Search Results (61)

This study investigates the occurrence, antimicrobial resistance (AMR) profiles, virulence factors, and plasmid composition of Enterococcus species isolated from salad ingredients in the United Arab Emirates (UAE). Four hundred salad vegetable items collected from local markets, over ten months through 2023, were screened, yielding an Enterococcus detection rate of 85.5% (342/400). E. casseliflavus was the most commonly identified species (50%), followed by E. faecium (20%) and E. faecalis (16%). Among 85 Enterococcus isolates tested for antimicrobial susceptibility, 55.3% displayed resistance to at least one agent, with 18.8% classified as multidrug-resistant (MDR). All isolates were not resistant to ampicillin, linezolid, teicoplanin, tigecycline, and high-level gentamicin. Intrinsic phenotypic resistance to vancomycin was found in E. gallinarum and E. casseliflavus, while low-level (<5%) ciprofloxacin and erythromycin resistance was sporadically detected in E. faecium and E. faecalis. Whole-genome sequencing (WGS) of 14 isolates (nine E. faecium, four E. faecalis, and one E. casseliflavus) unveiled a complex resistome. We report the first detection in salad vegetables of vancomycin resistance genes (vanC, vanXY-C2) in a vancomycin-susceptible E. faecalis isolate. Identifying tetM, ermB, and optrA genes in the studied isolates further underscored emerging resistance to tetracyclines, macrolides, and oxazolidinones. Concurrently, virulence gene analysis revealed 74 putative virulence factors, with E. faecalis harboring a higher diversity of biofilm-related and exoenzyme-encoding genes. One E. faecalis strain carried the cytolysin cluster (cylI, cylS, cylM), highlighting its pathogenic potential. Plasmid profiling identified 19 distinct plasmids, ranging from 3845 bp to 133,159 bp. Among the genome-sequenced isolates, mobilizable plasmids (47.3%) commonly carried AMR genes, especially tet(L) and tet(M), whereas conjugative plasmids (10.5%) did not harbor resistance determinants. These findings highlight that salad vegetables can still harbor and potentially transmit Enterococcus strains with clinically relevant resistance determinants and virulence traits. Enhancing foodborne AMR surveillance with WGS and targeted interventions is key to controlling its spread in the food. Full article

Perfringolysin O (PFO) is a prototypical member of a large family of pore-forming toxins (PFTs) that are potent virulence factors for many pathogenic bacteria. One of the most enigmatic properties of these PFTs is how structural changes are coordinated between different subunits within a single complex. Moreover, there are conflicting data in the literature, with gel electrophoresis results apparently showing that pores are only complete rings, whereas microscopy images clearly also show incomplete-ring pores. Here, we developed a novel multi-stack gel electrophoretic assay to finely separate PFO pore complexes and found that this assay indeed resolves both complete- and incomplete-ring pores. However, unexpectedly, we found that the stoichiometries of these complexes are predominantly integral multiples of six subunits. High-resolution atomic force microscopy images of PFO pore complexes also reveal a predominant hexameric-based stoichiometry. We also observed this hexameric-based stoichiometry at the prepore stage and identified a mutant that is kinetically trapped at a hexameric state. Thus, overall, these results reveal a previously unknown hexameric-based structural hierarchy in the PFO complexes. We suggest that the structural coordination within the hexamers is different than between the hexamers and is thus a critical feature of the structural coordination of the complex as a whole. Full article

Enterococcus faecalis (E. faecalis) is a ubiquitous microbe occurring in the environment and in the intestinal tract of poultry. E. faecalis has been identified in cases of egg infertility and/or decreased hatchability and can cause amyloid arthropathy in older laying chickens. E. faecalis produces cytolysin, a bacterial exotoxin that can cause lysis of erythrocytes. It has been difficult to demonstrate this virulence trait using conventional culture methods with sheep blood agar. A 96-well microplate hemolysis assay, along with a culture method incorporating glucose and L-arginine into the culture media, is described that demonstrates the production of cytolysin in E. faecalis isolates of avian origin. Additionally, the results show that horse and sheep erythrocytes were susceptible to lysis by the E. faecalis cytolysin, but cow and chicken erythrocytes were less susceptible. Full article

