Antibiotic synergist OM19r reverses aminoglycoside resistance in multidrug-resistant Escherichia coli.

Introduction: The continued emergence and spread of multidrug-resistant (MDR) bacterial pathogens require a new strategy to improve the efficacy of existing antibiotics. Proline-rich antimicrobial peptides (PrAMPs) could also be used as antibacterial synergists due to their unique mechanism of action. Methods: Utilizing a series of experiments on membrane permeability, In vitro protein synthesis, In vitro transcription and mRNA translation, to further elucidate the synergistic mechanism of OM19r combined with gentamicin. Results: A proline-rich antimicrobial peptide OM19r was identified in this study and its efficacy against Escherichia coli B2 (E. coli B2) was evaluated on multiple aspects. OM19r increased antibacterial activity of gentamicin against multidrug-resistance E. coli B2 by 64 folds, when used in combination with aminoglycoside antibiotics. Mechanistically, OM19r induced change of inner membrane permeability and inhibited translational elongation of protein synthesis by entering to E. coli B2 via intimal transporter SbmA. OM19r also facilitated the accumulation of intracellular reactive oxygen species (ROS). In animal models, OM19r significantly improved the efficacy of gentamicin against E. coli B2. Discussion: Our study reveals that OM19r combined with GEN had a strong synergistic inhibitory effect against multi-drug resistant E. coli B2. OM19r and GEN inhibited translation elongation and initiation, respectively, and ultimately affected the normal protein synthesis of bacteria. These findings provide a potential therapeutic option against multidrug-resistant E. coli.

Preparation, Characterization and Pharmacokinetic Study of N-Terminal PEGylated D-Form Antimicrobial Peptide OM19r-8.

Recently, new cationic antibacterial peptide OM19R has been designed with low minimum inhibitory concentration (MIC) values against some gram-negative bacteria, such as Escherichia coli, Salmonella, and Shigella. However, this hybrid peptide, like most antibacterial peptides, has low enzyme stability and short half-life, which, in turn, increases the drug's cost. In this study, an antibacterial peptide (OM19r-8) was obtained containing some D-Arg amino acids. The new preparations were carried out through the replacement of l-Arginine by d-Arginine and the addition of PEG chains. Firstly, eight OM19r series of antibacterial peptides were obtained by designing D-Arg. Then, a polyethylene glycol-modified product mPEG5-butyrALD-OM19r-8 (mPEG5-OM19r-8) was isolated and purified by reverse-phase high-performance liquid chromatography (RT-HPLC). The enzyme stability test showed that the resistance of antibacterial peptide OM19r-8 to protease degradation increased by 4-32-fold. Moreover, the Time-kill studies showed that the germicidal kinetics curves of mPEG5-OM19r-8 and OM19r-8 to Escherichia coli had a similar trend, thus suggesting that PEG modification has an acceptable effect on the activity of the original peptide. Furthermore, the elimination of half-life (28.09 ± 2.81min) of mPEG5-OM19r-8, and the area under the drug concentration-time curve (2686.48 ± 651.36min∗ug/ml) was significantly prolonged. The current study demonstrates an example that optimizes the AMP by utilizing L-to-D amino acid replacement and including PEG chains. These results provide useful data for the clinical application of the mPEG5-OM19r-8.

Genomic island type IV secretion system and transposons in genomic islands involved in antimicrobial resistance in Trueperella pyogenes.

Trueperella pyogenes (T. pyogenes) is a well-known opportunistic pathogen of many animal species. It can cause a variety of suppurative infections. The objective of this research was to get insight into the gene context and the location of the antimicrobial resistance determinants in the two multi-resistant T. pyogenes isolates TP3 and TP4. Comparative analysis of key factors leading to antimicrobial resistance was performed. Both isolates were resistant to erythromycin, azithromycin and tetracycline, and susceptible to ciprofloxacin, enrofloxacin, cefazolin and florfenicol. In addition, TP4 was resistant to amikacin and gentamicin. Whole-genome analyses revealed that both TP3 and TP4 contained two different genomic islands (TP3-GI1, TP3-GI5, TP4-GI5 and TP4-GI8) involved in multi-drug resistance. There is a common region in TP3-GI1 and TP4-GI5, containing the tetracycline resistance gene tet(W) and a series of genes involved in type IV secretion systems. Several genes located on TP3-GI5 and TP4-GI8 are highly homologous. Tetracycline-resistance gene tet(33) was potentially acquired by horizontal gene transfer via IS6100 located on 57,936 bp TP3-GI5. The macrolide resistance gene erm(X) was located near the end of the TP3-GI5. The sequence analysis of TP4-GI8 showed that two copies of erm(X) and two IS1634 elements located in the same orientation may have formed a composite transposon. GI-type T4SS, transposons and multiple resistance genes located on GIs play a key role in multiple drug resistance of TP3 and TP4.

