Major Pathogen Research Articles

The guideline for the treatment and prevention of lower respiratory tract infections caused by human respiratory syncytial virus (2024 edition)

Human respiratory syncytial virus (RSV) is a major pathogen causing lower respiratory tract infections in humans. It is particularly a common cause of hospitalization among high-risk groups such as infants, tots, and the old people, imposing a heavy burden on families and society. The standardized treatment and prevention of this disease are increasingly emphasized by healthcare professionals. However, there are currently no standardized guidelines in China for lower respiratory tract infections caused by RSV. Therefore, our group for this guideline has developed the "Guidelines for the treatment and prevention of lower respiratory tract infections caused by human respiratory syncytial virus (2024 edition)" based on the latest evidence-based medical evidence and guideline development methods. The aim is to provide guidance to relevant healthcare professionals and improve prevention and treatment levels.

Optimizing SureQuant for Targeted Peptide Quantification: a Technical Comparison with PRM and SWATH-MS Methods.

Bacterial infections are a major threat to human health worldwide. A better understanding of the properties and physiology of bacterial pathogens in human tissues is required to develop urgently needed novel control strategies. Mass spectrometry-based proteomics could yield such data, but identifying and quantifying scarce bacterial proteins against a preponderance of human proteins is challenging. Here, we explored the recently introduced SureQuant method for highly sensitive targeted mass spectrometry. Using a major human pathogen, the Gram-positive bacteria Staphylococcus aureus, as an example, we evaluated several parameters, including the number of targets and intensity thresholds, for optimal qualitative and quantitative protein analysis. By comparison, we found that SureQuant achieved the same quantitative performance as standard parallel reaction monitoring while allowing accurate and precise quantification of up to 400 targets. SureQuant also surpassed the sensitivity and quantification capabilities of global data-independent acquisition methods. Finally, to facilitate method development, we provide optimized MS parameters for the sensitive quantification of different peptide panel sizes. This study provides a foundation for the broader application of SureQuant in the analysis of clinical specimens containing trace amounts of bacterial proteins as well as other studies requiring ultrasensitive detection of low-abundant proteins.

Long-Term Lung Function and Pseudomonas aeruginosa Infection in Genotyped Primary Ciliary Dyskinesia.

Rationale: Primary ciliary dyskinesia is a rare genetic disorder characterized by progressive lung disease. Pseudomonas aeruginosa is a major pathogen in this disease, known to impact lung function. Previous genotype-phenotype studies have been limited by cross-sectional designs, isolated adult or pediatric populations, small numbers, or short follow-up durations. Objectives: We aimed to explore long-term lung function in primary ciliary dyskinesia grouped by genotypes and ultrastructural defects, considering the influence of P. aeruginosa. Methods: In this retrospective, observational study, we analyzed 43 years of spirometry and 20 years of microbiology data. Using linear mixed-effects models, we estimated FEV1 z-score trends and compared them at ages 10, 25, and 50 years, while Generalized Estimating Equations were used to assess P. aeruginosa prevalence between groups. In a secondary analysis, we matched spirometry and microbiology samples to evaluate the influence of P. aeruginosa on lung function. Measurements and Main Results: We included 127 genotyped subjects, 6,691 spirometries and 10,082 microbiology samples. CCDC39 and CCDC40 variants showed early onset and sustained decline in lung function, while DNAH11 and HYDIN variants demonstrated relative stability. Lung function in the proximity of positive P. aeruginosa cultures were on average 0.06 z-score lower. Despite this, differences between groups remained largely unaffected by P. aeruginosa. Conclusion: Long-term lung function in primary ciliary dyskinesia follows discrete genotype specific profiles and appear independent of P. aeruginosa infection. We confirm and extend previous findings of CCDC39 and CCDC40 as variants associated with early onset severe lung function impairment persisting in the long term.

Highly Effective Biocides against Pseudomonas aeruginosa Reveal New Mechanistic Insights Across Gram-Negative Bacteria.

