Nontyphoidal strains of are a major cause of foodborne illnesses, and infection with these bacteria results in inflammatory gastroenteritis. Polymorphonuclear leukocytes (PMNs), also known as neutrophils, are a dominant immune cell type found at the site of infection in infected individuals, but how they regulate infection outcome is not well understood. Here, we used a co-culture model of primary human PMNs and human intestinal organoids to probe the role of PMNs during infection with two of the most prevalent serovars: serovar Enteritidis and Typhimurium. Using a transcriptomics approach, we identified a dominant role for PMNs in mounting differential immune responses including production of pro-inflammatory cytokines, chemokines, and antimicrobial peptides. We also identified specific gene sets that were induced by PMNs in response to Enteritidis or Typhimurium infection. By comparing host responses to these serovars, we uncovered differential regulation of host metabolic pathways particularly induction of cholesterol biosynthetic pathways during Typhimurium infection and suppression of RNA metabolism during Enteritidis infection. Together, these findings provide insight into the role of human PMNs in modulating different host responses to pathogens that cause similar disease in humans.IMPORTANCENontyphoidal serovars of are known to induce robust recruitment of polymorphonuclear leukocytes (PMNs) in the gut during early stages of infection, but the specific role of PMNs in regulating infection outcome of different serovars is poorly understood. Due to differences in human infection progression compared to small animal models, characterizing the role of PMNs during infection has been challenging. Here, we used a co-culture model of human intestinal organoids with human primary PMNs to study the role of PMNs during infection of human intestinal epithelium. Using a transcriptomics approach, we define PMN-dependent reprogramming of the host response to , establishing a clear role in amplifying pro-inflammatory gene expression. Additionally, the host response driven by PMNs differed between two similar nontyphoidal serovars. These findings highlight the importance of building more physiological infection models to replicate human infection conditions to study host responses specific to individual pathogens.

is an obligate intracellular bacterial pathogen that replicates to high numbers in an acidified lysosome-derived vacuole. Intracellular replication requires the Dot/Icm type IVB secretion system, which translocates over 100 different effector proteins into the host cell. Screens employing random transposon mutagenesis have identified several effectors that play an important role in intracellular replication; however, the difficulty in conducting directed mutagenesis has been a barrier to the systematic analysis of effector mutants and to the construction of double mutants to assess epistatic interactions between effectors. Here, two CRISPR-Cas9 technology-based approaches were developed to study phenotypes resulting from targeted gene disruptions. CRISPRi was used to silence gene expression and demonstrated that silencing of effectors or Dot/Icm system components resulted in phenotypes similar to those of transposon insertion mutants. A CRISPR-Cas9-mediated cytosine base editing protocol was developed to generate targeted loss-of-function mutants through the introduction of premature stop codons into genes. Cytosine base editing successfully generated double mutants in a single step. A double mutant deficient in both and had a robust and additive intracellular replication defect when compared to either single mutant, which is consistent with Cig57 and Cig2 functioning in independent pathways that both contribute to a vacuole that supports replication. Thus, CRISPR-Cas9-based technologies expand the genetic toolbox for and will facilitate genetic studies aimed at investigating the mechanisms this pathogen uses to replicate inside host cells.

