Bactericidal Assay Research Articles

Capsular polysaccharide inhibits vaccine-induced O-antigen antibody binding and function across both classical and hypervirulent K2:O1 strains of Klebsiella pneumoniae.

Klebsiella pneumoniae presents as two circulating pathotypes: classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp). Classical isolates are considered urgent threats due to their antibiotic resistance profiles, while hvKp isolates have historically been antibiotic susceptible. Recently, however, increased rates of antibiotic resistance have been observed in both hvKp and cKp, further underscoring the need for preventive and effective immunotherapies. Two distinct surface polysaccharides have gained traction as vaccine candidates against K. pneumoniae: capsular polysaccharide and the O-antigen of lipopolysaccharide. While both targets have practical advantages and disadvantages, it remains unclear which of these antigens included in a vaccine would provide superior protection against matched K. pneumoniae strains. Here, we report the production of two bioconjugate vaccines, one targeting the K2 capsular serotype and the other targeting the O1 O-antigen. Using murine models, we investigated whether these vaccines induced specific antibody responses that recognize K2:O1 K. pneumoniae strains. While each vaccine was immunogenic in mice, both cKp and hvKp strains exhibited decreased O-antibody binding in the presence of capsule. Further, O1 antibodies demonstrated decreased killing in serum bactericidal assays with encapsulated strains, suggesting that the presence of K. pneumoniae capsule blocks O1-antibody binding and function. Finally, the K2 vaccine outperformed the O1 vaccine against both cKp and hvKp in two different murine infection models. These data suggest that capsule-based vaccines may be superior to O-antigen vaccines for targeting hvKp and some cKp strains, due to capsule blocking the O-antigen.

Involvement of ribonuclease Y in pilus production by M49 Streptococcus pyogenes strain via modulation of messenger RNA level of transcriptional regulator.

Streptococcus pyogenes displays a wide variety of pili, which is largely dependent on serotype. A distinct subset of S. pyogenes strains that possess the Nra transcriptional regulator demonstrates thermoregulated pilus production. Findings obtained in the present study of an Nra-positive serotype M49 strain revealed involvement of conserved virulence factor A (CvfA), also referred to as ribonuclease Y (RNase Y), in virulence factor expression and pilus production, while a cvfA deletion strain showed reduced pilus production and adherence to human keratinocytes as compared with wild-type and revertant strains. Furthermore, transcript levels of pilus subunits and srtC2 genes were decreased by cvfA deletion, which was remarkable at 25°C. Likewise, both messenger RNA (mRNA) and protein levels of Nra were remarkably decreased by cvfA deletion. Whether the expression of other pilus-related regulators, including fasX and CovR, was subject to thermoregulation was also examined. While the mRNA level of fasX, which inhibits cpa and fctA translation, was decreased by cvfA deletion at both 37°C and 25°C, CovR mRNA and protein levels, as well as its phosphorylation level were not significantly changed, suggesting that neither fasX nor CovR is necessarily involved in thermosensitive pilus production. Phenotypic analysis of the mutant strains revealed that culture temperature and cvfA deletion had varied effects on streptolysin S and SpeB activities. Furthermore, bactericidal assay data showed that cvfA deletion decreased the rate of survival in human blood. Together, the present findings indicate that CvfA is involved in regulation of pilus production and virulence-related phenotypes of the serotype M49 strain of S. pyogenes.

The propositive study: Immunogenicity and safety of a four-component recombinant protein-based vaccine against MenB and a quadrivalent conjugate MenACWY vaccine in people living with HIV.

