Gram-positive Pathogens Research Articles

Evaluation of Antibacterial (Antibiofilm) Activity Potential of ZnONPs Coated on Wound Dressing Cloth

Introduction: In light of the emergence of antibiotic-resistant bacteria and the necessity for efficient wound treatment, zinc oxide nanoparticles (ZnONPs) have garnered interest for their potent antibacterial and antibiofilm characteristics. This study examines the incorporation of green synthesized ZnONPs into wound dressing fabric to inhibit bacterial colonization and biofilm development, and significant obstacles in wound healing. The present study aims to assess the antibacterial efficacy of plant-mediated and pre-synthesized as well as characterized ZnONPs against opportunistic bacterial pathogens to create more effective wound dressings that facilitate expedited, infectionfree recovery. Methods: The antibacterial efficiency of this green-mediated ZnONPs coated wound dressing material against the opportunistic Gram-positive and negative bacterial pathogens were checked. Various concentrations (0.20, 0.40, and 0.60%) of ZnONPs were used to coat the dressing material. This ZnONPs antibacterial activity was analyzed quantitatively by various time intervals (4-24 hr) and incubated as per the standard bacterial growth conditions. Results: The findings show that 20 hr after incubation, Gram-negative bacterial growth was inhibited on dressing cloth coated with 0.60% ZnONPs, while Gram-positive bacteria inhibition was observed 24 hr after incubation on dressing cloth coated with 0.40% ZnONPs. These findings suggest that 0.40% and 0.60% ZnONPs significantly kill both groups of opportunistic pathogens. Discussion: Bacterial infections as well as biofilm formation on wound surfaces significantly impede effective healing, resulting in chronic wounds and elevated healthcare expenses. Conventional wound dressings frequently exhibit inadequate antimicrobial efficacy, particularly against antibiotic-resistant bacteria. ZnONPs have attracted interest owing to their strong antibacterial, antibiofilm, and biocompatibility characteristics. This study assesses the efficacy of ZnONPs-coated wound dressings in suppressing bacterial proliferation and biofilm development, potentially providing a remedy for infectionassociated complications in wound care. The results may facilitate the creation of more efficient wound dressings, thereby decreasing infection rates and enhancing patient outcomes in clinical environments. Conclusion: Thus, these ZnONPs could be employed as an antibiofilm/antibacterial coating material in wound dressing cloths to prevent secondary opportunistic bacterial infections.

Evaluation of Antibiotic Resistance Mechanisms in Gram-Positive Bacteria

The prevalence of resistance in Gram-positive bacterial infections is rapidly rising, presenting a pressing global challenge for both healthcare systems and economies. The WHO categorizes these bacteria into critical, high, and medium priority groups based on the urgency for developing new antibiotics. While the first priority pathogen list was issued in 2017, the 2024 list remains largely unchanged. Despite six years having passed, the progress that has been made in developing novel treatment approaches remains insufficient, allowing antimicrobial resistance to persist and worsen on a global scale. Various strategies have been implemented to address this growing threat by targeting specific resistance mechanisms. This review evaluates antimicrobial resistance (AMR) in Gram-positive bacteria, highlighting its critical impact on global health due to the rise of multidrug-resistant pathogens. It focuses on the unique cell wall structure of Gram-positive bacteria, which influences their identification and susceptibility to antibiotics. The review explores the mechanisms of AMR, including enzymatic inactivation, modification of drug targets, limiting drug uptake, and increased drug efflux. It also examines the resistance strategies employed by high-priority Gram-positive pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecium, as identified in the WHO’s 2024 priority list.

