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Antibiotic use can lead to the expansion of multi-drug-resistant pathobionts within the gut microbiome that can cause life-threatening infections. Selective alternatives to conventional antibiotics are in dire need. Here, we describe a Klebsiella PhageBank for the tailored design of bacteriophage cocktails to treat multi-drug-resistant Klebsiella pneumoniae. Using a transposon library in carbapenem-resistant K. pneumoniae , we identify host factors required for phage infection in major Klebsiella phage families. Leveraging the diversity of the PhageBank, we formulate phage combinations that eliminate K. pneumoniae with minimal phage resistance. Optimized cocktails selectively suppress the burden of K. pneumoniae in the mouse gut and drive the loss of key virulence factors that act as phage receptors. Phage-mediated diversification of bacterial populations in the gut leads to co-evolution of phage variants with higher virulence and broader host range. Altogether, the Klebsiella PhageBank charts a roadmap for phage therapy against a critical multidrug-resistant human pathogen. [Display omitted] • A diverse Klebsiella PhageBank is used to construct cocktails that prevent resistance • Host factors required by the taxonomically diverse Klebsiella phage are mapped • Phage cocktails suppress gut K. pneumoniae in mice and select for capsule loss • Antagonistic co-evolution drives phage and bacterial diversification in the gut Leveraging a well-characterized library of bacteriophages, Rotman et al. demonstrate that cocktails of phages can suppress the burden of carbapenem-resistant Klebsiella pneumoniae in the mouse gut microbiome. In the gut, phage-bacterial interactions select for bacterial mutants with lessened virulence and phage variants with broadened host range. [ABSTRACT FROM AUTHOR]

Introduction: Plasmids, the most important and versatile bacterial extrachromosomal DNA Molecules, has have been a center central topic for bacterial genetics and biology. However, the inability of short-read high-throughput sequencing methods to reliably assemble plasmids makes it difficult to investigate the diversity of plasmid structures and functions. Methods: In this work, we used the long-read Nanopore sequencing method to address this issue, by producing high quality whole genome sequences of 33 bacterial strains from 11 perianal abscess-suffering patients. Results and discussion: Successful high quality assemblies were generated with this method, including 20 perfect assemblies out of 33 genomes. A total of 47 plasmids were identified from the bacterial strains, including 12 unique, newly identified, high quality circular plasmids. These plasmids were further subject to structural analysis, leading to the finding of significant diversification from previously known plasmids, suggesting the diversity of plasmid structure and function. Particularly, two mcr-10.1-harboring conjugative plasmids were found from Citrobacter portucalensis and Enterobacter kobei, which were not previously reported. This works shows the feasibility of using long-read sequencing to identify plasmids, and the high diversity of plasmid structure and function that awaits further surveillance. [ABSTRACT FROM AUTHOR]

This article explores the relationship between clonality and resistance in Staphylococcus aureus (S. aureus) in the context of antibiotic use. S. aureus is a pathogen that can cause various infections and is a leading cause of death from bacterial infection globally. The article discusses the emergence, expansion, and disappearance of genetically identical hypervirulent clones of S. aureus, as well as the development of antibiotic resistance in specific clones. It also delves into the history of antibiotic resistance in S. aureus and the ongoing challenges it presents. The article concludes by mentioning efforts to prevent S. aureus infection by reducing or eliminating colonization. Additionally, it discusses different forms of antibiotic resistance in S. aureus, such as vancomycin intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA). VISA exhibits low-level resistance by trapping the antibiotic within a thickened cell wall, while VRSA demonstrates full resistance through the acquisition of the vanA gene cluster from enterococci. The article emphasizes the need for new approaches and strategies to combat this highly adaptable pathogen, including the development of vaccines and the potential for neutralizing staphylococcal toxins as a more effective strategy. [Extracted from the article]

Drugs intended to target mammalian cells can have broad off‐target effects on the human gut microbiota with potential downstream consequences for drug efficacy and side effect profiles. Yet, despite a rich literature on antibiotic resistance, we still know very little about the mechanisms through which commensal bacteria evade non‐antibiotic drugs. Here, we focus on statins, one of the most prescribed drug types in the world and an essential tool in the prevention and treatment of high circulating cholesterol levels. Prior work in humans, mice, and cell culture support an off‐target effect of statins on human gut bacteria; however, the genetic determinants of statin sensitivity remain unknown. We confirmed that simvastatin inhibits the growth of diverse human gut bacterial strains grown in communities and in pure cultures. Drug sensitivity varied between phyla and was dose‐dependent. We selected two representative simvastatin‐sensitive species for more in‐depth analysis: Eggerthella lenta (phylum: Actinobacteriota) and Bacteroides thetaiotaomicron (phylum: Bacteroidota). Transcriptomics revealed that both bacterial species upregulate genes in response to simvastatin that alter the cell membrane, including fatty acid biogenesis (E. lenta) and drug efflux systems (B. thetaiotaomicron). Transposon mutagenesis identified a key efflux system in B. thetaiotaomicron that enables growth in the presence of statins. Taken together, these results emphasize the importance of the bacterial cell membrane in countering the off‐target effects of host‐targeted drugs. Continued mechanistic dissection of the various mechanisms through which the human gut microbiota evades drugs will be essential to understand and predict the effects of drug administration in human cohorts and the potential downstream consequences for health and disease. [ABSTRACT FROM AUTHOR]

