Bactericidal Assay Research Articles

Intra-Laboratory Evaluation of Luminescence Based High-Throughput Serum Bactericidal Assay (L-SBA) to Determine Bactericidal Activity of Human Sera against Shigella.

Despite the huge decrease in deaths caused by Shigella worldwide in recent decades, shigellosis still causes over 200,000 deaths every year. No vaccine is currently available, and the morbidity of the disease coupled with the rise of antimicrobial resistance renders the introduction of an effective vaccine extremely urgent. Although a clear immune correlate of protection against shigellosis has not yet been established, the demonstration of the bactericidal activity of antibodies induced upon vaccination may provide one means of the functionality of antibodies induced in protecting against Shigella. The method of choice to evaluate the complement-mediated functional activity of vaccine-induced antibodies is the Serum Bactericidal Assay (SBA). Here we present the development and intra-laboratory characterization of a high-throughput luminescence-based SBA (L-SBA) method, based on the detection of ATP as a proxy of surviving bacteria, to evaluate the complement-mediated killing of human sera. We demonstrated the high specificity of the assay against a homologous strain without any heterologous aspecificity detected against species-related and non-species-related strains. We assessed the linearity, repeatability and reproducibility of L-SBA on human sera. This work will guide the bactericidal activity assessment of clinical sera raised against S. sonnei. The method has the potential of being applicable with similar performances to determine the bactericidal activity of any non-clinical and clinical sera that rely on complement-mediated killing.

Antibacterial activity of Xenorhabdus and Photorhabdus isolated from entomopathogenic nematodes against antibiotic-resistant bacteria.

Xenorhabdus and Photorhabdus, symbiotically associated with entomopathogenic nematodes (EPNs), produce a range of antimicrobial compounds. The objective of this study is to identify Xenorhabdus and Photorhabdus and their EPNs hosts, which were isolated from soil samples from Saraburi province, and study their antibacterial activity against 15 strains of drug-resistant bacteria. Fourteen isolates (6.1%), consisting of six Xenorhabdus isolates and eight Photorhabdus isolates, were obtained from 230 soil samples. Based on the BLASTN search incorporating the phylogenetic analysis of a partial recA gene, all six isolates of Xenorhabdus were found to be identical and closely related to X. stockiae. Five isolates of Photorhabdus were found to be identical and closely related to P. luminescens subsp. akhurstii. Two isolates of Photorhabdus were found to be identical and closely related to P. luminescens subsp. hainanensis. The remaining isolate of Photorhabdus was found to be identical to P. asymbiotica subsp. australis. The bacterial extracts from P. luminescens subsp. akhurstii showed strong inhibition the growth of S. aureus strain PB36 (MSRA) by disk diffusion, minimal inhibitory concentration, and minimal bactericidal concentration assay. The combination between each extract from Xenorhabdus/Photorhabdus and oxacillin or vancomycin against S. aureus strain PB36 (MRSA) exhibited no interaction on checkerboard assay. Moreover, killing curve assay of P. luminescens subsp. akhurstii extracts against S. aureus strain PB36 exhibited a steady reduction of 105 CFU/ml to 103 CFU/ml within 30 min. This study demonstrates that Xenorhabdus and Photorhabdus, showed antibacterial activity. This finding may be useful for further research on antibiotic production.

Ten-Year Antibody Persistence and Booster Response to MenACWY-TT Vaccine After Primary Vaccination at 1-10 Years of Age

ABSTRACT This phase 3B, open-label, extension study (NCT01962207) evaluated long-term persistence of antibodies induced by the quadrivalent meningococcal vaccine conjugated to tetanus toxoid (MenACWY-TT) compared with the meningococcal serogroup C vaccine conjugated to CRM (MenC-CRM) and the quadrivalent meningococcal polysaccharide vaccine (MenACWY-PS) 6 to 10 y after primary vaccination in toddlers (aged 1–<2 y; MenACWY-TT and MenC-CRM) and children (aged 2–<11 y; MenACWY-TT and MenACWY-PS). Antibody responses against meningococcal serogroups A, C, W, and Y were assessed by serum bactericidal antibody assays using rabbit (rSBA) or human (hSBA) complement. A MenACWY-TT booster dose at Year 10 was given to all eligible subjects regardless of the primary vaccine received. At Year 10, the percentages of subjects with rSBA titers ≥1:8 for serogroups A, C, W, and Y were as follows: MenACWY-TT (toddlers), 65.6%, 82.8%, 31.3%, 43.8%, respectively; MenC-CRM, 88.2% for serogroup C; MenACWY-TT (children), 88.9%, 84.1%, 67.1%, 65.9%; and MenACWY-PS, 28.6%, 81.0%, 23.8%, and 23.8%. Corresponding percentages for hSBA titers ≥1:4 were as follows: MenACWY-TT (toddlers), 31.1%, 91.9%, 44.4%, 41.4%; MenC-CRM, 93.8% for serogroup C; MenACWY-TT (children), 34.8%, 91.1%, 61.2%, 72.6%; and MenACWY-PS, 33.3%, 100.0%, 26.3%, and 44.4%. One month after the MenACWY-TT booster, the percentage of subjects with vaccine response ranged from 75.7% to 100.0% across serogroups in all study groups. Postbooster vaccine responses were generally comparable between groups across serogroups. No new safety signals were identified. Antibody responses persisted 10 y after MenACWY-TT vaccination. The MenACWY-TT booster dose was well tolerated and elicited robust immune responses.