Contamination of leafy greens with Staphylococcus spp. can occur at various supply chain stages, from farm to table. This study comprehensively analyzes the species diversity, antimicrobial resistance, and virulence factors of Staphylococci in salad vegetables from markets in the United Arab Emirates (UAE). A total of 343 salad items were sampled from three major cities in the UAE from May 2022 to February 2023 and tested for the presence of Staphylococcus spp. using standard culture-based methods. Species-level identification was achieved using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Antimicrobial susceptibility testing was conducted using the VITEK-2 system with AST-P592 cards. Additionally, whole genome sequencing (WGS) of ten selected isolates was performed to characterize antimicrobial resistance determinants and toxin-related virulence factors. Nine Staphylococcus species were identified in 37.6% (129/343) of the tested salad items, with coagulase-negative staphylococci (CoNS) dominating (87.6% [113/129]) and S. xylosus being the most prevalent (89.4% [101/113]). S. aureus was found in 4.6% (14/343) of the salad samples, averaging 1.7 log10 CFU/g. One isolate was confirmed as methicillin-resistant S. aureus, harboring the mecA gene. It belonged to multi-locus sequence type ST-672 and spa type t384 and was isolated from imported fresh dill. Among the characterized S. xylosus (n = 45), 13.3% tested positive in the cefoxitin screen test, and 6.6% were non-susceptible to oxacillin. WGS analysis revealed that the cytolysin gene (cylR2) was the only toxin-associated factor found in S. xylosus, while a methicillin-sensitive S. aureus isolate harbored the Panton-Valentine Leukocidin (LukSF/PVL) gene. This research is the first to document the presence of methicillin-resistant S. aureus in the UAE food chain. Furthermore, S. xylosus (a coagulase-negative staphylococcus not commonly screened in food) has demonstrated phenotypic resistance to clinically relevant antimicrobials. This underscores the need for vigilant monitoring of antimicrobial resistance in bacterial contaminants, whether pathogenic or commensal, at the human-food interface. Full article

Trueperella pyogenes is an important opportunistic pathogenic bacterium widely distributed in the environment. Pyolysin (PLO) is a primary virulence factor of T. pyogenes and capable of lysing many different cells. PLO is a member of the cholesterol-dependent cytolysin (CDC) family of which the primary structure only presents a low level of homology with other members from 31% to 45%. By deeply studying PLO, we can understand the overall pathogenic mechanism of CDC family proteins. This study established a mouse muscle tissue model infected with recombinant PLO (rPLO) and its single-point mutations, rPLO N139K and rPLO F240A, and explored its mechanism of causing inflammatory damage. The inflammatory injury abilities of rPLO N139K and rPLO F240A are significantly reduced compared to rPLO. This study elaborated on the inflammatory mechanism of PLO by examining its unit point mutations in detail. Our data also provide a theoretical basis and practical significance for future research on toxins and bacteria. Full article

A major Streptococcus pneumoniae pathogenic factor is the cholesterol-dependent cytolysin pneumolysin, binding membrane cholesterol and producing permanent lytic or transient pores. During brain infections, vascular damage with variable ischemia occurs. The role of ischemia on pneumolysin’s pore-forming capacity remains unknown. In acute brain slice cultures and primary cultured glia, we studied acute toxin lysis (via propidium iodide staining and LDH release) and transient pore formation (by analyzing increases in the intracellular calcium). We analyzed normal peripheral tissue glucose conditions (80 mg%), normal brain glucose levels (20 mg%), and brain hypoglycemic conditions (3 mg%), in combinations either with normoxia (8% oxygen) or hypoxia (2% oxygen). At 80 mg% glucose, hypoxia enhanced cytolysis via pneumolysin. At 20 mg% glucose, hypoxia did not affect cell lysis, but impaired calcium restoration after non-lytic pore formation. Only at 3 mg% glucose, during normoxia, did pneumolysin produce stronger lysis. In hypoglycemic (3 mg% glucose) conditions, pneumolysin caused a milder calcium increase, but restoration was missing. Microglia bound more pneumolysin than astrocytes and demonstrated generally stronger calcium elevation. Thus, our work demonstrated that the toxin pore-forming capacity in cells continuously diminishes when oxygen is reduced, overlapping with a continuously reduced ability of cells to maintain homeostasis of the calcium influx once oxygen and glucose are reduced. Full article

