Evaluating the Compatibility of New Recombinant Protein Antigens (Trivalent NRRV) with a Mock Pentavalent Combination Vaccine Containing Whole-Cell Pertussis: Analytical and Formulation Challenges.

Introducing new recombinant protein antigens to existing pediatric combination vaccines is important in improving coverage and affordability, especially in low- and middle-income countries (LMICs). This case-study highlights the analytical and formulation challenges encountered with three recombinant non-replicating rotavirus vaccine (NRRV) antigens (t-NRRV formulated with Alhydrogel® adjuvant, AH) combined with a mock multidose formulation of a pediatric pentavalent vaccine used in LMICs. This complex formulation contained (1) vaccine antigens (i.e., whole-cell pertussis (wP), diphtheria (D), tetanus (T), Haemophilus influenza (Hib), and hepatitis B (HepB), (2) a mixture of aluminum-salt adjuvants (AH and Adju-Phos®, AP), and (3) a preservative (thimerosal, TH). Selective, stability-indicating competitive immunoassays were developed to monitor binding of specific mAbs to each antigen, except wP which required the setup of a mouse immunogenicity assay. Simple mixing led to the desorption of t-NRRV antigens from AH and increased degradation during storage. These deleterious effects were caused by specific antigens, AP, and TH. An AH-only pentavalent formulation mitigated t-NRRV antigen desorption; however, the Hib antigen displayed previously reported AH-induced instability. The same rank-ordering of t-NRRV antigen stability (P[8] > P[4] > P[6]) was observed in mock pentavalent formulations and with various preservatives. The lessons learned are discussed to enable future multidose, combination vaccine formulation development with new vaccine candidates.

Functional Analysis of Phenazine Biosynthesis Genes in Burkholderia spp.

Burkholderia encompasses a group of ubiquitous Gram-negative bacteria that includes numerous saprophytes as well as species that cause infections in animals, immunocompromised patients, and plants. Some species of Burkholderia produce colored, redox-active secondary metabolites called phenazines. Phenazines contribute to competitiveness, biofilm formation, and virulence in the opportunistic pathogen Pseudomonas aeruginosa, but knowledge of their diversity, biosynthesis, and biological functions in Burkholderia is lacking. In this study, we screened publicly accessible genome sequence databases and identified phenazine biosynthesis genes in multiple strains of the Burkholderia cepacia complex, some isolates of the B. pseudomallei clade, and the plant pathogen B. glumae We then focused on B. lata ATCC 17760 to reveal the organization and function of genes involved in the production of dimethyl 4,9-dihydroxy-1,6-phenazinedicarboxylate. Using a combination of isogenic mutants and plasmids carrying different segments of the phz locus, we characterized three novel genes involved in the modification of the phenazine tricycle. Our functional studies revealed a connection between the presence and amount of phenazines and the dynamics of biofilm growth in flow cell and static experimental systems but at the same time failed to link the production of phenazines with the capacity of Burkholderia to kill fruit flies and rot onions.IMPORTANCE Although the production of phenazines in Burkholderia was first reported almost 70 years ago, the role these metabolites play in the biology of these economically important microorganisms remains poorly understood. Our results revealed that the phenazine biosynthetic pathway in Burkholderia has a complex evolutionary history, which likely involved horizontal gene transfers among several distantly related groups of organisms. The contribution of phenazines to the formation of biofilms suggests that Burkholderia, like fluorescent pseudomonads, may benefit from the unique redox-cycling properties of these versatile secondary metabolites.

Antimicrobial Activity of, and Cellular Pathways Targeted by, p-Anisaldehyde and Epigallocatechin Gallate in the Opportunistic Human Pathogen Pseudomonas aeruginosa.

Plant-derived aldehydes are constituents of essential oils that possess broad-spectrum antimicrobial activity and kill microorganisms without promoting resistance. In our previous study, we incorporated p-anisaldehyde from star anise into a polymer network called proantimicrobial networks via degradable acetals (PANDAs) and used it as a novel drug delivery platform. PANDAs released p-anisaldehyde upon a change in pH and humidity and controlled the growth of the multidrug-resistant pathogen Pseudomonas aeruginosa PAO1. In this study, we identified the cellular pathways targeted by p-anisaldehyde by generating 10,000 transposon mutants of PAO1 and screened them for hypersensitivity to p-anisaldehyde. To improve the antimicrobial efficacy of p-anisaldehyde, we combined it with epigallocatechin gallate (EGCG), a polyphenol from green tea, and demonstrated that it acts synergistically with p-anisaldehyde in killing P. aeruginosa We then used transcriptome sequencing to profile the responses of P. aeruginosa to p-anisaldehyde, EGCG, and their combination. The exposure to p-anisaldehyde altered the expression of genes involved in modification of the cell envelope, membrane transport, drug efflux, energy metabolism, molybdenum cofactor biosynthesis, and the stress response. We also demonstrate that the addition of EGCG reversed many p-anisaldehyde-coping effects and induced oxidative stress. Our results provide insight into the antimicrobial activity of p-anisaldehyde and its interactions with EGCG and may aid in the rational identification of new synergistically acting combinations of plant metabolites. Our study also confirms the utility of the thiol-ene polymer platform for the sustained and effective delivery of hydrophobic and volatile antimicrobial compounds.IMPORTANCE Essential oils (EOs) are plant-derived products that have long been exploited for their antimicrobial activities in medicine, agriculture, and food preservation. EOs represent a promising alternative to conventional antibiotics due to their broad-range antimicrobial activity, low toxicity to human commensal bacteria, and capacity to kill microorganisms without promoting resistance. Despite the progress in the understanding of the biological activity of EOs, our understanding of many aspects of their mode of action remains inconclusive. The overarching aim of this work was to address these gaps by studying the molecular interactions between an antimicrobial plant aldehyde and the opportunistic human pathogen Pseudomonas aeruginosa The results of this study identify the microbial genes and associated pathways involved in the response to antimicrobial phytoaldehydes and provide insights into the molecular mechanisms governing the synergistic effects of individual constituents within essential oils.

Using Aldehyde Synergism To Direct the Design of Degradable Pro-Antimicrobial Networks.

We describe the design and synthesis of degradable, dual-release, pro-antimicrobial poly(thioether acetal) networks derived from synergistic pairs of aromatic terpene aldehydes. Initially, we identified pairs of aromatic terpene aldehyde derivatives exhibiting a synergistic antimicrobial activity against Pseudomonas aeruginosa by determining fractional inhibitory concentrations. Synergistic aldehydes were converted into dialkene acetal monomers and copolymerized at various ratios with a multifunctional thiol via thiol-ene photopolymerization. The step-growth nature of the thiol-ene polymerization ensures every cross-link junction contains a degradable acetal linkage enabling a fully cross-linked polymer network to revert into its small molecule constituents upon hydrolysis, releasing the synergistic aldehydes as active antimicrobial compounds. A three-pronged approach was used to characterize the poly(thioether acetal) materials: (i) determination of the degradation/aldehyde release behavior, (ii) evaluation of the antimicrobial activity, and (iii) identification of the cellular pathways impacted by the aldehydes on a library of mutated bacteria. From this approach, a polymer network derived from a 40:60 p-bromobenzaldehyde/p-anisaldehyde monomer ratio exhibited potent antimicrobial action against Pseudomonas aeruginosa, a common opportunistic human pathogen. From a transposon mutagenesis assay, we showed that these aldehydes target porins and multidrug efflux pumps. The aldehydes released from the poly(thioether acetal) networks exhibited negligible toxicity to mammalian tissue culture cells, supporting the potential development of these materials as dual-release antimicrobial biomaterial platforms.