People living with HIV have been shown to have an increased risk of invasive meningococcal disease. In some countries, meningococcal vaccines are now routinely recommended to all people living with HIV, but no study has yet assessed the immunogenicity and safety of a meningococcal serogroup B vaccine or the co-administration of a MenB and MenACWY vaccine in people living with HIV. This phase IV open-label clinical trial investigated the immunogenicity and safety of two doses of a four-component recombinant protein-based MenB vaccine (4CMenB) and a quadrivalent conjugate polysaccharide MenACWY vaccine (MenACWY-CRM197) given 1 month apart in a population of people living with HIV. Immunogenicity analysis was performed before vaccination and 1 month after the second doses of 4CMenB and MenACWY. Primary outcome measures were serum bactericidal assay geometric mean titres against three MenB reference strains at baseline and 1 month post vaccination, the proportion of participants achieving a putative protective titre of ≥4, and the proportion of participants with a ≥4-fold rise in titre from baseline. Secondary outcome measures were serum bactericidal assay geometric mean titres against MenA, C, W, and Y reference strains at baseline and 1 month post vaccination, the proportion achieving a putative protective titre of ≥8, and the proportion with a ≥4-fold rise in titre from baseline. Safety outcomes were solicited and unsolicited adverse events in the 7 days following vaccination. The trial was registered with clinicaltrials.gov (NCT03682939). In total, 55 participants aged 20-45 years were enrolled. All participants (100%; 95% confidence interval [CI] 93-100) achieved putative protective titres for two of the three MenB strains and for MenA, W, and Y. A total of 98% (95% CI 89-100) achieved a protective titre for the third MenB strain and 94% (95% CI 83-99) for MenC. No serious adverse events were reported. 4CMenB and MenACWY were immunogenic and well-tolerated in a population of people living with HIV 1 month after two doses.

Effects of Organic Ligands on the Antibacterial Activity of Reduced Iron-Containing Clay Minerals: Higher Extracellular Hydroxyl Radical Production Yet Lower Bactericidal Activity.

Reduced iron-containing clay (RIC) minerals have been documented to exhibit antibacterial activity through a synergistic action of extracellular membrane attack and intracellular oxidation of cellular components. However, the relative importance between extracellular and intracellular processes has remained elusive. Here, metal-chelating organic ligands (lactate, oxalate, citrate, and ethylene diaminetetraacetic acid (EDTA)) were amended to the bactericidal assays such that the importance of the two processes could be evaluated. Reduced nontronite (rNAu-2) was used as a model clay mineral to produce extracellular hydroxyl radical (•OH) upon oxygenation. The presence of Fe-chelating ligands increased •OH yield by 3-5 times. Consequently, bacterial cell membrane attack was enhanced, yet the antibacterial activity of RIC diminished. Additional experiments revealed that the ligands inhibited soluble metal ions from adsorption onto the bacterial cell membrane and/or penetration into the cytoplasm. Consequently, intracellular Fe concentration for the ligand-treated group was nearly 2 orders of magnitude lower than that for no-ligand control, which greatly decreased intracellular accumulation of reactive oxygen species (ROS) and increased cell survival. These results highlight that destruction of intracellular contents (proteins and DNA) is more important than oxidative degradation of membrane lipids and cell envelope proteins in causing bacterial cell death by RIC.

Bactericidal synergism between phage endolysin Ply2660 and cathelicidin LL-37 against vancomycin-resistant Enterococcus faecalis biofilms

Antibiotic resistance and the ability to form biofilms of Enterococcus faecalis have compromised the choice of therapeutic options, which triggered the search for new therapeutic strategies, such as the use of phage endolysins and antimicrobial peptides. However, few studies have addressed the synergistic relationship between these two promising options. Here, we investigated the combination of the phage endolysin Ply2660 and the antimicrobial peptide LL-37 to target drug-resistant biofilm-producing E. faecalis. In vitro bactericidal assays were used to demonstrate the efficacy of the Ply2660–LL-37 combination against E. faecalis. Larger reductions in viable cell counts were observed when Ply2660 and LL-37 were applied together than after individual treatment with either substance. Transmission electron microscopy revealed that the Ply2660–LL-37 combination could lead to severe cell lysis of E. faecalis. The mode of action of the Ply2660–LL-37 combination against E. faecalis was that Ply2660 degrades cell wall peptidoglycan, and subsequently, LL-37 destroys the cytoplasmic membrane. Furthermore, Ply2660 and LL-37 act synergistically to inhibit the biofilm formation of E. faecalis. The Ply2660–LL-37 combination also showed a synergistic effect for the treatment of established biofilm, as biofilm killing with this combination was superior to each substance alone. In a murine peritoneal septicemia model, the Ply2660–LL-37 combination distinctly suppressed the dissemination of E. faecalis isolates and attenuated organ injury, being more effective than each treatment alone. Altogether, our findings indicate that the combination of a phage endolysin and an antimicrobial peptide may be a potential antimicrobial strategy for combating E. faecalis.

