Bacterial Virulence Research Articles

Prophage dynamics in gastric and enterohepatic environments: unraveling ecological barriers and adaptive transitions

Abstract Phage predation plays a critical role in shaping bacterial genetic diversity, with prophages playing a comparable role. However, the prevalence and genetic variability of prophages within the Helicobacter genus remain inadequately studied. Helicobacter species are clinically significant and occupy distinct digestive system regions, with gastric species (e.g., Helicobacter pylori) residing in the gastric mucosa and enterohepatic species colonizing the liver and intestines of various vertebrates. Here, we address this knowledge gap by analyzing prophage presence and diversity across 343 non-pylori Helicobacter genomes, mapping their distribution, comparing genomic features between gastric and enterohepatic prophages, and exploring their evolutionary relationships with hosts. We identified and analyzed a catalogue of 119 new complete and 78 incomplete prophages. Our analysis reveals significant differences between gastric and enterohepatic species. Gastric prophages exhibit high synteny, and cluster in a few groups, indicating a more conserved genetic structure. In contrast, enterohepatic prophages show greater diversity in gene order and content, reflecting their adaptation to varied host environments. Helicobacter cinaedi stands out, harboring a large number of prophages among the enterohepatic species, forming a distinct cohesive group. Phylogenetic analyses reveal a co-evolutionary relationship between several prophages and their bacterial hosts, though exceptions, such as the enterohepatic prophages from H. canis, Helicobacter equorum, H. jaachi, and the gastric prophage from H. himalayensis, suggesting more complex co-evolutionary dynamics like host jumps, recombination and horizontal gene transfer. The insights gained from this study enhance our understanding of prophage dynamics in Helicobacter, emphasizing their role in bacterial adaptation, virulence, and host specificity.

Targeting Helicobacter pylori Through the “Muco-Microbiotic Layer” Lens: The Challenge of Probiotics and Microbiota Nanovesicles

The muco-microbiotic layer represents a critical biological frontier in gastroenterology, emphasizing the intricate interplay between the protective mucus, its resident microbiota, and extracellular vesicles. This review explores the functional morphology of the gastric mucosa, focusing on the gastric muco-microbiotic layer, its role as a protective barrier, and its dynamic interaction with some of the most insidious pathogens such as Helicobacter pylori (H. pylori). Highlighting the multifaceted mechanisms of H. pylori pathogenesis, we have delved into bacterial virulence factors, host immune responses, and the microbiota’s regulatory effects. Novel therapeutic strategies for H. pylori eradication, including traditional antibiotic therapies and emerging adjuvant treatments like probiotics and probiotic-derived extracellular vesicles, are critically examined. These findings underscore the potential of targeting nanovesicular interactions in the gastric mucosa, proposing a paradigm shift in the management of H. pylori infections to improve patient outcomes while mitigating antibiotic resistance.

Role of CRISPR-Cas System on Virulence traits and Carbapenem Resistance in Clinical Klebsiella pneumoniae Isolates

Background and Objective(s)The bacterial adaptive immune system known as CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated protein) is engaged in defense against various mobile genetic elements (MGEs) such as plasmids and bacteriophages. The purpose of this study was to characterize the CRISPR-Cas systems in carbapenem-resistant Klebsiella pneumoniae isolates and assess any possible correlation between these systems with antibiotic susceptibility, biofilm formation, and bacterial virulence. Materials and MethodsA total of 156 CRKP isolates were collected from different specimens of the inpatients. Biofilm formation and antibiotic susceptibility testing were evaluated using standard methods. Furthermore, the CRISPR-Cas system subtype genes, 11 carbapenemase genes, and 17 virulence genes were identified using separate standard PCR reactions. The diversity of the isolates was determined by random amplified polymorphic DNA (RAPD)-PCR. ResultsThe development of biofilms and antibiotic susceptibility of several CRKP isolates were significantly correlated with the absence or presence of the CRISPR-Cas system. PCR analysis of carbapenemase genes revealed that the frequency of the blaNDM-1 gene was significantly higher in the isolates with the subtype I-E CRISPR-Cas system. Moreover, the isolates with the subtype I-E CRISPR-Cas system exhibited a propensity to possess more virulence genes such as allS, k2A, wcaG, aerobactin, rmpA, iroN, magA, rmpA2, kfu, iutA, iucB, ybtS, repA, and terW. ConclusionCRISPR-Cas systems could affect the antibiotic susceptibility, capacity for biofilm formation, and virulence of Klebsiella pneumoniae. Our findings showed that the isolates containing the CRISPR-Cas system were moderate or strong biofilm producers and had a higher frequency of virulence genes.