Coronavirus (CoV) possesses numerous functional cis-acting elements in its positive-strand genomic RNA. Although most of these RNA structures participate in viral replication, the functions of RNA structures in the genomic RNA of CoV in viral replication remain unclear. In this study, we investigated the functions of the higher-order RNA stem-loop (SL) structures SL5B, SL5C, and SL5D in the ORF1a coding region of Middle East respiratory syndrome coronavirus (MERS-CoV) in viral replication. Our approach, using reverse genetics of a bacterial artificial chromosome system, revealed that SL5B and SL5C play essential roles in the discontinuous transcription of MERS-CoV. In silico analyses predicted that SL5C interacts with a bulged stem-loop (BSL) in the 3′ untranslated region, suggesting that the RNA structure of SL5C is important for viral RNA transcription. Conversely, SL5D did not affect transcription, but mediated the synthesis of positive-strand genomic RNA. Additionally, the RNA secondary structure of SL5 in the revertant virus of the SL5D mutant was similar to that of the wild-type, indicating that the RNA structure of SL5D can finely tune RNA replication in MERS-CoV. Our data indicate novel regulatory mechanisms of viral RNA transcription and replication by higher-order RNA structures in the MERS-CoV genomic RNA. [ABSTRACT FROM AUTHOR]

To investigate how host and pathogen diversity govern immunity against Mycobacterium tuberculosis (Mtb), we performed a large-scale screen of vaccine-mediated protection against aerosol Mtb infection using three inbred mouse strains [C57BL/6 (B6), C3HeB/FeJ (C3H), Balb/c x 129/SvJ (C129F1)] and three Mtb strains (H37Rv, CDC1551, SA161) representing two lineages and distinct virulence properties. We compared three protective modalities, all of which involve inoculation with live mycobacteria: Bacillus Calmette-Gue'rin (BCG), the only approved TB vaccine, delivered either subcutaneously or intravenously, and concomitant Mtb infection (CoMtb), a model of pre-existing immunity in which a low-level Mtb infection is established in the cervical lymph node following intradermal inoculation. We examined lung bacterial burdens at early (Day 28) and late (Day 98) time points after aerosol Mtb challenge and histopathology at Day 98. We observed substantial heterogeneity in the reduction of bacterial load afforded by these modalities at Day 28 across the combinations and noted a strong positive correlation between bacterial burden in unvaccinated mice and the degree of protection afforded by vaccination. Although we observed variation in the degree of reduction in bacterial burdens across the nine mouse/bacterium strain combinations, virtually all protective modalities performed similarly for a given strain-strain combination. We also noted dramatic variation in histopathology changes driven by both host and bacterial genetic backgrounds. Vaccination improved pathology scores for all infections except CDC1551. However, the most dramatic impact of vaccination on lesion development occurred for the C3H-SA161 combination, where vaccination entirely abrogated the development of the large necrotic lesions that arise in unvaccinated mice. In conclusion, we find that substantial TB heterogeneity can be recapitulated by introducing variability in both host and bacterial genetics, resulting in changes in vaccine-mediated protection as measured both by bacterial burden as well as histopathology. These differences can be harnessed in future studies to identify immune correlates of vaccine efficacy. [ABSTRACT FROM AUTHOR]

The development of the Escherichia coli K‐12 laboratory strains JM83, JM109 and XL1‐Blue was instrumental in early gene technology. We report the comprehensive genome sequence analysis of JM83 and XL1‐Blue using Illumina and Oxford Nanopore technologies and a comparison with both the wild‐type sequence (MG1655) and the genome of JM109 deposited at GenBank. Our investigation provides insight into the way how the genomic background that allows blue/white colony selection—by complementing a functionally inactive ω‐fragment of β‐galactosidase (LacZ) with its α‐peptide encoded on the cloning vector—has been implemented independently in these three strains using classical bacterial genetics. In fact, their comparative analysis reveals recurrent motifs: (i) inactivation of the native enzyme via large deletions of chromosomal regions encompassing the lac locus, or a chemically induced frameshift deletion at the beginning of the lacZ cistron, and (ii) utilization of a defective prophage (ϕ80), or an F′‐plasmid, to provide the lacZ∆M15 allele encoding its ω‐fragment. While the genetic manipulations of the E. coli strains involved repeated use of mobile genetic elements as well as harsh chemical or physical mutagenesis, the individual modified traits appear remarkably stable as they can be found even in distantly related laboratory strains, beyond those investigated here. Our detailed characterization at the genome sequence level not only offers clues about the mechanisms of classical gene transduction and transposition but should also guide the future fine‐tuning of E. coli strains for gene cloning and protein expression, including phage display techniques, utilizing advanced tools for site‐specific genome engineering. [ABSTRACT FROM AUTHOR]

ABSTRACT Guanine nucleotides are required for growth and viability of cells due to their structural role in DNA and RNA, and their regulatory roles in translation, signal transduction, and cell division. The natural antibiotic mycophenolic acid (MPA) targets the rate-limiting step in de novo guanine nucleotide biosynthesis executed by inosine-5´-monophosphate dehydrogenase (IMPDH). MPA is used clinically as an immunosuppressant, but whether in vivo inhibition of bacterial IMPDH (GuaB) is a valid antibacterial strategy is controversial. Here, we describe the discovery of extremely potent small molecule GuaB inhibitors (GuaBi) specific to pathogenic bacteria with a low frequency of on-target spontaneous resistance and bactericidal efficacy in vivo against Acinetobacter baumannii mouse models of infection. The spectrum of GuaBi activity includes multidrug-resistant pathogens that are a critical priority of new antibiotic development. Co-crystal structures of A. baumannii, Staphylococcus aureus, and Escherichia coli GuaB proteins bound to inhibitors show comparable binding modes of GuaBi across species and identifies key binding site residues that are predictive of whole-cell activity across both Gram-positive and Gram-negative clades of Bacteria. The clear in vivo efficacy of these small molecule GuaB inhibitors in a model of A. baumannii infection validates GuaB as an essential antibiotic target.IMPORTANCEThe emergence of multidrug-resistant bacteria worldwide has renewed interest in discovering antibiotics with novel mechanism of action. For the first time ever, we demonstrate that pharmacological inhibition of de novo guanine biosynthesis is bactericidal in a mouse model of Acinetobacter baumannii infection. Structural analyses of novel inhibitors explain differences in biochemical and whole-cell activity across bacterial clades and underscore why this discovery may have broad translational impact on treatment of the most recalcitrant bacterial infections.