Pseudomonas aeruginosa is a major nosocomial pathogen that persists in healthcare settings despite rigorous disinfection protocols due to intrinsic mechanisms conferring resistance. We sought to systematically assess cationic biocide efficacy against this pathogen using a panel of multidrug-resistant P. aeruginosa clinical isolates. Our studies revealed widespread resistance to commercial cationic disinfectants that are the current standard of care, raising concerns about their efficacy. To address this shortcoming, we highlight a new class of quaternary phosphonium compounds that are highly effective against all members of the panel. To understand the difference in efficacy, mechanism of action studies were carried out, which identified a discrete inner-membrane selective target. Resistance selection studies implicated the SmvRA efflux system (a transcriptionally regulated, inner membrane-associated efflux system) as a major determinant of resistance. This system is also implicated in resistance to two commercial bolaamphiphile antiseptics, octenidine and chlorhexidine, which was further validated herein. In sum, this work highlights, for the first time, a discrete inner-membrane specific mechanism for the bolaamphiphile class of disinfectants that contrasts with the prevailing model of indiscriminate membrane interactions of commercial amphiphiles paving the way for future innovations in disinfectant research.

Development of bacterial resistance in Germany from 2008 to 2022 - major culprit pathogens, antibacterial drugs, and prescribing practices.

Rising bacterial resistance is a global threat, causing rising financial burdens on healthcare systems and endangering effective treatment of bacterial infections. To ensure the efficacy of antibacterial drugs, it is essential to identify the most dangerous pathogens and vulnerable antibacterial drugs. Previous research by our group suggested irrational outpatient prescribing practices in Germany, supporting a growing bacterial resistance. This study analyses developments and characteristics for the ten most prescribed antibacterial drugs in Germany from 2008 to 2022. Conclusions are based on the development of bacterial resistance levels and an analysis of correlations between pathogens. We identified cefuroxime axetil, sulfamethoxazole-trimethoprim and nitrofurantoin as the most problematic drugs. Particularly problematic pathogens include E. faecalis, E. faecium, K. pneumoniae, and P. mirabilis. Besides increasing bacterial resistance, they are characterised by a high proportion of significant positive correlations, indicating a high potential for mutually reinforcing resistance development. Alarmingly, most of the antibacterial drugs analysed showed a growing resistance to at least one of the analysed pathogens. In most cases, the best treatment option is threatened by increasing bacterial resistance. We also identified several differences between current bacterial resistance data and therapeutic guidelines. In aggregate, our findings support irrational prescribing behaviour and underscore the urgent need for improved prescribing practices to counter rising bacterial resistance in Germany. Moreover, therapeutic guidelines for bacterial infections, the "holy grail" of pharmacotherapy, must be updated more frequently.

Dual RNA-Seq Unveils Candidate Key Virulence Genes of Vibrio harveyi at the Early Stage of Infection in Hybrid Grouper (♀ Epinephelus polyphekadion × ♂ E. fuscoguttatus)

Vibrio harveyi is a major bacterial pathogen that causes disease in aquaculture animals worldwide. Although V. harveyi consistently harbors a range of traditional virulence genes, it remains unclear which specific genes are crucial for virulence at different infection stages. Dual RNA-seq is a cutting-edge RNA sequencing technology that is ideal for investigating the gene expression patterns of pathogens within the host, which is highly effective in identifying key virulence genes. In previous artificial infection experiments, we have identified the liver of hybrid grouper (♀ Epinephelus polyphekadion × ♂ E. fuscoguttatus) as the main target organ for pathogenic V. harveyi GDH11385 during the initial infection phase. To further explore the key virulence factors of V. harveyi at the early stage of infection, the liver of the hybrid grouper infected with strain GDH11385 was analyzed here by dual RNA-seq. The transcriptome data were compared with that of in vitro cultured bacteria. The results showed that 326 and 1140 DEGs (differentially expressed genes) were significantly up- and down-regulated, respectively, at 4 h post-infection (hpi). Further pathway enrichment analyses revealed that these up-regulated DEGs in vivo were mainly enriched in siderophore biosynthesis and transport, type VI secretion system (T6SS), flagellar assembly, glycolysis/gluconeogenesis, and ribosome. Notably, all genes involved in the metabolism and utilization of vibrioferrin (a carboxylate class of siderophore produced by Vibrio), and most of the genes within one of three T6SSs, were significantly up-regulated in vivo. This indicates that siderophore-dependent iron competition and T6SS-mediated delivery of virulence factors are vital for the successful colonization of V. harveyi at the early stage of infection. This study provides more precise clues to reveal the virulence mechanism of V. harveyi during the initial phase of infection.