Previous studies in Ghana indicated low prevalence of methicillin-resistant (MRSA) and predominance of ST152 methicillin-susceptible (MSSA) among clinical isolates. ST152 MRSA clones are associated with severe infections and epidemics. Using whole genome sequencing (WGS), 159 . isolated from clinical sources (wound, blood, urine, ear, abscess, umbilical cord, eye, vaginal samples, and others) from 10 hospitals across Ghana were investigated. (gene for methicillin resistance) was detected in 38% of the isolates. Panton-Valentine leucocidin toxin (PVL) gene occurred in 65% isolates, with 84% of the MRSA's harboring the PVL gene. ST152 was the major clone, with 74% harboring the gene. Other MRSA clones detected were ST5, ST5204, ST852, and ST1. MSSA clones included ST3249, ST152, ST5, ST1, and ST8. Twenty-three genes encoding resistance to 12 antimicrobial classes were observed with (97%) being the most prevalent. Other predominant resistance genes included (46%), (42%), and (36%) encoding resistance for tetracyclines, phenicols, and diaminopyrimidine, respectively. Virulence genes for enterotoxins, biofilms, toxic-shock-syndrome toxins, hemolysins and leukotoxins were also detected. Phylogenetic analysis revealed a shift in the dominant clone from MSSA ST152 to MRSA ST152 over the past decade. The study provides valuable insights into the genomic content of from clinical sources in Ghana. The finding of ST152 MRSA in high numbers suggests a shifting epidemiological landscape of these pathogens and continuous surveillance using robust tools like WGS is needed to monitor the rise and spread of these epidemic clones in the country.IMPORTANCESince its emergence in 1959, MRSA has been a significant public health concern, causing infections in both clinical and community settings. Patients with MRSA-related infections experience higher mortality rates due to its ability to evade antimicrobials and immune defenses. In Ghana, understanding the molecular epidemiology of MRSA has been hindered by the lack of appropriate laboratory infrastructure and the limited capacity for molecular data analysis. This study, the largest genomic study of in Ghana, addresses this gap by utilizing whole genome sequencing to examine the diversity of circulating strains from 10 hospitals. Our findings highlight the predominance of pandemic clones, particularly ST152, and the notable transition of ST152 MSSA to ST152 MRSA over the past decade. The findings from this study supports AMR surveillance efforts in Ghana and emphasize the importance of implementing genomic surveillance using WGS to comprehensively monitor the rise and spread of multi-drug-resitant organisms such as MRSA in the country.

Enzyme-IIA (EIIA, Crr) of the phosphotransferase system (PTS) connects the uptake of glucose-family sugars to the cAMP-Crp regulatory cascade; phosphorylated EIIA enhances cAMP-Crp activity, which then contributes to the antibiotic-mediated accumulation of reactive oxygen species (ROS) and cell death. Defects in PTS cause antibiotic and disinfectant tolerance. We report that mannitol, a carbon source whose uptake does not use EIIA, reduces antibiotic-mediated killing of without affecting antibiotic minimal inhibitory concentration. Thus, mannitol promotes antibiotic tolerance. The tolerance pathway was defined by the loss of ciprofloxacin lethality from the deletion of (first gene in PTS), (mannitol-specific Enzyme-II), (cAMP synthase), and (cAMP receptor protein) but not (EIIA). A mutant, which encodes a constitutively active Crp that bypasses the need for cAMP activation, also decreased mannitol-mediated antibiotic tolerance, as did exogenous cAMP. Thus, inhibition of antibiotic lethality by mannitol involves both PTS-mediated mannitol uptake and suppression of cAMP-Crp action, independent of EIIA. Mannitol suppressed the downstream antibiotic-mediated transcription of genes involved in NADH production and cellular respiration, expression of a superoxide reporter gene (), and accumulation of antibiotic-mediated ROS. Similar phenomena were observed with mannose and sorbitol, demonstrating that non-glucose PTS carbon sources can cause antibiotic tolerance by a novel path that reduces the ROS-promoting activity of cAMP-Crp. The work emphasizes that antibiotic tolerance, which contributes to disease relapse and the need for prolonged antibiotic treatment, can result from commonly consumed carbohydrates. This finding, plus mutations that interfere specifically with antibiotic lethality, makes tolerance a high probability event.IMPORTANCEBacterial tolerance constitutes a significant threat to anti-infective therapy and potentially to the use of disinfectants. Deficiency mutations that reduce glucose uptake, central carbon metabolism, and cellular respiration confer antibiotic/disinfectant tolerance by reducing the accumulation of reactive metabolites, such as reactive oxygen species. We identified novel environmental generators of tolerance by showing that non-glucose carbohydrates, such as mannitol, mannose, and sorbitol, generate tolerance to multiple antibiotic classes. Finding that these sugars inhibit a universal, stress-mediated death pathway emphasizes the potential danger of compounds that block the lethal response to severe stress. Immediate practical importance derives from mannitol being a popular food sweetener, a treatment for glaucoma, and a dehydrating agent for treating cerebral edema, including cases caused by bacterial infection: antibiotic tolerance could contra-indicate the use of mannitol and related carbohydrates during antibiotic treatment. Overall, the work shows that the presence of sugars must be considered during antimicrobial and perhaps disinfectant use.

