose, SP-Sepharose, and Q-Sepharose, and one gel-filtration step. The purified enzyme (TSL) was proved as a monomeric protein with a Mr of 59kDa based on SDS-PAGE and FPLC. Partial amino acid sequences were obtained by LC-MS/MS sharing considerably high sequence similarity with that of other laccases. It possessed optimal pH of 2.6 and temperature of 60°C using ABTS as the substrate. The Km of the laccase toward ABTS was estimated to 30.28µM at pH 2.6 and 40°C. TSL manifested considerably high oxidizing activity toward ABTS, but was avoid of degradative activity toward benzidine, caftaric acid, etc. It was effective in the decolorization of phenolic dyes - Bromothymol Blue and Malachite Green with decolorization rate higher than 60% after 24h of incubation. Adjunction of Cu(2+) with the final concentration of 2.0mmol/L significantly activated laccase production with a steady high level of 275.8-282.2U/mL in 96-144h. The high yield and short production period makes Trametes sp. LAC-01 and TSL potentially useful for industrial and environmental application and commercialization.