Multidrug resistance genes are associated with a 42-kb island TGI1 carrying a complex class 1 integron in Trueperella pyogenes.

OBJECTIVES: This research was conducted to ascertain the context and location of the antibiotic resistance determinants in a multiple antibiotic-resistant Trueperella pyogenes isolate TP1. METHODS: The genome was sequenced using PacBio RS II, and the filtered data were assembled using Canu. Sequences were annotated on the basis of those in GenBank, and the genomic island (GI) of the TP1 was predicted by IslandPath-DIMOB. RESULTS: TP1 as a multiple antibiotic-resistant isolate was recovered at Jilin Province (China) in 2017 from a dairy cow with pneumonia. TP1 exhibited resistance to aminoglycosides (gentamicin and amikacin), macrolides (erythromycin), lincosamides (clindamycin), sulfonamides (sulfamonomethoxine), tetracyclines (tetracycline and doxycycline) and chloramphenicols (chloramphenicol and florfenicol). An antibiotic resistance gene clustered together with the aadB, aadA1, cmlA5 and cmlA6 resistance genes located on a 7-kilobase (kb) multidrug-resistant (MDR) region, constituting a complex class 1 integron. The MDR region was located at one end of a 42-kb GI, and IS6100Δ1 mediated a genetic rearrangement with the complex class 1 integron-like SGI1 and formed a composite transposon. Furthermore, the tetW gene was located outside the four GIs consistent with tetracycline and doxycycline resistance. The ermD gene positioned in the front end of the 42-kb GI played an important role in mediating acquired erythromycin and clindamycin resistance. CONCLUSIONS: Multiple resistance genes are located in a complex class 1 integron within a 42-kb T. pyogenes genomic island (TGI1), leading to TP1 multiple drug resistance. In comparison with SG1 families, TGI1 possesses versatile gene distribution and specific gene context for it upstream and downstream, and it represents a new lineage of genomic resistance islands.

The complete mitochondrial genome of Tibetan hulless barley.

Hulless barley (Hordeum vulgar L. var. nudum) is one of the staple foods for Tibetans and an important livestock feed in the Tibetan Plateau. We report the complete mitochondrial genome of Tibetan hulless barely. The complete mitochondrial genome size is 416,675 bp. Hulless barely mitochondrial genome encode 34 protein-coding genes, 19 tRNA genes and three rRNA genes. The mitochondrial genome has 50 forward and palindrome repeats totally. Nucleotide sequence of coding region takes over 13.60%, GC content is 44.33%. The maximum-likelihood (ML) phylogenetic tree based on 11 protein-coding genes common to seven plant mitochondrial genomes using Arabidopsis thaliana as out-group support that hulless barely is close to Triticum species.

Transcriptome sequencing in a Tibetan barley landrace with high resistance to powdery mildew.

Hulless barley is an important cereal crop worldwide, especially in Tibet of China. However, this crop is usually susceptible to powdery mildew caused by Blumeria graminis f. sp. hordei. In this study, we aimed to understand the functions and pathways of genes involved in the disease resistance by transcriptome sequencing of a Tibetan barley landrace with high resistance to powdery mildew. A total of 831 significant differentially expressed genes were found in the infected seedlings, covering 19 functions. Either "cell," "cell part," and "extracellular region" in the cellular component category or "binding" and "catalytic" in the category of molecular function as well as "metabolic process" and "cellular process" in the biological process category together demonstrated that these functions may be involved in the resistance to powdery mildew of the hulless barley. In addition, 330 KEGG pathways were found using BLASTx with an E-value cut-off of <10(-5). Among them, three pathways, namely, "photosynthesis," "plant-pathogen interaction," and "photosynthesis-antenna proteins" had significant matches in the database. Significant expressions of the three pathways were detected at 24 h, 48 h, and 96 h after infection, respectively. These results indicated a complex process of barley response to powdery mildew infection.