Recombinant chimeric horsepox virus (TNX-801) is a preclinical vaccine in development against mpox and smallpox. In this report, we investigated the potential phenotypic differences in and models between TNX-801 and older vaccinia virus (VACV)-based vaccine strains (VACV-Lis and VACV-NYCBH) used in the eradication of smallpox as well as VACV-WR, VACV-IHD, and MVA. TNX-801 displayed a small plaque phenotype (~1-2 mm) in BSC-40 and Vero-E6 cells. Multi-step replication kinetics in immortalized nonhuman primate cell lines, and human primary cells from dermal and respiratory tracts yielded >10- to 100-fold lower infectious titers than the VACV strains. In addition, the infectious particle-to-genome copy ratio data suggests that TNX-801 genome packaging is ~10- to 100-fold less efficient than the VACV strains and the potential mechanism of TNX-801 attenuation is at the packaging/egress stage. Lastly, the susceptibility to VACV and TNX-801 infection of three new immunocompromised murine models (C56BL/6 , C56BL/6 , and C56BL/6 /) was investigated. VACV strains were able to produce severe disease including decrease in body weight and temperature, as well as lethality in murine models via the intraperitoneal or intranasal routes. In contrast to VACV strains, TNX-801 was unable to produce any disease in murine models. These data demonstrate that TNX-801 is >10- to 1,000-fold more attenuated compared to older VACV-based smallpox vaccine strains in human primary cell lines and immunocompromised mice.

The plasma membrane is critical for the virulence of the human fungal pathogen . In addition to functioning as a protective barrier, the plasma membrane plays dynamic roles in a wide range of functions needed for virulence including nutrient uptake, cell wall synthesis, morphogenesis, resistance to stress, and invasive hyphal growth. Screening a collection of mutants identified an understudied gene that is important for invasive hyphal growth, which we have termed (Cell Wall Regulatory kinase). A mutant strain lacking displayed defects in resisting stressful conditions that exacerbate cell wall defects. The Cwr1 protein shows strong similarity to protein kinases, suggesting it plays a regulatory role in coordinating plasma membrane and cell wall functions. A Cwr1-green fluorescent protein (GFP) fusion protein localized to punctate patches associated with the plasma membrane that partially overlapped Membrane Compartment of Can1 (MCC)/eisosome domains. In contrast to the static MCC/eisosome domains, the Cwr1-GFP patches were very dynamic. Truncation mutants lacking C-terminal sequences distal to the protein kinase domain failed to show detectable localization at the plasma membrane. Surprisingly, these mutant strains did not show the defects of a mutant, suggesting that localization to punctate patches associated with the plasma membrane is not essential for Cwr1 function. Altogether, these data indicate that Cwr1 contributes to the regulation of plasma membrane functions that promote proper morphogenesis and resistance to cell wall stress, both of which are important for virulence.

Motile flagella (also called "motile cilia") play a variety of important roles in lower and higher eukaryotes, including cellular motility and fertility. Flagellar motility is driven by several species of the gigantic motor-protein complexes, flagellar dyneins, that reside within these organelles. Among the flagellar-dynein species, a hetero-dimeric dynein called "IDA f/I1" has been shown to be particularly important in controlling the flagellar waveform, and defects in this dynein species in humans cause ciliopathies such as multiple morphological abnormalities of the flagella and asthenoteratozoospermia. IDA f/I1 is composed of many subunits, including two HCs (HCα and HCβ) and three ICs (IC140, IC138, and IC97), and among the three ICs of IDA f/I1, the exact molecular function(s) of IC97 in flagellar motility is not well understood. In this study, we isolated a mutant lacking IC97 and analyzed the phenotypes. The mutant phenocopied several aspects of the previously isolated IDA-f/I1-related mutants in and showed slow swimming compared to the wild type but retained the ability to phototaxis. Further analysis revealed that the mutant had low flagellar beat frequency and miscoordination between the two (- and -) flagella. In addition, the mutant cells swam in a comparatively straight path compared to the wild-type cells. Taken together, our results highlight the importance of proper assembly of IC97 in the IDA-f/I1 complex for the regulation of flagellar and cellular motility in and provide valuable insights into both the molecular functions of IC97 orthologs in higher eukaryotes and the pathogenetic mechanisms of human ciliopathies caused by IDA-f/I1 defects.