Streptococcus pneumoniae is a pathogen that causes community-associated infections in young children Whole-genome sequencing (WGS) has become a powerful tool for bacterial genotyping; costs have been decreasing as accessibility increases. [...]

This study investigates the in vitro activity of Nα-aroyl-N-aryl-phenylalanine amides (AAPs), previously identified as antimycobacterial RNA polymerase (RNAP) inhibitors, against a panel of 25 non-tuberculous mycobacteria (NTM). The compounds, including the hit compound MMV688845, were selected based on their structural diversity and previously described activity against mycobacteria. Bacterial strains, including the M. abscessus complex, M. avium complex, and other clinically relevant NTM, were cultured and subjected to growth inhibition assays. The results demonstrate significant activity against the most common NTM pathogens from the M. abscessus and M. avium complexes. Variations in activity were observed against other NTM species, with for instance M. ulcerans displaying high susceptibility and M. xenopi and M. simiae resistance to AAPs. Comparative analysis of RNAP β and β′ subunits across mycobacterial species revealed strain-specific polymorphisms, providing insights into differential compound susceptibility. While conservation of target structures was observed, differences in compound activity suggested influences beyond drug–target interactions. This study highlights the potential of AAPs as effective antimycobacterial agents and emphasizes the complex interplay between compound structure, bacterial genetics, and in vitro activity. [ABSTRACT FROM AUTHOR]

The Mycobacterium tuberculosis complex (MTBC) comprises nine human-adapted lineages that differ in their geographical distribution. Local adaptation of specific MTBC genotypes to the respective human host population has been invoked in this context. We aimed to assess if bacterial genetics governs MTBC pathogenesis or if local co-adaptation translates into differential susceptibility of human macrophages to infection by different MTBC genotypes. We generated macrophages from cryopreserved blood mononuclear cells of Tanzanian tuberculosis patients, from which the infecting MTBC strains had previously been phylogenetically characterized. We infected these macrophages ex vivo with a phylogenetically similar MTBC strain ("matched infection") or with strains representative of other MTBC lineages ("mismatched infection"). We found that L1 infections resulted in a significantly lower bacterial burden and that the intra-cellular replication rate of L2 strains was significantly higher compared the other MTBC lineages, irrespective of the MTBC lineage originally infecting the patients. Moreover, L4-infected macrophages released significantly greater amounts of TNF-α, IL-6, IL-10, MIP-1β, and IL-1β compared to macrophages infected by all other strains. While our results revealed no measurable effect of local adaptation, they further highlight the strong impact of MTBC phylogenetic diversity on the variable outcome of the host–pathogen interaction in human tuberculosis. [ABSTRACT FROM AUTHOR]

Gut microbes are closely related with human health, but remain much to learn. Clostridium symbiosum is a conditionally pathogenic human gut bacterium and regarded as a potential biomarker for early diagnosis of intestinal tumors. However, the absence of an efficient toolbox that allows diverse genetic manipulations of this bacterium limits its in-depth studies. Here, we obtained the complete genome sequence of C. symbiosum ATCC 14940, a representative strain of C. symbiosum. On this basis, we further developed a series of genetic manipulation methods for this bacterium. Firstly, following the identification of a functional replicon pBP1 in C. symbiosum ATCC 14940, a highly efficient conjugative DNA transfer method was established, enabling the rapid introduction of exogenous plasmids into cells. Next, we constructed a dual-plasmid CRISPR/Cas12a system for genome editing in this bacterium, reaching over 60 % repression for most of the chosen genes as well as efficient deletion (>90 %) of three target genes. Finally, this toolbox was used for the identification of crucial functional genes, involving growth, synthesis of important metabolites, and virulence of C. symbiosum ATCC 14940. Our work has effectively established and optimized genome editing methods in intestinal C. symbiosum, thereby providing strong support for further basic and application research in this bacterium. [ABSTRACT FROM AUTHOR]

Background: Multilocus sequence typing (MLST) is used to gain insight into the population genetics of bacteria in the form of sequence type (ST). MLST has been used to study the evolution and spread of virulent clones of Streptococcus pneumoniae in many parts of the world. Such data for S. pneumoniae are lacking for the countries of the Arabian Peninsula, including Kuwait. Methods: We determined the STs of all 31 strains of S. pneumoniae from invasive diseases received at a reference laboratory from various health centers in Kuwait during 2018 by MLST. The relationship among the isolates was determined by phylogenetic analysis. We also determined the serotypes by Quellung reaction, and antimicrobial susceptibility by Etest, against 15 antibiotics belonging to 10 classes. Results: There were 28 STs among the 31 isolates, of which 14 were new STs (45.2%) and 5 were rare STs (16.1%). Phylogenetic analysis revealed that 26 isolates (83.9%) were unrelated singletons, and the Kuwaiti isolates were related to those from neighboring countries whose information was gleaned from unpublished data available at the PubMLST website. Many of our isolates were resistant to penicillin, erythromycin, and azithromycin, and some were multidrug-resistant. Virulent serotype 8-ST53, and serotype 19A with new STs, were detected. Conclusions: Our study detected an unusually large number of novel STs, which may indicate that Kuwait provides a milieu for the evolution of novel STs. Novel STs may arise due to recombination and can result in capsular switching. This can impact the effect of vaccination programs on the burden of invasive pneumococcal disease. This first report from the Arabian Peninsula justifies the continuous monitoring of S. pneumoniae STs for the possible evolution of new virulent clones and capsular switching. [ABSTRACT FROM AUTHOR]