Dimethyl fumarate alleviates Staphylococcus pseudintermedius-induced cell damage by inhibiting pyroptosis and bacterial virulence.

The resistance of pathogenic bacteria to various clinical antibiotics is the major problem in treating bacterial keratitis. Dimethyl fumarate (DMF) has good anti-fungal and anti-inflammatory effects in fungal keratitis, but its effect on bacterial keratitis is unclear. This study aims to investigate DMF's anti-inflammatory and antibacterial effects. The pyroptosis model was constructed by intracellular infection of canine corneal epithelial cells (CCECs) with Staphylococcus pseudintermedius (S. pseudintermedius), and 200μM DMF was added to explore its function. Western blot, ELISA, immunostaining, flow cytometry, qRT-PCR, and bacterial counts were used to examine the expression of the NLRP3-GSDMD signaling pathway, virulence genes, and oxidant mediators. 111 clinical keratitis isolates or S. pseudintermedius were treated with different concentrations of DMF to detect bacterial growth and biofilm formation. Adding DMF resulted in the inhibition of the NLRP3-GSDMD pathway while activating the NRF2 pathway. This led to a decrease in pyroptosis rate, intracellular bacteria count, and ROS content. Additionally, DMF blocked the mRNA expression of virulence genes ebpS, hlgB, siet, lukS-I, PVL, icaA, icaD, spsD, and spsL associated with S. pseudintermedius infection. Furthermore, DMF demonstrated concentration-dependent inhibition of the growth of clinical isolates and the formation of S. pseudintermedius biofilm. In conclusion, our results indicate that DMF can inhibit pyroptosis and the growth of various clinical isolates, making it a novel ophthalmic drug with anti-inflammatory and antibacterial properties.

Functional characterization, transcriptome and metabolome analyses reveal that pacR possesses multifaceted physiological roles in Xanthomonas campestris pathovar campestris

Xanthomonas campestris pathovar campestris (Xcc) is the pathogen responsible for causing black rot in cruciferous plants. In this study, we show that mutation of AAW18_RS04175 (pacR, encodes a hypothetical protein containing a domain of unknown function, DUF1631) of Xcc strain Xc17 had decreased bacterial attachment, exopolysaccharide production, hypersensitive response and virulence. Furthermore, the pacR mutant exhibited reduced cell membrane integrity and outer membrane vesicle production. Transcriptomic analysis indicated that 225 genes were differentially expressed following pacR mutation. These genes can be classified into various functional categories, such as the type three secretion system and membrane component. Among them, genes associated with attachment, exopolysaccharide synthesis, the type three secretion system, and nucleotide metabolism were further verified by quantitative RT-PCR. Metabolomic analysis showed that 81 and 132 metabolites in positive and negative modes, respectively, were altered after pacR mutation. Among the identified metabolites, some are known to belong to different pathways, such as biosynthesis of secondary metabolites, microbial metabolism in diverse environments, and nucleotide and purine metabolism, while others have not been previously documented in microbial systems. Additionally, the transcription initiation point of pacR was mapped, and promoter analysis indicated that pacR expression is influenced by different conditions. Taken together, our findings advance the understanding of PacR function and expression in Xcc and offer new insights into the role of the DUF1631-containing hypothetical protein in bacterial physiology.

