Labile Research Articles

Combination of a novel bacteriophage and d-serine effectively controls Vibrio parahaemolyticus growth in seafood

Seafood is an important high-protein and low-fat food source prone to spoilage and may harbor food poisoning bacteria. Vibrio parahaemolyticus is a gram-negative halophilic bacterium that causes food poisoning. It is heat labile and can be eliminated through heating; however, V. parahaemolyticus may be present in seafood for raw consumption, such as sashimi. Recently, the use of bacteriophages or phages has been proposed as a non-thermal inactivation method for microbial control. Here, we characterized VF16, a novel phage that infects V. parahaemolyticus, and evaluated the effectiveness of the combination of VF16 with the antimicrobial amino acid d-serine in controlling V. parahaemolyticus through antimicrobial testing in broth cultures and raw scallops. The morphology of VF16 was examined using transmission electron microscopy, which demonstrated that it had a long, flexible tail characteristic of a siphovirus. Genome sequencing revealed that VF16 is a novel phage belonging to the family Demerecviridae, subfamily Ermolyevavirinae, and genus Vipunavirus. Analysis of adsorption and one-step growth curve in a liquid medium indicated that Ca2+ and Mg2+ are necessary for one or more subsequent replication processes of VF16 but are not essential for its adsorption. The combination of VF16 and d-serine completely inhibited the regrowth of V. parahaemolyticus in the liquid medium and effectively suppressed its growth in the scallop samples. No VF16-resistant mutants were isolated from the scallop samples treated with VF16 alone or in combination with d-serine. The combination of phages and d-amino acids is an effective non-thermal inactivation method for controlling foodborne pathogens.

Seasonal Variations, Origin, and Parameterization of Ice‐Nucleating Particles at a Mountain Station in Central France

AbstractIdentifying how aerosol particles interact with atmospheric water is critical to understand climate and precipitation. Ice‐nucleating particles (INP) trigger the formation of atmospheric ice crystals at higher temperatures than pure water. They are difficult to characterize because of their scarce occurrence, and variability, in the atmosphere, especially at temperatures above −20°C. It has been demonstrated that at these temperatures, biological aerosol particles can contribute significantly to INP number concentration. This study incorporates a series of offline, size‐segregated measurements of INPs collected at the Puy de Dôme station (PUY, 1,465 m a.s.l.) over a 6 month period from October to May, covering the transitions from autumn, winter, to spring. These measurements show a general trend of decreasing particle number concentrations during the winter months and higher concentration during autumn and spring. INP concentrations measured in the range of −5 and −18°C, had concentrations of 0.001 INP/Lair at the warmest temperatures, and between 0.01 and 0.1 INP/Lair at the coldest temperatures. The majority of INP measured at temperatures warmer than −15°C were heat labile, suggesting a biological or organic origin. The INP variability was compared with collocated aerosol physical and chemical properties, allowing us to associate highest INP concentrations with local and marine origins. Following these comparisons, we use aerosol total number concentration to develop a new parameterization. In addition, this parameterization is specifically optimized for warmer temperature INP measurements, and demonstrated a good performance when tested on independent data sets.

Vaccine-elicited IL-1R signaling results in Th17 TRM-mediated immunity

Lung tissue resident memory (TRM) cells are thought to play crucial roles in lung host defense. We have recently shown that immunization with the adjuvant LTA1 (derived from the A1 domain of E. coli heat labile toxin) admixed with OmpX from K. pneumoniae can elicit antigen specific lung Th17 TRM cells that provide serotype independent immunity to members of the Enterobacteriaceae family. However, the upstream requirements to generate these cells are unclear. Single-cell RNA-seq showed that vaccine-elicited Th17 TRM cells expressed high levels of IL-1R1, suggesting that IL-1 family members may be critical to generate these cells. Using a combination of genetic and antibody neutralization approaches, we show that Th17 TRM cells can be generated independent of caspase-1 but are compromised when IL-1α is neutralized. Moreover IL-1α could serve as a molecular adjuvant to generate lung Th17 TRM cells independent of LTA1. Taken together, these data suggest that IL-1α plays a major role in vaccine-mediated lung Th17 TRM generation.

