Severe Human Infections Research Articles

Comparative Analysis of Growth Media for Cultivating Acanthamoeba: Implications for Laboratory Diagnostics

Background Acanthamoeba, a genus of free-living amoebae found in soil, freshwater, and marine habitats, is crucial in environmental studies and medical microbiology due to its potential to cause severe human infections. Cultivating Acanthamoeba is crucial for diagnosing infections, guiding treatment, and assessing exposure risks to pathogenic strains. This study aims to compare different culture media for cultivating Acanthamoeba under laboratory conditions. Materials and Methods The study examined ten culture media for Acanthamoeba growth, including TSB, Trypticase soy agar (TSA), Trypticase soy yeast (TSY), Trypticase yeast iron-extracted (TYI), Trypticase, yeast maltose extract (TYM), Pepton Yeast Glucose Extract (PYG), Dulbecco's modified Eagle's medium (DMEM), RPMI 1640, Serum Casein Glucose Yeast Extract (SCGY), Coconut powder suspension in NNA medium, as well as non-nutrient agar (NNA) as the primary & control culture. Results The results showed varied growth rates of Acanthamoeba when cultured in different media, highlighting important trends in their adaptability and growth based on the nutritional composition of the media provided. In TSB, TSA, DMEM/F12 and SCGY media, no growth was observed, suggesting that the combination may lack essential nutrients or conditions necessary for fostering Acanthamoeba proliferation. TYI showed an excellent growth rate for Acanthamoeba, particularly notable during the first 24-72 hours. The use of maltose as a carbon source and FBS in the NNA environment appears to promote rapid proliferation of Acanthamoeba. Coconut powder suspension in NNA medium led to excellent growth rates of Acanthamoeba. Conclusion The effective cultivation of Acanthamoeba in TYI, TYM, and coconut powder highlights the significance of refining nutrient composition to enhance culturing methods.

Assessment of heat-killed E. coli expressing Chikungunya virus E2 protein as a candidate vaccine for dual protection against Chikungunya virus and E. coli.

The Chikungunya virus (CHIKV) is a mosquito-borne virus with a long history of recurring epidemics transmitted through Aedes mosquitoes. The rapid spread of CHIKV has intensified the need for potent vaccines. Escherichia coli (E.coli), a vital part of human gut microbiota, is utilized in recombinant DNA technology for cloning. However, its high adaptability can lead to severe infections in humans. This study aimed to develop the candidate dual vaccine against CHIKV and E. coli. For this, we expressed the CHIKV E2 protein in the E. coli Rosetta Bl21 cells and the protein expression was confirmed by western blotting. The IgG immune response of the candidate vaccine was determined against CHIKV and E. coli by ELISA. Further, the potential of antibodies to neutralize CHIKV was evaluated via Tissue Culture Infectious Dose 50 (TCID50). We observed that cells expressing E2 protein with alum immunized mice serum showed a five-fold higher IgG immune response against CHIKV, compared to control cells. The CHIKV neutralization assay results showed a two-fold decrease in CHIKV TCID50 value after 12 hours and a three-fold reduction after 120 hours. Similarly, the vaccine formulation also elicited a significantly higher IgG immune response against E. coli. The results suggested that expressing CHIKV E2 protein in E. coli is a potential approach for generating an IgG immune response against CHIKV and E. coli both. This study proposes a faster, safer, and cost-effective recombinant protein-based vaccine development.

Membrane-Active Singlet Oxygen Photogenerators as a Paradigm for Broad-Spectrum Antivirals: The Case of Halogenated (BOron)-DIPYrromethenes.

Enveloped viruses, such as flaviviruses and coronaviruses, are pathogens of significant medical concern that cause severe infections in humans. Some photosensitizers are known to possess virucidal activity against enveloped viruses, targeting their lipid bilayer. Here we report a series of halogenated difluoroboron-dipyrromethene (BODIPYs) photosensitizers with strong virus-inactivating activity. Our structure-activity relationship analysis revealed that BODIPY scaffolds with a heavy halogen atom demonstrate significant efficacy against both tick-borne encephalitis virus (TBEV; Flaviviridae family) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; Coronaviridae family) along with high singlet oxygen quantum yields. Moreover, select compounds also inactivated other enveloped viruses, such as herpes simplex virus type 1 and monkeypox virus. The nature and length of the alkyl side chain notably influenced the virus-inactivating activity of BODIPY molecules. Furthermore, molecular dynamics studies highlighted the critical importance of the positioning of the chromophore moiety within the lipid bilayer. As membrane-targeting photosensitizers, BODIPYs interact directly with virus particles, causing damage to the viral envelope membranes. Thus, TBEV pretreated with BODIPY was completely noninfective for lab mice. Consequently, BODIPY-based photosensitizers hold potential either as broad-spectrum virus-inactivating antivirals against a variety of phylogenetically unrelated enveloped viruses or as potent inactivators of viruses for the development of vaccines for preventing life-threatening emerging viral diseases.