Mpox disease, caused by the monkeypox virus (MPXV), was recently classified as a public health emergency of international concern due to its high lethality and pandemic potential. MPXV is a zoonotic disease that emerged and is primarily spread by small rodents. Historically, it was considered mainly zoonotic and not likely to sustain human-to-human transmission. However, the worldwide outbreak of Clade IIb MPXV from 2020 to 2022 and ongoing Clade I MPXV epidemics in the Democratic Republic of the Congo and surrounding areas are a warning that human-adapted MPXVs will continually arise. Understanding the viral genetic determinants of host range, pathogenesis, and immune evasion is imperative for developing control strategies and predicting the future of Mpox. Here, we delve into the MPXV genome to detail genes involved in host immune evasion strategies for this zoonotic rodent-borne and human-circulating virus. We compare MPXV gene content to related Orthopoxviruses, which have narrow host ranges, to identify potential genes involved in species-specific pathogenesis and host tropism. In addition, we cover the key virulence factor differences that distinguish the MPXV clade lineages. Finally, we dissect how genomic reduction of Orthopoxviruses, through various molecular mechanisms, is contributing to the generation of novel MPXV lineages with increased human adaptation. This review aims to highlight gene content that defines the MPXV species, MPXV clades, and novel MPXV lineages that have culminated in this virus being elevated to a public health emergency of national concern.

Bacteria assume two distinct lifestyles: the planktonic and biofilm modes of growth. Additionally, dispersion has emerged as a third phenotype, accompanied by the distinct phenotypes and the unique expression of >600 genes. Here, we asked whether the distinct phenotype of dispersed cells is already apparent within minutes of egressing from the biofilm. We used RNA-seq to show that the physiology of freshly dispersed cells from biofilms is highly different from those of planktonic and biofilm cells, apparent by dispersed cells uniquely expressing 194 genes. Unique and differentially expressed genes relative to planktonic or biofilm cells include genes associated with type IV pili, pyoverdine, type III and type VI secretion systems, and antibiotic resistance that are downregulated in dispersed cells, whereas the transcript abundance of genes involved in swimming motility, Hxc type II secretion system and various other virulence factors, and metabolic and energy-generating pathways are increased, indicative of dispersion coinciding with an awakening and re-energizing of dispersed cells, and a switch in virulence, further apparent by freshly dispersed cells significantly subverting engulfment by macrophages. The findings suggest that dispersed cells display a distinct phenotype within minutes of egressing from the biofilm, with freshly dispersed cells already capable of efficiently evading phagocytosis.

is an opportunistic human fungal pathogen that causes superficial mucosal and life-threatening bloodstream infections in immunocompromised individuals. Remodeling in cell wall components has been extensively exploited by fungal pathogens to adapt to host-derived stresses, as well as immune evasion. How this process contributes to pathogenicity is less understood. Here, we applied RNA sequencing and an invasive infection model to elucidate the prompt response of during infection. Fungal transcriptomes show a dramatic alteration in the expression of Srp1/Tip1-family cell wall structural proteins during systemic infection. Deletion of all six genes in this family ( and ) that are upregulated during infection leads to a significantly lower fungal burden in organs, as well as an attenuated virulence in the dextran sulfate sodium-induced colitis model. The sextuple mutant does not display any defect in response to host-derived stresses. Rather, deletion of all these six genes results in a lower cell surface exposure of an adhesin Epa1, which could contribute to its reduced adhesion to epithelial cells and cytotoxicity, as well as attenuated virulence. Our study reveals that cell wall remodeling triggered by the alteration in the expression of structural proteins is a key virulence attribute in that facilitates this fungus adhering to host cells and persisting in organs.IMPORTANCE is one of the most frequent causes of candidiasis after . While has been extensively studied, the mechanisms of infection and invasion of have not been fully elucidated. Using an infection model of systemic candidiasis and RNA sequencing, we show that there is a dramatic change in the expression of Srp1/Tip1-family genes during infection. Deletion of all six Srp1/Tip1-family genes that are upregulated during infection decreases the amount of cell wall-localized Epa1, probably reflecting the reduced adherence to epithelial cells and attenuated virulence in the sextuple mutant. These data suggest that alterations in the expression of Srp1/Tip1-family structural proteins trigger cell wall remodeling that increases the cell surface exposure of adhesins, such as Epa1, to promote virulence. Our study provides a pathogenic mechanism associated with in ensuring its sustenance and survival during infection.