Effect of extrusion process conditions on extrudates enriched with carotenoids encapsulated by different methods using gum arabic and vegetable fat as carriers

Bioactive compounds into extruded foods enhance their nutritional value but they are heat and shear labile and prone to oxidation. This study was aimed to examine the impacts of distinct encapsulation methods on the stability of carotenoids under typical extrusion conditions. The study presents innovative encapsulation methods and investigates the protection efficacy of carotenoids degradation, as well as the effects on the physicochemical characteristics of carotenoid-rich products. Thus, spray drying, spray chilling, and their combination were compared based on their ability to protect carotenoids. Processing temperatures were 110 °C and 140 °C, and shear rates 500 and 2000 1/s. Carotenoid retention was determined, β- and α-carotene retention ranged from 17 to 44 % and 18 to 48 %, respectively. Upon storage at room temperature, the carotenoid content was stable for 15 days, followed by a marked reduction after 30 days. Extrudates enriched microparticles produced by spray chilling and the combined methods exhibited higher carotenoid protection during storage. They also showed better quality attributes, notably bulk density, high water absorption index, color properties, and carotenoid retention. These findings suggest that encapsulation can protect carotenoids during extrusion, and the protection can be tailored to optimize the attributes of the final products.

Physicochemical characterization and source apportionment of Arctic ice-nucleating particles observed in Ny-Ålesund in autumn 2019

Abstract. Ice-nucleating particles (INPs) initiate primary ice formation in Arctic mixed-phase clouds (MPCs), altering cloud radiative properties and modulating precipitation. For atmospheric INPs, the complexity of their spatiotemporal variations, heterogeneous sources, and evolution via intricate atmospheric interactions challenge the understanding of their impact on microphysical processes in Arctic MPCs and induce an uncertain representation in climate models. In this work, we performed a comprehensive analysis of atmospheric aerosols at the Arctic coastal site in Ny-Ålesund (Svalbard, Norway) from October to November 2019, including their ice nucleation ability, physicochemical properties, and potential sources. Overall, INP concentrations (NINP) during the observation season were approximately up to 3 orders of magnitude lower compared to the global average, with several samples showing degradation of NINP after heat treatment, implying the presence of proteinaceous INPs. Particle fluorescence was substantially associated with INP concentrations at warmer ice nucleation temperatures, indicating that in the far-reaching Arctic, aerosols of biogenic origin throughout the snow- and ice-free season may serve as important INP sources. In addition, case studies revealed the links between elevated NINP and heat lability, fluorescence, high wind speeds originating from the ocean, augmented concentration of coarse-mode particles, and abundant organics. Backward trajectory analysis demonstrated a potential connection between high-latitude dust sources and high INP concentrations, while prolonged air mass history over the ice pack was identified for most scant INP cases. The combination of the above analyses demonstrates that the abundance, physicochemical properties, and potential sources of INPs in the Arctic are highly variable despite its remote location.

T helper cell responses in adult diarrheal patients following natural infection with enterotoxigenic Escherichia coli are primarily of the Th17 type.

Infection with enterotoxigenic Escherichia coli (ETEC) gives rise to IgA antibodies against both the heat labile toxin (LT) and colonization factors (CFs), which are considered to synergistically protect against ETEC diarrhea. Since the development of ETEC-specific long lived plasma cells and memory B cells is likely to be dependent on T helper (Th) cells, we investigated if natural ETEC diarrhea elicits ETEC-specific Th cells and their relation to IgA responses. Th cell subsets were analyzed in adult Bangladeshi patients hospitalized due to ETEC diarrhea by flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) isolated from blood collected day 2, 7, 30 and 90 after hospitalization as well as in healthy controls. The LT- and CF-specific Th responses were determined by analysis of IL-17A and IFN-γ in antigen stimulated PBMC cultures using ELISA. ETEC-specific IgA secreted by circulating antibody secreting cells (plasmablasts) were analyzed by using the antibodies in lymphocyte supernatants (ALS) ELISA-based method and plasma IgA was also measured by ELISA. ETEC patients mounted significant ALS and plasma IgA responses against LTB and CFs on day 7 after hospitalization. ETEC patients had significantly elevated proportions of memory Th cells with a Th17 phenotype (CCR6+CXCR3-) in blood compared to controls, while frequencies of Th1 (CCR6-CXCR3+) or Th2 (CCR6-CXCR3-) cells were not increased. Antigen stimulation of PBMCs revealed IL-17A responses to LT, most clearly observed after stimulation with double mutant heat labile toxin (dmLT), but also with LT B subunit (LTB), and to CS6 in samples from patients with LT+ or CS6+ ETEC bacteria. Some individuals also mounted IFN-γ responses to dmLT and LTB. Levels of LTB specific IgA antibodies in ALS, but not plasma samples correlated with both IL-17A (r=0.5, p=0.02) and IFN-γ (r=0.6, p=0.01) responses to dmLT. Our results show that ETEC diarrhea induces T cell responses, which are predominantly of the Th17 type. The correlations between IL-17A and IFN-g and intestine-derived plasmablast responses support that Th responses may contribute to the development of protective IgA responses against ETEC infection. These observations provide important insights into T cell responses that need to be considered in the evaluation of advanced ETEC vaccine candidates.

