Bridging upstream and downstream for improved adenovirus 5 bioprocess.

Adenoviruses are well-known viral vectors that have been previously used in gene therapy and as a vaccine-delivery vehicle for humans and animals. During the COVID-19 pandemic, it gained renewed attention, but at the same time, it raised concerns due to side effects observed with some of the resulting vaccines administered to patients. It has been indicated that these side effects might be attributed to impurities present in the final product. Therefore, constant enhancement of the vaccine purity and further improvement of impurity detection methods are needed. In this work, we showcase an example of industry-relevant adenovirus bioprocess optimization. Our data show the effect of upstream parameters on the bioburden introduced to the downstream process. We provide an example of process optimization using a combination of the PATfix analytical method, ddPCR, infectivity, total DNA, and total protein analyses to optimize cell density, multiplicity of infection, and length of production. Additionally, we provide data illustrating the robustness of the convective interaction media quaternary amine monolithic chromatography step. This anion exchange strategy was shown to remove over 99% of protein and DNA impurities, including those unable to be addressed by tangential flow filtration, while maintaining high adenovirus recoveries.

Resistance to bile salts and sodium deoxycholate in macrolide- and fluoroquinolone-susceptible and resistant Campylobacter jejuni and Campylobacter coli strains.

Campylobacter are the most commonly reported bacterial causes of human gastroenteritis, and they are becoming increasingly resistant to antibiotics, including macrolides and fluoroquinolones, those most frequently used for the treatment of campylobacteriosis. Active efflux mechanisms are involved in resistance of Campylobacter to a broad spectrum of antimicrobials, and are also essential for Campylobacter colonization in the animal intestine, through mediation of bile resistance. Acquisition of antibiotic resistance through resistance-conferring mutations can impose a fitness cost of Campylobacter. The aim of the present study was to determine whether macrolide and fluoroquinolone resistance in Campylobacter affects their tolerance to bile salts and sodium deoxycholate through the most frequent resistance-conferring mutations. Antimicrobial efflux was studied on the basis of restored sensitivity in the presence of the efflux-pump inhibitors (EPIs) phenylalanine-arginine beta-naphthylamide (PAßN) and 1-(1-naphthylmethyl)-piperazine. In the 15 Campylobacter jejuni and 23 Campylobacter coli strains examined here, both of these EPIs partially reversed the resistance to bile salts and sodium deoxycholate. Erythromycin-sensitive C. coli strains were more resistant to bile salts and sodium deoxycholate than erythromycin-resistant strains. PAßN had greater effects on bile salt and sodium deoxycholate resistance in these erythromycin-resistant strains compared to erythromycin-sensitive strains. However, no differences were seen between the ciprofloxacin-sensitive and ciprofloxacin-resistant strains.

Development of antimicrobial resistance in Campylobacter jejuni and Campylobacter coli adapted to biocides.

The potential for adaptive resistance of Campylobacter jejuni and Campylobacter coli after step-wise exposure to increasing sub-inhibitory concentrations of five biocides as triclosan, benzalkonium chloride, cetylpyridinium chloride, chlorhexidine diacetate and trisodium phosphate, was investigated, to identify the mechanisms underlying resistance. The biocide resistance and cross-resistance to the antimicrobials erythromycin and ciprofloxacin, and to sodium dodecyl sulphate, were examined according to the broth microdilution method. The presence of active efflux was studied on the basis of restored sensitivity in the presence of the efflux pump inhibitors phenylalanine-arginine beta-naphthylamide, 1-(1-naphthylmethyl)-piperazine, cyanide 3-chlorophenylhydrazone, verapamil and reserpine. Changes in the outer membrane protein profiles and morphological changes in adapted strains were studied, as compared with the parent strains. Repeated exposure of C. jejuni and C. coli to biocides resulted in partial increases in tolerance to biocides itself, to other biocides and antimicrobial compounds. The developed resistance was stable for up to 10 passages in biocide-free medium. More than one type of active efflux was identified in adapted strains. These adapted strains showed different alterations to their outer membrane protein profiles, along with morphological changes. The data presented here suggest that different mechanisms are involved in adaptation to biocides and that this adaptation is unique to each strain of Campylobacter and does not result from a single species-specific mechanism.

Effects of efflux-pump inducers and genetic variation of the multidrug transporter cmeB in biocide resistance of Campylobacter jejuni and Campylobacter coli.