Muramyl dipeptide potentiates Staphylococcus aureus lipoteichoic acid-induced nitric oxide production via TLR2/NOD2/PAFR signaling pathways

Lipoteichoic acid (LTA) and peptidoglycan (PGN) are considered as key virulence factors of Staphylococcus aureus, which is a representative sepsis-causing Gram-positive pathogen. However, cooperative effect of S. aureus LTA and PGN on nitric oxide (NO) production is still unclear despite the pivotal roles of NO in initiation and progression of sepsis. We here evaluated the cooperative effects of S. aureus LTA (SaLTA) and muramyl dipeptide (MDP), the minimal structure of PGN, on NO production in both a mouse macrophage-like cell line, RAW 264.7 and mouse bone marrow-derived macrophages (BMMs). Although MDP alone did not affect NO production, MDP potently enhanced SaLTA-induced NO production via the expression of inducible NO synthases. The enhanced NO production was ameliorated in BMMs from TLR2-, CD14-, MyD88-, and NOD2-deficient mice. Moreover, the augmented SaLTA-induced NO production by MDP was attenuated by inhibitors specific for PAFR and MAP kinases. Furthermore, MDP also potently increased SaLTA-induced activities of STAT1, NF-κB, and AP-1 transcription factors, and specific inhibitors for these transcription factors suppressed the elevated NO production. Collectively, these results demonstrated that MDP potentiates SaLTA-induced NO production via TLR2/NOD2/PAFR, MAP kinases signaling axis, resulting in the activation of NF-κB, AP-1 and STAT1 transcription factors.

Distribution and Antimicrobial Susceptibility Profiles of Pathogens in Patients With Esophageal Cancer From 2013 to 2022: A Retrospective Study.

Pathogenic microbial infections are closely related to the development and prognosis of esophageal cancer. The distribution and resistance of pathogens in different diseases are regional and gradually change over time. This study aimed to determine the distribution and drug resistance of pathogens isolated from patients with esophageal cancer and provide a reference for the rational use of antibiotics. The results of strain identification and antimicrobial susceptibility testing of pathogens in patients with esophageal cancer from January 2013 to December 2022 at our hospital were retrospectively analyzed. SPSS Statistics 26.0 (IBM) and R software 4.3.1 were used for data analysis. In total, 2322 non-repetitive pathogens were isolated from 14,037 samples. Of all strains, 1713 (73.77%) were Gram-negative bacteria, 483 (20.80%) were Gram-positive bacteria, and 126 (5.43%) were fungi. The top 10 pathogens were Pseudomonas aeruginosa (19.81%), Stenotrophomonas maltophilia (12.88%), A. baumannii (9.91%), Klebsiella pneumoniae (9.82%), Staphylococcus aureus (7.54%), Candida albicans (3.92%), Staphylococcus epidermidis (3.19%), Escherichia coli (3.14%), Enterococcus faecalis (2.97%), and Serratia marcescens (2.15%). The isolation rate of S. maltophilia showed an upward trend (p < 0.05). The resistance rates of P. aeruginosa, S. maltophilia, A. baumannii, and Enterobacteriaceae bacteria to some common antibiotics showed a tendency to change (p < 0.05), and 2019 became a turning point to some extent. All common Gram-positive pathogens were sensitive to vancomycin, except for three Enterococcus spp. isolates that showed intrinsic resistance. The prevalence of MRSA was 65.14% (114/175) in this study. In addition, the resistance rates of MRSA and MSSA to moxifloxacin, ciprofloxacin, levofloxacin, erythromycin, clindamycin, and penicillin were significantly different (p < 0.001). Pathogens are diverse in patients with esophageal cancer, with the most common being P. aeruginosa, followed by S. maltophilia. The pathogens exhibited different patterns of resistance. Antibiotics should be used rationally according to pathogen resistance patterns.

Case report: Relaps of Streptococcus suis infection in patient with Meningoencephalitis at Prof. Ngoerah Hospital, Bali