Short communication: Effects of moringa extract on adhesion and invasion of Escherichia coli O55 in bovine mammary epithelial cells

The objective of this study was to evaluate the antibacterial activities of extract derived from moringa leaves. In particular, the effect of moringa extract (Mor) on adhesion and invasion of Escherichia coli O55, Enterococcus faecalis, Staphylococcus simulans, and Serratia liquefaciens was evaluated in bovine mammary epithelial cells (MAC-T). Broth microdilution method, minimum inhibitory concentration and minimum bactericidal concentration assays, adhesion and invasion assays, and real-time PCR were performed. The minimum inhibitory concentration and minimum bactericidal concentration of Mor ranged from 12.5 to 50 mg/mL on 18 out of 27 tested isolates. Treatment of E. coli O55 with Mor (100 and 200 μg/mL) inhibited the adhesion and invasion on MAC-T cells via downregulation of adhesion factors (i.e., papC, f17c-A, and eaeA). Also, when MAC-T cells were pretreated with Mor (200 μg/mL, 12 h) and incubated with E. coli O55, Enterococcus faecalis, Staphylococcus simulans, or Serratia liquefaciens, both E. coli O55 and Enterococcus faecalis showed a significant decrease in adhesion and invasion. Staphylococcus simulans exhibited decreased adhesion and increased invasion. Serratia liquefaciens showed increased adhesion and decreased invasion. In addition, Mor increased mRNA expression of antioxidant enzymes (e.g., heme oxygenase-1, NAD(P)H:quinone oxidoreductase-1, and thioredoxin reductase 1) in MAC-T cells. In conclusion, 12.5 to 50 mg/mL of Mor exhibited antibacterial activity against 18 out of 27 tested isolates. Also, pretreatment of 200 μg/mL of Mor to MAC-T cells modulated adhesion and invasion of E. coli O55 and other mastitis-associated pathogens. Furthermore, Mor increased antioxidant capacities in MAC-T cells, but further in vivo studies are needed.

Safety and Immunogenicity of a Quadrivalent Meningococcal Conjugate Vaccine (MenACWY-D) in Children 9–23 Months of Age in Russian Federation: Results of Russian Part of International Study in Russia and India

Aim. Assessment of tolerability, safety and immunogenicity of the 4-valent conjugated meningococcal MenACYW-D vaccine, administered 2 times to children 1–2 years of age in the Russian Federation.Study participants. 100 children aged 9–17 months not previously vaccinated against meningococcal infection.Methods. Two doses of the MenACYW-D vaccine were administered intramuscularly at intervals of 3–6 months. Antibody titers for meningococci of serogroups A, C, W, and Y were determined using a serum bactericidal assay in the presence of human complement before the 1st vaccination and 28 days after the 2nd vaccination. Participants with titers ≥ 1:8 were considered protected from meningococcal infection caused by the corresponding serogroup of meningococci.Results. After two vaccinations, the level of seroprotection in relation to these four serogroups of meningococci was achieved in 92.9–99.0% of vaccinated children. No immediate adverse events were reported after any of the 2 doses of the vaccine studied. The frequency of local and general expected adverse reactions after any of the 2 doses of the vaccine was 45% and 40%, respectively, in terms of severity they were mostly weak and disappeared within 3 days. In general, there was no increase in the reactogenicity of the vaccine after administration of the 2nd dose compared to the 1st dose. Unexpected adverse events were recorded in 10% of the study participants, of which only one (diarrhea that stopped within one day) at the conclusion of the research physician was a causally related with the vaccine. None of the adverse events led to the early termination of participation in the study. One serious adverse event has been reported, which the physician has identified as not having a causal relationship with the vaccine being studied.Conclusions. Two dose immunization with the MenACWY-D vaccine in children 1–2 years of age in the Russian Federation was safe, well tolerated, and induced a pronounced bactericidal humoral immune response against meningococci of serogroups A, C, W, and Y.

