Mona Kashef

Microbiome projects are currently booming around the globe, enabled by advances in culture-independent microbial community analysis and high-throughput sequencing. One emerging application of microbiome science involves exploring microbial diversity in built environments, and one unexplored built environment is the pharmaceutical factory, notably factories producing antibiotics, as they could be enriched in antibiotic-resistant microbes. To examine the drug factory microbiome, we launched this interdisciplinary hypothesis-generating study to benchmark culture-independent microbiome analysis in drug manufacturing units producing antibiotics and nonantibiotic drugs, against traditional microbial identification and quantification techniques. Over a course of 4 months, we prospectively collected 234 samples from antibiotic (kanamycin and amoxicillin) and nonantibiotic (acetaminophen) production clean rooms within a pharmaceutical factory in Egypt. All samples were analyzed by traditional culture-based methods, and microbial communities of representative samples were profiled by16S rRNA gene sequencing. In addition, antibiotic resistance profiles of some samples were determined, and representative resistance genes were screened. The 16S rRNA analysis revealed a typical predominance of Proteobacteria (36%), Firmicutes (31%), and Bacteroidetes (16%). The microbial composition of the samples was highly affected by the use of water, environmental conditions during the production process, the presence of personnel, and the type of the product. The effect of these factors was confirmed by total aerobic microbial counts and identification of biomarker microbes. In conclusion, these observations can aid in the future for optimal design and management of pharmaceutical manufacturing units, and speak to a greater need for implementing microbiome research in the quality assurance of built environments.

The emergence of microbial resistance to the available antibiotics is a major public health concern, especially with the limited rate of developing new antibiotics. The utilization of anti-virulence agents is a non-conventional approach that can be used to combat microbial infection. In , many virulence factors are regulated by the Agr-mediated quorum sensing (QS). We developed a chemical compound that acts a potential Agr-inhibitor without reducing bacterial viability. The compound was designated staquorsin for S QS inhibitor. analyses confirmed the binding of staquorsin to the AgrA active site with an absolute binding score comparable to savirin, a previously described AgrA inhibitor. However, staquorsin turned out to be superior over savarin in not affecting the viability in concentrations up to 600 μM. On the other hand, savirin inhibited growth in concentrations as low as 25 μM. Moreover, staquorsin proved to be a potent inhibitor of the Agr system by inhibiting hemolysins, lipase production, and affecting biofilms formation and detachment. On the molecular level it significantly inhibited the effector transcript RNA III. testing, using the murine skin abscess model, confirmed the ability of staquorsin to modulate virulence by effectively controlling the infection. Twenty passages of in the presence of 40 μM staquorsin have not resulted in loss of activity as evidenced by maintaining its ability to reduce hemolysin production and RNA III transcript levels. In conclusion, we hereby describe a novel anti-virulence compound inhibiting the Agr-system and its associated virulence factors. It is active both and , and its frequent use does not lead to the development of resistance. These findings model staquorsin as a promising drug candidate to join the fierce battle against the formidable pathogen .

Enterococcus faecalis is a troublesome nosocomial pathogen that acquired resistance to most available antimicrobial agents. Antivirulence agents represent an unconventional treatment approach. Here, transcription factor decoy (TFD)-loaded cationic liposomes (TLL) were developed as an inhibitor of the Fsr quorum-sensing system and its associated virulence traits, in E. faecalis. The consensus sequence of the FsrA binding site was found conserved among 651 E. faecalis annotated genomes. The TFD was synthesized as an 82 bp DNA duplex, containing the conserved binding sequence, and loaded onto cationic liposomes. The optimum loading capacity, mean particle size, and zeta potential of the TLL were characterized. The developed TLL lacked any effect on E. faecalis growth and significantly inhibited the in vitro production of the proteolytic enzymes controlled by the Fsr system; gelatinase and serine protease, in a concentration-dependent manner. This inhibition was accompanied by a significant reduction in the transcription levels of FsrA-regulated genes (fsrB, gelE, and sprE). The developed TLL were safe as evidenced by the nonhemolytic effect on human RBCs and the negligible cytotoxicity on human skin fibroblast cells. Moreover, in the larvae infection model, TLL displayed a significant abolish in the mortality rates of Galleria mellonella larvae infected with E. faecalis. In conclusion, the developed TLL offer a new safe strategy for combating E. faecalis infection through the inhibition of quorum-sensing-mediated virulence; providing a platform for the development of similar agents to combat many other pathogens.

AIMS: The current work aims to fully characterize a new antimicrobial agent against Acinetobacter baumannii, which continues to represent a growing threat to healthcare settings worldwide. With minimal treatment options due to the extensive spread of resistance to almost all the available antimicrobials, the hunt for new antimicrobial agents is a high priority.

METHODS AND RESULTS: An Egyptian soil-derived bacterium strain NHM-077B proved to be a promising source for a new antimicrobial agent. Bio-guided fractionation of the culture supernatants of NHM-077B followed by chemical structure elucidation identified the active antimicrobial agent as 1-hydroxy phenazine. Chemical synthesis yielded more derivatives, including dihydrophenazine (DHP), which proved to be the most potent against A. baumannii, yet it exhibited a marginally safe cytotoxicity profile against human skin fibroblasts. Proteomics analysis of the cells treated with DHP revealed multiple proteins with altered expression that could be correlated to the observed phenotypes and potential mechanism of the antimicrobial action of DHP. DHP is a multipronged agent that affects membrane integrity, increases susceptibility to oxidative stress, interferes with amino acids/protein synthesis, and modulates virulence-related proteins. Interestingly, DHP in subinhibitory concentrations re-sensitizes the highly virulent carbapenem-resistant A. baumannii strain AB5075 to carbapenems providing great hope in regaining some of the benefits of this important class of antibiotics.

CONCLUSIONS: This work underscores the potential of DHP as a promising new agent with multifunctional roles as both a classical and nonconventional antimicrobial agent that is urgently needed.