A three-way interface of the Nipah virus phosphoprotein X-domain coordinates polymerase movement along the viral genome.

Mononegaviruses comprise major human pathogens such as the Ebola virus, rabies virus, respiratory syncytial virus, measles virus, and Nipah virus (NiV). For replication and transcription, their polymerase complexes must negotiate a protein-encapsidated RNA genome, which requires the highly coordinated continuous formation and resolution of protein-protein interfaces as the polymerase advances along the template. The viral P protein assumes a central role in this process, but the molecular mechanism of ensuring polymerase mobility is poorly understood. Studying NiV polymerase complexes, we applied functional and biochemical assays to map three distinct interfaces in the NiV P XD and identified transient interactions between XD and the nucleocapsid core as instrumental in coordinating polymerase advance. These results define a conserved molecular principle regulating paramyxovirus polymerase dynamics and illuminate a promising druggable target for the structure-guided development of broad-spectrum polymerase inhibitors.

Shiga toxin-producing Escherichia coli in the milk production chain: Evaluation of virulence genes and clonal diversity of O157:H7 strains

Microbial contamination of milk and dairy products is a significant issue due to sensory changes and the risk of foodborne diseases. Shiga toxin-producing Escherichia coli (STEC), particularly the O157:H7, are major pathogens transmitted by dairy products. This study aimed to evaluate the microbiological quality of bovine milk at various stages of the production chain in a dairy cooperative and its suppliers in Rio de Janeiro, Brazil. Additionally, the presence of STEC in milk and faeces from dairy cattle was investigated, along with the isolation of STEC O157:H7 strains and the evaluation of virulence genes and clonal diversity. These findings were compared with previously isolated STEC O157:H7 strains from the same region. A total of 100 raw milk samples (54 farm gallon (FG) samples, 25 community bulk tank milk (CBTM) samples, 14 isothermal car-tanker milk (ICTM) samples, and 7 industry tank milk (ITM) samples) and 10 pasteurized milk samples, along with 63 bovine faeces samples, were analysed. The stx gene, a marker for STEC, was detected in 65% of raw milk samples and 88.9% of faecal samples, indicating a high risk of contamination throughout the production chain. None of the pasteurized milk samples tested positive for the stx gene. The prevalence of stx in faeces and raw milk underscores the need for rigorous controls to prevent contamination. While pasteurization was effective, the microbiological quality of raw milk remained unsatisfactory, highlighting the importance of good hygiene practices and continuous monitoring to ensure dairy safety.

A simple and cost-effective transformation system for Porphyromonas gingivalis via natural competence

Porphyromonas gingivalis is a major oral bacterial pathogen responsible for severe periodontal diseases. Numerous studies have used genetic approaches to elucidate the molecular mechanisms underlying its pathogenicity. Typically, electroporation and conjugation are utilized for mutagenesis of P. gingivalis; however, these techniques require specialized equipment such as high-voltage electroporators, conjugative plasmids and donor strains. In this study, we present a simple, cost-effective transformation method for P. gingivalis without any special equipment by exploiting its natural DNA competence. P. gingivalis ATCC 33277 was grown to the early-exponential phase and mixed with a donor DNA cassette. This mixture was then spotted onto a BHI-HM blood-agar plate and incubated for one day to promote colony biofilm formation. The resulting colony biofilm was suspended in a liquid medium and spread onto antibiotic-containing agar plates. Transformants appeared within 4 to 5 days, achieving a maximum efficiency of 7.7 × 106 CFU/μg. Although we optimized the transformation conditions using a representative strain ATCC 33277, but the method was also effective for other P. gingivalis strains, W83 and TDC60. Additionally, we discovered that deletion of PGN_0421 or PGN_0519, encoding putative ComEA and ComEC, abolished competency, indicating that these gene products are essential for the natural competence.

Gut Microbiota and New Microbiome-Targeted Drugs for Clostridioides difficile Infections.