Safety and Immunogenicity of a Quadrivalent Meningococcal Conjugate Vaccine (MenACWY-D) in Children 9–23 Months of Age in Russian Federation: Results of Russian Part of International Study in Russia and India

Aim. Assessment of tolerability, safety and immunogenicity of the 4-valent conjugated meningococcal MenACYW-D vaccine, administered 2 times to children 1–2 years of age in the Russian Federation.Study participants. 100 children aged 9–17 months not previously vaccinated against meningococcal infection.Methods. Two doses of the MenACYW-D vaccine were administered intramuscularly at intervals of 3–6 months. Antibody titers for meningococci of serogroups A, C, W, and Y were determined using a serum bactericidal assay in the presence of human complement before the 1st vaccination and 28 days after the 2nd vaccination. Participants with titers ≥ 1:8 were considered protected from meningococcal infection caused by the corresponding serogroup of meningococci.Results. After two vaccinations, the level of seroprotection in relation to these four serogroups of meningococci was achieved in 92.9–99.0% of vaccinated children. No immediate adverse events were reported after any of the 2 doses of the vaccine studied. The frequency of local and general expected adverse reactions after any of the 2 doses of the vaccine was 45% and 40%, respectively, in terms of severity they were mostly weak and disappeared within 3 days. In general, there was no increase in the reactogenicity of the vaccine after administration of the 2nd dose compared to the 1st dose. Unexpected adverse events were recorded in 10% of the study participants, of which only one (diarrhea that stopped within one day) at the conclusion of the research physician was a causally related with the vaccine. None of the adverse events led to the early termination of participation in the study. One serious adverse event has been reported, which the physician has identified as not having a causal relationship with the vaccine being studied.Conclusions. Two dose immunization with the MenACWY-D vaccine in children 1–2 years of age in the Russian Federation was safe, well tolerated, and induced a pronounced bactericidal humoral immune response against meningococci of serogroups A, C, W, and Y.

Efficacy and safety of a booster dose of the meningococcal A, C, W, Y-tetanus toxoid conjugate vaccine administered 10 years after primary vaccination and long-term persistence of tetanus toxoid conjugate or polysaccharide vaccine

ABSTRACT A previous phase 3, randomized, multicenter study showed the immunogenicity of a primary vaccination of subjects aged 11 to 17 years with the quadrivalent meningococcal vaccine conjugated to tetanus toxoid (MenACWY-TT) or the quadrivalent meningococcal polysaccharide vaccine (MenACWY-PS). This extension study evaluated the safety and immunogenicity of a MenACWY-TT booster 10 years after receiving a primary dose of either MenACWY-TT or MenACWY-PS. The primary immunogenicity endpoint was booster response, evaluated using serum bactericidal antibody assays with rabbit complement (rSBA), 1 month postbooster. Safety endpoints included the percentage of subjects experiencing local and general adverse events (AEs) ≤4 days after MenACWY-TT booster. Of 229 subjects enrolled, 169 and 58 in the MenACWY-TT and MenACWY-PS groups, respectively, completed the booster phase. The 1 month postbooster response for each serogroup ranged from 81.5% to 95.7% for MenACWY-TT and 66.7% to 94.1% for MenACWY-PS. Similar percentages of MenACWY-TT and MenACWY-PS recipients had a booster response to serogroups A, W, and Y, whereas more MenACWY-TT recipients than MenACWY-PS recipients had a booster response to serogroup C. For the MenACWY-TT and MenACWY-PS groups, respectively, the MenACWY-TT booster elicited rSBA titers ≥1:8 in 100% and ≥98.0% of subjects across all serogroups; 100% and ≥96.1% of all subjects had titers ≥1:128. No new safety signals were observed during the booster phase. In conclusion, a MenACWY-TT booster dose after receiving either a primary dose of MenACWY-TT or MenACWY-PS elicited robust immune responses and was well tolerated. Functional antibody responses last up to 10 years after primary MenACWY-TT vaccination.