Background: S. suis is a facultative anaerobic Gram-positive bacterium that causes human infection. It is a primary health problem in the swine industry. Ceftriaxone is a therapeutic option for meningitis besides Ampicillin. Recently, the incidence of resistance to ceftriaxone was reported. The recurrence of S. suis in this patient is thought to be caused by ceftriaxone resistance due to inadequate initial therapy. Case Presentation: A 43-year-old male patient came to the ER with headaches in the head and neck. The patient had a history of meningitis due to S. suis twice before. While in the ER, the patient was given ceftriaxone while awaiting culture results. Blood cultures showed S. suis infection using VITEK 2 COMPACT (Biomeriuex®) and ceftriaxone resistance. The patient was successfully managed with Ampicillin following these culture results, leading to good outcomes. Discussion: S. suis is a gram-positive bacterial pathogen in pigs that can cause severe human infection, including meningitis, septicemia, endocarditis and deafness the most common complications. The patient experienced three relapses due to S. suis. On the third visit, blood culture results identified ceftriaxone-resistant S. suis. The patient's recurrent relapses could be attributed to several factors, including inadequate antibiotic administration, antibiotic resistance, weak immune system, contact with the source of infection and environmental variables, highlighting the complexity and challenges in treating S. suis infection. Conclusion: The use of antibiotics in relapse cases has the potential to cause antibiotic resistance, so it’s necessary to make guidelines for antibiotic therapy in S. suis infection to prevent recurrent infections.

Rapid inference of antibiotic susceptibility phenotype of uropathogens using metagenomic sequencing with neighbor typing.

Urinary tract infections (UTIs) are a common diagnosis in hospitals and are often treated empirically with broad-spectrum antibiotics. These broad-spectrum agents can select for resistance in these bacteria and co-colonizing organisms. The use of narrow-spectrum agents is desirable as an antibiotic stewardship measure; however, it is counterbalanced by the need for adequate therapy. Identification of causative organisms and their antibiotic susceptibility can help direct treatment; however, conventional testing requires days to produce actionable results. Methods to quickly and accurately predict susceptibility phenotypes for pathogens causing UTI could thus improve both patient outcomes and antibiotic stewardship. Here, expanding on previous work showing accurate prediction for certain Gram-positive pathogens, we demonstrate how the use of RASE from metagenomic sequencing can provide informative and rapid phenotype prediction results for common Gram-negative pathogens in UTI, highlighting the future potential of this method to be used in clinical settings to guide empiric antibiotic selection.

Discovery and optimization of tetrahydroacridine derivatives as a novel class of antibiotics against multidrug-resistant Gram-positive pathogens by targeting type I signal peptidase and disrupting bacterial membrane

Increasing antimicrobial resistance underscores the urgent need for new antibiotics with unique mechanisms. Type I signal peptidase (SPase I) is crucial for bacterial survival and a promising target for antibiotics. Herein we designed and synthesized innovative tetrahydroacridine-9-carboxylic acid derivatives by optimizing the initial hit compound SP11 based on virtual screening. Structure-activity relationship (SAR) studies and bioactivity assessments identified compound C09 as a standout, showing excellent in vitro antimicrobial activity against MRSA and other multidrug-resistant Gram-positive pathogens. C09 targets SPase I with a favorable affinity, disrupts bacterial cell membranes, and eradicates biofilms, reducing resistance risk. In vivo tests in a murine MRSA skin infection model demonstrated significant efficacy. Additionally, C09 has good liver microsome metabolic stability, safe hemolytic activity and mammalian cytotoxicity, as well as a good in vivo safety profile. Overall, our findings highlight the potential of tetrahydroacridine-9-carboxylic acid derivatives as a novel class of antibiotics against multidrug-resistant Gram-positive bacteria.

Durlobactam to boost the clinical utility of standard of care β-lactams against Mycobacterium abscessus lung disease.

β-Lactams present several desirable pharmacodynamic features leading to the rapid eradication of many bacterial pathogens. Imipenem (IPM) and cefoxitin (FOX) are injectable β-lactams recommended during the intensive treatment phase of pulmonary infections caused by Mycobacterium abscessus (Mab). However, their potency against Mab is many-fold lower than against Gram-positive and Gram-negative pathogens for which they were optimized, putting into question their clinical utility. Here, we show that adding the recently approved durlobactam-sulbactam (DUR-SUL) pair to either IPM or FOX achieves growth inhibition, bactericidal, and cytolytic activity at concentrations that are within those achieved in patients and below the clinical breakpoints established for each agent. Synergies between DUR-SUL and IPM or FOX were confirmed across a large panel of clinical isolates. Through in vitro resistant mutant selection, we also show that adding DUR-SUL abrogates acquired resistance to IPM and FOX. Since the use of β-lactam injectables is firmly grounded in clinical practice during the intensive treatment phase of Mab pulmonary disease, their potentiation by FDA-approved DUR-SUL to bring minimum inhibitory concentration distributions within achievable concentration ranges could offer significant short-term benefits to patients, while novel β-lactam combinations are optimized specifically against Mab pulmonary infections, for which no reliable cure exists.