Species identification, antibiotic resistance, and virulence in Enterobacter cloacae complex clinical isolates from South Korea.

This study aimed to identify the species of Enterobacter cloacae complex (ECC) isolates and compare the genotype, antibiotic resistance, and virulence among them. A total of 183 ECC isolates were collected from patients in eight hospitals in South Korea. Based on partial sequences of hsp60 and phylogenetic analysis, all ECC isolates were identified as nine species and six subspecies. Enterobacter hormaechei was the predominant species (47.0%), followed by Enterobacter kobei, Enterobacter asburiae, Enterobacter ludiwigii, and Enterobacter roggenkampii. Multilocus sequence typing analysis revealed that dissemination was not limited to a few clones, but E. hormaechei subsp. xiangfangensis, E. hormaechei subsp. steigerwaltii, and E. ludwigii formed large clonal complexes. Antibiotic resistance rates were different between the ECC species. In particular, E. asburiae, E. kobei, E. roggenkampii, and E. cloacae isolates were highly resistant to colistin, whereas most E. hormaechei and E. ludwigii isolates were susceptible to colistin. Virulence was evaluated through serum bactericidal assay and the Galleria mellonella larvae infection model. Consistency in the results between the serum resistance and the G. mellonella larvae infection assay was observed. Serum bactericidal assay showed that E. hormaechei, E. kobei, and E. ludwigii were significantly more virulent than E. asburiae and E. roggenkampii. In this study, we identified the predominant ECC species in South Korea and observed the differences in antibiotic resistance and virulence between the species. Our findings suggest that correct species identification, as well as continuous monitoring is crucial in clinical settings.

Super effective antimicrobial silver-sputtered coatings on poly(lactic acid) against bacteria and omicron SARS-CoV-2

Poly(lactic acid) (PLA) is a biopolymer with properties potentially suitable for fabricating packaging, medical devices, and healthcare products in a more friendly environmental way because this polymer presents biodegradability, compostability, low carbon footprint, and recyclability. However, PLA does not present intrinsic antimicrobial properties. Antimicrobial materials are highly desirable for manufacturing smart packaging and personal protective equipment to secure food and health professionals against pathogenic microorganisms. In this work, we evaluated the antimicrobial performance of (Ag)-coated PLA against Escherichia coli, Bacillus subtilis, and Omicron severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). PLA was rapidly coated with metallic Ag by pulsed direct current magnetron sputtering (pDCMS) for 5, 10, and 20 s. Atomic force microscopy indicates that the Ag coating grows predominantly on the PLA surface via a bulk diffusion mechanism. According to bactericidal and quantitative reverse transcription polymerase chain reaction assays, Ag-coated PLA was capable of inhibiting bacterial biofilm formation and disrupting the genetic material of the Omicron SARS-CoV-2. X-ray high-resolution photoelectron and nuclear magnetic resonance results suggest no polymer chain scission in the PLA bulk due to plasma thermal stress effects during Ag sputtering.

How Does Time Affect the Antimicrobial Activity of Super-Oxidized Commercial Antiseptic Solutions? An In Vitro Test.

This study aimed to identify variation in the minimum biocidal concentration (MBC) over time, comparing three commercial super-oxidized solutions with different chemical compositions. In the bactericidal assay, the following bacteria were tested: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923), and for each ATCC, one wild-type strain was used. In vitro experiments were performed in triplicate at 0, 60, and 120 days of follow up. A commercial formulation based on sodium and chloride ions (SCSS) was tested using a standard accelerated aging protocol. Data were analyzed with the Friedman and Wilcoxon signed-rank tests. The results showed that super-oxidized solution bases of 20 ppm of sodium (SSS) had a significant change in MBC at 120 days (p < 0.001), whereas SCSS remained stable during the same period (p = 0.18). However, after accelerated aging treatment, the MBC of SCSS increased (p < 0.001). With our proposed approach, the two SSS showed MBC variation at 120 days, whereas SCSS showed stability over time, similar to chlorhexidine, but lost its bactericidal properties after accelerated aging treatment.

Genetic, Functional, and Immunogenic Analyses of the O-Linked Protein Glycosylation System in Neisseria meningitidis Serogroup A ST-7 Isolates.