Clostridioides difficile is a major causative pathogen for antibiotic-associated diarrhea and C. difficile infections (CDIs) may lead to life-threatening diseases in clinical settings. Most of the risk factors for the incidence of CDIs, i.e., antibiotic use, treatment by proton pump inhibitors, old age, and hospitalization, are associated with dysbiosis of gut microbiota and associated metabolites and, consequently, treatment options for CDIs include normalizing the composition of the intestinal microbiome. In this review, with an introduction to the CDI and its global epidemiology, CDI-associated traits of the gut microbiome and its metabolites were reviewed, and microbiome-targeting treatment options were introduced, which was approved recently as a new drug by the United States Food and Drug Administration (U.S. FDA), rather than a medical practice.

Sarocladium kiliense: A first report of phytopathogenic fungus causing maize stalk rot in India

Post-flowering stalk rot (PFSR) disease drastically affects maize yields, leading to substantial economic losses. Fusarium verticillioides was widely recognized as a major pathogen responsible for maize PFSR disease. Astonishingly, apart from F. verticillioides, another pathogen was also observed infecting the stalk rot and thus, further pathogen purification was initiated. In this study, we revealed the first report of Sarocladium kiliense, as a maize stalk rot pathogen. This research study was validated by isolation, purification, and morphological examination, which revealed the variability among S. kiliense isolates in traits such as pigmentation, colony color, mycelial characteristics, and conidial morphology. Notably, isolates exhibited distinct pigmentation patterns: 3 were orange and 3 were yellow, with consistent white to dirty white colony colors. Pathogenicity assays using toothpick inoculation demonstrated that all the S. kiliense isolates caused significant damage to the maize stalks, with brown discoloration, conversion of pith into a powdery texture, and subsequent stalk lodging. Isolate PKSK55 exhibited the highest pathogenicity, producing the longest lesions among tested isolates. Molecular characterization using ITS rDNA and RPB-2 genes confirmed the identity of S. kiliense across all isolates. Conclusively, this research uncovered and documents the first report of S. kiliense causing stalk rot in maize in India.

Pathogenicity of Citrobacter freundii Causing Mass Mortalities of Macrobrachium rosenbergii and Its Induced Host Immune Response.

Citrobacter freundii is an opportunistic pathogen of freshwater aquatic animals, which severely restricts the sustainable development of the aquaculture industry. In this study, a dominant strain, named FSNM-1, was isolated from the hepatopancreas of diseased Macrobrachium rosenbergii. This strain was identified as C. freundii based on a comprehensive analysis of its morphological, physiological, and biochemical features and molecular identification. Challenge experiments were conducted to assess the pathogenicity of C. freundii to M. rosenbergii. The results showed that the FSNM-1 strain had high virulence to M. rosenbergii with a median lethal dose (LD50) of 1.1 × 106 CFU/mL. Histopathological analysis revealed that C. freundii infection caused different degrees of inflammation in the hepatopancreas, gills, and intestines of M. rosenbergii. The detection of virulence-related genes revealed that the FSNM-1 strain carried colonization factor antigen (cfa1, cfa2), ureases (ureG, ureF, ureD, ureE), and outer membrane protein (ompX), and virulence factor detection showed that the FSNM-1 strain had lecithinase, amylase, lipase, gelatinase, and hemolysin activities but did not produce protease and DNase activities. To investigate the immune response of M. rosenbergii to C. freundii, the expression levels of ALF3, MyD88, SOD, proPO, TRAF6, and TNF immune-related genes were monitored at different points of time in the hepatopancreas, gills, intestines, and hemocytes of M. rosenbergii after infection. The results demonstrated a significant upregulation in the expression levels of the ALF3, MyD88, SOD, proPO, TRAF6, and TNF genes in M. rosenbergii at the early stage of C. freundii infection. This study highlights C. freundii as a major pathogen causing mass mortality in M. rosenbergii and provides valuable insights into its virulence mechanisms and the host's immune response.

Transcriptomic Analysis of the Effect of Glabridin on Biofilm Formation in Staphylococcus Aureus.