Supramolecular chemistry enables vat photopolymerization 3D printing of novel water-soluble tablets

Vat photopolymerization has garnered interest from pharmaceutical researchers for the fabrication of personalised medicines, especially for drugs that require high precision dosing or are heat labile. However, the 3D printed structures created thus far have been insoluble, limiting printable dosage forms to sustained-release systems or drug-eluting medical devices which do not require dissolution of the printed matrix. Resins that produce water-soluble structures will enable more versatile drug release profiles and expand potential applications. To achieve this, instead of employing cross-linking chemistry to fabricate matrices, supramolecular chemistry may be used to impart dynamic interaction between polymer chains. In this study, water-soluble drug-loaded printlets (3D printed tablets) are fabricated via digital light processing (DLP) 3DP for the first time. Six formulations with varying ratios of an electrolyte acrylate monomer, [2-(acryloyloxy)ethyl]trimethylammonium chloride (TMAEA), and a co-monomer, 1-vinyl-2-pyrrolidone (NVP), were prepared to produce paracetamol-loaded printlets. 1H NMR spectroscopy analysis confirmed the integration of TMAEA and NVP in the polymer, and residual TMAEA monomers were found to be present only in trace amounts (0.71 – 1.37 %w/w). The apparent molecular mass of the photopolymerised polymer was found to exceed 300,000 Da with hydrodynamic radii of 15 – 20 nm, estimated based on 1H DOSY NMR measurements The loaded paracetamol was completely released from the printlets between 45 minutes to 5 hours. In vivo single-dose acute toxicity studies in rats suggest that the printlets did not cause any tissue damage. The findings reported in this study represent a significant step towards the adoption of vat photopolymerization-based 3DP to produce personalised medicines.

Improving expression and assembly of difficult-to-express heterologous proteins in Saccharomyces cerevisiae by culturing at a sub-physiological temperature

BackgroundEscherichia coli heat labile toxin B subunit (LTB) is one of the most popular oral vaccine adjuvants and intestine adsorption enhancers. It is often expressed as a fusion partner with target antigens to enhance their immunogenicity as well as gut absorbability. However, high expression levels of a fusion protein are critical to the outcome of immunization experiments and the success of subsequent vaccine development efforts. In order to improve the expression and functional assembly of LTB-fusion proteins using Saccharomyces cerevisiae, we compared their expression under culture conditions at a sub-physiological temperature 20 °C with their expression under a standard 30 °C.ResultsThe assembled expression of LTB-EDIII2 (LTB fused to the envelope domain III (EDIII) of Dengue virus serotype 2), which was expressed at the level of 20 µg/L in our previous study, was higher when the expression temperature was 20 °C as opposed to 30 °C. We also tested whether the expression and functional assembly of a difficult-to-express LTB fusion protein could be increased. The assembled expression of the difficult-to-express LTB-VP1 fusion protein (LTB fused to VP1 antigen of Foot-and-Mouth Disease Virus) dramatically increased, although the total amount of expressed protein was still lower than that of LTB-EDIII2. Slight but significant increase in the expression of well-known reporter protein eGFP, which has previously been shown to be increased by cultivation at 20 °C, was also observed in our expression system. As no significant changes in corresponding transcripts levels and cell growth were observed between 20 °C and 30 °C, we infer that translation and post-translational assembly are responsible for these enhancements.ConclusionsThe effects of lowering the expression temperature from 30 °C to 20 °C on protein expression and folding levels in S. cerevisiae, using several proteins as models, are reported. When heterologous proteins are expressed at 20 °C, a greater amount of (specially, more assembled) functional proteins accumulated than at 30 °C. Although further studies are required to understand the molecular mechanisms, our results suggest that lowering the expression temperature is a convenient strategy for improving the expression of relatively complexly structured and difficult-to-express proteins in S. cerevisiae.