Immunogenicity and safety of a quadrivalent meningococcal tetanus toxoid-conjugate vaccine (MenACYW-TT) in ≥56-year-olds: A Phase III randomized study

BackgroundInvasive meningococcal disease has a high mortality rate in individuals aged ≥56 years, but no vaccine is currently licensed in the USA for this age group. This study assessed the safety and immunogenicity of an investigational quadrivalent meningococcal tetanus toxoid conjugate vaccine (MenACYW-TT) compared with a meningococcal quadrivalent polysaccharide vaccine (MPSV4) in this age group. MethodsThis was a Phase III, modified double-blind, randomized, non-inferiority study (NCT02842866) across 35 clinical sites in the USA and Puerto Rico in individuals aged ≥56 years. A single dose of the MenACYW-TT (n = 451) or MPSV4 vaccine (n = 455) was administered on Day 0. A serum bactericidal assay with human (hSBA) and baby rabbit (rSBA) complement was used to measure antibodies against serogroups A, C, W, and Y test strains at baseline and Day 30. Safety data were collected up to six months post-vaccination. ResultsThe seroresponse to MenACYW-TT was non-inferior to MPSV4 for each of the serogroups (A: 58.2% vs. 42.5%; C: 77.1% vs. 49.7%; W: 62.6% vs. 44.8%, Y: 74.4% vs. 43.4%, respectively). At Day 30, participants achieving hSBA titers ≥1:8 were higher for all serogroups after MenACYW-TT vs. MPSV4 (77.4–91.7 vs. 63.1–84.2%, respectively). No safety concerns were identified for either vaccine. ConclusionMenACYW-TT was well-tolerated and immunogenic in ≥56-year-olds, offering the potential to replace MPSV4 in this age group.

Nonencapsulated Streptococcus pneumoniae causes Bacteremia in C1q-Deficient Mice

Abstract Since the introduction of the pneumococcal conjugate vaccines, the frequency of infections by nonencapsulated Streptococcus pneumoniae (NESp) has steadily increased. We previously demonstrated that NESp expressing AliC and AliD instead of the canonical capsule synthesis genes in the cps locus are able to survive killing in a whole blood bactericidal assay. Also, NESp expressing AliC and AliD are associated with invasive pneumococcal disease (IPD). The purpose of this study was to determine if NESp expressing AliC and AliD evade complement-mediated killing. Flow cytometry was used to examine binding of C3b and factor H (FH) to NESp in vitro. We used a chinchilla model of otitis media (OM) and mouse models of infection to examine complement interaction with NESp in vivo. Deletion of aliC and aliD resulted in reduced expression of choline binding protein AC (CbpAC), a variant of pneumococcal surface protein C (PspC). There was a four-fold increase in C3b deposition on a CbpAC mutant compared to the wild-type. Deletion of cbpA significantly reduced virulence in OM in which complement is known to play a role. When C1q-deficient mice were intravenously challenged with the wild-type strain, 60% of the mice failed to survive compared to 100% survival of mice challenged with the CbpA deficient mutant. Further, the wild-type NESp burden in the blood of C1q-deficient mice reached 1×107CFU/ml, and bacteremia persisted for at least five days. In contrast, the CbpAC mutant was not detectable in the blood by 48 hours post-infection. Overall, the ratio of CbpA and deposited C3b on the NESp surface appears to be crucial for NESp to counteract complement deposition and evade host response. Thus, NESp are able to initiate bacteremia and persist within the host.

Bactericidal Activity and Structural Studies of the Steviol Derivative 17- Hydroxy-16-hydroxyiminobayeran-19-oic Acid

Background: According to the World Health Organization (WHO), the routine use of antibiotics has led to the increase of microbial resistance. Thus, the search for new compounds that present antimicrobial activity must be constant. This study reports the bactericidal activity assay of the steviol derivative 17-hydroxy-16-hydroxyiminobayeran-19-oic acid against various bacteria and structural studies by quantum chemistry and molecular dynamics. Methods: Bactericidal activity assays of the steviol derivative 17-hydroxy-16-hydroxyiminobayeran-19- oic acid against Salmonella typhimurium [ATCC 14028], Staphylococcus aureus [ATCC 6538], Bacillus cereus [ATCC 11778], Helicobacter pylori [ATCC 26695], Pseudomonas aeruginosa [ATCC 27853], Escherichia coli [ATCC 25922] and Bacillus subtilis [ATCC 23857] were performed, as well as structural studies by quantum chemistry and molecular dynamics. Results: The results show that the compound exhibits activity towards S. typhimurium, what makes it an interesting compound for future studies on the development of antibiotics against this bacteria. An intramolecular hydrogen bond does not seem to be maintained in solution, therefore, corresponding moieties should be prone to interactions with their surroundings. Conclusions: The results indicate that the title compound exhibits activity towards S. typhimurium, what sums up to similar results from other steviol derivatives and stevioside, thus reinforcing the potential of these compounds for future studies on the development of antibiotics against this bacteria. The potential energy surface for the selected torsion angles and molecular dynamics have revealed that an intramolecular hydrogen bond, though slightly energetically favorable, does not seem to be maintained in solution; therefore, corresponding moieties should be prone to interactions with their surroundings, an important feature in further studies involving inhibitor/drug design from this compound.