Multidrug efflux pumps, such as CmeABC and CmeDEF, are involved in the resistance of Campylobacter to a broad spectrum of antimicrobials. The aim of this study was to analyse the effects of two putative efflux-pump inducers, bile salts and sodium deoxycholate, on the resistance of Campylobacter to biocides (triclosan, benzalkonium chloride, chlorhexidine diacetate, cetylpyridinium chloride and trisodium phosphate), SDS and erythromycin. The involvement of the CmeABC and CmeDEF efflux pumps in this resistance was studied on the basis of the effects of bile salts and sodium deoxycholate in Campylobacter cmeB, cmeF and cmeR mutants. The genetic variation in the cmeB gene was also examined, to see whether this polymorphism is related to the function of the efflux pump. In 15 Campylobacter jejuni and 23 Campylobacter coli strains, bile salts and sodium deoxycholate increased the MICs of benzalkonium chloride, chlorhexidine diacetate, cetylpyridinium chloride and SDS, and decreased the MICs of triclosan, trisodium phosphate and erythromycin. Bile salts and sodium deoxycholate further decreased or increased the MICs of biocides and erythromycin in the cmeF and cmeR mutants. For cmeB polymorphisms, 17 different cmeB-specific PCR-RFLP patterns were identified: six within C. jejuni only, nine within C. coli only and two in both species. In conclusion, bile salts and sodium deoxycholate can increase or decrease bacterial resistance to structurally unrelated antimicrobials. The MIC increases in the cmeF and cmeR mutants indicated that at least one non-CmeABC efflux system is involved in resistance to biocides. These results indicate that the cmeB gene polymorphism identified is not associated with biocide and erythromycin resistance in Campylobacter.

Chemical properties and antioxidant and antimicrobial activities of Slovenian propolis.

The chemical composition as well as the antioxidant and antimicrobial activities of two EtOH extracts of propolis (PEEs) from Slovenia were determined. EtOH was used as extracting solvent at 70 and 96%, providing the extracts PEE70 and PEE96, respectively. The extraction with 70% EtOH was more efficient than that with 96% EtOH, as the PEE70 was richer in total phenolic compounds than the PEE96. The Slovenian propolis was characterized by different phenolic acids and flavonoids. The PEE96 was slightly richer in three specific compounds, i.e., caffeic acid, ferulic acid, and luteolin, while all other substances detected showed higher contents in the PEE70. The PEE70 showed a stronger reducing power and ability to scavenge free radicals and metal ions than the PEE96. Both PEEs were in the main more effective against Gram-positive bacteria than against fungi and Gram-negative bacteria like Salmonella and Escherichia coli, with the exception of Campylobacter. The PEE96 decreased the intracellular oxidation in Saccharomyces cerevisiae in a dose-dependent manner. The antimicrobial activities and antioxidant properties were related to the total phenolic contents. The two PEEs have the potential for use as natural antimicrobial and antioxidant additives in foods.

Involvement of efflux mechanisms in biocide resistance of Campylobacter jejuni and Campylobacter coli.

Active efflux has an important role in the antimicrobial resistance of Campylobacter jejuni and Campylobacter coli. The effects of two putative efflux pump inhibitors (EPIs), phenylalanine-arginine ß-naphthylamide and 1-(1-naphthylmethyl)-piperazine, and the effects of inactivation of the cmeB,cmeF and cmeR genes on resistance to a broad range of antimicrobials were studied using the broth microdilution method. The antimicrobials tested in C. jejuni and C. coli were the biocides triclosan, benzalkonium chloride, chlorhexidine diacetate, cetylpyridinium chloride and trisodium phosphate, along with the anionic surfactant SDS and the antibiotics erythromycin and ciprofloxacin. Both EPIs partially reversed the resistance to all of these antimicrobials. Differences between these EPIs were seen for substrate preference and reductions in MIC. The MICs of the antimicrobials were reduced in the cmeB and cmeF mutants and increased in the cmeR mutant, with few exceptions. Both of these putative EPIs further decreased the MICs of the antimicrobials in these mutant strains. These data confirm that active efflux is an important mechanism in biocide resistance in C. jejuni and C. coli. At least one non-CmeABC efflux system or reduced uptake is responsible for resistance to biocides.