Development of a novel encystment medium: Enhancing diagnostic potential of Acanthamoeba spp.

Background and Aim: Acanthamoeba spp. are pathogenic microorganisms linked to severe infections in humans and animals, requiring a deeper understanding of their encystation process for effective diagnostics and research. This study focused on developing a novel encystment medium to induce synchronized encystation of Acanthamoeba spp. efficiently and rapidly. Materials and Methods: The study employed response surface methodology with a central composite design to optimize the encystment medium formulation. The key components included Tris-HCl, NaCl, glucose, and MgCl2. The optimized liquid medium was spray-dried to produce a dehydrated powder for practical application. The encystation efficiency of different Acanthamoeba strains was assessed using hemocytometry and fluorescence microscopy. Results: The optimized medium, comprising 3.152 g/L Tris-HCl, 5.55 g/L NaCl, 8% (w/v) glucose, and 5.0 mM MgCl2 at pH 9.0, demonstrated exceptional encystation efficiency with rates ranging from 99% to 100%. A spray-dried powdered version of this medium was equally effective, achieving a 98.77% encystation rate for A. castellanii American Type Culture Collection 50739 in glucose-free conditions. Notably, optimal glucose concentrations varied among Acanthamoeba strains, with certain strains reaching maximum encystation at 6–8% glucose. Conclusion: This study successfully developed an innovative encystment medium that promotes rapid and efficient cyst production in Acanthamoeba spp. The medium enhances laboratory research and diagnostic capabilities, paving the way for future advancements in understanding and managing Acanthamoeba infections. Keywords: Acanthamoeba, diagnostic tools, encystation, medium optimization, response surface methodology.

Identification and characterization of the functional tetrameric UDP-glucose pyrophosphorylase from Klebsiella pneumoniae.

In all kingdoms of life, the enzyme uridine diphosphate-glucose pyrophosphorylase (UGP) occupies a central role in metabolism, as its reaction product uridine diphosphate-glucose (UDP-Glc) is involved in various crucial cellular processes. Pathogens, including fungi, parasites, and bacteria, depend on UGP for the synthesis of virulence factors; in particular, various bacterial species utilize UDP-Glc and its derivatives for the synthesis of lipopolysaccharides, capsular polysaccharides, and biofilm exopolysaccharides. UGPs have, therefore, gained attention as anti-bacterial drug target candidates, prompting us to study their structure-function relationships to provide a basis for the rational development of specific inhibitors. UGP function is tied to its oligomeric state, and the majority of bacterial homologs have been described as tetramers encoded by the galU gene. Uniquely, enterobacterial species harbor a second gene, galF, encoding a protein with high homology to UGP, whose function is somewhat controversial. Here, we show that the galF gene of the opportunistic pathogen Klebsiella pneumoniae encodes a dimeric protein that has lost UGP activity, likely due to a combination of active site mutations and an inability to tetramerize, whereas the functional K. pneumoniae UGP, encoded by galU, is an active tetramer. Our AlphaFold-assisted structure-function relationship studies underline that tetramerization is essential for bacterial UGP function and is facilitated by a common mechanism utilizing conserved key residues. Targeting the respective molecular interfaces, which are absent in human UGP, could provide a means of selectively inhibiting the bacterial virulence factor UGP and potentially rendering pathogenic species avirulent.IMPORTANCEThe enzyme uridine diphosphate-glucose pyrophosphorylase (UGP) is important for the virulence of bacterial pathogens and, therefore, a potential drug target. In this study, we identify the gene encoding the functional UGP in Klebsiella pneumoniae, a bacterium notoriously causing severe antibiotic-resistant infections in humans, and reveal structural and functional features that may aid in the development of new antibiotics.