Neisseria meningitidis exhibits a general O-linked protein glycosylation system in which pili and other extracytoplasmic proteins are glycosylated. To investigate glycan antigenicity in humans and the significance of high glycan diversity on immune escape mechanisms, we exploited serogroup A meningococcal strains and serum samples obtained from laboratory-confirmed Ethiopian patients with meningococcal disease. The 37 meningococcal isolates were sequenced, and their protein glycosylation (pgl) genotypes and protein glycosylation phenotypes were investigated in detail. An insertion sequence (IS1655) element in pglH reduced glycan variability in the majority of isolates, while phase variation strengthened glycan variability and microheterogeneity. Homologous recombination events within the pgl genes were identified in eight of the 37 isolates, and the phenotypic consequences ranged from none detected to altered glycoforms in two of the isolates in which the whole pgl locus was exchanged. Immunoblotting of sera against a complete panel of glycan-expressing mutant strains demonstrated that most of these patient sera had IgG antibodies against various neisserial protein glycan antigens. Furthermore, using a bactericidal assay comparing a wild-type meningococcal A strain and a glycosylation-null variant strain, we showed that these protein glycan antigens interfere with bactericidal killing by antibodies in patient sera. Altogether, we were largely able to link pgl genotype with glycosylation phenotype. Our study reveals that protein glycans seem to contribute to the ability of N. meningitidis to resist the bactericidal activity of human serum, possibly by masking protein epitopes important for bactericidal killing and thus protection against meningococcal disease. IMPORTANCE Bacterial meningitis is a serious global health problem, and one of the major causative organisms is Neisseria meningitidis. Extensive variability in protein glycan structure and antigenicity is due to phase variation of protein glycosylation genes and polymorphic gene content and function. The exact role(s) of glycosylation in Neisseria remains to be determined, but increasing evidence, supported by this study, suggests that glycan variability can be a strategy to escape the human immune system. The complexity of the O-linked protein glycosylation system requires further studies to fully comprehend how these bacteria utilize variation in pgl genes to produce such high glycoform diversity and to evade the human immune response.

An “intelligent -responsive” bactericidal system based on OSA-starch Pickering emulsion

Pickering emulsion based on OSA-starch was developed in this study as an intelligent delivery system for the application of thymol against foodborne pathogens. Morphology and microstructure characterization showed that the Pickering emulsion was an O/W type emulsion and stayed stable at starch concentration of 200 mg/mL and oil fraction at 30 % with particle size of 10 μm and absolute Zeta potential of −12.5 mV. Low field nuclear magnetic resonance and rheology experiments indicated that a denser network structure was formed in this stable Pickering emulsion. Besides, the Pickering emulsion could endure long-time storage, low pH (3,5) and additional NaCl (50, 100, 200, 400 mM) and it showed enhanced bactericidal effects against Escherichia coli, Staphylococcus aureus (thymol =1.48 μmol/L) and Aspergillus flavus (thymol = 0.624 μmol/L) by inducing ROS eruption, membrane lipid peroxidation and cell shrink. Moreover, the bactericidal assay demonstrated that thymol could be intelligently released and a considerable 75 % timely bactericidal effect was detected after 9 days' intermittently exposing to E. coli, S. aureus and A. flavus in vitro, by comparison thymol alone showed only 20 % bactericidal effect due to its volatility. The results are of great importance to offer an intelligent delivery system of bio-actives defending foodborne pathogens.