Quorum sensing is a mechanism of bacterial cell-cell communication that relies on the production and detection of small molecule autoinducers, which facilitate the synchronous expression of genes involved in group behaviors, such as virulence factor production and biofilm formation. The Pseudomonas aeruginosa quorum sensing network consists of multiple interconnected transcriptional regulators, with the transcription factor, RhlR, acting as one of the main drivers of quorum sensing behaviors. RhlR is a LuxR-type transcription factor that regulates its target genes when bound to its cognate autoinducer, C4-homoserine lactone, which is synthesized by RhlI. RhlR function is also regulated by the metallo-β-hydrolase enzyme, PqsE. We recently showed that PqsE binds RhlR to alter its affinity for promoter DNA, a new mechanism of quorum-sensing receptor activation. Here, we perform ChIP-seq analyses of RhlR to map the binding of RhlR across the P. aeruginosa genome, and to determine the impact of C4-homoserine lactone and PqsE on RhlR binding to different sites across the P. aeruginosa genome. We identify 40 RhlR binding sites, all but three of which are associated with genes known to be regulated by RhlR. C4-homoserine lactone is required for maximal binding of RhlR to many of its DNA sites. Moreover, C4-homoserine lactone is required for maximal RhlR-dependent transcription activation from all sites, regardless of whether it impacts RhlR binding to DNA. PqsE is required for maximal binding of RhlR to many DNA sites, with similar effects on RhlR-dependent transcription activation from those sites. However, the effects of PqsE on RhlR specificity are distinct from those of C4-homoserine lactone, and PqsE is sufficient for RhlR binding to some DNA sites in the absence of C4-homoserine lactone. Together, C4-homoserine lactone and PqsE are required for RhlR binding at the large majority of its DNA sites. Thus, our work reveals three distinct modes of activation by RhlR: i) when RhlR is unbound by autoinducer but bound by PqsE, ii) when RhlR is bound by autoinducer but not bound by PqsE, and iii) when RhlR is bound by both autoinducer and PqsE, establishing a stepwise mechanism for the progression of the RhlR-RhlI-PqsE quorum sensing pathway in P. aeruginosa. Author summary: Pseudomonas aeruginosa represents a serious threat to public health in the United States because of its intrinsic mechanisms of antimicrobial resistance. One of the primary drivers of its antimicrobial resistance profile is biofilm formation. Biofilms are multicellular communities that are formed in an ordered mechanism by the collective. In the case of P. aeruginosa, biofilm formation is controlled by a cell-cell communication process called quorum sensing. Quorum sensing underpins transcriptional changes in a bacterium, allowing for the transition from individual, planktonic behaviors to group, sessile behaviors. Group behaviors are behaviors that are only beneficial to engage in once a critical threshold of kin population is reached. The transition to group behaviors is mediated by quorum sensing through an interconnected regulatory system, with the transcription factor receptor RhlR regulating the expression of hundreds of genes. RhlR-dependent transcription relies on the detection of a ligand produced by its partner synthase, RhlI, and an accessory binding protein that alters its affinity for promoter DNA, PqsE, resulting in a dual regulatory mechanism that was not previously observed in quorum sensing. The main goal of this study was to determine the molecular basis for promoter selection by RhlR using bacterial genetics and DNA-based sequencing methods for measuring site-specific protein binding. Our approach established the entirety of the RhlR direct regulon and the contributions of each of the known regulators to RhlR-dependent promoter binding and gene regulation. [ABSTRACT FROM AUTHOR]

Cotton crop yields are largely affected by infestations of Anthonomus grandis, which is its main pest. Although Bacillus thuringiensis (Bt) derived proteins can limit insect pest infestations, the diverse use of control methods becomes a viable alternative in order to prolong the use of technology in the field. One of the alternative methods to Bt technology has been the utilization of certain Pseudomonas species highly efficient in controlling coleopteran insects have been used to produce highly toxic insecticidal proteins. This study aimed to evaluate the toxicity of IPD072Aa and PIP-47Aa proteins, isolated from Pseudomonas spp., in interaction with Cry1Ia10, Cry3Aa, and Cry8B proteins isolated from B. thuringiensis, to control A. grandis in cotton crops. The genes IPD072Aa and PIP-47Aa were synthesized and cloned into a pET-SUMO expression vector. Moreover, Cry1Ia10, Cry3Aa, and Cry8B proteins were obtained by inducing recombinant E. coli clones, which were previously acquired by our research group from the Laboratory of Bacteria Genetics and Applied Biotechnology (LGBBA). These proteins were visualized in SDS-PAGE, quantified, and incorporated into an artificial diet to estimate their lethal concentrations (LC) through individual or combined bioassays. The results of individual toxicity revealed that IPD072Aa, PIP-47Aa, Cry1Ia10, Cry3Aa, and Cry8B were efficient in controlling A. grandis, with the latter being the most toxic. Regarding interaction assays, a high synergistic interaction was observed between Cry1Ia10 and Cry3Aa. All interactions involving Cry3Aa and PIP-47Aa, when combined with other proteins, showed a clear synergistic effect. Our findings highlighted that the tested proteins in combination, for the most part, increase toxicity against A. grandis neonate larvae, suggesting possible constructions for pyramiding cotton plants to the manage and the control boll weevils. [ABSTRACT FROM AUTHOR]