Xanthomonas citri subsp. citri requires a polyketide cyclase to activate the type III secretion system for virulence

BackgroundXanthomonas citri subsp. citri is the causal agent of citrus canker, which causes substantial losses in citrus production. Here, we report the role of a polyketide cyclase (PKC) on the virulence in X. citri subsp. citri.MethodsThe structure of PKC was precisely predicted using Alphafold3. Promoter GUS fusion constructs and real-time quantitative reverse transcription (qRT-PCR) were employed to study the pattern of expression of the polyketide gene. A deletion mutation was created to explore the role of PKC in virulence and metabolic change.ResultsThe PKC was determined to have a signal peptide, a START/RHO_alpha_C/PITP/Bet_v1/CoxG/CalC (SRPBCC) domain, and a GyrI-like small molecule binding domain. The expression of the PKC gene was induced in planta, as well as under stress by CuSO4 and SDS. An in-frame deletion mutation resulted in a loss of virulence on the citrus hosts, which was restored by the SRPBCC domain. Furthermore, there as a remarkable reduction in the expression of type III genes, such as hrpG and hrpX. In the mutant carrying the pkc deletion, ketoleucine and acetone cyanohydrin were downregulated, and four metabolites, including d-ribose, creatine, polyoxyethylene dioleate, and cohibin C, were upregulated.ConclusionsThe overall data indicate that the PKC affects bacterial virulence by modulating the type III secretion system, possibly through the biosynthesis of particular metabolites.

A novel two-component system contributing the catabolism of c-di-GMP influences virulence in Aeromonas veronii

IntroductionResponse regulators from diverse two-component systems often function as diguanylate cyclases or phosphodiesterases, thereby enabling precise regulation of intracellular c-di-GMP levels to control bacterial virulence and motility. However, the regulatory mechanisms of c-di-GMP require further elucidation.MethodsThis study confirmed that ArrS and ArrR form a two-component system via structural analysis, two-hybrid, and phosphodiesterase activity detection. To evaluate the impact of ArrS/ArrR on intracellular c-di-GMP levels, biofilm detection, motility detection, fluorescence reporter plasmids, and LC-MS/MS analysis were employed. One-hybrid, EMSA, and RT-qPCR were used to demonstrate the function of ArgR on arrSR promoter. The roles of ArrS/ArrR in Aeromonas veronii were investigated using RT-qPCR, murine model, and proteomics.ResultsArrS and ArrR constituted a two-component system in Aeromonas veronii and were transcriptionally repressed by ArgR. ArrR exhibited phosphodiesterase activity, which is inhibited through phosphorylation mediated by ArrS. In Aeromonas veronii, ArrS/ArrR significantly altered the intracellular c-di-GMP levels. In a murine model, ΔarrS exhibited increased pathogenicity, leading to elevated TNF-α and IFN-γ levels in serum, and severer toxicity to spleen and kidney. These effects might be elucidated by the upregulated inflammation-associated proteins in ΔarrS. Moreover, the exonuclease RecB was also up-regulated in ΔarrS.DiscussionWe elucidated the regulatory mechanism of ArrS/ArrR on intracellular c-di-GMP levels and its impact on the virulence in Aeromonas veronii, and discussed the intricate relationship between c-di-GMP metabolism and arginine metabolism.

Incidence of Tooth decay and Its Percentage Bacterial Load in Mbaitolu Imo State of Nigeria.