Long-term antibody persistence after a booster dose of quadrivalent meningococcal ACWY-tetanus toxoid conjugate vaccine in healthy 5-year-old children

BackgroundTo provide continuing protection, available meningococcal vaccines must provide long-term persistence of circulating functional antibodies against prevalent serogroups causing invasive meningococcal disease (IMD). This study assessed antibody persistence and safety of the quadrivalent meningococcal vaccine conjugated to tetanus toxoid (MenACWY-TT) and the meningococcal serogroup C vaccine conjugated to Corynebacterium diphtheriae CRM197 protein (MenC-CRM) for up to 6 years after booster dosing in children. MethodsIn the primary vaccination study, children were vaccinated at age 12 to 23 months. In the first extension study, children who completed the primary study received a booster dose 4 years later with the same primary vaccine. The current study assessed antibody persistence at 2 to 6 years postbooster against each of the 4 meningococcal serogroups using serum bactericidal assays using rabbit (rSBA) or human (hSBA) complement with antibody titer thresholds of ≥1:8 or ≥1:4, respectively, and geometric mean titers (GMTs). Safety evaluations during this period included serious adverse events (SAEs) related to vaccination and any event related to lack of vaccine efficacy. ResultsA total of 184 subjects were enrolled (MenACWY-TT = 159; MenC-CRM = 25). For MenACWY-TT, the percentages of subjects with rSBA titers ≥1:8 ranged from 96.7% to 100% across serogroups at 2 years postbooster and 71.6% to 94.0% at 6 years postbooster; rSBA GMTs decreased from Year 2 to 4 and generally remained stable thereafter. The percentages of subjects in the MenACWY-TT group with hSBA titers ≥1:4 were 70.0% to 100% across serogroups at 2 years postbooster and 58.5% to 98.5% at 6 years postbooster. No lack of efficacy, SAEs, or vaccine-related adverse events were reported. ConclusionsThe persistence of rSBA and hSBA antibodies was shown up to 6 years postbooster (10 years postprimary vaccination) with either MenACWY-TT or MenC-CRM, suggesting that this schedule may provide long-term protection against IMD. Clinicaltrials.gov: NCT01900899.

Immunogenicity and safety of a quadrivalent meningococcal tetanus toxoid-conjugate vaccine (MenACYW-TT) in healthy toddlers: a Phase II randomized study

ABSTRACT Neisseria meningitidis can lead to invasive meningococcal disease to which young children are particularly vulnerable. We assessed the immunogenicity and safety of Sanofi Pasteur’s investigational quadrivalent (serogroups A, C, Y, and W) meningococcal tetanus-toxoid conjugate vaccine, MenACYW-TT, as a single dose, in healthy meningococcal vaccine-naïve toddlers versus a licensed conjugate vaccine MCV4-TT (NCT03205358). In this Phase II study conducted in Finland, 188 toddlers aged 12–24 months were randomized 1:1 to MenACYW-TT or MCV4-TT. Serum bactericidal antibody assays using human complement (hSBA) and baby rabbit complement (rSBA) measured antibodies against each serogroup before and 30 days after vaccination. Participants were monitored for immediate adverse events (AEs) and post-vaccination AEs for 30 days. All analyses were descriptive. All 188 participants completed the study. The Day 30 hSBA seroresponses (hSBA titer <8 at baseline and post-vaccination titer ≥8, or ≥8 at baseline and ≥4-fold increase post-vaccination) were comparable between participants receiving MenACYW-TT (96.7–100%), and MCV4-TT (86.0–100.0%) for each serogroup. Most unsolicited AEs were of Grade 1 or Grade 2 intensity. There were no immediate hypersensitivity reactions, and no AEs or serious AEs leading to discontinuation from the study. In this exploratory study, MenACYW-TT vaccine was well tolerated and immunogenic. If confirmed in Phase III, a single dose of the MenACYW-TT vaccine may show promise as an alternative vaccine option for toddlers receiving meningococcal vaccination for the first time.