Genetic differences among mammalian hosts and among strains of Mycobacterium tuberculosis (Mtb) are well-established determinants of tuberculosis (TB) patient outcomes. The advent of recombinant inbred mouse panels and next-generation transposon mutagenesis and sequencing approaches has enabled dissection of complex host-pathogen interactions. To identify host and pathogen genetic determinants of Mtb pathogenesis, we infected members of the highly diverse BXD family of strains with a comprehensive library of Mtb transposon mutants (TnSeq). Members of the BXD family segregate for Mtb-resistant C57BL/6J (B6 or B) and Mtb-susceptible DBA/2J (D2 or D) haplotypes. The survival of each bacterial mutant was quantified within each BXD host, and we identified those bacterial genes that were differentially required for Mtb fitness across BXD genotypes. Mutants that varied in survival among the host family of strains were leveraged as reporters of "endophenotypes," each bacterial fitness profile directly probing specific components of the infection microenvironment. We conducted quantitative trait loci (QTL) mapping of these bacterial fitness endophenotypes and identified 140 host-pathogen QTL (hpQTL). We located a QTL hotspot on chromosome 6 (75.97-88.58 Mb) associated with the genetic requirement of multiple Mtb genes: Rv0127 (mak), Rv0359 (rip2), Rv0955 (perM), and Rv3849 (espR). Together, this screen reinforces the utility of bacterial mutant libraries as precise reporters of the host immunological microenvironment during infection and highlights specific host-pathogen genetic interactions for further investigation. To enable downstream follow-up for both bacterial and mammalian genetic research communities, all bacterial fitness profiles have been deposited into GeneNetwork.org and added into the comprehensive collection of TnSeq libraries in MtbTnDB. [ABSTRACT FROM AUTHOR]

The overprescribing and misuse of antibiotics have led to the rapid development of multidrug-resistant bacteria, such as those that cause UTIs. UTIs are the most common outpatient infections and are mainly caused by Escherichia coli and Klebsiella spp., although some Gram-positive bacteria, such as Pseudomonas aeruginosa, have been isolated in many cases. The rise of antimicrobial-resistant bacteria is a major public health concern, as it is predicted to lead to increased healthcare costs and poor patient outcomes and is expected to be the leading cause of global mortality by 2050. Antibiotic resistance among bacterial species can arise from a myriad of factors, including intrinsic and acquired resistance mechanisms, as well as mobile genetic elements, such as transposons, integrons, and plasmids. Plasmid-mediated resistance is of major concern as drug-resistance genes can quickly and efficiently spread across bacterial species via horizontal gene transfer. The emergence of extended-spectrum β-lactamases (ESBLs) such as NDM-1, OXA, KPC, and CTX-M family members has conferred resistance to many commonly used antibiotics in the treatment of UTIs, including penicillins, carbapenems, cephalosporins, and sulfamethoxazole. This review will focus on plasmid-mediated bacterial genes, especially those that encode ESBLs, and how they contribute to antibiotic resistance. Early clinical detection of these genes in patient samples will provide better treatment options and reduce the threat of antibiotic resistance. [ABSTRACT FROM AUTHOR]

Background: Streptococcus mutans (S. mutans) is a pivotal cariogenic pathogen contributing to its multiple virulence factors, one of which is synthesizing exopolysaccharides (EPS). VicK, a sensor histidine kinase, plays a major role in regulating genes associated with EPS synthesis and adhesion. Here we first identified an antisense vicK RNA (ASvicK) bound with vicK into double-stranded RNA (dsRNA). Objective: This study aims to investigate the effect and mechanism of ASvicK in the EPS metabolism and cariogenesis of S. mutans. Methods: The phenotypes of biofilm were detected by scanning electron microscopy (SEM), gas chromatography-mass spectrometery (GC-MS), gel permeation chromatography (GPC), transcriptome analysis and Western blot. Co-immunoprecipitation (Co-ip) assay and enzyme activity experiment were adopted to investigate the mechanism of ASvicK regulation. Caries animal models were developed to study the relationship between ASvicK and cariogenicity of S. mutans. Results: Overexpression of ASvicK can inhibit the growth of biofilm, reduce the production of EPS and alter genes and protein related to EPS metabolism. ASvicK can adsorb RNase III to regulate vicK and affect the cariogenicity of S. mutans. Conclusions: ASvicK regulates vicK at the transcriptional and post-transcriptional levels, effectively inhibits EPS synthesis and biofilm formation and reduces its cariogenicity in vivo. [ABSTRACT FROM AUTHOR]

This is a systematic review and meta-analysis that evaluated the prevalence of Escherichia coli antibiotic-resistant genes (ARGs) in animals, humans, and the environment in South Africa. This study followed Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines to search and use literature published between 1 January 2000 to 12 December 2021, on the prevalence of South African E. coli isolates' ARGs. Articles were downloaded from African Journals Online, PubMed, ScienceDirect, Scopus, and Google Scholar search engines. A random effects meta-analysis was used to estimate the antibiotic-resistant genes of E. coli in animals, humans, and the environment. Out of 10764 published articles, only 23 studies met the inclusion criteria. The obtained results indicated that the pooled prevalence estimates (PPE) of E. coli ARGs was 36.3%, 34.4%, 32.9%, and 28.8% for blaTEM-M-1, ampC, tetA, and blaTEM, respectively. Eight ARGs (blaCTX-M, blaCTX-M-1, blaTEM, tetA, tetB, sul1, sulII, and aadA) were detected in humans, animals and the environmental samples. Human E. coli isolate samples harboured 38% of the ARGs. Analyzed data from this study highlights the occurrence of ARGs in E. coli isolates from animals, humans, and environmental samples in South Africa. Therefore, there is a necessity to develop a comprehensive "One Health" strategy to assess antibiotics use in order to understand the causes and dynamics of antibiotic resistance development, as such information will enable the formulation of intervention strategies to stop the spread of ARGs in the future. [ABSTRACT FROM AUTHOR]