Alcohol-associated liver disease (ALD) is a major global health issue, contributing significantly to morbidity and mortality worldwide. Among the ALD subtypes, alcohol-associated hepatitis poses a severe and urgent medical challenge with high short-term mortality rates. Despite extensive research, the current therapeutic approaches for alcohol-associated hepatitis have limited efficacy, necessitating novel interventions. Recent studies have highlighted the crucial role of the gut microbiota in ALD pathogenesis, particularly Enterococcus faecalis (E. faecalis) and its cytolysin exotoxin. This study presents the development of a standardized real-time quantitative polymerase chain reaction (RT-qPCR) assay to detect and quantify cytolysin in fecal samples from patients with alcohol-associated hepatitis. The diagnostic assay allows for an association analysis between cytolysin-positive E. faecalis and disease severity as well as mortality. This assay was developed to standardize the identification of cytolysin-positive patients who can be selected for clinical trials. Full article

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. The pore-forming cholesterol-dependent cytolysin (CDC) pneumolysin (PLY) and the physiological metabolite hydrogen peroxide (H₂O₂) can greatly increase the virulence of pneumococci. Although most studies have focused on the contribution of both virulence factors to the course of pneumococcal infection, it is unknown whether or how H₂O₂ can affect PLY activity. Of note, S. pneumoniae exploits endogenous H₂O₂ as an intracellular signalling molecule to modulate the activity of several proteins. Here, we demonstrate that H₂O₂ negatively affects the haemolytic activity of PLY in a concentration-dependent manner. Prevention of cysteine-dependent sulfenylation upon substitution of the unique and highly conserved cysteine residue to serine in PLY significantly reduces the toxin’s susceptibility to H₂O₂ treatment and completely abolishes the ability of DTT to activate PLY. We also detect a clear gradual correlation between endogenous H₂O₂ generation and PLY release, with decreased H₂O₂ production causing a decline in the release of PLY. Comparative transcriptome sequencing analysis of the wild-type S. pneumoniae strain and three mutants impaired in H₂O₂ production indicates enhanced expression of several genes involved in peptidoglycan (PG) synthesis and in the production of choline-binding proteins (CPBs). One explanation for the impact of H₂O₂ on PLY release is the observed upregulation of the PG bridge formation alanyltransferases MurM and MurN, which evidentially negatively affect the PLY release. Our findings shed light on the significance of endogenous pneumococcal H₂O₂ in controlling PLY activity and release. Full article

Enterococcus species are known for their ability to form biofilms, which contributes to their survival in extreme environments and involvement in persistent bacterial infections, especially in the case of multi-drug-resistant strains. This review aims to provide a comprehensive understanding of the mechanisms underlying biofilm formation in clinically important species such as Enterococcus faecalis and the less studied but increasingly multi-drug-resistant Enterococcus faecium, and explores potential strategies for their eradication. Biofilm formation in Enterococcus involves a complex interplay of genes and virulence factors, including gelatinase, cytolysin, Secreted antigen A, pili, microbial surface components that recognize adhesive matrix molecules (MSCRAMMs), and DNA release. Quorum sensing, a process of intercellular communication, mediated by peptide pheromones such as Cob, Ccf, and Cpd, plays a crucial role in coordinating biofilm development by targeting gene expression and regulation. Additionally, the regulation of extracellular DNA (eDNA) release has emerged as a fundamental component in biofilm formation. In E. faecalis, the autolysin N-acetylglucosaminidase and proteases such as gelatinase and serin protease are key players in this process, influencing biofilm development and virulence. Targeting eDNA may offer a promising avenue for intervention in biofilm-producing E. faecalis infections. Overall, gaining insights into the intricate mechanisms of biofilm formation in Enterococcus may provide directions for anti-biofilm therapeutic research, with the purpose of reducing the burden of Enterococcus-associated infections. Full article