Structural basis of different neutralization capabilities of monoclonal antibodies against H7N9 virus.

Neutralizing antibodies (nAbs) are important for the treatment of emerging viral diseases and for effective vaccine development. In this study, we generated and evaluated three nAbs (1H9, 2D7, and C4H4) against H7N9 influenza viruses and found that they differ in their ability to inhibit viral attachment, membrane fusion, and egress. We resolved the cryo-electron microscopy (cryo-EM) structures of H7N9 hemagglutinin (HA) alone and in complex with the nAb antigen-binding fragments (Fabs) and identified the HA head-located epitope for each nAb, thereby revealing the molecular basis and key residues that determine the differences in these nAbs in neutralizing H7N9 viruses. Moreover, we found that the humanized nAb CC4H4 provided complete protection in mice against death caused by a lethal H7N9 virus infection, even when nAb was given 3 days after the mice were infected. These findings provide new insights into the neutralizing mechanism and structural basis for the rational design of H7N9 virus vaccines and therapeutics.IMPORTANCEH7N9 viruses have caused severe infections in both birds and humans since their emergence in early 2013 in China. Their persistent presence and variation in avian populations pose a significant threat to both poultry and humans. There are no treatments for human infections. In this study, we thoroughly investigated the neutralization mechanisms, structural basis, and therapeutic effects of three nAbs (1H9, 2D7, and C4H4) against H7N9 viruses. We revealed the molecular determinants underlying the varied performances of the three nAbs in neutralizing H7N9 viruses and protecting H7N9-infected mice. These insights provide a solid foundation for the rational design of vaccines and therapeutics against H7N9 viruses.

Case report: Relaps of Streptococcus suis infection in patient with Meningoencephalitis at Prof. Ngoerah Hospital, Bali

Background: S. suis is a facultative anaerobic Gram-positive bacterium that causes human infection. It is a primary health problem in the swine industry. Ceftriaxone is a therapeutic option for meningitis besides Ampicillin. Recently, the incidence of resistance to ceftriaxone was reported. The recurrence of S. suis in this patient is thought to be caused by ceftriaxone resistance due to inadequate initial therapy. Case Presentation: A 43-year-old male patient came to the ER with headaches in the head and neck. The patient had a history of meningitis due to S. suis twice before. While in the ER, the patient was given ceftriaxone while awaiting culture results. Blood cultures showed S. suis infection using VITEK 2 COMPACT (Biomeriuex®) and ceftriaxone resistance. The patient was successfully managed with Ampicillin following these culture results, leading to good outcomes. Discussion: S. suis is a gram-positive bacterial pathogen in pigs that can cause severe human infection, including meningitis, septicemia, endocarditis and deafness the most common complications. The patient experienced three relapses due to S. suis. On the third visit, blood culture results identified ceftriaxone-resistant S. suis. The patient's recurrent relapses could be attributed to several factors, including inadequate antibiotic administration, antibiotic resistance, weak immune system, contact with the source of infection and environmental variables, highlighting the complexity and challenges in treating S. suis infection. Conclusion: The use of antibiotics in relapse cases has the potential to cause antibiotic resistance, so it’s necessary to make guidelines for antibiotic therapy in S. suis infection to prevent recurrent infections.

The two-component system ArlRS is essential for wall teichoic acid glycoswitching in Staphylococcus aureus.