Exploring northeast India’s culturable soil Actinomycetia for potent antibacterial agents against gram-positive bacterial pathogens of clinical importance

This study investigated the isolation and bioactivity of Actinomycetia from the soil of Northeast India, a region rich in microbial diversity. A total of 187 presumptive Actinomycetia isolates were obtained and 53 were found to exhibit antimicrobial properties. Phylogenetic analysis based on 16 S rRNA gene sequencing revealed that the isolates were predominantly from the genus Streptomyces. Among these, the strain Streptomyces sp. NP14 (ANP14ARS) demonstrated specific and significant antimicrobial activity against gram-positive bacterial pathogens. The strain was further assayed against a panel of clinically important bacterial pathogens including Methicillin-resistant Staphylococcus aureus (MRSA). Its antimicrobial activity was confirmed using disc diffusion and membrane disruption assay, and its minimum inhibitory concentration was determined to be ≥ 3.12 ± 0.5 µg/ml against MRSA. Chemical analyses using FTIR and GC-MS identified key bioactive compounds, including Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- and Phenol 3,5-bis(1,1-dimethylethyl), known for their antimicrobial properties. Toxicity studies against animal liver cell lines indicated that the ethyl acetate extract was non-toxic at concentrations below 125 µg/mL, compared to Vancomycin, which was found to be hepatotoxic at similar concentrations. These findings highlight the potential of ANP14ARS as a source of bioactive natural products that is specific towards priority pathogens such as MRSA.

Repurposing Benzbromarone as an Antibacterial Agent against Gram-Positive Bacteria.

The rise of antibiotic-resistant Gram-positive pathogens, particularly methicillin-resistant Staphylococcus aureus (MRSA), presents a significant challenge in clinical settings. There is a critical need for new antibacterial agents to combat these resistant strains. Our study reveals that the uricosuric drug Benzbromarone (Benz) exhibits potent antibacterial activity against Gram-positive pathogens, with minimum inhibitory concentrations (MICs) ranging from 8 to 32 μg/mL and minimum bactericidal concentrations (MBCs) ranging from 32 to 128 μg/mL against clinical isolates of S. aureus, S. epidermidis, Enterococcus faecalis, and Streptococcus agalactiae. Furthermore, Benz significantly inhibits biofilm formation at subinhibitory concentrations and eradicates mature biofilms at higher concentrations. Benz also suppresses the hemolytic activity of S. aureus, indicating its potential to reduce virulence. Proteomic and in vitro induced resistance analyses indicate that Benz inhibits protein synthesis and turnover. Additionally, Benz induces membrane depolarization and increases membrane permeability, likely by targeting the membrane phospholipid phosphatidylethanolamine (PE). In the mouse wound infection model, Benz promotes wound healing and significantly reduces bacterial load. These findings suggest that Benz is a promising candidate for developing new antibacterial therapies against Gram-positive bacterial infections.

In vitro activities of eravacycline against clinical bacterial isolates: a multicenter study in Guangdong, China.