is a genus of saprophytic fungi with global distribution. Two species complexes, and , pose health risks to humans and animals. Cryptococcal infections result from inhalation of aerosolized spores and/or desiccated yeasts from terrestrial reservoirs such as soil and trees. More recently, has been implicated in infections in marine mammals, suggesting that inhalation of cells from the air-water interface is also an important, yet understudied, mode of respiratory exposure. Based on historical records and epidemiological factors, water transport has been hypothesized to play a role in the spread of from tropical to temperate environments. However, the dynamics of fungal persistence and transport in water have not been fully studied. The size of the cryptococcal capsule was previously shown to reduce cell density and increase buoyancy. Here, we demonstrate that cell buoyancy is also impacted by the salinity of the solution in which cells are suspended, with the formation of a halocline significantly slowing the rate of settling and resulting in persistence of within 1 cm of the water surface for over 60 min and for 4-6 h. During the culture of three strains of in yeast peptone dextrose media, we also identified aggregates of extracellular polysaccharide with complex structures, which we hypothesize from rafts that entrap cells and augment buoyancy. These findings illustrate new mechanisms by which cryptococcal cells may persist in aquatic environments, with important implications for aqueous transport and pathogen exposure.

Genes encoding lipid A modifying phosphoethanolamine transferases (PETs) are genetically diverse and can confer resistance to colistin and antimicrobial peptides. To better understand the functional diversity of PETs, we characterized three canonical mobile colistin resistance () alleles (, , ), one intrinsic (), and two -like genes (, ) in . Using an isogenic expression system, we show that and confer similar phenotypes of decreased colistin susceptibility with low fitness costs. , which is phylogenetically closely related to , and only provide fitness advantages in the presence of sub-inhibitory concentrations of colistin and significantly reduce fitness in media without colistin. PET-B and PET-C were phenotypically distinct from bonafide PETs; neither impacted colistin susceptibility nor caused considerable fitness cost. Strikingly, we found for the first time that different PETs selectively modify different phosphates of lipid A; MCR-1, MCR-3, and PET-C selectively modify the 4'-phosphate, whereas MCR-9 and EptA modify the 1-phosphate. However, 4'-phosphate modifications facilitated by MCR-1 and -3 are associated with lowered colistin susceptibility and low toxicity. Our results suggest that PETs have a wide phenotypic diversity and that increased colistin resistance is associated with specific lipid A modification patterns that have been largely unexplored thus far.

Inhalation of prions into the nasal cavity is an efficient route of infection. Following inhalation of infectious prions, animals develop disease with a similar incubation period compared with per os exposure, but with greater efficiency. To identify the reason for this increased efficiency, we identified neural structures that uniquely innervate the nasal cavity and neural structures known to mediate neuroinvasion following oral infection and used immunohistochemistry to determine the temporal and spatial accumulation of prions from hamster tissue sections containing cell bodies and axons at 2-week intervals following prion exposure. Prions were identified in the trigeminal ganglion, the spinal trigeminal tract in the brainstem, the intermediolateral cell column of the thoracic spinal cord, and the dorsal motor nucleus of the vagus/solitary nucleus complex months prior to detection of prions in the olfactory bulb or superior cervical ganglion. These results indicate that the trigeminal nerve, but not the olfactory nerve or sympathetic nerves, are involved in neuroinvasion following inhalation of prions into the nasal cavity. The detection of prions in the intermediolateral cell column of the thoracic spinal cord and dorsal motor nucleus of the vagus nerve 14 weeks following inhalation is consistent with inoculum crossing the alimentary wall and infecting the enteric nervous system via this route of infection. Neuroinvasion via the trigeminal nerve, in combination with entry into the central nervous system via autonomic innervation of the enteric nervous system, may contribute to increased efficiency of nasal cavity exposure to prions compared with per os exposure in hamsters.IMPORTANCEInhalation of prions into the nasal cavity is thought to be a route of infection in naturally acquired prion diseases. Experimental studies indicate that inhalation of prions is up to two orders of magnitude more efficient compared with ingestion. The mechanisms underlying this observation are poorly understood. We found a previously unreported direct route of neuroinvasion from the nasal cavity to the nervous system. Importantly, the peripheral ganglia involved may be a useful tissue to sample for prion diagnostics. Overall, identification of a new route of neuroinvasion following prion infection may provide an anatomical basis to explain the increased efficiency of infection following prion inhalation.