Staphylococcus aureus is among the leading causes of hospital-acquired infections. Critical to S. aureus biology and pathogenesis are the cell wall-anchored glycopolymers wall teichoic acids (WTA). Approximately one-third of S. aureus isolates decorates WTA with a mixture of α1,4- and β1,4-N-acetylglucosamine (GlcNAc), which requires the dedicated glycosyltransferases TarM and TarS, respectively. Environmental conditions, such as high salt concentrations, affect the abundance and ratio of α1,4- and β1,4-GlcNAc WTA decorations, thereby impacting biological properties such as antibody binding and phage infection. To identify regulatory mechanisms underlying WTA glycoswitching, we screened 1,920 S. aureus mutants (Nebraska Transposon Mutant Library) by immunoblotting for differential expression of WTA-linked α1,4- or β1,4-GlcNAc using specific monoclonal antibody Fab fragments. Three two-component systems (TCS), GraRS, ArlRS, and AgrCA, were among the 230 potential hits. Using isogenic TCS mutants, we demonstrated that ArlRS is essential for WTA β1,4-GlcNAc decoration. ArlRS repressed tarM expression through the transcriptional regulator MgrA. In bacteria lacking arlRS, the increased expression of tarM correlated with the absence of WTA β1,4-GlcNAc, likely by outcompeting TarS enzymatic activity. ArlRS was responsive to Mg2+, but not Na+, revealing its role in the previously reported salt-induced WTA glycoswitch from α1,4-GlcNAc to β1,4-GlcNAc. Importantly, ArlRS-mediated regulation of WTA glycosylation affected S. aureus interaction with the innate receptor langerin and lysis by β1,4-GlcNAc-dependent phages. Since WTA represents a promising target for future immune-based treatments and vaccines, our findings provide important insight to align strategies targeting S. aureus WTA glycosylation patterns during infection.IMPORTANCEStaphylococcus aureus is a common colonizer but can also cause severe infections in humans. The development of antibiotic resistance complicates the treatment of S. aureus infections, increasing the need for antibiotic alternatives such as vaccines and therapies with bacterial viruses also known as phages. Wall teichoic acids (WTA) are abundant glycosylated structures of the S. aureus cell wall that have gained attention as a promising target for new treatments. Importantly, WTA glycosylation patterns show variation depending on environmental conditions, thereby impacting phage binding and interaction with host factors, such as antibodies and innate pattern-recognition receptors. Here, we show that the two-component system ArlRS is involved in the regulation of WTA glycosylation by responding to environmental changes in Mg2+ concentration. These findings may support the design of new treatment strategies that target WTA glycosylation patterns of S. aureus during infection.

Construction and validation of a mouse model for studying severe human adenovirus infections

Human adenoviruses (HAdVs) are highly contagious pathogens with various genotypes implicated in acute respiratory disease (ARD) and linked to fatality, especially in immunosuppressed patients, young children, and military recruits. Currently, no vaccines or specific drugs are approved for clinical use. The hosts of adenoviruses are strictly species-specific, which strongly limits the development of vaccines and drugs against HAdVs. In this study, immunocompetent BALB/c mice were challenged with different doses of human adenovirus type 5 (HAdV-5) via tail intravenous injection (i.v.). All mice challenged with a high dose of HAdV-5 (3.2 ​× ​1010 TCID50/kg) died within 3–5 days, while those receiving a low dose of HAdV-5 (8 ​× ​109 or 4 ​× ​109 TCID50/kg) survived. Interestingly, among the mice receiving a medium dose of HAdV-5 (1.6 ​× ​1010 TCID50/kg), 60% (n ​= ​3/5) of male mice died, while all female mice survived. This suggests that male mice may be more susceptible to HAdV-5 infection than female mice, consistent with clinical findings in children. HAdV-5 DNA was mainly distributed in the liver, followed by the spleen and lung. Pathological changes were observed in the lung, liver, and spleen, with severity increasing in correlation with the virus challenge dosage. Transcriptome and qPCR analyses of the liver indicated that the down-regulated expression of the H2-Aa, H2-Ea-ps, CD74, and H2-Eb1 genes in male mice, as well as the AHR gene in female mice, may contribute to the observed higher mortality rates in male mice. Therefore, this effective, feasible, and cost-efficient mouse model could serve as a candidate for evaluating HAdV vaccines and anti-adenovirus therapeutics.

Characterization, Antibiotic Susceptibility, and Clonal Analysis of Carbapenem-Resistant Klebsiella pneumoniae From Different Clinical Cases.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is recognized for its great ability to resist prescription drugs and its association with severe infections in humans. This study was designed to evaluate the characteristic resistance spectrum, to characterize the implicated carbapenem-resistant genes (CRGs), and to determine the extent of genetic diversity among Klebsiella pneumoniae isolates from human clinical cases in Duhok province. Methodology: The VITEK-2 system was used to investigate the phenotypic antibiotic susceptibility of 23 K. pneumoniae isolated from distinct human clinical situations, multiplex PCR was used to assign the key common carbapenem-resistant genes (IMP, OXA48-like, bla-NDM, and KPC) in phenotypically carbapenem-resistant isolates, and the Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) assay was utilized to ascertain the clonal associations among those isolates. Phenotypic resistance analysis revealed high resistance rates to various antibiotics, with all isolates exhibiting multidrug resistance (MDR). Coronavirus disease 2019 (COVID-19) patient isolates demonstrated significantly higher resistance compared to other sources. In addition, all isolates showed complete phenotypic resistance to carbapenems, PCR screening for CRGs identified blaOXA-48 as the predominant gene, present in all isolates. Genetic fingerprinting revealed diverse genotypes, with COVID-19 patient isolates exhibiting high similarity, contrasting with maximum diversity in non-COVID-19 clinical isolates.