Hypochlorous acid inactivates oral pathogens and a SARS-CoV-2-surrogate

BackgroundDroplets and aerosols produced during dental procedures are a risk factor for microbial and viral transmission. Unlike sodium hypochlorite, hypochlorous acid (HOCl) is nontoxic to tissues but still exhibits broad microbicidal effect. HOCl solution may be applicable as a supplement to water and/or mouthwash. This study aims to evaluate the effectiveness of HOCl solution on common human oral pathogens and a SARS-CoV-2 surrogate MHV A59 virus, considering the dental practice environment.MethodsHOCl was generated by electrolysis of 3% hydrochloric acid. The effect of HOCl on human oral pathogens, Fusobacterium nucleatum, Prevotella intermedia, Streptococcus intermedius, Parvimonas micra, and MHV A59 virus was studied from four perspectives: concentration; volume; presence of saliva; and storage. HOCl solution in different conditions was utilized in bactericidal and virucidal assays, and the minimum inhibitory volume ratio that is required to completely inhibit the pathogens was determined.ResultsIn the absence of saliva, the minimum inhibitory volume ratio of freshly prepared HOCl solution (45–60 ppm) was 4:1 for bacterial suspensions and 6:1 for viral suspensions. The presence of saliva increased the minimum inhibitory volume ratio to 8:1 and 7:1 for bacteria and viruses, respectively. Applying a higher concentration of HOCl solution (220 or 330 ppm) did not lead to a significant decrease in the minimum inhibitory volume ratio against S. intermedius and P. micra. The minimum inhibitory volume ratio increases in applications of HOCl solution via the dental unit water line. One week of storage of HOCl solution degraded HOCl and increased the minimum growth inhibition volume ratio.ConclusionsHOCl solution (45–60 ppm) is still effective against oral pathogens and SAR-CoV-2 surrogate viruses even in the presence of saliva and after passing through the dental unit water line. This study indicates that the HOCl solution can be used as therapeutic water or mouthwash and may ultimately reduce the risk of airborne infection in dental practice.

Antibiotic resistance and virulence characteristics of four carbapenem-resistant Klebsiella pneumoniae strains coharbouring blaKPC and blaNDM based on whole genome sequences from a tertiary general teaching hospital in central China between 2019 and 2021.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection is a worldwide health issue that poses a serious threat to public health. This study summarizes the clinical features of four patients with CRKP coproducing NDM and KPC infections and further analyses the molecular typing, resistance and virulence factors of the four CRKP strains. Of the twenty-two CRKP isolates, four strains coharbouring blaKPC and blaNDM isolated from four patients were screened by Sanger sequencing between October 2019 and April 2021. Demographics, clinical and pathological data of the four patients were collected through electronic medical records. Antimicrobial susceptibility testing, biofilm formation assays and serum bactericidal assays were performed on the four isolates. The antibiotic resistance and virulence genes were investigated by whole-genome sequencing. Sequence types (STs) were determined by multilocus sequence typing, and serotypes were identified by wzi gene sequencing. Three patients recovered, and one patient stopped treatment. Four strains were multiple carbapenemase producers: KPC-2, NDM-4, SME-5 and IMI-4 coproducer; KPC-2, NDM-1 and SME-3 coproducer; KPC-2, NDM-1 and IMI-3 coproducer; KPC-2 and NDM-5 coproducer. They also harboured ESBL genes and mutations in the efflux pump regulator genes. They were multidrug resistant but sensitive to tigecycline and colistin. Four isolates had moderate biofilm-forming abilities and carried various virulence genes, including siderophores, type 1 fimbriae and E. coli common pilus. Only the NO. 3 strain was resistant to the serum. The STs and serotypes of the four strains were ST11 and KL64, ST337 and none, ST307 and KL102KL149KL155, and ST29 and K54, respectively. Four CRKP strains coharbouring blaKPC and blaNDM also carried other carbapenemase genes. Notably, the NO. 1 isolate carrying four carbapenemase genes has not been reported globally until now. Four strains exhibited a high level of resistance to multiple antibiotics. Additionally, three of the four patients were exposed to invasive medical devices that provided an environment for biofilm formation. Meanwhile, three strains with adhesion genes as moderate biofilm formers might form biofilms resulting in long hospital stays, increasing therapeutic difficulty, and even treatment failure. This study reminds clinicians that CRKP strains with multiple carbapenemase genes emerged in our hospital, and stronger measures should be taken to the control of nosocomial infections.

Application of Phage Therapy in a Case of a Chronic Hip-Prosthetic Joint Infection due to Pseudomonas aeruginosa: An Italian Real-Life Experience and In Vitro Analysis.