Eravacycline (ERV), a novel tetracycline derivative, exhibits broad-spectrum antibacterial activity, but data on the bacterial activity against Chinese bacterial isolates are very scarce. This study aims to evaluate the activity of eravacycline against the common Gram-positive and Gram-negative bacteria isolates in Guangdong, China. The clinical isolates were collected from four centers between 1 November 2023 and 31 January 2024, and the susceptibility of eravacycline (MIC50, MIC90, and MIC) was determined using broth microdilution as a reference method and E-TEST strips to evaluate their consistency. A total of 594 strains were collected from the four centers, including Staphylococcus aureus (n = 126), Enterococcus faecalis (n = 58), Enterococcus faecium (n = 29), Klebsiella pneumoniae (n = 136), Escherichia coli (n = 187), and Acinetobacter baumannii (n = 58). The MIC50 and MIC90 (mg/L) of eravacycline were 0.12 and 1 for S. aureus, 0.06 and 0.12 for E. faecalis, 0.06 and 0.5 for E. faecium, 0.25 and 0.5 for E. coli, 0.5 and 2 for K. pneumoniae, and 0.25 and 2 for A. baumannii. Based on the FDA and EUCAST breakpoints, the susceptibility of eravacycline against S. aureus was 46.03% vs. 83.33%, 56.90% vs. 94.93% against E. faecalis, and 62.07% vs. 79.31% in E. faecium. The susceptibility rates of E. coli and K. pneumoniae were 90.37% and 58.09, respectively. To evaluate the performance between the broth microdilution test (BMD) and ETEST methods, we compared essential agreement (EA), categorical agreement (CA), very major error (VME), and major error (ME). The results demonstrated that compared with BMD, eravacycline measured by ETEST had higher VME and ME referring to FDA breakpoints than EUCAST breakpoints in the Gram-positive isolates. Since there were no intermediate breakpoints for the eravacycline, the MIC values measured by the ETEST method might result in lower CA and higher VME and ME. This study provides MIC values of eravacycline against Gram-positive and Gram-negative pathogens in four hospitals in Guangdong Province, and eravacycline is an effective therapeutic candidate for common bacteria.

Phage-Derived Endolysins Against Resistant Staphylococcus spp.: A Review of Features, Antibacterial Activities, and Recent Applications.

Antimicrobial resistance is a significant global public health issue, and the dissemination of antibiotic resistance in Gram-positive bacterial pathogens has significantly increased morbidity, mortality rates, and healthcare costs. Among them, Staphylococcus, especially methicillin-resistant Staphylococcus aureus (MRSA), causes a wide range of diseases due to its diverse pathogenic factors and infection strategies. These bacteria also present significant issues in veterinary medicine and food safety. Effectively managing staphylococci-related problems necessitates a concerted effort to implement preventive measures, rapidly detect the pathogen, and develop new and safe antimicrobial therapies. In recent years, there has been growing interest in using endolysins to combat bacterial infections. These enzymes, which are also referred to as lysins, are a unique class of hydrolytic enzymes synthesized by double-stranded DNA bacteriophages. They possess glycosidase, lytic transglycosylase, amidase, and endopeptidase activities, effectively destroying the peptidoglycan layer and resulting in bacterial lysis. This unique property makes endolysins powerful antimicrobial agents, particularly against Gram-positive organisms with more accessible peptidoglycan layers. Therefore, considering the potential benefits of endolysins compared to conventional antibiotics, we have endeavored to gather and review the characteristics and uses of endolysins derived from staphylococcal bacteriophages, as well as their antibacterial effectiveness against Staphylococcus spp. based on conducted experiments and trials.

Deciphering supramolecular arrangements, micellization patterns, and antimicrobial potential of bacterial rhamnolipids under extreme treatments of temperature and electrolyte.

The micellization properties of rhamnolipids (RLs) in extreme electrolyte concentrations and temperatures have gained considerable attention due to their broad industrial applications. In this study, the aggregation behavior, specifically the micellization pattern (critical micelle concentration (CMC)) of RLs produced from a newly isolated thermophilic strain of Pseudomonas aeruginosa from a harsh environment of an oil field, was investigated by a spectrophotometric method at various temperatures (293-393 K) and electrolyte concentrations (NaCl: 2-20%). The result indicated that the CMC values (0.267-0.140 mM⋅dm-3) were both electrolyte- and temperature-dependent exhibiting a U-shaped trend as temperature and NaCl concentration increased. Variations in NaCl concentration and temperature also affected the standard Gibbs free energy (ΔGo mic ), enthalpy (ΔHo mic ), and entropy (ΔSo mic ) of micellization. The molecule also showed stability at a broad range of temperatures, pH, and NaCl concentrations. Thin-layer chromatography (TLC) and Fourier-transform infrared (FTIR) analysis confirmed the similarity in composition between the crude extract and the commercial RL with Rf values of 0.72 for mono-rhamnolipids and 0.28 for di-rhamnolipids. FTIR analysis confirmed the chemical nature particularly key aliphatic functional groups present in the fatty acid tail of RLs and the -COC- bond in the structure of the rhamnose moiety. Additionally, LC-ESI-QTOF analysis confirmed corresponding ionic fragments of mono- and di-rhamnolipids congeners. Furthermore, the antimicrobial potential was determined against different human pathogens in the absence and presence of NaCl by measuring zones of inhibition. The result revealed enhanced inhibitory effects against Gram-positive pathogens (S. aureus, S. epidermidis, and L. monocytogene), with zones of inhibition of 26, 30, and 20 mm in the presence of NaCl. These findings underline the role of NaCl in the micellization of RL molecules and highlight their importance in environmental applications, pharmaceuticals, and various life science sectors.