Oral streptococci are gram-positive facultative anaerobic bacteria that are normal inhabitants of the human oral cavity and play an important role in maintaining oral microecological balance and pathogenesis. Transposon mutagenesis is an effective genetic manipulation strategy for studying the function of genomic features. In order to study cariogenic related genes and crucial biological element genes of oral Streptococcus, transposon mutagenesis was widely used to identify functional genes. With the advent of next-generation sequencing (NGS) technology and the development of transposon random mutation library construction methods, transposon insertion sequencing (TIS) came into being. Benefiting from high-throughput advances in NGS, TIS was able to evaluate the fitness contribution and essentiality of genetic features in the bacterial genome. The application of transposon mutagenesis, including TIS, to oral streptococci provided a massive amount of valuable detailed linkage data between genetic fitness and genetic backgrounds, further clarify the processes of colonization, virulence, and persistence and provides a more reliable basis for investigating relationships with host ecology and disease status. This review focuses on transposon mutagenesis, including TIS, and its applicability in oral streptococci. [ABSTRACT FROM AUTHOR]

This is a correction to: Genetics of Bacteria: a call for papers, G3 Genes|Genomes|Genetics, Volume 14, Issue 7, July 2024, jkae133, https://doi.org/10.1093/g3journal/jkae133This article has been corrected to update the deadline for submissions of papers on the Genetics of Bacteria to December 2, 2024. [Extracted from the article]

ABSTRACT Enzymes involved in rescuing stalled ribosomes and recycling translation machinery are ubiquitous in bacteria and required for growth. Peptidyl tRNA drop-off is a type of abortive translation that results in the release of a truncated peptide that is still bound to tRNA (peptidyl tRNA) into the cytoplasm. Peptidyl tRNA hydrolase (Pth) recycles the released tRNA by cleaving off the unfinished peptide and is essential in most bacteria. We developed a sequencing-based strategy called copper sulfate-based tRNA sequencing (Cu-tRNAseq) to study the physiological role of Pth in Mycobacterium tuberculosis (Mtb). While most peptidyl tRNA species accumulated in a strain with impaired Pth expression, peptidyl prolyl-tRNA was particularly enriched, suggesting that Pth is required for robust peptidyl prolyl-tRNA turnover. Reducing Pth levels increased Mtb’s susceptibility to tRNA synthetase inhibitors that are in development to treat tuberculosis (TB) and rendered this pathogen highly susceptible to macrolides, drugs that are ordinarily ineffective against Mtb. Collectively, our findings reveal the potency of Cu-tRNAseq for profiling peptidyl tRNAs and suggest that targeting Pth would open new therapeutic approaches for TB. IMPORTANCE Peptidyl tRNA hydrolase (Pth) is an enzyme that cuts unfinished peptides off tRNA that has been prematurely released from a stalled ribosome. Pth is essential in nearly all bacteria, including the pathogen Mycobacterium tuberculosis (Mtb), but it has not been clear why. We have used genetic and novel biochemical approaches to show that when Pth levels decline in Mtb, peptidyl tRNA accumulates to such an extent that usable tRNA pools drop. Thus, Pth is needed to maintain normal tRNA levels, most strikingly for prolyl-tRNAs. Many antibiotics act on protein synthesis and could be affected by altering the availability of tRNA. This is certainly true for tRNA synthetase inhibitors, several of which are drug candidates for tuberculosis. We find that their action is potentiated by Pth depletion. Furthermore, Pth depletion results in hypersensitivity to macrolides, drugs that are not active enough under ordinary circumstances to be useful for tuberculosis.

Oral streptococci are gram-positive facultative anaerobic bacteria that are normal inhabitants of the human oral cavity and play an important role in maintaining oral microecological balance and pathogenesis. Transposon mutagenesis is an effective genetic manipulation strategy for studying the function of genomic features. In order to study cariogenic related genes and crucial biological element genes of oral Streptococcus, transposon mutagenesis was widely used to identify functional genes. With the advent of next-generation sequencing (NGS) technology and the development of transposon random mutation library construction methods, transposon insertion sequencing (TIS) came into being. Benefiting from high-throughput advances in NGS, TIS was able to evaluate the fitness contribution and essentiality of genetic features in the bacterial genome. The application of transposon mutagenesis, including TIS, to oral streptococci provided a massive amount of valuable detailed linkage data between genetic fitness and genetic backgrounds, further clarify the processes of colonization, virulence, and persistence and provides a more reliable basis for investigating relationships with host ecology and disease status. This review focuses on transposon mutagenesis, including TIS, and its applicability in oral streptococci.

Background: Milk yield and fine composition in sheep depend on the volatile and long-chain fatty acids, microbial proteins, vitamins produced through feedstuff digestion by the rumen microbiota. In cattle, the host genome has been shown to have a low to moderate genetic control on rumen microbiota abundance but a high control on dairy traits with heritabilities higher than 0.30. There is little information on the genetic correlations and quantitative trait loci (QTL) that simultaneously affect rumen microbiota abundance and dairy traits in ruminants, especially in sheep. Thus, our aim was to quantify the effect of the host genetics on rumen bacterial abundance and the genetic correlations between rumen bacterial abundance and several dairy traits, and to identify QTL that are associated with both rumen bacterial abundance and milk traits. Results: Our results in Lacaune sheep show that the heritability of rumen bacterial abundance ranges from 0 to 0.29 and that the heritability of 306 operational taxonomic units (OTU) is significantly different from 0. Of these 306 OTU, 96 that belong mainly to the Prevotellaceae, Lachnospiraceae and Ruminococcaceae bacterial families show strong genetic correlations with milk fatty acids and proteins (absolute values ranging from 0.33 to 0.99). Genome-wide association studies revealed a QTL for alpha-lactalbumin concentration in milk on Ovis aries chromosome (OAR) 11, and six QTL for rumen bacterial abundances i.e., for two OTU belonging to the genera Prevotella (OAR3 and 5), Rikeneleaceae_RC9_gut_group (OAR5), Ruminococcus (OAR5), an unknown genus of order Clostridia UCG-014 (OAR10), and CAG-352 (OAR11). None of these detected regions are simultaneously associated with rumen bacterial abundance and dairy traits, but the bacterial families Prevotellaceae, Lachnospiraceae and F082 show colocalized signals on OAR3, 5, 15 and 26. Conclusions: In Lacaune dairy sheep, rumen microbiota abundance is partially controlled by the host genetics and is poorly genetically linked with milk protein and fatty acid compositions, and three main bacterial families, Prevotellaceae, Lachnospiraceae and F082, show specific associations with OAR3, 5, 15 and 26. [ABSTRACT FROM AUTHOR]