Staphylococcus aureus (S. aureus) is among the major skin infection-causing pathogens in animals and humans. Its ability to form biofilms has become a foremost cause of bacterial infections and the extensive spread of drug resistance, which poses a great difficulty in clinical treatment. Glabridin (Glb), an extract of licorice with antibacterial and anti-infective properties, has a partially understood biofilm-inhibitory mechanism. This study investigated the inhibitory and antibiofilm activities of subinhibitory concentrations of Glb against S. aureus. The crystal violet assay revealed that Glb significantly suppressed biofilm expression. Scanning electron microscopy observations unveiled that Glb reduced S. aureus adhesion and accumulation by disrupting the spatial structure of the biofilm. In vitro extracellular DNA (eDNA) inhibition assays demonstrated that Glb inhibited biofilm formation by S. aureus by suppressing eDNA secretion. In total, 184 differentially expressed genes were obtained through transcriptomic (RNA-seq) sequencing, of which 81 and 103 genes were upregulated and downregulated, respectively. Glb regulated the transcript levels of biofilm-related genes through the phosphatase transfer system, two-component regulatory system, and nitrogen metabolism. The qPCR analysis was performed to confirm whether Glb interfered with the expression of regulatory genes involved in S. aureus biofilm formation (SarA, ArlR, FnbA, ClfA, icaD, and icaR) as well as the virulence gene Hla. In conclusion, this study demonstrates that Glb has a significant inhibitory effect on biofilm activity and is expected to be a good antibiofilm drug.

Bloodstream infections: mechanisms of pathogenesis and opportunities for intervention.

Bloodstream infections (BSIs) are common in hospitals, often life-threatening and increasing in prevalence. Microorganisms in the blood are usually rapidly cleared by the immune system and filtering organs but, in some cases, they can cause an acute infection and trigger sepsis, a systemic response to infection that leads to circulatory collapse, multiorgan dysfunction and death. Most BSIs are caused by bacteria, although fungi also contribute to a substantial portion of cases. Escherichia coli, Staphylococcus aureus, coagulase-negative Staphylococcus, Klebsiella pneumoniae and Candida albicans are leading causes of BSIs, although their prevalence depends on patient demographics and geographical region. Each species is equipped with unique factors that aid in the colonization of initial sites and dissemination and survival in the blood, and these factors represent potential opportunities for interventions. As many pathogens become increasingly resistant to antimicrobials, new approaches to diagnose and treat BSIs at all stages of infection are urgently needed. In this Review, we explore the prevalence of major BSI pathogens, prominent mechanisms of BSI pathogenesis, opportunities for prevention and diagnosis, and treatment options.

Long-read transcriptomics of Ostreid herpesvirus 1 uncovers a conserved expression strategy for the capsid maturation module and pinpoints a mechanism for evasion of the ADAR-based antiviral defence

Abstract Ostreid herpesvirus 1 (OsHV-1), a member of the family Malacoherpesviridae (order Herpesvirales), is a major pathogen of bivalves. However, the molecular details of the malacoherpesvirus infection cycle and its overall similarity to the replication of mammalian herpesviruses (family Orthoherpesviridae) remain obscure. Here, to gain insights into the OsHV-1 biology, we performed long read sequencing of infected blood clams, Anadara broughtonii, which yielded over one million OsHV-1 long reads. This data enabled the annotation of the viral genome with 78 gene units and 274 transcripts, of which 67 were polycistronic mRNAs, 35 ncRNAs and 20 natural antisense transcripts (NATs). Transcriptomics and proteomics data indicate preferential transcription and independent translation of the capsid scaffold protein as an OsHV-1 capsid maturation protease isoform. The conservation of this transcriptional architecture across Herpesvirales likely indicates its functional importance and ancient origin. Moreover, we traced RNA editing events using short read sequencing and supported the presence of inosine nucleotides in native OsHV-1 RNA, consistent with the activity of ADAR1. Our data suggests that, whereas RNA hyper-editing is concentrated in specific regions of the OsHV-1 genome, single nucleotide editing is more dispersed along the OsHV-1 transcripts. In conclusion, we reveal the existence of a conserved pan-Herpesvirales transcriptomic architecture of the capsid maturation module and uncover a transcription-based viral counter defence mechanism, which presumably facilitates the evasion of the host ADAR antiviral system.

