Typhoid Fever in the Eastern Mediterranean Region: A Systematic Review, 1990-2021.

The occurrence and antimicrobial resistance (AMR) of typhoid fever in the WHO Eastern Mediterranean Region (EMR) are poorly characterized. Robust surveillance data are needed to inform strategies for typhoid control and prevention in the region. We conducted a systematic review of typhoid fever occurrence, complications, and AMR patterns in EMR countries. We identified 70 studies published from 1990 to 2021, including a total of 44,541 cases with blood culture confirmed typhoid fever in 12 EMR countries, with 48 (69%) studies and 42,008 cases from Pakistan. Among 56 studies with AMR data, fluroquinolone (68% of 13,013 tested isolates), and multidrug resistance (40% of 15,765 tested isolates) were common. Forty (57%) of the 56 studies were from Pakistan, and all reports of extensively drug resistant Salmonella Typhi (48% of 9,578 tested isolates) were from studies in Pakistan. Our findings support the need for continued efforts to strengthen surveillance and laboratory capacity for blood-culture detection of typhoid fever in the region, including data from an ongoing collaboration among CDC, the American University of Beirut, and the WHO EMR office.

Regio- and Stereoselective Epoxidation and Acidic Epoxide Opening of Antibacterial and Antiplasmodial Chlorotonils Yield Highly Potent Derivatives.

The rise of antimicrobial resistance poses a severe threat to public health. The natural product chlorotonil was identified as a new antibiotic targeting multidrug resistant Gram-positive pathogens and Plasmodium falciparum. Although chlorotonil shows promising activities, the scaffold is highly lipophilic and displays potential biological instabilities. Therefore, we strived towards improving its pharmaceutical properties by semisynthesis. We demonstrated stereoselective epoxidation of chlorotonils and epoxide ring opening in moderate to good yields providing derivatives with significantly enhanced solubility. Furthermore, in vivo stability of the derivatives was improved while retaining their nanomolar activity against critical human pathogens (e.g. methicillin-resistant Staphylococcus aureus and P. falciparum). Intriguingly, we showed further superb activity for the frontrunner molecule in a mouse model of S. aureus infection.

The antibacterial activity of Libanstin from Ilex paraguariensis (Yerba Mate).

Infectious diseases are reported to be one of the major causes of death in the world. The World Health Organization (WHO) warns of an increase in the deaths number because of antibacterial resistance. Lately, a trend towards searching for new active antibacterial compounds in plants has been observed. Ilex paraguariensis, known as Yerba Mate, is a plant known to be rich in numerous bioactive compounds that have an important role in human health. In this study, Yerba Mate was extracted with acetone: water (1:1) and further fractionated with hexane, chloroform and ethyl acetate. The obtained fractions were tested for antibacterial activity against Staphylococcus aureus and Salmonella species. The minimum inhibitory concentration (MIC) values on S. aureus ranged from 1.56 to 3.12 mg/mL for both the chloroform and ethyl acetate fractions. Whereas for the water fraction, the MIC values ranged from 0.78 to 3.12 mg/mL on S. aureus and ranged from 1.56 mg/mL to 3.12 mg/mL on Salmonella species. The aqueous fraction was further treated with different enzymes to mimic in vivo digestion and the fractions obtained were then tested for antibacterial activity. Furthermore, the Yerba Mate aqueous fraction was run on High Performance Liquid Chromatography (HPLC) and collected fractions were tested for antibacterial activity, to identify the active metabolite. Fraction 3 was tested on different strains of S. aureus and the MIC values ranged from 0.19 to 1.56 µg/mL. A novel pyrazinone, Libanstin, from Ilex paraguariensis was identified using NMR spectroscopy.

Drosophila melanogaster as a Model System to Assess the Effect of Epstein-Barr Virus DNA on Inflammatory Gut Diseases.

The Epstein-Barr virus (EBV) commonly infects humans and is highly associated with different types of cancers and autoimmune diseases. EBV has also been detected in inflamed gastrointestinal mucosa of patients suffering from prolonged inflammation of the digestive tract such as inflammatory bowel disease (IBD) with no clear role identified yet for EBV in the pathology of such diseases. Since we have previously reported immune-stimulating capabilities of EBV DNA in various models, in this study we investigated whether EBV DNA may play a role in exacerbating intestinal inflammation through innate immune and regeneration responses using the Drosophila melanogaster model. We have generated inflamed gastrointestinal tracts in adult fruit flies through the administration of dextran sodium sulfate (DSS), a sulfated polysaccharide that causes human ulcerative colitis- like pathologies due to its toxicity to intestinal cells. Intestinal damage induced by inflammation recruited plasmatocytes to the ileum in fly hindguts. EBV DNA aggravated inflammation by enhancing the immune deficiency (IMD) pathway as well as further increasing the cellular inflammatory responses manifested upon the administration of DSS. The study at hand proposes a possible immunostimulatory role of the viral DNA exerted specifically in the fly hindgut hence further developing our understanding of immune responses mounted against EBV DNA in the latter intestinal segment of the D. melanogaster gut. These findings suggest that EBV DNA may perpetuate proinflammatory processes initiated in an inflamed digestive system. Our findings indicate that D. melanogaster can serve as a model to further understand EBV-associated gastroinflammatory pathologies. Further studies employing mammalian models may validate the immunogenicity of EBV DNA in an IBD context and its role in exacerbating the disease through inflammatory mediators.