CD59 is a GPI-anchored cell surface receptor that serves as a gatekeeper to controlling pore formation. It is the only membrane-bound inhibitor of the complement membrane attack complex (MAC), an immune pore that can damage human cells. While CD59 blocks MAC pores, the receptor is co-opted by bacterial pore-forming proteins to target human cells. Recent structures of CD59 in complexes with binding partners showed dramatic differences in the orientation of its ectodomain relative to the membrane. Here, we show how GPI-anchored CD59 can satisfy this diversity in binding modes. We present a PyLipID analysis of coarse-grain molecular dynamics simulations of a CD59-inhibited MAC to reveal residues of complement proteins (C6:Y285, C6:R407 C6:K412, C7:F224, C8β:F202, C8β:K326) that likely interact with lipids. Using modules of the MDAnalysis package to investigate atomistic simulations of GPI-anchored CD59, we discover properties of CD59 that encode the flexibility necessary to bind both complement proteins and bacterial virulence factors. Full article

The plasma membrane (PM) protects cells from extracellular threats and supports cellular homeostasis. Some pathogens produce pore-forming toxins (PFTs) that disrupt PM integrity by forming transmembrane pores. High PFT concentrations cause massive damage leading to cell death and facilitating infection. Sub-lytic PFT doses activate repair mechanisms to restore PM integrity, support cell survival and limit disease. Shedding of extracellular vesicles (EVs) has been proposed as a key mechanism to eliminate PFT pores and restore PM integrity. We show here that cholesterol-dependent cytolysins (CDCs), a specific family of PFTs, are at least partially eliminated through EVs release, and we hypothesize that proteins important for PM repair might be included in EVs shed by cells during repair. To identify new PM repair proteins, we collected EVs released by cells challenged with sub-lytic doses of two different bacterial CDCs, listeriolysin O and pneumolysin, and determined the EV proteomic repertoire by LC-MS/MS. Intoxicated cells release similar EVs irrespectively of the CDC used. Also, they release more and larger EVs than non-intoxicated cells. A cluster of 70 proteins including calcium-binding proteins, molecular chaperones, cytoskeletal, scaffold and membrane trafficking proteins, was detected enriched in EVs collected from intoxicated cells. While some of these proteins have well-characterized roles in repair, the involvement of others requires further study. As proof of concept, we show here that Copine-1 and Copine-3, proteins abundantly detected in EVs released by intoxicated cells, are required for efficient repair of CDC-induced PM damage. Additionally, we reveal here new proteins potentially involved in PM repair and give new insights into common mechanisms and machinery engaged by cells in response to PM damage. Full article

Artificial membrane systems can serve as models to investigate molecular mechanisms of different cellular processes, including transport, pore formation, and viral fusion. However, the current, such as SUVs, GUVs, and the supported lipid bilayers suffer from issues, namely high curvature, heterogeneity, and surface artefacts, respectively. Freestanding membranes provide a facile solution to these issues, but current systems developed by various groups use silicon or aluminum oxide wafers for fabrication that involves access to a dedicated nanolithography facility and high cost while conferring poor membrane stability. Here, we report the development, characterization and applications of an easy-to-fabricate suspended lipid bilayer (SULB) membrane platform leveraging commercial track-etched porous filters (PCTE) with defined microwell size. Our SULB system offers a platform to study the lipid composition-dependent structural and functional properties of membranes with exceptional stability. With dye entrapped in PCTE microwells by SULB, we show that sphingomyelin significantly augments the activity of pore-forming toxin, Cytolysin A (ClyA) and the pore formation induces lipid exchange between the bilayer leaflets. Further, we demonstrate high efficiency and rapid kinetics of membrane fusion by dengue virus in our SULB platform. Our suspended bilayer membrane mimetic offers a novel platform to investigate a large class of biomembrane interactions and processes. Full article