The Role of Nucleocapsid Protein (NP) in the Immunology of Crimean-Congo Hemorrhagic Fever Virus (CCHFV).

Crimean-Congo hemorrhagic fever virus (CCHFV) is an orthonairovirus from the Bunyavirales order that is widely distributed geographically and causes severe or fatal infections in humans. The viral genome consists of three segmented negative-sense RNA molecules. The CCHFV nucleocapsid protein (CCHFV NP) is encoded by the smallest segment of the virus. CCHFV NP, the primary function of which is the encapsidation of viral RNA molecules, plays a critical role in various mechanisms important for viral replication and pathogenesis. This review is an attempt to revisit the literature available on the highly immunogenic and highly conserved CCHFV NP, summarizing the multifunctional roles of this protein in the immunology of CCHFV. Specifically, the review addresses the impact of CCHFV NP on innate, humoral, and cellular immune responses, epitopes recognized by B and T cells that limit viral spread, and its role as a target for diagnostic tests and for vaccine design. Based on the extensive information generated by many research groups, it could be stated that NP constitutes a significant and critical player in the immunology of CCHFV.

Molecular-phylogenetic analyses of Babesia and Theileria species from small mammals and their ticks in northern China suggest new reservoirs of bovine and equine piroplasms

Babesia and Theileria species (Apicomplexa: Piroplasmida) are tick-borne protozoan parasites that can cause mild to severe infection in humans, wildlife, livestock and companion animals. To date, reports on the molecular study of piroplasms from wild living small mammals and their ticks are still limited, especially in Asia. This study encompassed an extensive survey involving 907 liver samples and 145 ixodid ticks from 16 different species of small mammals (Rodentia, Lagomorpha, Eulipotyphla). These were collected in 13 cities and counties in northern China. DNA extracts from these samples were screened for the presence of piroplasm 18S rRNA gene. Samples that tested positive were further evaluated for other genetic markers of piroplasms, including the cox1 gene and the ITS1-5.8S rDNA-ITS2 region. Several piroplasm species were identified, including Babesia sp. tavsan2, Babesia occultans, Theileria sp. Xinjiang, Theileria equi, and Theileria sp. Kalecik. Among these, Theileria sp. Xinjiang was shown to be the most prevalent. Importantly, Babesia sp. tavsan2 was identified in the tick Rhipicephalus sanguineus from the Yarkand hare and Theileria sp. Kalecik in Hyalomma asiaticum from the long-eared hedgehog, in line with the detection of these pathogens in tissue samples of the relevant hosts. This study further disclosed the presence of DNA from B. occultans and T. equi, typically found in cattle and horses respectively, with an additional discovery in small mammals. Moreover, Theileria sp. Kalecik, which was first detected in small-sized mammals, and Babesia sp. tavsan2, were both reported for the first time in China.

Molecular Characterization of Listeria monocytogenes in the Food Chain of the Republic of Kosovo from 2016 to 2022.

The present study describes the genetic characterization of L. monocytogenes strains found in the Republic of Kosovo's food chain. From 2016 to 2022, 995 samples were collected. Overall, 648 samples were from ready-to-eat (RTE) food products, 281 from food products consumed cooked (FPCC), 60 from raw materials, and 6 from environmental samples. Overall, 11.76% (117 out of 995) of the samples were contaminated by L. monocytogenes, comprising 6.33% (41 out of 648) from RTE products, 14.95% (42 out of 281) from FPCC, 55.00% (33 out of 60) from raw materials, and 16.66% (1 out of 6) from environmental samples. All isolates were subjected to molecular serotyping and clonal complex (CC) identification by using real-time PCR, as well as multilocus sequence typing. All isolates were grouped into four molecular serotypes, IIa (34.19%), IIb (3.48%), IIc (32.48%), and IVb (29.91%), as well as Lineage I (33.33%) and Lineage II (66.66%). In total, 14 CCs were identified from 41 RTE isolates; however, CC29 (7), CC2 (6), and CC6 (6) were the most dominant. By contrast, CC9 was by far the most represented CC in both FPCC (21) and RM (14). Moreover, 30 isolates expressed CC1, CC2, CC4, or CC6, which are particularly associated with severe human infections.