Engineering Atypical Tetracycline Formation in Amycolatopsis sulphurea for the Production of Modified Chelocardin Antibiotics.

To combat the increasing spread of antimicrobial resistance and the shortage of novel anti-infectives, one strategy for the development of new antibiotics is to optimize known chemical scaffolds. Here, we focus on the biosynthetic engineering of Amycolatopsis sulphurea for derivatization of the atypical tetracycline chelocardin and its potent broad-spectrum derivative 2-carboxamido-2-deacetyl-chelocardin. Heterologous biosynthetic genes were introduced into this chelocardin producer to modify functional groups and generate new derivatives. We demonstrate cooperation of chelocardin polyketide synthase with tailoring enzymes involved in biosynthesis of oxytetracycline from Streptomyces rimosus. An interesting feature of chelocardin, compared with oxytetracycline, is the opposite stereochemistry of the C4 amino group. Genes involved in C4 transamination and N,N-dimethylation of oxytetracycline were heterologously expressed in an A. sulphurea mutant lacking C4-aminotransferase. Chelocardin derivatives with opposite stereochemistry of the C4 amino group, as N,N-dimethyl- epi-chelocardin and N,N-dimethyl-2-carboxamido-2-deacetyl- epi-chelocardin, were produced only when the aminotransferase from oxytetracycline was coexpressed with the N-methyltransferase OxyT. Surprisingly, OxyT exclusively accepted intermediates carrying an S-configured amino group at C4 in chelocardin. Applying medicinal chemistry approaches, several 2-carboxamido-2-deacetyl- epi-chelocardin derivatives modified at C4 were produced. Analysis of the antimicrobial activities of the modified compounds demonstrated that the primary amine in the R configuration is a crucial structural feature for activity of chelocardin. Unexpectedly, C10 glycosylated chelocardin analogues were identified, thus revealing the glycosylation potential of A. sulphurea. However, efficient glycosylation of the chelocardin backbone occurred only after engineering of a dimethylated amino group at the C4 position in the opposite S configuration, which suggests some evolutionary remains of chelocardin glycosylation.

Genome-wide mutant profiling predicts the mechanism of a Lipid II binding antibiotic.

Identifying targets of antibacterial compounds remains a challenging step in the development of antibiotics. We have developed a two-pronged functional genomics approach to predict mechanism of action that uses mutant fitness data from antibiotic-treated transposon libraries containing both upregulation and inactivation mutants. We treated a Staphylococcus aureus transposon library containing 690,000 unique insertions with 32 antibiotics. Upregulation signatures identified from directional biases in insertions revealed known molecular targets and resistance mechanisms for the majority of these. Because single-gene upregulation does not always confer resistance, we used a complementary machine-learning approach to predict the mechanism from inactivation mutant fitness profiles. This approach suggested the cell wall precursor Lipid II as the molecular target of the lysocins, a mechanism we have confirmed. We conclude that docking to membrane-anchored Lipid II precedes the selective bacteriolysis that distinguishes these lytic natural products, showing the utility of our approach for nominating the antibiotic mechanism of action.

Total Biosynthesis of the Pyrrolo[4,2]benzodiazepine Scaffold Tomaymycin on an In Vitro Reconstituted NRPS System.

In vitro reconstitution and biochemical analysis of natural product biosynthetic pathways remains a challenging endeavor, especially if megaenzymes of the nonribosomal peptide synthetase (NRPS) type are involved. In theory, all biosynthetic steps may be deciphered using mass spectrometry (MS)-based analyses of both the carrier protein-coupled intermediates and the free intermediates. We here report the "total biosynthesis" of the pyrrolo[4,2]benzodiazepine scaffold tomaymycin using an in vitro reconstituted NRPS system. Proteoforms were analyzed by liquid chromatography (LC)-MS to decipher every step of the biosynthesis on its respective megasynthetase with up to 170 kDa in size. To the best of our knowledge, this is the first report of a comprehensive analysis of virtually all chemical steps involved in the biosynthesis of nonribosomally synthesized natural products. The study includes experiments to determine substrate specificities of the corresponding A-domains in competition assays by analyzing the adenylation step as well as the transfer to the respective carrier protein domain.

The generation of "unnatural" products: synthetic biology meets synthetic chemistry.

Natural product analogue generation is important, providing tools for chemical biology, enabling structure activity relationship determination and insight into the way in which natural products interact with their target biomolecules. The generation of analogues is also often necessary in order to improve bioavailability and to fine tune compounds' activity. This review provides an overview of the catalogue of approaches available for accessing series of analogues. Over the last few years there have been major advances in genome sequencing and the development of tools for biosynthetic pathway engineering; it is therefore becoming increasingly easy to combine molecular biology and synthetic organic chemistry in order to enable expeditious access to series of natural products. This review outlines the various ways of combining biology and chemistry that have been applied to analogue generation, drawing upon a series of examples to illustrate each approach.