Immunogenicity and safety of a quadrivalent meningococcal tetanus toxoid-conjugate vaccine (MenACYW-TT) in adults 56 years of age and older: a Phase II randomized study

ABSTRACT MenACYW-TT is an investigational quadrivalent meningococcal conjugate vaccine intended for the prevention of invasive meningococcal disease (IMD) caused by serogroups A, C, W, and Y in individuals aged 6 weeks and above. This Phase II, randomized, open-label, multicenter, exploratory study assessed the safety and immunogenicity of MenACYW-TT compared with a quadrivalent meningococcal polysaccharide vaccine (MPSV4) in 301 healthy adults aged ≥56 y in the US (NCT01732627). Participants were randomized 2:1 to receive MenACYW-TT or MPSV4. Serum bactericidal assays using human (hSBA) or baby rabbit (rSBA) complement were used to measure functional antibodies against meningococcal serogroups A, C, W, and Y at baseline and 30 d post-vaccination. Safety data were collected up to 30 d post-vaccination. Proportions of study participants with hSBA titers ≥1:8 against serogroups A, C, W, and Y were increased at Day 30 compared with baseline in both vaccine groups. The proportions of participants with hSBA titers ≥1:8 after MenACYW-TT vaccination were comparable to those after MPSV4 vaccination for serogroups A and C (A: 93.8% vs. 85.1%; C: 74.9% vs. 62.8%) and distinctly higher than after MPSV4 for serogroups W and Y (W: 79.5% vs. 60.6%; Y: 80.5% vs. 59.6%). Proportions of participants with rSBA titers ≥1:8 were comparable between vaccine groups for all four serogroups. The reactogenicity profiles of both vaccines were similar. Most unsolicited adverse events (AEs) were of Grade 1 or Grade 2 intensity, and no serious AEs were reported. The MenACYW-TT conjugate vaccine was well tolerated and immunogenic in adults aged ≥56 y.

Immunogenicity and safety of a booster dose of a quadrivalent meningococcal tetanus toxoid-conjugate vaccine (MenACYW-TT) in adolescents and adults: a Phase III randomized study

ABSTRACT The quadrivalent meningococcal tetanus toxoid-conjugate vaccine (MenACYW-TT) was assessed as a booster in this Phase III trial (NCT02752906). Quadrivalent meningococcal conjugate vaccine (MCV4)-primed individuals aged ≥15 y (n = 810) were randomized 1:1 to receive a single booster dose of MenACYW-TT (n = 403) or a licensed MCV4 (Menactra®; MCV4-DT [n = 407]). Serum bactericidal antibody assay with human complement (hSBA) was used to measure functional antibodies against serogroups A, C, W, and Y at baseline and Day 30 post-vaccination. Proportions of participants achieving seroresponse (post-vaccination titer ≥1:16 for those with baseline titer <1:8 or ≥4-fold increase in post-vaccination titer for those with baseline titer ≥1:8) were determined. Safety data were collected for 180 d post-vaccination. Non-inferiority of the immune response was demonstrated for MenACYW-TT compared with MCV4-DT based on the proportion of participants achieving hSBA vaccine seroresponse for each of the meningococcal serogroups at Day 30. Moreover, ≥99% of participants in both study groups had hSBA titers ≥1:8 for the four meningococcal serogroups at Day 30. Reactogenicity profiles were comparable between groups. These Phase III data in adolescents and adults show that MenACYW-TT boosts the immune response in those primed with MCV4 vaccines 4–10 y previously, irrespective of whether MCV4-DT or MCV4-CRM was used for priming.

Chlorine Induces Physiological and Morphological Changes on Chicken Meat Campylobacter Isolates.

Broiler chickens frequently become colonized by Campylobacter species. As a consequence, Campylobacter, can enter the poultry meat supply chain and represents a significant risk for human public health. A number of on-farm biosecurity and processing measures are used to mitigate the load of Campylobacter on chicken meat. In many countries, chlorine is commonly used as a biocide in processing plants to reduce bacterial loads on poultry carcasses but there is limited evidence of its effectiveness on Campylobacter. In this study, 116 Campylobacter isolates (89 C. jejuni and 27 C. coli) were isolated from poultry meat carcasses prior to the inside/outside wash step and used in in vitro assays exploring the efficacy of chlorine. A high proportion of isolates exhibited MIC and MBC values of 128 ppm but organic material present in the broth likely affected this result. Thus, additional bactericidal assays (time kill and chlorine inactivation) were used to characterize the response of C. jejuni isolates to different concentrations of chlorine. At 106 CFU, C. jejuni was found to be highly sensitive to concentrations of chlorine and was inhibited at low concentrations (0.2–2.0 ppm). At a higher bacterial load (108 CFU), variation in the response of different C. jejuni isolates was observed. One isolate was growth inhibited at 1.8 ppm while another required 16 ppm. At 108 CFU, C. jejuni could be resuscitated following exposure to chlorine highlighting a potential limitation of chlorine use. Analysis of UV leakage indicated that high chlorine concentrations resulted in increased 280 nm absorbance values suggesting bacterial membrane damage. Scanning electron and transmission electron microscopy were performed to characterize the morphological effects of chlorine exposure. Some effects of chlorine exposure included changes in shape (coccoid, or elongated), cellular degeneration, and shriveled bacterial cells. Interestingly, C. jejuni cells with normal morphology were also observed in the chlorine exposed group and represent a population of cells that could be resuscitated. This study is useful for the chicken meat industry and provides data for future optimization of chlorine use in reducing Campylobacter loads.