In Vivo Efficacy of a Nanoconjugated Glycopeptide Antibiotic in Silkworm Larvae Infected by Staphylococcus aureus.

To contrast the rapid spread of antibiotic resistance in bacteria, new alternative therapeutic options are urgently needed. The use of nanoparticles as carriers for clinically relevant antibiotics represents a promising solution to potentiate their efficacy. In this study, we used Bombyx mori larvae for the first time as an animal model for testing a nanoconjugated glycopeptide antibiotic (teicoplanin) against Staphylococcus aureus infection. B. mori larvae might thus replace the use of mammalian models for preclinical tests, in agreement with the European Parliament Directive 2010/63/EU. The curative effect of teicoplanin (a last resort antibiotic against Gram-positive bacterial pathogens) conjugated to iron oxide nanoparticles was assessed by monitoring the survival rate of the larvae and some immunological markers (i.e., hemocyte viability, phenoloxidase system activation, and lysozyme activity). Human physiological conditions of infection were reproduced by performing the experiments at 37 °C. In this condition, nanoconjugated teicoplanin cured the bacterial infection at the same antibiotic concentration of the free counterpart, blocking the insect immune response without causing mortality of silkworm larvae. These results demonstrate the value and robustness of the silkworm as an infection model for testing the in vivo efficacy of nanoconjugated antimicrobial molecules.

Development of Alginate Hydrogels Incorporating Essential Oils Loaded in Chitosan Nanoparticles for Biomedical Applications.

A hybrid alginate hydrogel-chitosan nanoparticle system suitable for biomedical applications was prepared. Chitosan (CS) was used as a matrix for the encapsulation of lavender (Lavandula angustifolia) essential oil (LEO) and Mentha (Mentha arvensis) essential oil (MEO). An aqueous solution of an acidic Natural Deep Eutectic Solvent (NADES), namely choline chloride/ascorbic acid in a 2:1 molar ratio, was used to achieve the acidic environment for the dissolution of chitosan and also played the role of the ionic gelator for the preparation of the chitosan nanoparticles (CS-NPs). The hydrodynamic diameter of the CS-MEO NPs was 130.7 nm, and the size of the CS-LEO NPs was 143.4 nm (as determined using Nanoparticle Tracking Analysis). The CS-NPs were incorporated into alginate hydrogels crosslinked with CaCl2. The hydrogels showed significant water retention capacity (>80%) even after the swollen sample was kept in the aqueous HCl solution (pH 1.2) for 4 h, indicating a good stability of the network. The hydrogels were tested (a) for their ability to absorb dietary lipids and (b) for their antimicrobial activity against Gram-positive and Gram-negative foodborne pathogens. The antimicrobial activity of the hybrid hydrogels was comparable to that of the widely used food preservative sodium benzoate 5% w/v.

Bacterial profile and antimicrobial susceptibility patterns of common neonatal sepsis pathogens in Gulf Cooperation Council countries: A systematic review and meta-analysis.