Rift Valley Fever vaccine strategies: Enhanced stability of RVF Clone 13

Rift Valley Fever virus (RVFV) causes the zoonotic RVF disease, which results in substantial economic losses in livestock industries. Regular vaccination of livestock against RVF is necessary to generate long-term immunity and avoid the loss of livestock. The live attenuated vaccine based on Clone 13 virus strain has been used to reduce the negative impact of RVF disease. The vaccine strain is heat labile and requires stringent conditions for storage and handling. This research evaluated lactose and sucrose-based stabilizers coupled with lyophilisation to enhance stability of the RVF Clone 13 vaccine strain. The glass transition temperature (Tg) of the sucrose-RVF vaccine was 97.0 °C with average residual moisture of below 2 %. The lactose formulation was characterised with Tg of 83.5 °C and residual moisture of above 2 %. The RVF Clone 13 sucrose-based formulation maintained higher antigen titres during lyophilisation compared to the lactose-formulated vaccine. Cellular-mediated and humoral immunity was evaluated and compared for the two newly formulated vaccines. Pheroid® technology was also investigated as a potential adjuvant and its ability to further enhance the immunogenicity conferred by the RVF Clone 13 vaccine formulations in Merino sheep. No adverse reactions were observed following injection of the vaccine formulations in mice, guinea pigs and Merino sheep. Comparable protective humoral immune responses against RVF were obtained for all animals vaccinated with the lactose and sucrose-based stabilisers with and without the Pheroid® adjuvant. No proliferation of CD8+ and CD4+ T-cells as well as expression of IFN-γ was observed for all animals group vaccinated with Pheroid® only. Specific CD8+ IFN-γ+T–cells were expressed at higher levels compared to the CD4+ IFN-γ+T–cells in the RVF Clone 13 vaccines, suggesting that cellular immunity against RVF is through the Class I antigen presentation pathway.

Evaluation of biochemical changes in wheat varieties as influenced by terminal heat stress under varying environments

Climate is considered as the major uncontrollable factor in crop production. Alternation in the climate worldwide is predicted to have critical impact on crop production. High temperature negatively affects growth process of wheat by upsetting various physiological and biochemical processes and thereby affecting the nutritional quality of the crop. Heat stress during reproductive growth stages of wheat is detrimental to chloroplast activity, reducing activity of source organs and thereby reducing its sink capacity. Reduction in starch content is the major reason leading to yield loss during heat stress and this is because of accumulation of starch is associated with activities of various enzymes like sucrose synthase, soluble starch synthase, etc which are highly heat labile. Metabolic pathways are most altered during heat stress followed by secondary metabolite biosynthesis pathways. A field experiment was conducted during the Rabi season of 2020-21 at Student’s Instructional Farm of Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya, Uttar Pradesh to evaluate the biochemical changes in wheat varieties as influenced by terminal heat stress. The treatment for the experiment consisted of sowing on three different dates i.e. D1 (30th November), D2 (15th December), D3 (30th December). It was observed that delayed sowing decreased substantially total chlorophyll content, total soluble sugar content in leaves of V1 (PBW-343) and V2 (HD-2967). This reduction was caused due to onset of high temperature during reproductive stage of wheat crop. Variety V3 (Halna) reduced the detrimental effect of heat stress and thus showed greater total chlorophyll content, total soluble sugar content. Proline content in leaves increased with age of crop up to 75 DAS in all the sowing dates this varying dates of sowing because proline is thought to play an adaptive role in mediating osmotic adjustment and protecting sub cellular structure in stressed plants.