Mycobacteria, including the human pathogen Mycobacterium tuberculosis, grow by inserting new cell wall material at their poles. This process and that of division are asymmetric, producing a phenotypically heterogeneous population of cells that respond non-uniformly to stress (Aldridge et al., 2012; Rego et al., 2017). Surprisingly, deletion of a single gene - lamA - leads to more symmetry, and to a population of cells that is more uniformly killed by antibiotics (Rego et al., 2017). How does LamA create asymmetry? Here, using a combination of quantitative time-lapse imaging, bacterial genetics, and lipid profiling, we find that LamA recruits essential proteins involved in cell wall synthesis to one side of the cell - the old pole. One of these proteins, MSMEG_0317, here renamed PgfA, was of unknown function. We show that PgfA is a periplasmic protein that interacts with MmpL3, an essential transporter that flips mycolic acids in the form of trehalose monomycolate (TMM), across the plasma membrane. PgfA interacts with a TMM analog suggesting a direct role in TMM transport. Yet our data point to a broader function as well, as cells with altered PgfA levels have differences in the abundance of other lipids and are differentially reliant on those lipids for survival. Overexpression of PgfA, but not MmpL3, restores growth at the old poles in cells missing lamA. Together, our results suggest that PgfA is a key determinant of polar growth and cell envelope composition in mycobacteria, and that the LamA-mediated recruitment of this protein to one side of the cell is a required step in the establishment of cellular asymmetry. [ABSTRACT FROM AUTHOR]

Despite dramatic advances in genomics, connecting genotypes to phenotypes is still challenging. Sexual genetics combined with linkage analysis is a powerful solution to this problem but generally unavailable in bacteria. We build upon a strong negative selection system to invent mass allelic exchange (MAE), which enables hybridization of arbitrary (including pathogenic) strains of Escherichia coli. MAE reimplements the natural phenomenon of random cross-overs, enabling classical linkage analysis. We demonstrate the utility of MAE with virulence-related gain-of-function screens, discovering that transfer of a single operon from a uropathogenic strain is sufficient for enabling a commensal E. coli to form large intracellular bacterial collections within bladder epithelial cells. MAE thus enables assaying natural allelic variation in E. coli (and potentially other bacteria), complementing existing loss-of-function genomic techniques. [ABSTRACT FROM AUTHOR]

Xanthomonas citri pv. citri (Xcc), a gram-negative bacterium, is the causal agent of citrus canker, one of the most devastating diseases threatening the citrus industry worldwide. Understanding the diversity and population structure of Xcc is a prerequisite for disease epidemiological monitoring and effective disease management. Recent characterization of the clustered regularly interspaced short palindromic repeats (CRISPR)/cas (CRISPR-associated proteins genes) system with a highly variable repeat number among species provides a new molecular typing method for bacterial genetic analysis. In this study, we performed systematic in silico analyses of 28 Xcc genomes and identified a credible CRISPR/cas in Xcc strains. Further analysis of CRISPR polymorphisms (repeat number and spacer types) in 129 Xcc A strains collected from six provinces in China identified 15 types of CRISPR arrays with 25 spacers. Phylogenetic analysis of Xcc strains based on the CRISPR locus produced a more reliable and accurate typing result compared to the commonly used loci. In addition, seven associated cas genes--cas 1, cas 2, cas 3, cas 4, cas 5, cas 7 (csd 2), and cas 8 (csd 1)--were found located adjacent to the CRISPR array. BLAST results showed > 99% similarity of seven cas genes among Xcc strains. Homology analysis of spacer sequences showed that six spacers had possible phage/prophage origin. The characterization of the CRISPR/cas system among Xcc strains provided an updated strain typing method for Xcc diversity analysis and yielded a panoramic view of CRISPR evolution for further studies of Xcc -phage interactions. [ABSTRACT FROM AUTHOR]

The presence of hydrocarbons in the soil is considered one of the main problems of pollution. In our current study, eight samples isolated from soil saturated with hydrocarbons were taken from different areas of Baghdad, Iraq. In this study, 5 isolates belonging to Pseudomonas aeruginosa by 99%, 4 isolates to Klebsiella pneumoniae by 98%, and 3 isolates to Enterobacter hormaechei by 97% were diagnosed in different ways. A molecular examination was also conducted by 16sRNA. We recorded P. aeruginosa, K. Pneumoniae and E. hormaechei as new local isolates in NCBI. In addition, a comparison was made between our isolates and the global isolates to determine the degree of convergence in the evolutionary line. The genes alkB and nahAc7 were diagnosed in P. aeruginosa capable of degradation hydrocarbons. The aim of this study was to identify the bacterial species that resist the presence of hydrocarbons in the soil and also to diagnose some genes in the bacteria responsible for degradation of hydrocarbons in order to find the biological treatment methods. [ABSTRACT FROM AUTHOR]