A toothache is mild to severe pain in or around the teeth and jaws. The occasional sight of persons holding the jaw and groaning of aches necessitated this study. Thirty (30) subjects consisting of male and female persons identified as having toothache and visiting dental centres were selected for this study to evaluate the percentage bacterial load present in the diseases. Many methods were adopted in identification of various bacteria in the samples of patients collected with swab sticks from site of the infected tooth. The samples were incubated for 24 to 48hrs and after that, colonies were isolated and treated further with HLR-S (High level resistant to streptomycin) agar, agglutination, immunofluorescence a dot blot immunoassay procedures and electron microscopic tests to identify the bacteria among the mixed colonies. Streptococcus mutans was identified as the highest (60%) occurring bacteria in tooth decay followed by Actinomyces specie (30%) and others such as Lactobacillus (5%) S. Sobrinus (3%) and Atopium Specie (2%). In periodontal disease the highest occurring virulent bacteria is Treponema denticola (55%) followed by P.gingivalis (30%) peptostreptocaccus (10%) and others. Aggregatibacta (3%) and F. Mucleatum (2%). It could be deduced that the highest percentages occurrence of T.dentcola and S. mutans in tooth problem could necessitate the aches and necrosis involved in tooth aches.

Environmental desiccation stress induces viable but non culturable state in Neisseria meningitidis.

Environmental factors play a crucial role in bacterial virulence. During transmission, in a non-host environment bacteria are exposed to various environmental stress which could alter bacterial physiology and virulence. N. meningitidis is transmitted from person to person through direct contact. However, the role of environmental desiccation in the virulence of bacterial pathogens is not clearly understood. Therefore, the effect of environmental desiccation on survival, transmission, and virulence needs further investigation.We demonstrate that N. meningitidis was sensitive to desiccation stress. The viable counts reduced significantly (p < 0.05) after desiccation. It was found that desiccation induces a viable but non-culturable state (VBNC) in N. meningitidis. We considered cells to be in VBNC when no viable counts were obtained on growth media and live cells were detected after live-dead staining. After resuscitation, N. meningitidis retained virulence characteristics which indicate thatitcan transit between the host in VBNC state. Furthermore, the relative expression of capsule increased significantly after 12 and 24h of desiccation. The observations indicate that the environmental desiccation might induce capsule biosynthesis in N. meningitidis, leading to enhanced virulence and survival in macrophages.

P-667. The Impact of Delay in the Start of Effective Antibiotics on the Course of Pseudomonas aeruginosa Pneumonia: a Retrospective Study

Abstract Background The decision to target up to Pseudomonas aeruginosa at the start of antibiotic treatment of bacterial pneumonia is a major turning point. Since P. aeruginosa itself is a mildly virulent bacterium, we experience that P. aeruginosa pneumonia is improved even targeting P. aeruginosa after its detection. We studied the differences in mortality among P. aeruginosapneumonia cases depending on whether antibiotics with anti-P. aeruginosa activity were administered from the first time (empirical group, group E) or after the detection of P. aeruginosa (definitive group, group D). Methods We retrospectively studied patients who received antibiotics with anti-P. aeruginosa activity for P. aeruginosa pneumonia at our hospital from October 2016 to December 2023. The primary endpoint was 30-day mortality, and the secondary endpoints were total duration of treatment, length of hospital stay, and adverse events. This study was conducted with the approval of the Ethics Committee of our hospital (approve number: 23-075). Results There were 95 applicable participants. The group E had 62 patients and the group D had 33 patients. In the background between the two groups, clinical frailty scale was significantly worse, and impaired consciousness and hypotension. The 30-day mortality was not significantly different between the two groups (group E vs. group D, 1.6% vs. 3.0%, p &amp;gt; 0.99). And there were also no significant differences in mean total duration of antibiotics treatment, mean length of hospital stays, or incidence of adverse events between the two groups (13.2 days vs. 12.9 days, 83.8 days vs. 58.5 days, 54.8% vs. 63.6% in the group E vs. group D, respectively). Conclusion There was no statistical difference in mortality by the delay in targeting P. aeruginosa for treatment of P. aeruginosapneumonia. It was suggested that this could lead to appropriate use of broad-spectrum antibiotics that target even P. aeruginosa. Disclosures All Authors: No reported disclosures