The KdpD/KdpE two-component system contributes to the motility and virulence of avian pathogenic Escherichia coli

Two-component systems (TCSs) are widespread regulatory systems which can help bacteria to control their cellular functions and respond to a diverse range of stimuli. The KdpD/KdpE system had been well studied for regulating potassium transport and identified as an adaptive regulator involved in the virulence of some pathogenic bacteria, but its role in avian pathogenic Escherichia coli (APEC) was still unknown. In this study, the mutant strain AE17ΔKdpDE was obtained successfully of a clinical APEC isolation AE17 using the lambda Red recombinase system and performed the transcriptional sequencing of the wild type strain AE17 and the mutant strain AE17ΔKdpDE. The transcriptional sequencing results revealed that the KdpD/KdpE two-component system mainly influenced the expression of the genes covering metabolic pathways, flagellar assembly, global transcription regulator. The expression of some flagellar-related genes detecting by quantitative real-time PCR was consistent with the results of transcriptional sequencing. Importantly, fewer flagellum of the mutant strain AE17ΔKdpDE was observed than AE17 using the transmission electron microscope and a decreased motility circle of AE17ΔKdpDE appeared in the semisolid medium. In addition, the serum bactericidal assay was carried out with the specific-pathogen-free chicken in different dilution and the survival ability in the serum of AE17ΔKdpDE was also obviously lower than that of AE17. These results suggested that in APEC, the KdpD/KdpE two-component system mainly influenced the expression of flagella-related genes, the flagellum formation, the motility and antiserum bactericidal activity.

A Phase II, randomized, immunogenicity and safety study of a quadrivalent meningococcal conjugate vaccine, MenACYW-TT, in healthy adolescents in the United States

BackgroundMenACYW-TT is an investigational quadrivalent meningococcal conjugate vaccine intended for use in individuals ≥6 weeks of age. We evaluated the safety and immunogenicity of MenACYW-TT when compared to a licensed quadrivalent conjugate meningococcal vaccine (Menveo®; MCV4-CRM; GlaxoSmithKline, Italy), and when co-administered with tetanus, diphtheria, acellular pertussis (Tdap) and human papilloma virus (HPV4) vaccines in healthy meningococcal vaccine-naïve adolescents (10–17 years old) in the United States of America. MethodsIn this pivotal Phase II, open-label, multicenter study, 1715 participants were randomized to receive MenACYW-TT, MCV4-CRM, MenACYW-TT co-administered with Tdap and HPV4, or Tdap and HPV4 vaccines alone (NCT02199691).The primary objective was to evaluate whether antibody responses to MenACYW-TT antigens were non-inferior to antibody responses after MCV4-CRM administration. Meningococcal antibody titers were determined using human complement serum bactericidal assay (hSBA) with titers measured at baseline, and 30 days post vaccination (D30). A vaccine seroresponse was defined as baseline titers <1:8 with post-vaccination titers ≥1:8 or baseline titers ≥1:8 with a ≥4-fold increase at post-vaccination. Safety data were collected up to six months post-vaccination. ResultsNon-inferiority was demonstrated for MenACYW-TT vs MCV4-CRM (primary endpoint), and for MenACYW-TT co-administered with Tdap and HPV4 vs MenACYW-TT alone (secondary endpoint). The vaccine seroresponse rate was higher with MenACYW-TT than with MCV4-CRM, for each serogroup: A: 75.6% vs 66.4%; C: 97.2% vs 72.6%; W: 86.2% vs 66.6%; Y: 97.0% vs 80.8%. The safety profiles of MenACYW-TT, MCV4-CRM, and Tdap and HPV4 vaccines, administered with or without MenACYW-TT, were comparable. There were no vaccine-related serious adverse events. ConclusionsThe MenACYW-TT vaccine was well tolerated and generated an immune response that was non-inferior to the licensed MCV4-CRM vaccine. Immunogenicity and safety profiles were comparable when MenACYW-TT was administered with or without Tdap and HPV4 vaccines in meningococcal vaccine-naïve adolescents.

Evaluation of protective immunity responses against pneumococcal PhtD and its C-terminal in combination with outer-membrane vesicles as adjuvants.