Characterization and Pathogenicity of Soilborne Pathogens in Gloriosa superba: Effects of Single- and Multiple-Pathogen Coinfection on Disease Responses.

Glory lily (Gloriosa superba), an ornamental climbing plant, contains the bioactive compound colchicine, attracting attention from the pharmaceutical industry. However, soilborne pathogens have emerged as a serious threat to the cultivation of glory lily, leading to substantial economic losses in the southern parts of India. Among these, the three major pathogens are Macrophomina phaseolina, Fusarium oxysporum, and Agroathelia rolfsii, causing dry root rot (also referred to as charcoal rot), wilt, and stem rot, respectively. Here, we characterized these pathogens using morphological characteristics and phylogenetic analysis of DNA sequences related to the internal transcribed spacer of ribosomal DNA, calmodulin (CAL), and translation elongation factor 1-alpha (TEF-1α). Furthermore, in the pathogenicity tests, the inoculation of M. phaseolina alone resulted in lesions measuring 7.54 ± 0.01 mm on tubers and 90% seedling mortality. This severity was comparable to the simultaneous inoculation of all three pathogens, indicating the prominence of dry root rot among soilborne diseases. This study marks the first detailed investigation of soilborne pathogens combined infection in G. superba, contributing to the understanding of fungal disease complexity in medicinal plants.

Escherichia coli heat-labile enterotoxin B subunit as an adjuvant of mucosal immune combined with GCRV-II VP6 triggers innate immunity and enhances adaptive immune responses following oral vaccination of grass carp (Ctenopharyngodon idella)

The grass carp reovirus (GCRV) is the most major pathogen that has threatened the grass carp (Ctenopharyngodon idella) industry of China for years. Though the oral vaccine has many advantages, the current vaccines still do not provide complete protection. Therefor the exploration of new preventive strategies is urgently needed. In this study, heat-labile enterotoxin B subunit of Escherichia coli (LTB) was combined with VP6 from GCRV type II (GCRV-II) via Lactococcus lactis expression system to form a potent oral vaccine and determines if fusion of LTB to the protective vaccine antigen can enhance protection in the fish. The expression of recombinant protein was confirmed by Western-blotting and enzyme-linked immunosorbent assay. The rare minnow was set as the model for the evaluation of the experiment administrated orally. The immune response including the antibody titer and the immune-related gene expression, and the protective efficacy which included the virus loaded and the relative protection, were thoroughly investigated after the trial. The results indicated that LTB can significantly elicit a higher neutralizing antibody responses and enhanced T-cell priming, activities and proliferation in mononuclear cells from intestine, spleen and kidney tissues when compared to the VP6 vaccine alone. Moreover, the combined adjuvant can significantly up-regulate type I interferon signaling in different immune organs, especially the mucosa associated lymphoidtissue which could not be induced by VP6 along, result in the contribution of the improvement in adaptive immune responses of the fish. In addition, challenge study showed that LTB combined VP6 could greatly improve the relative percent survival of the fish during the virus infection. These results highlight that LTB has the potential value to be a mucosal adjuvant of the fish, approaching for improving the efficacy of vaccination against GCRV-II, which does elicit both non-specific and specific immune responses.

Recombinant Lactococcus lactis secreting FliC protein nanobodies for resistance against Salmonella enteritidis invasion in the intestinal tract

Salmonella Enteritidis is a major foodborne pathogen throughout the world and the increase in antibiotic resistance of Salmonella poses a significant threat to public safety. Natural nanobodies exhibit high affinity, thermal stability, ease of production, and notably higher diversity, making them widely applicable for the treatment of viral and bacterial infections. Recombinant expression using Lactococcus lactis leverages both acid resistance and mucosal colonization properties of these bacteria, allowing the effective expression of exogenous proteins for therapeutic effects. In this study, nine specific nanobodies against the flagellar protein FliC were identified and expressed. In vitro experiments demonstrated that FliC-Nb-76 effectively inhibited the motility of S. Enteritidis and inhibited its adhesion to and invasion of HIEC-6, RAW264.7, and chicken intestinal epithelial cells. Additionally, a recombinant L. lactis strain secreting the nanobody, L. lactis-Nb76, was obtained. Animal experiments confirmed that it could significantly reduce the mortality rates of chickens infected with S. Enteritidis, together with alleviating the inflammatory response caused by the pathogen. These results provide a novel strategy for the treatment of antibiotic-resistant S. Enteritidis infection in the intestinal tract.Graphical

High-throughput transposon mutagenesis in the family Enterobacteriaceae reveals core essential genes and rapid turnover of essentiality.