Natural Phenolics Disrupt Microbial Communication by Inhibiting Quorum Sensing

Quorum sensing, a bacterial cell-to-cell communication mechanism, plays a key role in bacterial virulence and biofilm formation. Targeting quorum-sensing pathways represents a promising strategy for the development of novel antibacterial agents. This study evaluated the anti-quorum-sensing activities of 18 natural compounds, including cannabinoids, arylbenzofurans, flavonoids, caffeine, and chlorogenic acid, using the luminescent biosensor strain Vibrio harveyi MM30. V. harveyi MM30, a mutant strain deficient in the production of autoinducer-2 (AI-2) but responsive to exogenous AI-2, was used to assess the activity of test compounds on the AI-2 receptor pathway. Test compounds were incubated in AI-2-containing media, and luminescence was measured to evaluate quorum-sensing inhibition. Comparisons were made in the absence of AI-2 to determine AI-2-independent inhibitory activity. The most active compounds were further tested on methicillin-resistant Staphylococcus aureus (MRSA 7112) to determine their effects on AI-2 production in spent media. Among the tested compounds, the non-prenylated arylbenzofuran moracin M and the prenylated arylbenzofuran moracin C exhibited significant quorum-sensing inhibitory activity in the AI-2-mediated pathway. None of the test compounds significantly inhibited quorum sensing in the absence of AI-2. Five compounds (cannabigerol, cannabidiol, cannabigerolic acid, moracin M, and moracin C) were selected for further investigation in MRSA 7112 cultures. The spent media from MRSA 7112 cultures treated with moracin M (16, 32, 64 µg/mL) and cannabigerolic acid (16 µg/mL) showed significant inhibition of AI-2 production when transferred to V. harveyi MM30 cultures. Moracin M and cannabigerolic acid demonstrated potential as quorum-sensing inhibitors by targeting AI-2 production and signalling pathways in MRSA 7112 and V. harveyi. These findings suggest their potential for further development as antibacterial agents targeting quorum-sensing mechanisms.

Repurposing Diflunisal as an Anti-Virulence Agent Against Staphylococcus aureus

Abstract Infections refractory to standard antibiotic therapy are contributing to adverse treatment outcomes in patients suffering from deep-seated bacterial infections caused by increasingly resistant pathogens. Adjunctive strategies targeting bacterial virulence factors have been considered to supplement the host immune response in fighting the infection. Previous studies suggest that the FDA-approved anti-inflammatory drug diflunisal inhibits Staphylococcus aureus (SA) α-toxin expression by its interaction with the response regulator AgrA. We investigated the broader anti-virulence properties of diflunisal against pathogenic strains of SA and established proof-of-concept for its efficacy in blocking SA virulence. Our studies reveal that diflunisal inhibits α-toxin production, sensitizes SA to cationic antibiotics and human antimicrobial peptides, inhibits the production of the golden pigment staphyloxanthin, and reduces biofilm formation. Molecular docking simulations revealed potential interactions between diflunisal and AgrA binding sites. In addition, sequence alignment of the SA AgrA response regulator demonstrated similarities to other response regulators involved in controlling virulence factor expression. Appreciation of the antivirulence properties of diflunisal supports a therapeutic strategy distinct from structurally similar compounds, such as salicylic acid. The repurposing of diflunisal may mitigate disease severity and provide a unique adjunctive tool in the treatment of SA infection.

Understanding the Molecular Interactions Between Influenza A Virus and Streptococcus Proteins in Co-Infection: A Scoping Review