Long-term occurrence of multiple antimicrobial drug resistant Klebsiella pneumoniae isolates harboring virulent potential in a tertiary hospital from Brazil.

Klebsiella pneumoniae strains are globally associated with a plethora of opportunistic and severe human infections and are known to spread genes conferring antimicrobial resistance. Some strains harbor virulence determinants that enable them to cause serious disease in any patient, both in the hospital and in the community. The aim of this study was to determine the frequency of antimicrobial resistance and virulence traits (by gene detection and string test) among 83K. pneumoniae isolates obtained from patient cultures of a scholar tertiary hospital in the Midwestern Brazil (Brasília, DF). Antimicrobial susceptibility analysis showed that 94% (78/83) of the isolates presented one of the following resistance profiles: resistant (R, 39), multidrug-resistant (MDR, 29), or extensively drug-resistant (XDR, 10). Several MDR and XDR strains harbored multiple virulence genes and displayed hypermucoviscous phenotype. These characteristics were observed among isolates obtained throughout all the sample collection period (2013 - 2017). The K2 serotype gene, a molecular marker of hypervirulence, was detected in three isolates, one of which classified as XDR. Sequence typing revealed the occurrence of isolates belonged to high-risk (ST13) and multiple resistance-spreading clones (ST105). Thus, our findings showed the occurrence of virulent potential isolates that also presented MDR/XDR phenotypes from 2013 to 2015. This study also indicates the probable convergence of virulence and resistance since at least 2013 in Brazil.

An attachment glycoprotein nanoparticle elicits broadly neutralizing antibodies and protects against lethal Nipah virus infection

Nipah virus (NiV) is a zoonotic emergent paramyxovirus that can cause severe encephalitis and respiratory infections in humans, with a high fatality rate ranging from 40% to 75%. Currently, there are no approved human vaccines or antiviral drugs against NiV. Here, we designed a ferritin-based self-assembling nanoparticle displaying the NiV G head domain on the surface (NiV G-ferritin) and assessed immune responses elicited by the soluble NiV G head domain (NiV sG) or NiV G-ferritin. Immunization with NiV G-ferritin or NiV sG conferred complete protection against lethal NiV challenge without detection of viral RNA in Syrian golden hamsters. Compared to NiV sG, NiV G-ferritin induced significantly faster, broader, and higher serum neutralizing responses against three pathogenic henipaviruses (NiV-Malaysia, NiV-Bangladesh, and Hendra virus). Moreover, NiV G-ferritin induced a durable neutralizing immunity in mice as antisera potently inhibited NiV infection even after six months of the third immunization. Additionally, we isolated a panel of 27 NiV G-binding monoclonal antibodies (mAbs) from NiV G-ferritin immunized mice and found that these mAbs targeted four distinct antigenic sites on NiV G head domain with two sites that have not been defined previously. Notably, 25 isolated mAbs have potent neutralizing activity with 50% inhibitory concentrations less than 10 ng/mL against NiV pseudovirus. Collectively, these findings provide new insights into the immunogenicity of NiV G protein and reveal that NiV G-ferritin is a safe and highly effective vaccine candidate against Nipah virus infection.

A genome-scale metabolic model of a globally disseminated hyperinvasive M1 strain of Streptococcus pyogenes.

