To address the increasing demand for safe and effective treatment options for pelvic organ prolapse (POP) due to the worldwide ban of the traditional polypropylene meshes, this study introduced degradable polycaprolactone (PCL)/polyethylene glycol (PEG) composite meshes fabricated with melt-electrowriting (MEW). Two PCL/PEG mesh groups: 90:10 and 75:25 (PCL:PEG, wt%) were fabricated and characterized for their degradation rate and mechanical properties, with PCL meshes used as a control. The PCL/PEG composites showed controllable degradation rates by adjusting the PEG content and produced mechanical properties, such as maximal forces, that were higher than PCL alone. The antibacterial properties of the meshes were elicited by coating them with a commonly used antibiotic: azithromycin. Two dosage levels were used for the coating: 0.5 mg and 1 mg per mesh, and both dosage levels were found to be effective in suppressing the growth of S. aureus bacteria. The biocompatibility of the meshes was assessed using human immortalized adipose derived mesenchymal stem cells (hMSC). In vitro assays were used to assess the cell viability (LIVE/DEAD assay), cell metabolic activity (alamarBlue assay) and cell morphology on the meshes (fluorescent and electron microscopy). The cell attachment was found to decrease with increased PEG content. The freshly drug-coated meshes showed signs of cytotoxicity during the cell study process. However, when pre-released for 14 days in phosphate buffered saline, the initial delay in cell attachment on the drug-coated mesh groups showed full recovery at the 14-day cell culture time point. These results indicated that the PCL/PEG meshes with antibiotics coating will be an effective anti-infectious device when first implanted into the patients, and, after about 2 weeks of drug release, the mesh will be supporting cell attachment and proliferation. These meshes demonstrated a potential effective treatment option for POP that may circumvent the issues related to the traditional polypropylene meshes.

Escherichia coli K-12 is a model organism for bacteriology and has served as a workhorse for molecular biology and biochemistry for over a century since its first isolation in 1922. However, Escherichia coli K-12 strains are phenotypically devoid of an O antigen (OAg) since early reports in the scientific literature. Recent studies have reported the presence of independent mutations that abolish OAg repeating-unit (RU) biogenesis in E. coli K-12 strains from the same original source, suggesting unknown evolutionary forces have selected for inactivation of OAg biogenesis during the early propagation of K-12. Here, we show for the first time that restoration of OAg in E. coli K-12 strain MG1655 synergistically sensitises bacteria to vancomycin with bile salts (VBS). Suppressor mutants surviving lethal doses of VBS primarily contained disruptions in OAg biogenesis. We present data supporting a model where the transient presence and accumulation of lipid-linked OAg intermediates in the periplasmic leaflet of the inner membrane interfere with peptidoglycan sacculus biosynthesis, causing growth defects that are synergistically enhanced by bile salts. Lastly, we demonstrate that continuous bile salt exposure of OAg-producing MG1655 in the laboratory, can recreate a scenario where OAg disruption is selected for as an evolutionary fitness benefit. Our work thus provides a plausible explanation for the long-held mystery of the selective pressure that may have led to the loss of OAg biogenesis in E. coli K-12; this opens new avenues for exploring long-standing questions on the intricate network coordinating the synthesis of different cell envelope components in Gram-negative bacteria.

The study of clinically relevant bacterial pathogens relies on molecular and genetic approaches. However, the generally low transformation frequency among natural isolates poses technical hurdles to widely applying common methods in molecular biology, including transformation of large constructs, chromosomal genetic manipulation, and dense mutant library construction. Here we demonstrate that culturing clinical isolates in the presence of polymyxin B nonapeptide (PMBN) improves their transformation frequency via electroporation by up to 100-fold in a dose-dependent and reversible manner. The effect was observed for PMBN-binding uropathogenicEscherichia coli(UPEC) andSalmonella entericastrains but not naturally polymyxin resistantProteus mirabilis. Using our PMBN electroporation method we show efficient delivery of large plasmid constructs into UPEC, which otherwise failed using a conventional electroporation protocol. Moreover, we show a fivefold increase in the yield of engineered mutant colonies obtained inS. entericawith the widely used lambda-Red recombineering method, when cells are cultured in the presence of PMBN. Lastly, we demonstrate that PMBN treatment can enhance the delivery of DNA-transposase complexes into UPEC and increase transposon mutant yield by eightfold when constructing Transposon Insertion Sequencing (TIS) libraries. Therefore, PMBN can be used as a powerful electropermeabilisation adjuvant to aid the delivery of DNA and DNA–protein complexes into clinically important bacteria.

Shigella species cause bacillary dysentery, especially among young individuals. Shigellae target the human colon for invasion; however, the initial adhesion mechanism is poorly understood. The Shigella surface protein IcsA, in addition to its role in actin-based motility, acts as a host cell adhesin through unknown mechanism(s). Here we confirmed the role of IcsA in cell adhesion and defined the region required for IcsA adhesin activity. Purified IcsA passenger domain was able block S. flexneri adherence and was also used as a molecular probe that recognised multiple components from host cells. The region within IcsA’s functional passenger domain (aa 138–148) was identified by mutagenesis. Upon the deletion of this region, the purified IcsA^Δ138–148 was found to no longer block S. flexneri adherence and had reduced ability to interact with host molecules. Furthermore, S. flexneri expressing IcsA^Δ138–148 was found to be significantly defective in both cell adherence and invasion. Taken together, our data identify an adherence region within the IcsA functional domain and provides useful information for designing therapeutics for Shigella infection.

The O-antigen, a long polysaccharide that constitutes the distal part of the outer membrane-anchored lipopolysaccharide, is one of the critical components in the protective outer membrane of Gram-negative bacteria. Most species produce one of the structurally diverse O-antigens, with nearly all the polysaccharide components having complex structures made by the Wzx/Wzy pathway.

Gram-negative pathogens are a major concern for global public health due to increasing rates of antibiotic resistance and the lack of new drugs. A major contributing factor toward antibiotic resistance in Gram-negative bacteria is their formidable outer membrane, which acts as a permeability barrier preventing many biologically active antimicrobials from reaching the intracellular targets and thus limiting their efficacy.