Influenza A virus infections are known to predispose infected individuals to bacterial infections of the respiratory tract that result in co-infection with severe disease outcomes. Co-infections involving influenza A viruses and streptococcus bacteria result in protein–protein interactions that can alter disease outcomes, promoting bacterial colonisation, immune evasion, and tissue damage. Focusing on the synergistic effects of proteins from different pathogens during co-infection, this scoping review evaluated evidence for protein–protein interactions between influenza A virus proteins and streptococcus bacterial proteins. Of the 2366 studies initially identified, only 32 satisfied all the inclusion criteria. Analysis of the 32 studies showed that viral and bacterial neuraminidases (including NanA, NanB and NanC) are key players in desialylating host cell receptors, promoting bacterial adherence and colonisation of the respiratory tract. Virus hemagglutinin modulates bacterial virulence factors, hence aiding bacterial internalisation. Pneumococcal surface proteins (PspA and PspK), bacterial M protein, and pneumolysin (PLY) enhance immune evasion during influenza co-infections thus altering disease severity. This review highlights the importance of understanding the interaction of viral and bacterial proteins during influenza virus infection, which could provide opportunities to mitigate the severity of secondary bacterial infections through synergistic mechanisms.

Arsenic-induced modulation of virulence and drug resistance in Pseudomonas aeruginosa.

Arsenic contamination of water sources, whether from natural or industrial origins, represents a significant risk to human health. However, its impact on waterborne pathogens remains understudied. This research explores the effects of arsenic exposure on the opportunistic pathogen Pseudomonas aeruginosa, a bacterium found in diverse environments. The arsenic exposure at concentrations of 0.12-20 mg/L As(III) resulted in rapid growth arrest of P. aeruginosa. Moreover, arsenic exposure significantly reduced the production of key virulence factors such as elastase (by 1.48- to 9.24-fold), pyocyanin, and flagella while increasing siderophore and extracellular polysaccharide production (by 1.44-1.75 and 1.36-2.59 times, respectively). Proteomic analysis revealed that both low (0.12 mg/L) and high (1.2 mg/L) As(III) levels activated an antioxidant defense response, with upregulation of Fnr-2, TrxB2, and Ohr. Furthermore, arsenic-induced the overexpression of multidrug resistance efflux proteins MexAB-OprM, MexCD-OprJ, and MexEF-OprN. At the same time, proteins associated with quorum sensing (QS), type III secretion system (T3SS), pyocyanin biosynthesis, and flagellar assembly were downregulated. In vitro assays confirmed that arsenic reduced bacterial virulence and significantly enhanced survival and proliferation under antibiotic treatment. These results indicate that arsenic exposure modulates the virulence and antibiotic resistance of P. aeruginosa, raising concerns about the public health risks posed by the convergence of arsenic-contaminated water and multidrug-resistant bacteria.

Klebsiella pneumoniae T6SS impact in IBDs

Abstract Background and aims Our hypothesis is that Klebsiella pneumoniae (Kp) Type VI Secretion System (T6SS), normally used for bacteria competition during host colonization have a collateral impact on the host inflammatory pathway (Fig1). Our specific aims are to: (i) Characterize the impact of bacterial type VI Secretion System (T6SS) on host gut inflammation (ii) Develop a screen to identify T6SS inhibitors. Methods In vivo experiments involve infecting mice with Kp wild type (WT) and T6SS mutants to evaluate gut inflammation and immune response. Metagenomic analysis of IBD and CRC patients' microbiota will be conducted to assess T6SS gene enrichment. The molecular mechanism of T6SS activity and LPS release will be explored using various imaging and biochemical techniques. High-throughput screening of small molecule inhibitors targeting T6SS assembly will be performed, followed by validation in cellular models. The role of bacteria in tumorigenesis is increasingly recognized, yet our understanding of the underlying mechanisms remains incomplete, hindering the identification of new therapeutic strategies. It is therefore critical to decipher the bacterial mechanisms and their impact on the host to identify pathways that can be targeted from both sides. Anticipated impact Our preliminary data have uncovered a novel role of Type VI Secretion System (T6SS) role in gut inflammation. Building on our extensive expertise of this specific system, we will employ a multidisciplinary approach to inhibit T6SS. Inhibiting the T6SS could prevent both gut inflammation and Enterobacteria competition advantage in the gut, therefore reducing dysbiosis. Moreover, inhibition of bacterial virulence factors is an attractive strategy to complement antibiotic and anti-inflammatory treatments already in use.