Incidences of fluconazole (FLC) resistance among clinical isolates are a growing issue in clinics. The pleiotropic drug response network in confers azole resistance and is defined primarily by the ZnCys zinc cluster-containing transcription factor Pdr1 and target genes such as , which encodes an ATP-binding cassette transporter protein thought to act as an FLC efflux pump. Mutations in the gene that render the transcription factor hyperactive are the most common cause of fluconazole resistance among clinical isolates. The phenothiazine class drug fluphenazine and a molecular derivative, CWHM-974, which both exhibit antifungal properties, have been shown to induce the expression of Cdr1 in spp. We have used a firefly luciferase reporter gene driven by the promoter to demonstrate two distinct patterns of promoter activation kinetics: gradual promoter activation kinetics that occur in response to ergosterol limitations imposed by exposure to azole and polyene class antifungals and a robust and rapid induction occurring in response to the stress imposed by fluphenazines. We can attribute these different patterns of induction as proceeding through the promoter region of this gene since this is the only segment of the gene included in the luciferase reporter construct. Genetic analysis indicates that the signaling pathways responsible for phenothiazine and azole induction of overlap but are not identical. The short time course of phenothiazine induction suggests that these compounds may act more directly on the Pdr1 protein to stimulate its activity.

Coral microbiomes play a crucial role in maintaining the health and functionality of holobionts. Disruption in the equilibrium of holobionts, including bacteria, fungi, and archaea, can result in the bleaching of coral. However, little is known about the viruses that can infect holobionts in coral, especially bacteriophages. Here, we employed a combination of amplicon and metagenomic analyses on and to investigate the diversity and functionality of viruses in healthy and bleached corals. Analysis showed that the alpha diversity of holobionts (bacteria, eukaryotes, zooxanthellae, and lysogenic and lytic viruses) was higher in bleached corals than that in healthy corals. Meanwhile, bleached corals exhibited a relatively higher abundance of specific viral classes, including Revtraviricetes, Arfiviricetes, Faserviricetes, Caudoviricetes, Herviviricetes, and Tectiliviricetes; moreover, we found that the expression levels of functional genes involved in carbon and sulfur metabolism were enriched. An increase in abundance has been reported as a notable factor in coral bleaching; our analysis also revealed an increased abundance of in bleached coral. Finally, bleached corals contained a higher abundance of phages and encoded more virulence factor genes to increase the competitiveness of after coral bleaching. In conclusion, we attempted to understand the causes of coral bleaching from the perspective of phage-bacteria-coral tripartite interaction.

Porcine epidemic diarrhea virus (PEDV), the major causative pathogen of porcine epidemic diarrhea, poses a severe threat to the swine industry, particularly affecting neonatal piglets. Maternal milk-derived IgA antibody is crucial for protecting piglets from PEDV infection. Despite the effectiveness of current intramuscularly administered PEDV vaccines in inducing strong systemic immune responses, their ability to generate high levels of maternal milk IgA is limited. This study explores the potential of Astragalus polysaccharide (APS) to enhance PEDV vaccine efficacy, specifically focusing on maternal milk IgA levels. We first evaluated anti-PEDV antibody levels in the blood and colostrum of sows vaccinated with PEDV or subjected to feedback feeding. Our results indicated that while vaccination induced robust serum PEDV-specific IgG and IgA, milk IgA levels were lower compared to the feedback group. To address this limitation, APS was administered orally to sows before PEDV vaccination. APS supplementation significantly increased both serum and milk PEDV-specific IgA levels and enhanced cellular immune responses, as evidenced by elevated cytokine levels. Further analysis demonstrated that APS improved intestinal immune function and homeostasis in piglets. Overall, APS supplementation proved to be an effective immune booster, enhancing PEDV vaccine-induced mucosal immunity and providing a promising strategy for improving maternal immunity and piglet protection against PEDV.

Bacterial symbionts are critical members of many marine sponge holobionts. Some sponge-associated bacterial lineages, such as Poribacteria, sponge-associated unclassified lineage (SAUL), and Tethybacterales, appear to have broad-host ranges and associate with a diversity of sponge species, while others are more species-specific, having adapted to the niche environment of their host. Host-associated spirochete symbionts that are numerically dominant have been documented in several invertebrates including termites, starfish, and corals. However, dominant spirochete populations are rare in marine sponges, having thus far been observed only in and various species within the Latrunculiidae family, where they are co-dominant alongside Tethybacterales symbionts. This study aimed to characterize these spirochetes and their potential role in the host sponge. Analysis of metagenome-assembled genomes from eight latrunculid sponges revealed that these unusual spirochetes are relatively recent symbionts and are phylogenetically distinct from other sponge-associated spirochetes. Functional comparative analysis suggests that the host sponge may have selected for these spirochetes due to their ability to produce terpenoids and/or possible structural contributions.IMPORTANCESouth African latrunculid sponges are host to co-dominant Tethybacterales and Spirochete symbionts. While the Tethybacterales are broad-host range symbionts, the spirochetes have not been reported as abundant in any other marine sponge except . However, spirochetes are regularly the most dominant populations in marine corals and terrestrial invertebrates where they are predicted to serve as beneficial symbionts. Here, we interrogated eight metagenome-assembled genomes of the latrunculid-associated spirochetes and found that these symbionts are phylogenetically distinct from all invertebrate-associated spirochetes. The symbiosis between the spirochetes and their sponge host appears to have been established relatively recently.