Neonatal sepsis (NS) is a major healthcare burden in Gulf Cooperation Council (GCC) countries, with a prevalence higher than the global average. Microbial drug resistance has major implications for mortality and morbidity from NS. To synthesize data regarding the patterns of causative bacteria of NS in the GCC and their antimicrobial susceptibility profiles. Following the exploration of four electronic databases, i.e., EBSCOhost, ProQuest, PubMed/MEDLINE, and ScienceDirect, eligible studies were identified (i.e., published between 2013 and 2023 and reported bacterial profile and/or antimicrobial susceptibility patterns). The outcomes included the pooled prevalence of bacteria and their susceptibility patterns. Proportion meta-analysis was performed for each outcome of interest. Fifteen studies were eligible (total positive cases = 2,473). Coagulase-negative Staphylococci (CoNS) (28.1%) were the most common gram-positive causative pathogen, followed by group B Streptococcus (GBS) (16.2%) and Staphylococcus aureus (9.9%); for gram-negative, Escherichia coli (12.7%) and Klebsiella species (11.4%) were most common. The susceptibility rates of these bacteria to first-line antibiotics were high; gram-positive bacteria had the highest susceptibility to ampicillin (72.8-98%), and gram-negative bacteria was most susceptible to amikacin (94.6-98%). Additionally, both gram-positive (67-77%) and negative (87-93%) bacteria exhibited high susceptibility to gentamicin. The most common pathogens among NS patients were gram-positive. The pathogens, irrespective of stain test, were susceptible to the current antibiotic therapy. We recommend the judicious use of empirical antibiotic therapy to prevent the growing risk of antimicrobial resistance.

Discovery of a family of menaquinone-targeting cyclic lipodepsipeptides for multidrug-resistant Gram-positive pathogens

Menaquinone (MK) in bacterial membrane is an attractive target for the development of novel therapeutic agents. Mining the untapped chemical diversity encoded by Gram-negative bacteria presents an opportunity to identify additional MK-binding antibiotics (MBAs). By MK-binding motif searching of bioinformatically predicted linear non-ribosomal peptides from 14,298 sequenced genomes of 45 underexplored Gram-negative bacterial genera, here we identify a novel MBA structural family, including silvmeb and pseudomeb, using structure prediction-guided chemical synthesis. Both MBAs show rapid bacteriolysis by MK-dependent membrane depolarization to achieve their potent activities against a panel of Gram-positive pathogens. Furthermore, both MBAs are proven to be effective against methicillin-resistant Staphylococcus aureus in a murine peritonitis-sepsis model. Our findings suggest that MBAs are a kind of structurally diverse and still underexplored antibacterial lipodepsipeptide class. The interrogation of underexplored bacterial taxa using synthetic bioinformatic natural product methods is an appealing strategy for discovering novel biomedically relevant agents to confront the crisis of antimicrobial resistance.

Novel motif associated with carbon catabolite repression in two major Gram-positive pathogen virulence regulatory proteins.

Carbon catabolite repression (CCR) is a widely conserved regulatory process that ensures enzymes and transporters of less-preferred carbohydrates are transcriptionally repressed in the presence of a preferred carbohydrate. This phenomenon can be regulated via a CcpA-dependent or CcpA-independent mechanism. The CcpA-independent mechanism typically requires a transcriptional regulator harboring a phosphotransferase regulatory domain (PRD) that interacts with phosphotransferase system (PTS) components. PRDs contain a conserved histidine residue that is phosphorylated by the PTS-associated HPr-His15~P protein. PRD-containing regulators often harbor additional domains that resemble PTS-associated EIIB protein domains with a conserved cysteine residue that can be phosphorylated by cognate PTS components. We noted that Mga, the PRD-containing central virulence regulator of Streptococcus pyogenes, has an EIIBGat domain containing a cysteine that, based on the presence of a similar motif in glycerol kinase, could be a target for phosphorylation. Using site-directed mutagenesis, we constructed phospho-ablative and phospho-mimetic substitutions of this cysteine and found that these substitutions modify the CCR of the Rgg2/3 quorum-sensing system. Moreover, we provide genetic evidence that the phospho-donor of this cysteine residue is likely to be ManL, the EIIA/B subunit of the mannose PTS system. Interestingly, a structurally distinct virulence gene regulator, PrfA of Listeria monocytogenes, harbors a similar cysteine-containing motif, and phospho-ablative and phospho-mimetic substitutions of the cysteine-altered CCR of PrfA-dependent virulence gene expression. Collectively, our data suggest that phosphorylation of a cysteine within the shared novel motif in Mga and PrfA may be a heretofore missing link between cellular metabolism and virulence.IMPORTANCEIn this study, we identified a novel cysteine-containing motif within the amino acid sequence of two structurally distinct transcriptional regulators of virulence in two Gram-positive pathogens that appears to link carbon metabolism with virulence gene expression. The results also highlight the potential post-translational modification of cysteine in bacterial species, a rare and understudied modification.