Self-Immobilizing Fluorogenic Probe for In Situ Labeling of Granzyme B Activity in Host Immune Response to Bacterial Infections.

Bacterial infections, particularly those caused by drug-resistant bacteria, represent a pressing global health challenge. During the interaction between pathogen infection and host defense, bacterial infections initiate the host's immune response, which involves the activation of proteases that play a critical role in antibacterial defense. Granzyme B (GzmB), a key immune-related biomarker associated with cytotoxic T lymphocytes (CTLs), plays a pivotal role in this process. Therefore, detecting the activity of GzmB is crucial for understanding the host immune response to bacterial infections and for developing therapeutic strategies to overcome bacterial virulence. In this study, we designed and synthesized three granzyme B-activated near-infrared molecular probes. Among them, the probe HCy-F demonstrates in situ imaging capability, enabling precise quantification of GzmB activity. This development offers a valuable tool for monitoring immune responses and optimizing immunotherapy approaches for combating drug-resistant pathogens.

P0055 Impact of Indole-3-Propionic Acid on Bacterial Invasion Potential of Klebsiella Strains Isolated from Pediatric Ulcerative Colitis Patients

Abstract Background Indole-3-propionic acid (IPA), a tryptophan metabolite produced by microbes, has demonstrated beneficial effects, including reversing dysbiosis, balancing bacterial populations, and reducing inflammation.1,2,3 However, the mechanisms behind these effects and their impact on pathogenic microbes remain unclear. This study aimed to evaluate the effects of IPA on bacterial invasion in vitro, using potential pathobionts (Klebsiella variicola and Klebsiella quasipneumoniae) isolated from non-inflamed sections of the colon in pediatric ulcerative colitis (UC) patients. We hypothesized that IPA could reduce virulence traits, promoting a more homeostatic environment, which could lower inflammation in pediatric UC patients. Methods Caco-2 cells (colonic epithelial cell line) were infected with Klebsiella strains, with or without 0.5 mM IPA. Bacterial invasion was assessed via gentamicin protection assay, and qPCR measured Interleukin-8, IL-10, and TNF-α expression in infected cells. Transepithelial electrical resistance (TEER) assay evaluated barrier integrity, and qPCR analyzed the expression of bacterial virulence genes, including entB (siderophores), manC (capsule), and hcp1 (type VI secretion) in IPA-treated and untreated cultures. Additionally, siderophore production was quantified using a Chrome-Azurol Sulfonate (CAS) assay, and Anthony’s stain visualized the bacterial capsule. Results IPA-treated Caco-2 cells showed significantly reduced bacterial invasion, with further reductions when bacteria were also exposed to IPA. qPCR revealed a significant decrease in IL-8 expression in IPA-treated cells during infection. IL-10 levels decreased non-significantly, and TNF-α expression showed variable results. A trend toward improved tight junction integrity was observed in the TEER assay with IPA treatment, although no significant differences were detected. IPA treatment increased the expression of entB, manC, and hcp1 genes, except for K. variicola, where manC expression remained unchanged. IPA significantly reduced the capsule thickness of K. variicola and resulted in a significant reduction in K. quasipneumoniae siderophore production. Conclusion IPA reduces bacterial invasion and modulates immune responses in Caco-2 cells infected with Klebsiella pathobiont strains. It reduces the virulence of K. quasipneumoniae by targeting siderophore production and diminishes the virulence of K. variicola by reducing capsule formation, suggesting its potential as an anti-virulence agent against these pathobionts. These findings highlight IPA’s potential as an immunomodulatory agent for managing IBD. However, further research is needed into other Klebsiella virulence pathways and host-microbiota interactions.