Prosthetic joint infection (PJI) caused by Pseudomonas aeruginosa represents a severe complication in orthopedic surgery. We report the case of a patient with chronic PJI from P. aeruginosa successfully treated with personalized phage therapy (PT) in combination with meropenem. A 62-year-old woman was affected by a chronic right hip prosthesis infection caused by P. aeruginosa since 2016 . The patient was treated with phage Pa53 (I day 10 mL q8h, then 5 mL q8h via joint drainage for 2 weeks) in association with meropenem (2gr q12h iv) after a surgical procedure. A 2-year clinical follow up was performed. An in vitro bactericidal assay of the phage alone and in combination with meropenem against a 24-hour-old biofilm of bacterial isolate was also carried out. No severe adverse events were observed during PT. Two years after suspension, there were no clinical signs of infection relapse, and a marked leukocyte scan showed no pathological uptake areas. In vitro studies showed that the minimum biofilm eradicating concentration of meropenem was 8 µg/mL. No biofilm eradication was observed at 24 hours incubation with phages alone (108 plaque-forming units [PFU]/mL). However, the addition of meropenem at suberadicating concentration (1 µg/mL) to phages at lower titer (103 PFU/mL) resulted in a synergistic eradication after 24 hours of incubation. Personalized PT, in combination with meropenem, was found to be safe and effective in eradicating P. aeruginosa infection. These data encourage the development of personalized clinical studies aimed at evaluating the efficacy of PT as an adjunct to antibiotic therapy for chronic persistent infections.

Photodynamic activity enhanced by in situ biosynthetic BC/CQDs@PCN-224 membranes through FRET strategy

Porphyrin-based metal organic frameworks (MOFs) with efficient bactericidal performance have increasingly attracted attention in photodynamic inactivation materials. However, low reactive oxygen species (ROS) yield and drug residue hazards of current porphyrin-MOFs materials lead to unsatisfactory clinical therapeutic effects. In this paper, carbon quantum dots (CQDs) were encapsulated into PCN-224, which enhanced the photodynamic activity of the MOFs through fluorescence resonance energy transfer (FRET) process. Singlet oxygen (1O2) detection confirmed that the photodynamic activity of CQDs-doped PCN-224 (CQDs@PCN-224) was enhanced than that of pristine PCN-224 under illumination. Furthermore, the CQDs@PCN-224 were firmly embedded into bacterial cellulose (BC) nanofibrous membranes by using an eco-friendly biosynthetic approach, efficiently preventing MOFs leakage during use. The results of bactericidal assays demonstrated that BC/CQDs@PCN-224 membrane with higher photodynamic activity causes more severe disruption to bacterial structure and possesses better antibacterial efficiency (>99.99 % reduction of both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli O157:H7 within 30 min) than BC/PCN-224 membrane under visible light illumination (500 W, 15 cm height, λ ≥ 420 nm). In addition, the biosynthesized BC/CQDs@PCN-224 membrane showed good hemocompatibility and low cytotoxicity, revealing that the BC- and MOFs-based material with enhanced PDI efficiency and satisfying safety has great potential in medical fields.

Uracil restores susceptibility of methicillin-resistant Staphylococcus aureus to aminoglycosides through metabolic reprogramming.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) has now become a major nosocomial pathogen bacteria and resistant to many antibiotics. Therefore, Development of novel approaches to combat the disease is especially important. The present study aimed to provide a novel approach involving the use of nucleotide-mediated metabolic reprogramming to tackle intractable methicillin-resistant S. aureus (MRSA) infections. Objective: This study aims to explore the bacterial effects and mechanism of uracil and gentamicin in S. aureus. Methods: Antibiotic bactericidal assays was used to determine the synergistic bactericidal effect of uracil and gentamicin. How did uracil regulate bacterial metabolism including the tricarboxylic acid (TCA) cycle by GC-MS-based metabolomics. Next, genes and activity of key enzymes in the TCA cycle, PMF, and intracellular aminoglycosides were measured. Finally, bacterial respiration, reactive oxygen species (ROS), and ATP levels were also assayed in this study. Results: In the present study, we found that uracil could synergize with aminoglycosides to kill MRSA (USA300) by 400-fold. Reprogramming metabolomics displayed uracil reprogrammed bacterial metabolism, especially enhanced the TCA cycle to elevate NADH production and proton motive force, thereby promoting the uptake of antibiotics. Furthermore, uracil increased cellular respiration and ATP production, resulting the generation of ROS. Thus, the combined activity of uracil and antibiotics induced bacterial death. Inhibition of the TCA cycle or ROS production could attenuate bactericidal efficiency. Moreover, uracil exhibited bactericidal activity in cooperation with aminoglycosides against other pathogenic bacteria. In a mouse mode of MRSA infection, the combination of gentamicin and uracil increased the survival rate of infected mice. Conclusion: Our results suggest that uracil enhances the activity of bactericidal antibiotics to kill Gram-positive bacteria by modulating bacterial metabolism.