Intensification and diversification of pasturelands in Brazil: Patterns and driving factors in the soil carbon stocks

The extensive system of pasture management in Brazil can reduce soil C stocks and increase CO2 emissions to the atmosphere. However, the adoption of sustainable management systems can change these conditions and enact modern practices of natural resource management as defined by the concept of ecosystem services. A field study was therefore carried out to assess the effects of adopting more intensive and diversified systems of pasture management on the quality and stocks of soil C. Treatments included fertilized pasture (FP), integrated crop-livestock (ICL), and integrated livestock-forest (ILF) and were compared to conventional management systems (CS) under different soil and climate conditions (tropical humid, subtropical dry and subtropical humid). Soil carbon stocks and soil organic matter (SOM) fractions were determined in the top one-meter layer. With the SOM fractions, we calculated the C lability (LI), pool (CPI) and management (CMI) indices. The results showed that adopting ICL systems under the conditions of a tropical humid and subtropical humid climate produced increases in soil C stocks of 0.75 Mg ha−1 yr−1 (P < 0.01) and 0.15 Mg ha−1 yr−1 (P < 0.05), respectively, relative to the CS. Similarly, the conversion of a CS to FP (tropical humid climate) and ILF (subtropical dry climate) led to increases in soil C stocks of 2 and 0.55 Mg ha−1 yr−1 (P < 0.05), respectively. In general, the adoption of more intensive and diversified systems of pasture management can also afford increases in the LI, CPI and CMI indices. These results can serve as a scientific basis for government initiatives that focus on recovering degraded pastures in Brazil through the use of more sustainable management systems and can also help to sequester more C in soils.

Safety assessment of Mpp75Aa1.1, a new ETX_MTX2 protein from Brevibacillus laterosporus that controls western corn rootworm.

The recently discovered insecticidal protein Mpp75Aa1.1 from Brevibacillus laterosporus is a member of the ETX_MTX family of beta-pore forming proteins (β-PFPs) expressed in genetically modified (GM) maize to control western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte). In this manuscript, bioinformatic analysis establishes that although Mpp75Aa1.1 shares varying degrees of similarity to members of the ETX_MTX2 protein family, it is unlikely to have any allergenic, toxic, or otherwise adverse biological effects. The safety of Mpp75Aa1.1 is further supported by a weight of evidence approach including evaluation of the history of safe use (HOSU) of ETX_MTX2 proteins and Breviballus laterosporus. Comparisons between purified Mpp75Aa1.1 protein and a poly-histidine-tagged (His-tagged) variant of the Mpp75Aa1.1 protein demonstrate that both forms of the protein are heat labile at temperatures at or above 55°C, degraded by gastrointestinal proteases within 0.5 min, and have no adverse effects in acute mouse oral toxicity studies at a dose level of 1920 or 2120 mg/kg body weight. These results support the use of His-tagged proteins as suitable surrogates for assessing the safety of their non-tagged parent proteins. Taken together, we report that Mpp75Aa1.1 is the first ETX-MTX2 insecticidal protein from B. laterosporus and displays a similar safety profile as typical Cry proteins from Bacillus thuringiensis.

QUANTITATIVE ESTIMATION OF SOME ANTIOXIDANTS AND ANTI NUTRITIONAL CONTENT OF GLINUSOPPOSITIFOLIUS

Glinusoppositifolius(L.)Aug.DC (Molluginaceae), known as gimashak and it contains linear toovate,opposite leaves and greenish flowers. (1).This study was performed to measure theantioxidants and anti nutritional contents of the aqueous paste of Glinusoppositifolius(ediblepart). Some of these phytochemicals are heat labile, and reduced after cooking .So all parameterswerealsomeasuredafterheattreatment.Resultshowedadifferencebetweentherawsampleandcookedsampleofthese allparameters.Studyfoundthatafterboilingtherewas17.74%,8.36%,8.8%,0.44%,60%,11.11%,52.17%,1.90%18.33%,4.34%and5.71%reductionoftotalphenol,flavonoid,DPPH(IC50),FRAPassay,totalalkaloid,oxalate,phytate,tannin,totalsaponin,steroidalsaponinandcardiac glycosidesrespectively.

Field evaluation of a novel, rapid diagnostic assay, and molecular epidemiology of enterotoxigenic E. coli among Zambian children presenting with diarrhea.