The Enterobacteriaceae are a scientifically and medically important clade of bacteria, containing the model organism Escherichia coli, as well as major human pathogens including Salmonella enterica and Klebsiella pneumoniae. Essential gene sets have been determined for several members of the Enterobacteriaceae, with the Keio E. coli single-gene deletion library often regarded as a gold standard. However, it remains unclear how gene essentiality varies between related strains and species. To investigate this, we have assembled a collection of 13 sequenced high-density transposon mutant libraries from five genera within the Enterobacteriaceae. We first assess several gene essentiality prediction approaches, investigate the effects of transposon density on essentiality prediction, and identify biases in transposon insertion sequencing data. Based on these investigations, we develop a new classifier for gene essentiality. Using this new classifier, we define a core essential genome in the Enterobacteriaceae of 201 universally essential genes. Despite the presence of a large cohort of variably essential genes, we find an absence of evidence for genus-specific essential genes. A clear example of this sporadic essentiality is given by the set of genes regulating the σE extracytoplasmic stress response, which appears to have independently acquired essentiality multiple times in the Enterobacteriaceae. Finally, we compare our essential gene sets to the natural experiment of gene loss in obligate insect endosymbionts that have emerged from within the Enterobacteriaceae. This isolates a remarkably small set of genes absolutely required for survival and identifies several instances of essential stress responses masked by redundancy in free-living bacteria.IMPORTANCEThe essential genome, that is the set of genes absolutely required to sustain life, is a core concept in genetics. Essential genes in bacteria serve as drug targets, put constraints on the engineering of biological chassis for technological or industrial purposes, and are key to constructing synthetic life. Despite decades of study, relatively little is known about how gene essentiality varies across related bacteria. In this study, we have collected gene essentiality data for 13 bacteria related to the model organism Escherichia coli, including several human pathogens, and investigated the conservation of essentiality. We find that approximately a third of the genes essential in any particular strain are non-essential in another related strain. Surprisingly, we do not find evidence for essential genes unique to specific genera; rather it appears a substantial fraction of the essential genome rapidly gains or loses essentiality during evolution. This suggests that essentiality is not an immutable characteristic but depends crucially on the genomic context. We illustrate this through a comparison of our essential genes in free-living bacteria to genes conserved in 34 insect endosymbionts with naturally reduced genomes, finding several cases where genes generally regarded as being important for specific stress responses appear to have become essential in endosymbionts due to a loss of functional redundancy in the genome.

The current riboswitch landscape in Clostridioides difficile.

Riboswitches are 5' RNA regulatory elements that are capable of binding to various ligands, such as small metabolites, ions and tRNAs, leading to conformational changes and affecting gene transcription or translation. They are widespread in bacteria and frequently control genes that are essential for the survival or virulence of major pathogens. As a result, they represent promising targets for the development of new antimicrobial treatments. Clostridioides difficile, a leading cause of antibiotic-associated nosocomial diarrhoea in adults, possesses numerous riboswitches in its genome. Accumulating knowledge of riboswitch-based regulatory mechanisms provides insights into the potential therapeutic targets for treating C. difficile infections. This review offers an in-depth examination of the current state of knowledge regarding riboswitch-mediated regulation in C. difficile, highlighting their importance in bacterial adaptability and pathogenicity. Particular attention is given to the ligand specificity and function of known riboswitches in this bacterium. The review also discusses the recent progress that has been made in the development of riboswitch-targeting compounds as potential treatments for C. difficile infections. Future research directions are proposed, emphasizing the need for detailed structural and functional analyses of riboswitches to fully harness their regulatory capabilities for developing new antimicrobial strategies.