Synergistic antibacterial activity of tetrandrine combined with colistin against MCR-mediated colistin-resistant Salmonella.

It has been recognized that colistin resistance is a growing problem that seriously impairs the clinical efficacy of colistin against bacterial infections. One strategy that has been proven to have therapeutic effect is to overcome the widespread emergence of antibiotic-resistant pathogens by combining existing antibiotics with promising non-antibiotic agents. In this work, antibiotic susceptibility testing, checkerboard assays and time-kill curves were used to investigate the antibacterial activity of the individual drugs and the potential synergistic activity of the combination. The molecular mechanisms of tetrandrine in combination with colistin were analyzed using fluorometric assay and Real-time PCR. To predict possible interactions between tetrandrine and MCR-1, molecular docking assay was taken. Finally, we evaluated the in vivo efficacy of tetrandrine in combination with colistin against MCR-positive Salmonella. Overall, the combination of tetrandrine and colistin showed significant synergistic activity. In-depth mechanistic analysis showed that the combination of tetrandrine with colistin enhances the membrane-damaging ability of colistin, undermines the functions of proton motive force (PMF) and efflux pumps in MCR-positive bacteria. The results of molecular docking and RT-PCR analyses showed that tetrandrine not only affects the expression of mcr-1 but is also an effective MCR-1 inhibitor. Compared with colistin monotherapy, the combination of tetrandrine with colistin significantly reduced the bacterial load in vivo. Our findings demonstrated that tetrandrine serves as a potential colistin adjuvant against MCR-positive Salmonella.

Double deletion of cpxR and tolC significantly increases the susceptibility of Salmonella enterica serovar Typhimurium to colistin.

BACKGROUND: The increasing use of colistin causes a serious breach in our last line of defence against MDR Gram-negative pathogens. Our previous study showed that CpxR overexpression increases the susceptibility of acrB and cpxR double-deleted Salmonella enterica serovar Typhimurium to colistin. OBJECTIVES: To identify the mechanism of CpxAR and efflux pumps that synergistically enhance the susceptibility of S. Typhimurium to colistin. METHODS: A series of cpxR- and tolC-deleted mutants and a cpxR-complemented strain from a multidrug-susceptible standard strain of S. Typhimurium (JS) were generated in our previous study. Herein, we investigated the susceptibility of these strains to colistin through the broth microdilution method, time-kill curves and survival assays. Growth curves were measured by OD600 in LB broth, tryptone-soy broth (TSB) and M9-glucose (0.2%) minimal media. Finally, molecular mechanisms underlying the mode of action were elucidated by transcriptomic analysis. RESULTS: We found that in contrast to JS (0.8 mg/L), the MIC of colistin for JSΔtolC::kan showed a 16-fold decrease (0.05 mg/L). Notably, JSΔcpxRΔtolC and JSΔcpxRΔtolC/pcpxR were associated with a 256-fold decrease (0.0031 mg/L) compared with JS. Growth curves identified that JSΔcpxRΔtolC and JSΔcpxRΔtolC/pcpxR displayed a markedly lower growth rate and poorer adaptability. In addition, time-kill curves and survival assays showed that JSΔcpxRΔtolC and JSΔcpxRΔtolC/pcpxR were more susceptible to colistin. Lastly, double deletion of cpxR and tolC enhanced oxidative damage through promoting oxidative phosphorylation, the tricarboxylic acid (TCA) cycle and trimethylamine N-oxide (TMAO) respiration. CONCLUSIONS: Our findings revealed that double deletion of cpxR and tolC significantly increases the susceptibility of S. Typhimurium to colistin.

Response of a novel selenium-dependent glutathione peroxidase from thick shell mussel Mytilus coruscus exposed to lipopolysaccharide, copper and benzo[α]pyrene.

Glutathione peroxidase (GPx) plays an important antioxidant role in cellular defense against environmental stress. In the present study, a novel selenium-dependent glutathione peroxidase termed McSeGPx firstly identified in thick shell mussel Mytilus coruscus. McSeGPx consists of 197 amino acid residues, characterized with one selenocysteine residue encoded by an opal stop codon TGA, one selenocysteine insertion sequence (SECIS) in the 3' untranslated region (UTR), two active site motifs and one signature sequence motif. McSeGPx transcripts were constitutively expressed in all examined tissues, and were significantly induced in gills and digestive glands with the stimulations of lipopolysaccharide (LPS), copper (Cu) and benzo[α]pyrene (B[α]P). Additionally, rough increases in McSeGPx activity were detected in both tissues under the challenge of LPS, Cu and B[α]P. Collectively, these results suggested that McSeGPx affiliate to selenocysteine dependent GPx (SeGPx) family and might play an important role in mediating the environmental stressors and antioxidant response in M. coruscus.

Identification and functional characterization of three myeloid differentiation factor 88 (MyD88) isoforms from thick shell mussel Mytilus coruscus.

Myeloid differentiation factor 88 (MyD88) is a pivotal adapter protein that involved in interleukin-1 receptor/toll-like receptor (IL-1R/TLR) signal transduction, which could spur downstream cascades and eventually drawn into innate immune response. MyD88 has been extensively studied in vertebrates, however, the information ascribe to MyD88 in invertebrates is still very scarce especially its function annotation remains extremely obscure. At here, three novel MyD88 isoforms termed McMyD88a, McMyD88b and McMyD88c were firstly cloned from thick shell mussel Mytilus coruscus. McMyD88a, McMyD88b and McMyD88c shared domain topology containing the Death domain (DD) and TIR domain (TIR) with its counterparts in mammals. All three McMyD88s were ubiquitously expressed in examined tissues in thick shell mussel, with the higher expression levels in immune-related tissues such as haemocytes, gills and digestive glands. Upon Vibrio alginolyticus, polyinosine-polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) challenge, McMyD88a, McMyD88b and McMyD88c transcripts were significantly induced in haemocytes despite of differential expression levels and responsive time points. Overexpression of McMyD88a, McMyD88b and McMyD88c showed a dose-dependent induction to NF-κB or ISRE in mammalian cell lines. Taken together, these results suggested that McMyD88a, McMyD88b and McMyD88c are members of MyD88 family and play potential roles in innate immune response to pathogenic invasions in thick shell mussel. Moreover, these results suggested indirectly the existence of a MyD88-dependent signaling pathway in thick shell mussel, and provide insight into the immunoregulatory effect in molluscs.

Effects of low concentrations copper on antioxidant responses, DNA damage and genotoxicity in thick shell mussel Mytilus coruscus.

For the aim to study potential detrimental effects induced by Cu exposure at low, environmentally relevant concentrations, the in vivo activities at different levels of biological organisations of thick shell mussel Mytilus coruscus exposed to two levels of copper were assessed. Cu-induced stresses were evaluated through antioxidant responses, DNA damage and genotoxicity. The results revealed significant higher SOD and CAT activities, and MDA concentration in haemocytes of M. coruscus with Cu exposure at 8 µg/L, while only significant accumulation in CAT activity with Cu exposure at 2 µg/L and no significant changes with SOD activity and MDA concentration at this level of Cu exposure. Copper exposure induced DNA damage as induction of OTM value in a time- and concentration-dependent manner. In addition, copper exposure could significantly induced the expressions of MT-10, Hsp70, Hsp90 and C3. The present results deepen the mussels as a suitable model marine invertebrate species to study potential detrimental effects induced by possible toxicants, in combinations at different levels of biological organisations.