Phytochemical properties of Cyrtocarpa edulis peel exert antimicrobial activity and enhance immunobiological parameters in Almaco jack Seriola rivoliana cells

Fish aquaculture has increased but also disease problems related to it, along with antimicrobial resistance. The use of medicinal plants containing phytochemicals with antimicrobial, antioxidant, and immunomodulatory activities has been proposed. In this study, C. edulis “peel” was investigated for its phytochemical composition, antioxidant capacity, antimicrobial activities, and immunostimulant properties in leukocytes from Almaco Jack, S. rivoliana. The proximal composition of peel indicated 4.10 % protein 5.37 % ash and 6.93 % crude fiber, whereas total phenolic and flavonoid contents were 180 mg and 50 mg per gram of dry sample, respectively. Superoxide radical scavenging, total antioxidant, and iron chelation activities were also detected. In vitro, Almaco Jack S. rivoliana leukocytes treated with peel extract showed no cytotoxicity, whereas respiratory burst, myeloperoxidase, and SOD activities, as well as nitric oxide production, were enhanced. Most bacterial strains were inhibited 70 % at a concentration of 1 mg/mL C. edulis peel extract, except for Salmonella Enteritidis (40 %) and Vibrio parahaemolyticus (25 %). In conclusion, peel has phytochemicals with antioxidant capacity, immunostimulatory effects for Almaco Jack, Seriola rivoliana, and antimicrobial activity against Gram-positive and Gram-negative pathogens. Future experiments in fish must be conducted using C. edulis peel in diets to confirm their immunostimulant, antioxidant, and antimicrobial properties.

Metabolic engineering of Streptomyces roseosporus for increased production of clinically important antibiotic daptomycin.

Daptomycin (DAP), a novel cyclic lipopeptide antibiotic produced by Streptomyces roseosporus, is clinically important for treatment of infections caused by multidrug-resistant Gram-positive pathogens, but the low yield hampers its large-scale industrial production. Here, we describe a combination metabolic engineering strategy for constructing a DAP high-yielding strain. Initially, we enhanced aspartate (Asp) precursor supply in S. roseosporus wild-type (WT) strain by separately inhibiting Asp degradation and competitive pathway genes using CRISPRi and overexpressing Asp synthetic pathway genes using strong promoter kasOp*. The resulting strains all showed increased DAP titre. Combined inhibition of acsA4, pta, pyrB, and pyrC increased DAP titre to 167.4 μg/mL (73.5% higher than WT value). Co-overexpression of aspC, gdhA, ppc, and ecaA led to DAP titre 168 μg/mL (75.7% higher than WT value). Concurrently, we constructed a chassis strain favourable for DAP production by abolishing by-product production (i.e., deleting a 21.1 kb region of the red pigment biosynthetic gene cluster (BGC)) and engineering the DAP BGC (i.e., replacing its native dptEp with kasOp*). Titre for the resulting chassis strain reached 185.8 μg/mL. Application of our Asp precursor supply strategies to the chassis strain further increased DAP titre to 302 μg/mL (2.1-fold higher than WT value). Subsequently, we cloned the engineered DAP BGC and duplicated it in the chassis strain, leading to DAP titre 274.6 μg/mL. The above strategies, in combination, resulted in maximal DAP titre 350.7 μg/mL (2.6-fold higher than WT value), representing the highest reported DAP titre in shake-flask fermentation. These findings provide an efficient combination strategy for increasing DAP production and can also be readily applied in the overproduction of other Asp-related antibiotics.