Immunogenicity and Safety of a Quadrivalent Meningococcal Tetanus Toxoid­-Conjugate Vaccine (MenACYW­-TT) Administered Concomitantly with Pneumococcal Conjugate Vaccine in Healthy Toddlers in the Russian Federation: a Phase III Randomized Study

Relevance. Invasive meningococcal disease (IMD) has high morbidity and mortality, with infants and young children among those at greatest risk.Materials &amp; Methods. A phase III, open-­label, randomized study in toddlers aged 12–23 months evaluated the immunogenicity and safety of MenACYW­TT, a tetanus toxoid conjugated vaccine against meningococcal serogroups A, C, W, and Y, when coadministered with paediatric vaccines (measles, mumps and rubella [MMR]; varicella [V] in South Korea and Thailand; 6­in­1 combination vaccine against diphtheria, tetanus, pertussis, polio, hepatitis B and Haemophilus influenzae type b [DTaP­IPVHepB­Hib] in Mexico and pneumococcal conjugate vaccine [PCV13]) in the Russian Federation (NCT03205371). This manuscript reports the outcome of the part of the study conducted in the Russian Federation using PCV13 as the co­administered vaccine. Immunogenicity to each meningococcal serogroup was assessed by serum bactericidal antibody assay using human complement (hSBA) and, for a subset of subjects, baby rabbit complement (rSBA). Vaccine safety profiles were described up to 30 days postvaccination.Results. A total of 1,183 participants were enrolled in the study, out of which 400 were from the Russian Federation. The proportion with seroprotection (hSBA ≥1:8) to each meningococcal serogroup at Day 30 was comparable between the MenACYW­-TT and MenACYW-­TT + PCV13 groups (≥91% and ≥84%, respectively). The safety profiles of MenACYW­-TT and PCV13, when given alone or concomitantly, were generally comparable.Conclusion. Coadministration of MenACYW­-TT with pneumococcal conjugate vaccine in toddlers had no clinically relevant effect on the immunogenicity and safety of any of the vaccines.

Immunogenicity and safety of a quadrivalent meningococcal conjugate vaccine (MenACYW-TT) administered as a booster to adults aged ≥59 years: A phase III randomized study

ABSTRACT This 2-stage Phase III study (NCT04142242) of a recently licensed quadrivalent meningococcal tetanus toxoid-conjugate vaccine (MenACYW-TT) assessed the safety and immunogenicity of a booster dose in older adults (≥59 years) primed with either MenACYW-TT or a quadrivalent meningococcal polysaccharide vaccine (MPSV4). Immune persistence of MenACYW-TT and MPSV4 after primary vaccination was also evaluated. During Stage I, the participants administered MPSV4 (n = 165) or MenACYW-TT (n = 236) 3 years previously were randomized 9:2 to receive either a MenACYW-TT booster or to have blood drawn for persistence only. Participants primed with MPSV4 or MenACYW-TT 6–7 years previously had blood drawn for antibody persistence only. A serum bactericidal assay using human complement was used to measure functional antibodies against each serogroup at baseline and, for those receiving a booster, 30 days post-vaccination (D30). Proportions of participants with seroresponse (post-vaccination titers ≥1:16 when baseline titers <1:8 or ≥ 4-fold increase when baseline titers ≥1:8) were determined. Safety data were collected up to D30. Seroresponse rates for all serogroups at D30 ranged from 49.2% to 60.8% in the MPSV4-primed group, and 79.3–93.1% in the MenACYW-TT-primed group. MenACYW-TT induced sufficient seroresponses in each primed group. Geometric mean titers (GMTs) for serogroups C, W, and Y remained or trended higher than pre-vaccination levels at both 3 and 6–7 years after primary vaccination, indicating immune persistence. Safety outcomes were comparable between groups. A MenACYW-TT booster was immunogenic and well tolerated in participants aged ≥59 years regardless of previous quadrivalent meningococcal vaccine received. The greatest immune responses occurred in those primed with MenACYW-TT.

Immunization with recombinant DcaP-like protein and AbOmpA revealed protections against sepsis infection of multi-drug resistant Acinetobacter baumannii ST2Pas in a C57BL/6 mouse model.