This study aims at an in vitro characterization of the acid and bile tolerance of Lactobacillus fermentum InaCC B1295 (LFB1295) encapsulated with hydrogel cellulose microfibers (CMF) from oil palm empty fruit bunches (OPEFBs). The viability at different storage temperatures was assessed. The experimental design used in this research was an in vitro trial. The microencapsulated probiotic was stored at 25 °C and 4 °C for 28 days. LFB1295 encapsulated with cellulose microfiber hydrogel from OPEFB showed a stable viability of probiotic bacteria at pH 2 and 0.5% (m/v) oxgall. In addition, the microencapsulation maintained the viability at 25 °C and 4 °C at 0, 14, and 28 days. The characterization of the encapsulant CMF-OPEFB showed that the thickness of CMF was in the range of 5–15 μm, and XRD patterns showed that CMF was of the cellulose I type with a crystallinity index of 77.08%. Based on its resistance to hydrogen peroxide, ability to scavenge DPPH radicals, and activity in scavenging hydroxyl radicals, LFB1295 encapsulated with CMF hydrogel of OPEFB exhibits antioxidant properties as good as the scavenging ability of DPPH radicals with IC₅₀ of 36.880, 188.530, and 195.358 µg/mL, respectively, during storage for 0, 14, and 28 days at room and refrigerated temperature. Furthermore, hydroxyl radicals (HR)-scavenging activity showed an increased inhibition along with the increasing concentration of the Fenton reaction and decreasing concentration of cell-free supernatant (CFS) during storage time. In vitro safety tests, including hemolytic activity, biogenic amines, cytolysin, and gelatinase production, showed that the encapsulated LFB1295 was safe to use as a probiotic. The results of the inhibitory activity against hydrogen peroxide LFB1295 show that the higher the concentration of H₂O₂, the lower the inhibition value during 28 days of storage. Based on the storage temperature, the inhibition of LAB against H₂O₂ based on different storage temperatures showed a better level of the inhibition at cold temperatures compared to at room temperature. Full article

The foodborne transfer of resistant genes from enterococci to humans and their tolerance to several commonly used antimicrobials are of growing concern worldwide. Linezolid is a last-line drug for managing complicated illnesses resulting from multidrug-resistant Gram-positive bacteria. The optrA gene has been reported in enterococci as one of the acquired linezolid resistance mechanisms. The present study uses whole-genome sequencing analysis to characterize the first reported isolates of linezolid-resistant E. faecium (n = 6) and E. faecalis (n = 10) harboring the optrA gene isolated from samples of supermarket broiler meat (n = 165) in the United Arab Emirates (UAE). The sequenced genomes were used to appraise the study isolates’ genetic relatedness, antimicrobial resistance determinants, and virulence traits. All 16 isolates carrying the optrA gene demonstrated multidrug-resistance profiles. Genome-based relatedness classified the isolates into five clusters that were independent of the isolate sources. The most frequently known genotype among the isolates was the sequence type ST476 among E. faecalis (50% (5/10)). The study isolates revealed five novel sequence types. Antimicrobial resistance genes (ranging from 5 to 13) were found among all isolates that conferred resistance against 6 to 11 different classes of antimicrobials. Sixteen different virulence genes were found distributed across the optrA-carrying E. faecalis isolates. The virulence genes in E. faecalis included genes encoding invasion, cell adhesion, sex pheromones, aggregation, toxins production, the formation of biofilms, immunity, antiphagocytic activity, proteases, and the production of cytolysin. This study presented the first description and in-depth genomic characterization of the optrA-gene-carrying linezolid-resistant enterococci from retail broiler meat in the UAE and the Middle East. Our results call for further monitoring of the emergence of linezolid resistance at the retail and farm levels. These findings elaborate on the importance of adopting a One Health surveillance approach involving enterococci as a prospective bacterial indicator for antimicrobial resistance spread at the human–food interface. Full article

The Brucella species is the causative agent of brucellosis in humans and animals. So far, brucellosis has caused considerable economic losses and serious public health threats. Furthermore, Brucella is classified as a category B bioterrorism agent. Although the mortality of brucellosis is low, the pathogens are persistent in mammalian hosts and result in chronic infection. Brucella is a facultative intracellular bacterium; hence, it has to invade different professional and non-professional phagocytes through the host phagocytosis mechanism to establish its lifecycle. The phagocytosis of Brucella into the host cells undergoes several phases including Brucella detection, formation of Brucella-containing vacuoles, and Brucella survival via intracellular growth or being killed by host-specific bactericidal activities. Different host surface receptors contribute effectively to recognize Brucella including non-opsonic receptors (toll-like receptors and scavenger receptor A) or opsonic receptors (Fc receptors and complement system receptors). Brucella lacks classical virulence factors such as exotoxin, spores, cytolysins, exoenzymes, virulence plasmid, and capsules. However, once internalized, Brucella expresses various virulence factors to avoid phagolysosome fusion, bypass harsh environments, and establish a replicative niche. This review provides general and updated information regarding Brucella phagocytosis mediated by pathogen-host interactions and their intracellular survival in host cells. Full article