Introduction. Streptococcus pneumoniae is a significant bacterial pathogen in humans. Currently, there are two types of pneumococcal vaccines, but there are concerns regarding their application.Aim. Since many pneumococcal proteins are serotype-independent, polyhistidine triad protein D (PhtD) has been selected as a vaccine candidate.Methodology. We prepared recombinant PhtD and its C-terminal fragment (PhtD-C) using alum and outer-membrane vesicles (OMVs) as adjuvants. The combinations were injected intraperitoneally into mice, and then total immunoglobulin G (IgG) and specific IgG, IgG1 and IgG2a were measured. A serum bactericidal assay and opsonophagocytosis were also performed as complementary tests. Meningococcal OMVs were used as an adjuvant.Results. The levels of specific IgG and IgG1 against combinations of PhtD and its C-terminal with OMVs and alum as adjuvants increased at the time of the third mouse immunization on day 35. Forty per cent and 60% of S. pneumoniae ATCC 6303 (serotype 3) as a virulent pneumococcal strain, respectively, were killed in the opsonophagocytosis test and these results could also be observed in the serum bactericidal assay. Mice mmunized iwith PhtD and its C-terminal with OMVs and alum as adjuvants survived after 10 days of pneumococcal challenge.Conclusion. The combination of PhtD and PhtD-C with alum produced optimal results, but the combination of PhtD and PhtD-C with OMVs produced minimal results by comparison. The survival rates were also measured, and these corresponded with the results of the immunological assessments. Our findings showed that mice receiving PhtD and PhtD-C plus OMV and alum had higher survival rates than the mice in the other groups.

Nitro-imidazole-based ruthenium complexes with antioxidant and anti-inflammatory activities

Inflammation is a physiological process triggered in response to tissue damage, and involves events related to cell recruitment, cytokines release and reactive oxygen species (ROS) production. Failing to control the process duration lead to chronification and may be associated with the development of various pathologies, including autoimmune diseases and cancer. Considering the pharmacological potential of metal-based compounds, two new ruthenium complexes were synthesized: cis-[Ru(NO2)(bpy)2(5NIM)]PF6 (1) and cis-[RuCl(bpy)2(MTZ)]PF6 (2), where bpy = 2,2′-bipyridine, 5NIM = 5-nitroimidazole and MTZ = metronidazole. Both products were characterized by spectroscopic techniques, followed by Density Functional Theory (DFT) calculations in order to support experimental findings. Afterwards, their in vitro cytotoxic, antioxidant and anti-inflammatory activities were investigated. Compounds 1 and 2 presented expressive in vitro antioxidant activity, reducing lipid peroxidation and decreasing intracellular ROS levels with comparable effectiveness to the standard steroidal drug dexamethasone or α-tocopherol. These complexes showed no noticeable cytotoxicity on the tested cancer cell lines. Bactericidal assay against metronidazole-resistant Helicobacter pylori, a microorganism able to disrupt oxidative balance, unraveled compound 1 moderate activity over that strain. Besides this, it was able to inhibit interleukin-6 (IL-6) and tumor necrosis factor-α (TNF- α) production as well as interleukin-1β (IL-1β) and cyclooxygenase-2 (COX-2) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. This latter activity is remarkable, which has not been reported for other ruthenium-based complexes. Altogether, these results suggest cis-[Ru(NO2)(bpy)2(5NIM)]PF6 complex has potential pharmacological application as an anti-inflammatory agent that deserve further biological investigation.

Effect of Nonthermal Processing on Human Milk Bactericidal Activity Against Escherichia coli.

Nonthermal methods are more efficient at preserving various biological properties of human milk, as compared with holder pasteurization (HoP), which is the most common preservation method. This study was performed to assess the effects of nonthermal processing on bactericidal activity against Escherichia coli in human milk. Milk samples obtained from the Regional Human Milk Bank in Warsaw at Holy Family Hospital were processed by HoP, irradiated with ultraviolet-C (UV-C) for 5, 10, and 15 minutes (6720 J/L each minute), subjected to 2 variations of high-pressure processing (HPP): 450 MPa for 15 minutes and 200 MPa for 10 minutes + 400 MPa for 10 min, with a 10-minutes break. The samples were then evaluated by a bactericidal assay (raw untreated human milk was used as a control). The bactericidal capacity after HoP was preserved in 12.1% of samples, showing a significant reduction in bactericidal properties compared with in raw milk (P < 0.05). The differences between samples preserved by nonthermal methods and raw milk were not significant (P > 0.05). Nonthermal methods of human milk treatment better preserve the bactericidal capacity compared with holder pasteurisation. Those alternative technologies to HoP can be proposed after further investigation for milk processing for Human Milk Banks facilities.