Streptococcus pyogenes is responsible for a range of diseases in humans contributing significantly to morbidity and mortality. Among more than 200 serotypes of S. pyogenes, serotype M1 strains hold the greatest clinical relevance due to their high prevalence in severe human infections. To enhance our understanding of pathogenesis and discovery of potential therapeutic approaches, we have developed the first genome-scale metabolic model (GEM) for a serotype M1 S. pyogenes strain, which we name iYH543. The curation of iYH543 involved cross-referencing a draft GEM of S. pyogenes serotype M1 from the AGORA2 database with gene essentiality and autotrophy data obtained from transposon mutagenesis-based and growth screens. We achieved a 92.6% (503/543 genes) accuracy in predicting gene essentiality and a 95% (19/20 amino acids) accuracy in predicting amino acid auxotrophy. Additionally, Biolog Phenotype microarrays were employed to examine the growth phenotypes of S. pyogenes, which further contributed to the refinement of iYH543. Notably, iYH543 demonstrated 88% accuracy (168/190 carbon sources) in predicting growth on various sole carbon sources. Discrepancies observed between iYH543 and the actual behavior of living S. pyogenes highlighted areas of uncertainty in the current understanding of S. pyogenes metabolism. iYH543 offers novel insights and hypotheses that can guide future research efforts and ultimately inform novel therapeutic strategies.IMPORTANCEGenome-scale models (GEMs) play a crucial role in investigating bacterial metabolism, predicting the effects of inhibiting specific metabolic genes and pathways, and aiding in the identification of potential drug targets. Here, we have developed the first GEM for the S. pyogenes highly virulent serotype, M1, which we name iYH543. The iYH543 achieved high accuracy in predicting gene essentiality. We also show that the knowledge obtained by substituting actual measurement values for iYH543 helps us gain insights that connect metabolism and virulence. iYH543 will serve as a useful tool for rational drug design targeting S. pyogenes metabolism and computational screening to investigate the interplay between inhibiting virulence factor synthesis and growth.

Synthesized Bis-Triphenyl Phosphonium-Based Nano Vesicles Have Potent and Selective Antibacterial Effects on Several Clinically Relevant Superbugs.

The increasing emergence of multidrug-resistant (MDR) pathogens due to antibiotic misuse translates into obstinate infections with high morbidity and high-cost hospitalizations. To oppose these MDR superbugs, new antimicrobial options are necessary. Although both quaternary ammonium salts (QASs) and phosphonium salts (QPSs) possess antimicrobial effects, QPSs have been studied to a lesser extent. Recently, we successfully reported the bacteriostatic and cytotoxic effects of a triphenyl phosphonium salt against MDR isolates of the Enterococcus and Staphylococcus genera. Here, aiming at finding new antibacterial devices possibly active toward a broader spectrum of clinically relevant bacteria responsible for severe human infections, we synthesized a water-soluble, sterically hindered quaternary phosphonium salt (BPPB). It encompasses two triphenyl phosphonium groups linked by a C12 alkyl chain, thus embodying the characteristics of molecules known as bola-amphiphiles. BPPB was characterized by ATR-FTIR, NMR, and UV spectroscopy, FIA-MS (ESI), elemental analysis, and potentiometric titrations. Optical and DLS analyses evidenced BPPB tendency to self-forming spherical vesicles of 45 nm (DLS) in dilute solution, tending to form larger aggregates in concentrate solution (DLS and optical microscope), having a positive zeta potential (+18 mV). The antibacterial effects of BPPB were, for the first time, assessed against fifty clinical isolates of both Gram-positive and Gram-negative species. Excellent antibacterial effects were observed for all strains tested, involving all the most concerning species included in ESKAPE bacteria. The lowest MICs were 0.250 µg/mL, while the highest ones (32 µg/mL) were observed for MDR Gram-negative metallo-β-lactamase-producing bacteria and/or species resistant also to colistin, carbapenems, cefiderocol, and therefore intractable with currently available antibiotics. Moreover, when administered to HepG2 human hepatic and Cos-7 monkey kidney cell lines, BPPB showed selectivity indices > 10 for all Gram-positive isolates and for clinically relevant Gram-negative superbugs such as those of E. coli species, thus being very promising for clinical development.

Advances in Bioreceptor Layer Engineering in Nanomaterial-based Sensing of Pseudomonas Aeruginosa and its Metabolites.