Functional analysis of the type II toxin-antitoxin system ParDE in Streptococcus suis serotype 2

Streptococcus suis (S. suis) is a major pathogen in swine and poses a potential zoonotic threat, which may cause serious diseases. Many toxin-antitoxin (TA) systems have been discovered in S. suis, but their functions have not yet been fully elucidated. In this study, an auto-regulating type II TA system, ParDE, was identified in S. suis serotype 2 strain ZY05719. We constructed a mutant strain, ΔparDE, to explore its functions in bacterial virulence, various stress responses, and biofilm formation capabilities. The toxicity exerted by the toxin ParE can be neutralized by the antitoxin ParD. The β-galactosidase activity analysis indicated that ParDE has an autoregulatory function. An electrophoretic mobility shift assay (EMSA) confirmed that the antitoxin ParD bound to the promoter of ParDE as dimers. In the mouse infection model, the deletion of ParDE in ZY05719 significantly attenuated virulence. ΔparDE also exhibited a reduced anti-oxidative stress ability, and ΔparDE was more susceptible to phagocytosis and killing by macrophages. Moreover, the biofilm formation ability of the ΔparDE strain was significantly enhanced compared to ZY05719. Taken together, these findings indicate that the type II TA system ParDE plays a significant role in the pathogenesis of S. suis, providing new insights into its pathogenic mechanisms.

NupR Is Involved in the Control of PlcR: A Pleiotropic Regulator of Extracellular Virulence Factors.

NupR is a nucleoside permease regulator belonging to the GntR family, mainly regulating nucleoside transport in Bacillus thuringiensis. A conserved binding site for NupR was found in the promoter region of plcR. This study aimed to investigate the regulation of the virulence regulator PlcR by NupR and its impact on Bt virulence. We demonstrated that NupR can directly repress the expression of plcR. The expression of plcR can be induced by glucose and nucleosides. Glucose impacts the expression of plcR mainly through Spo0A, while the induction effect of nucleosides may be due to the production of ribose through nucleoside catabolism. In addition, NupR regulates the expression of the PlcR regulon, including hemolysin, phospholipase C, papR, and oligopeptide permease, which could result in the culture supernatant of BMB171 being less virulent to sf9 cells compared to the nupR knockout strain. The results combine the nutritional status of cells with virulence to form a regulatory loop, providing new ideas and research foundations for the study of bacterial virulence.

The Sub-inhibitory Effect of Azithromycin and Ciprofloxacin on &lt;i&gt;virF&lt;/i&gt; Gene Expression in Enteroinvasive &lt;i&gt;Escherichia coli&lt;/i&gt; and &lt;i&gt;Shigella flexner&lt;/i&gt;i

Background: Bacillary dysentery is an invasive bacterial gastroenteritis that damages the colon epithelium and leads to bloody diarrhea. Enteroinvasive Escherichia coli (EIEC) and Shigella flexneri are two major etiologic agents of the disease. The virF gene is a transcriptional regulator of the virulence genes involved in the invasion of these bacteria. Previous studies have shown that sub-minimum inhibitory concentrations (sub-MICs) of antibiotics have significant effects on bacterial virulence. Objectives: This study aimed to evaluate the effects of sub-MICs of ciprofloxacin and azithromycin on S. flexneri and EIEC. Methods: Prototype strains of both bacteria were treated with sub-MICs of 1/2, 1/4, and 1/8 of ciprofloxacin and azithromycin antibiotics. Changes in the expression of the virF gene in antibiotic-treated samples compared to control samples were analyzed using relative real-time polymerase chain reaction (PCR). Results: The mean expression of the virF gene in all sub-MICs of ciprofloxacin was increased in both S. flexneri and EIEC, while a down-regulation was observed in sub-MICs of azithromycin. These gene expression changes were dose-dependent. Conclusions: The results demonstrated that the virulence of S. flexneri and EIEC is affected by sub-MICs of azithromycin and ciprofloxacin. Given that azithromycin, unlike ciprofloxacin, reduces the severity of infection at sub-MICs, it is a more appropriate choice with a lower risk for treating acute infections caused by these bacteria.