Comparative genomics of predatory bacteria is important to understand their ecology and evolution and explore their potential to treat drug-resistant infections. We compared chromosomes of 18 obligate predators from phylum (16 intraperiplasmic, two epibiotic) and 15 non-predatory bacteria. Phylogenetics of conserved single-copy genes and analysis of genome-wide average amino acid identity provide evidence for at least five species and support recent reclassifications of predatory taxa. To define shared and differential genome content, we grouped predicted protein sequences into gene clusters based on sequence similarity. Few gene clusters are shared by all 33 bacteria or all 18 predatory bacteria; however, we identified gene clusters conserved within lineages, such as intraperiplasmic , and not found in other bacteria. Many of these are predicted to function in cell envelope biogenesis, signal transduction, and other roles important for predatory lifestyles. Among intraperiplasmic , we detected high abundance of gene clusters predicted to encode transglycosylases, endopeptidases, and lysozymes, and we identified six gene clusters (amidase, L,D-transpeptidase, four transglycosylases) with evidence of recent gene duplication and gene family expansion. Focusing on peptidoglycan metabolism, we defined a suite of gene clusters that include peptidoglycan-degrading and -modifying enzymes and occur only in predatory bacteria, suggesting these proteins may have evolved activities specific to predation. Our analyses highlight key genome content differences between obligate predatory bacteria and non-predatory relatives and identify gene clusters that may encode enzymes adapted to predatory lifestyles. These lineage-specific proteins are strong candidates for functional characterization to clarify their role in predation.IMPORTANCEEvolution of predation as a bacterial lifestyle involves selective pressure on and adaptation of enzymes that contribute to killing and digestion of prey bacteria, in some cases from within the prey itself. Such enzymes are a hallmark of obligate predatory bacteria belonging to phylum , which includes the well-studied predator . By comparing protein sequences of obligate predatory bacteria and their non-predatory relatives, we define key genome content differences that distinguish bacterial predators and identify lineage-specific enzymes that may have evolved unique activities due to selective pressures related to a predatory lifestyle. In addition to providing insights into the ecology and evolution of predatory bacteria, comparative genomics studies, like this, can inform efforts to develop predatory bacteria and/or their enzymes as potential biocontrol agents to combat drug-resistant bacterial infections.

Acquired antimicrobial resistance and metabolic changes are central for bacterial host adaptation during the long-term hospitalization of patients. We aimed to analyze the genomic and phenotypic evolution of enteric populations in long-term intensive care unit (ICU) patients. Weekly rectal swabs were prospectively collected from all patients admitted to the ICU in a teaching hospital from December 2018 to February 2019. The inclusion criterion for patients was hospitalization for more than 15 days in the ICU without any history of hospitalization or antibiotic treatment for the 3 months prior to admission. Among them, enteric species complex (KpSC) populations were detected. For each isolate, extensive antimicrobial resistance profiles were determined using the disk diffusion method, and the whole genome was sequenced using an Illumina platform. typing methods, such as Multilocus Sequence Typing (MLST), core-genome MLST, SNP typing, resistome characterization and mutation point detection, were applied. During the study period, 471 patients were admitted to ICUs. Among them, 21 patients met the inclusion criteria, and only 5 patients (24%) carried unique and distinct KpSC populations during 2-10 weeks in the gut that as detected at admission and excluding acquisition during the ICU stay. One patient showed a rare ST1563 persistent carriage for 7 consecutive weeks, which displayed important antimicrobial resistance phenotype changes in the 2 last weeks. In-depth characterization and RNA sequencing of these strains revealed a mutation within the transcriptional regulator resulting in overexpression of the regulator and decreased expression of , which controls antibiotic efflux. This mutation also impacts tolerance to biliary salts. This study revealed the importance of endogenous colonization of KpSC populations in the gut throughout the patient's long-term ICU stay and highlighted the role of in drug susceptibility.