The prevalence of infections associated with multi-drug resistant (MDR) Acinetobacter baumannii is increasing worldwide. Therefore, the introduction of effective vaccines against this bacterium seems necessary. AbOmpA and DcaP-like protein were selected as promising and putative immunogenic candidates based on previous in silico studies. Three formulations including AbOmpA, DcaP-like protein, and AbOmpA+DcaP-like protein were injected into C57BL/6 mice three times with Alum adjuvant. The specific production of IgG antibodies (e.g. total IgG, IgG1 and IgG2c) and cytokines (e.g. IL-4, IL-6, and IL-17A), were evaluated. LD50% of MDR A. baumannii ST2Pas was measured using Probit's method. After the challenge with bacteria, a decrease in bacterial loads (DLs) in the lung and spleen of mice was measured. Then serum bactericidal assay was performed to determine the function of antibodies on day 42. In addition, histopathological examinations of the spleen and lung, the number of macrophage and neutrophil, as well as the rate of lymphocyte infiltration were assessed. The highest level of total IgG was reported in the group immunized with DcaP-like protein on day 42. The survival rate of mice was 80% in the AbOmpA immunized group and 100% for the rest of two groups. DLs in the spleen of mice immunized with AbOmpA, DcaP-like protein, and combination form were 3.5, 3, and 3.4 Log10 (CFU/g), respectively. While in the lung, the DLs were 7.5 Log10 (CFU/g) for the AbOmpA group and 5 for the rest of two groups. The levels of IL-6, IL-4, and IL-17A were significantly decreased in all immunized groups after the bacterial challenge (except for IL-17A in the group of AbOmpA). The bactericidal effect of antibodies against DcaP-like protein was more effective. No histopathological damage was observed in the combination immunized group. The DcaP-like protein was more effective in neutrophil and macrophage deployment and decreased lymphocyte infiltration. The results of immunization with AbOmpA+DcaP-like protein induced a protective reaction against the sepsis infection of MDR A. baumannii. It seems that in the future, these proteins can be considered as promising components in the development of the A. baumannii vaccine.

Production and Characterization of Bacterial Ghost Vaccine against Neisseria meningitidis.

Bacterial ghosts (BGS) are empty non-living envelopes produced either genetically or chemically. This study investigated a novel chemical protocol for the production of Neisseria meningitidis ghost vaccine using tween 80 followed by a pH reduction with lactic acid. For our vaccine candidate, both safety and immunogenicity aspects were evaluated. The ghost pellets showed no sign of growth upon cultivation. BGS were visualized by scanning electron microscopy, illustrating the formation of trans-membrane tunnels with maintained cell morphology. Gel electrophoresis showed no distinctive bands of the cytoplasmic proteins and DNA, assuring the formation of ghost cells. In animal model, humoral immune response significantly increased when compared to commercial vaccine (p < 0.01). Moreover, serum bactericidal assay (SBA) recorded 94.67% inhibition compared to 64% only for the commercial vaccine after three vaccination doses. In conclusion, this is the first N. meningitidis ghost vaccine candidate, proven to be effective, economic, and with significant humoral response and efficient SBA values; however, clinical studies should be performed.

Development and Characterization of a Luminescence-Based High-Throughput Serum Bactericidal Assay (L-SBA) to Assess Bactericidal Activity of Human Sera against Nontyphoidal Salmonella.

Salmonella Typhimurium and Salmonella Enteritidis are leading causative agents of invasive nontyphoidal Salmonella (iNTS) disease, which represents one of the major causes of death and morbidity in sub-Saharan Africa, still partially underestimated. Large sero-epidemiological studies are necessary to unravel the burden of disease and guide the introduction of vaccines that are not yet available. Even if no correlate of protection has been determined so far for iNTS, the evaluation of complement-mediated functionality of antibodies generated towards natural infection or elicited upon vaccination may represent a big step towards this achievement. Here we present the setup and the intra-laboratory characterization in terms of repeatability, intermediate precision, linearity, and specificity of a high-throughput luminescence-based serum bactericidal assay (L-SBA). This method could be useful to perform sero-epidemiological studies across iNTS endemic countries and for evaluation of antibodies raised against iNTS vaccine candidates in upcoming clinical trials.