Therapeutic advances in the 20th century significantly reduced tuberculosis (TB) mortality. Nonetheless, TB still poses a massive global health challenge with significant annual morbidity and mortality that has been amplified during the COVID-19 pandemic. Unlike most common bacterial infectious diseases, successful TB treatment requires months-long regimens, which complicates the ability to treat all cases quickly and effectively. Improving TB chemotherapy by reducing treatment duration and optimizing combinations of drugs is an important step to reducing relapse. In this review, we outline the limitations of current multidrug regimens against TB and have reviewed the genetic tools available to improve the identification of drug targets. The rational design of regimens that sterilize diverse phenotypic subpopulations will maximize bacterial killing while minimizing both treatment duration and infection relapse. Importantly, the TB field currently has all the necessary genetic and analytical tools to screen for and prioritize drug targets in vitro based on the vulnerability of essential and nonessential genes in the Mtb genome and to translate these findings in in vivo models. Combining genetic methods with chemical screens offers a formidable strategy to redefine the preclinical design of TB therapy by identifying powerful new targets altogether, as well as targets that lend new efficacy to existing drugs. [ABSTRACT FROM AUTHOR]

Guanine nucleotides are required for growth and viability of cells due to their structural role in DNA and RNA, and their regulatory roles in translation, signal transduction, and cell division. The natural antibiotic mycophenolic acid (MPA) targets the rate-limiting step in de novo guanine nucleotide biosynthesis executed by inosine-5´-monophosphate dehydrogenase (IMPDH). MPA is used clinically as an immunosuppressant, but whether in vivo inhibition of bacterial IMPDH (GuaB) is a valid antibacterial strategy is controversial. Here, we describe the discovery of extremely potent small molecule GuaB inhibitors (GuaBi) specific to pathogenic bacteria with a low frequency of on-target spontaneous resistance and bactericidal efficacy in vivo against Acinetobacter baumannii mouse models of infection. The spectrum of GuaBi activity includes multidrug-resistant pathogens that are a critical priority of new antibiotic development. Co-crystal structures of A. baumannii, Staphylococcus aureus , and Escherichia coli GuaB proteins bound to inhibitors show comparable binding modes of GuaBi across species and identifies key binding site residues that are predictive of whole-cell activity across both Gram-positive and Gram-negative clades of Bacteria. The clear in vivo efficacy of these small molecule GuaB inhibitors in a model of A. baumannii infection validates GuaB as an essential antibiotic target., Importance: The emergence of multidrug-resistant bacteria worldwide has renewed interest in discovering antibiotics with novel mechanism of action. For the first time ever, we demonstrate that pharmacological inhibition of de novo guanine biosynthesis is bactericidal in a mouse model of Acinetobacter baumannii infection. Structural analyses of novel inhibitors explain differences in biochemical and whole-cell activity across bacterial clades and underscore why this discovery may have broad translational impact on treatment of the most recalcitrant bacterial infections., Competing Interests: All authors were employed by Genentech, Inc., a member of the Roche Group.

The flagellar motors of Campylobacter jejuni (C. jejuni) and related Campylobacterota (previously epsilonproteobacteria) feature 100-nm-wide periplasmic "basal disks" that have been implicated in scaffolding a wider ring of additional motor proteins to increase torque, but the size of these disks is excessive for a role solely in scaffolding motor proteins. Here, we show that the basal disk is a flange that braces the flagellar motor during disentanglement of its flagellar filament from interactions with the cell body and other filaments. We show that motor output is unaffected when we shrink or displace the basal disk, and suppressor mutations of debilitated motors occur in flagellar-filament or cell-surface glycosylation pathways, thus sidestepping the need for a flange to overcome the interactions between two flagellar filaments and between flagellar filaments and the cell body. Our results identify unanticipated co-dependencies in the evolution of flagellar motor structure and cell-surface properties in the Campylobacterota., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Therapeutic advances in the 20th century significantly reduced tuberculosis (TB) mortality. Nonetheless, TB still poses a massive global health challenge with significant annual morbidity and mortality that has been amplified during the COVID-19 pandemic. Unlike most common bacterial infectious diseases, successful TB treatment requires months-long regimens, which complicates the ability to treat all cases quickly and effectively. Improving TB chemotherapy by reducing treatment duration and optimizing combinations of drugs is an important step to reducing relapse. In this review, we outline the limitations of current multidrug regimens against TB and have reviewed the genetic tools available to improve the identification of drug targets. The rational design of regimens that sterilize diverse phenotypic subpopulations will maximize bacterial killing while minimizing both treatment duration and infection relapse. Importantly, the TB field currently has all the necessary genetic and analytical tools to screen for and prioritize drug targets in vitro based on the vulnerability of essential and non-essential genes in the Mtb genome and to translate these findings in in vivo models. Combining genetic methods with chemical screens offers a formidable strategy to redefine the preclinical design of TB therapy by identifying powerful new targets altogether, as well as targets that lend new efficacy to existing drugs.

Bifidobacterium is a commensal bacterial genus ubiquitous in the human gastrointestinal tract, which is associated with a range of health benefits. The advent of CRISPR based genome editing technologies provides opportunities to investigate the genetics of important bacteria and transcend the lack of genetic tools in bifidobacteria to study the basis for their health-promoting attributes. Here, we repurpose the endogenous type I-G CRISPR-Cas system and adopt an exogenous CRISPR base editor for genome engineering in B. animalis subsp. lactis, demonstrating that both genomic and epigenetic contexts drive editing outcomes across strains. We reprogrammed the endogenous type I-G system to screen for naturally occurring large deletions up to 27 kb and to generate a 500-bp deletion in tetW to abolish tetracycline resistance. A CRISPR-cytosine base editor was optimized to install C•G-to-T•A amber mutations to resensitize multiple B. lactis strains to tetracycline. Remarkably, we uncovered epigenetic patterns that are distributed unevenly among B. lactis strains, despite their genomic homogeneity, that may contribute to editing efficiency variability. Insights were also expanded to Bifidobacterium longum subsp. infantis to emphasize the broad relevance of these findings. This study highlights the need to develop individualized CRISPR-based genome engineering approaches for distinct bacterial strains and opens avenues for engineering of next generation probiotics. [ABSTRACT FROM AUTHOR]