Pseudomonas aeruginosa is a pathogen that infects wounds and burns and causes severe infections in immunocompromised humans. The high virulence, the rise of antibiotic-resistant strains, and the easy transmissibility of P.aeruginosa necessitate its fast detection and control. The gold standard for detecting P.aeruginosa, the plate culture method, though reliable, takes several days to complete. Therefore, developing accurate, rapid, and easy-to-use diagnostic tools for P.aeruginosa is highly desirable. Nanomaterial-based biosensors are at the forefront of detecting P.aeruginosa and its secondary metabolites. This review summarises the biorecognition elements, biomarkers, immobilisation strategies, and current state-of-the-art biosensors for P.aeruginosa. The review highlights the underlying principles of bioreceptor layer engineering and the design of optical, electrochemical, mass-based, and thermal biosensors based on nanomaterials. The advantages and disadvantages of these biosensors and their future point-of-care applications are also discussed. This review outlines significant advancements in biosensors and sensors for detecting P.aeruginosa and its metabolites. Research efforts have identified biorecognition elements specific and selective towards P.aeruginosa. The stability, ease of preparation, cost-effectiveness, and integration of these biorecognition elements onto transducers are pivotal for their application in biosensors and sensors. At the same time, when developing sensors for clinically significant analytes such as P.aeruginosa, virulence factors need to be addressed, such as the sensor's sensitivity, reliability, and response time in samples obtained from patients. The point-of-care applicability of the developed sensor may be an added advantage since it enables onsite determination. In this context, optical methods developed for P.aeruginosa offer promising potential.

The socioeconomic impacts of Rift Valley fever: A rapid review.

Rift Valley fever (RVF) is a neglected vector-borne disease which is endemic in many countries across Africa and has seen recent geographical expansions into the Arabian Peninsula. RVF can cause severe infections in both animals and humans. RVF infections in livestock can lead to mass fatalities. In humans, the symptoms are nonspecific and can often lead to misdiagnosis. However, a small proportion progresses to haemorrhagic infection with a significantly higher mortality rate. The culmination of this can cause severe socioeconomic impacts. This review aims to identify the main socioeconomic impacts caused by RVF outbreaks as well as existing knowledge gaps. Ninety-three academic and grey papers were selected, covering 19 countries and 10 methodological approaches. A variety of socioeconomic impacts were found across all levels of society: Livestock trade disruptions consequently impacted local food security, local and national economies. Most livestock farmers in endemic countries are subsistence farmers and so rely on their livestock for sustenance and income. RVF outbreaks resulted in a variety of socioeconomic impacts, e.g., the inability to pay for school fees. Main barriers to vaccine uptake in communities were lack of access, funds, interest along with other social aspects. The occupational risks for women (and pregnant women) are largely unknown. To our knowledge, this is the first review on RVF to highlight the clear knowledge gap surrounding the potential gender differences on risks of RVF exposure, as well as differences on occupational health risk in pastoral communities. Further work is required to fill the gaps identified in this review and inform control policies.

Next-Generation Sequencing for Characterizing Respiratory Tract Virome and Improving Detection of Viral Pathogens in Children With Pneumonia.

Pneumonia is typically caused by a variety of pathogenic microorganisms. Traditional research often focuses on the infection of a few microorganisms, whereas metagenomic studies focus on the impact of the bacteriome and mycobiome on respiratory diseases. Reports on the virome characteristics of pediatric pneumonia remain relatively scarce. We employed de novo assembly and combined homology- and feature-based methods to characterize the respiratory virome in whole-genome DNA sequencing samples from oropharynx (OP) swabs, nasopharynx (NP) swabs, and bronchoalveolar lavage fluids (BALF) of children with pneumonia. Significant differences were observed in the alpha and beta diversity indexes, as well as in the composition of the oropharyngeal virome, between pneumonia cases and controls. We identified 1137 viral operational taxonomic units (vOTUs) with significant differences, indicating a preference of pneumonia-reduced vOTUs for infecting Prevotella, Neisseria, and Veillonella, whereas pneumonia-enriched vOTUs included polyomavirus, human adenovirus, and phages targeting Staphylococcus, Streptococcus, Granulicatella, and Actinomyces. Comparative analysis revealed higher relative abundances and prevalence rates of pneumonia-enriched OP vOTUs in NP and BALF samples compared to pneumonia-reduced vOTUs. Additionally, virome analysis identified six pediatric patients with severe human adenovirus or polyomavirus infections, five of whom might have been undetected by targeted polymerase chain reaction (PCR)-based testing. This study offers insights into pediatric pneumonia respiratory viromes, highlighting frequent transmission of potentially pathogenic viruses and demonstrating virome analysis as a valuable adjunct for pathogen detection.