BackgroundEnterotoxigenic Escherichia coli (ETEC) is one of the top aetiologic agents of diarrhea in children under the age of 5 in low-middle income countries (LMICs). The lack of point of care diagnostic tools for routine ETEC diagnosis results in limited data regarding the actual burden and epidemiology in the endemic areas. We evaluated performance of the novel Rapid LAMP based Diagnostic Test (RLDT) for detection of ETEC in stool as a point of care diagnostic assay in a resource-limited setting.MethodsWe conducted a cross-sectional study of 324 randomly selected stool samples from children under 5 presenting with moderate to severe diarrhea (MSD). The samples were collected between November 2012 to September 2013 at selected health facilities in Zambia. The RLDT was evaluated by targeting three ETEC toxin genes [heat labile toxin (LT) and heat stable toxins (STh and STp)]. Quantitative PCR was used as the “gold standard” to evaluate the diagnostic sensitivity and specificity of RLDT for detection of ETEC. We additionally described the prevalence and seasonality of ETEC.ResultsThe study included 324 participants, 50.6% of which were female. The overall prevalence of ETEC was 19.8% by qPCR and 19.4% by RLDT. The children between 12 to 59 months had the highest prevalence of 22%. The study determined ETEC toxin distribution was LT 28/321(9%), ST 18/321(6%) and LT/ST 16/321(5%). The sensitivity and specificity of the RLDT compared to qPCR using a Ct 35 as the cut-off, were 90.7% and 97.5% for LT, 85.2% and 99.3% for STh and 100% and 99.7% for STp, respectively.ConclusionThe results of this study suggest that RLDT is sufficiently sensitive and specific and easy to implement in the endemic countries. Being rapid and simple, the RLDT also presents as an attractive tool for point-of-care testing at the health facilities and laboratories in the resource-limited settings.

USEFULNESS OF THERMO LABILE ALKALINE PHOSPHATASE AS A BIOMARKER IN DIAGNOSIS OF POST-MENOPAUSAL OSTEOPOROSIS

Background: Osteoporosis is characterized by demineralization of bones and it is a complication of women in post-menopausal life. Bone alkaline phosphatase is an isoenzyme of alkaline phosphatase which helps in the mineralization of bones and it is heat labile in nature. Its measurement facilitates the clinical diagnosis of Osteoporosis. Objectives: To assess the status of total alkaline phosphatase and heat-labile isoenzyme fraction (bone fraction) in serum of pre and post-menopausal women and to relate the findings to the clinical condition /final diagnosis to make out the correlation between the pre and postmenopausal women. Methodology:A total of 70 subjects, consisting of 35 pre-menopausal women (38.11 ± 4.3 years of age) and 35 post-menopausal women (54.40 ± 4.6 years) were constituted for the study. Their total alkaline phosphatase was measured directly and the heat stable alkaline phosphatase was measured after heating the serum at 56℃ for 10 minutes. Spectrophotometric P- Nitro-Phenyl Phosphate method was followed for the estimation. Heat labile alkaline phosphatase levels were obtained by subtracting the heat stable fraction from total Alkaline phosphatase. Result: The measured serum total alkaline phosphatase level was slightly increased in post menopause compared to premenopausal women but it was not statistically significant. The levels of heat-labile Alkaline phosphatase showed significant difference between pre and post-menopausal women by ***: p &lt; 0.01: ***: p &lt; 0.001 respectively. Conclusion: Heat labile alkaline phosphatase can be used as a biomarker to diagnose Osteoporosis in post-menopausal women.

Diarrhoeagenic E. coli occurrence and antimicrobial resistance of Extended Spectrum Beta-Lactamases isolated from diarrhoea patients attending health facilities in Accra, Ghana.

IntroductionDiarrhoea accounts for high morbidity and mortality in children and adults worldwide. Extended Spectrum Beta-Lactamase-Producing Enterobacteriaceae (ESBL-PE) and Diarrhoeagenic Escherichia coli (DEC) contribute to prolonged hospitalization because of their resistance and virulence properties aiding in the spread of diarrhoeal disease and delayed treatment.AimTo determine DEC and the antimicrobial resistance of ESBL-PE isolated among diarrhoea patients attending two health facilities in Ghana.MethodsStool samples were collected from 122 diarrhoeal patients who attended Maamobi General Hospital and Kaneshie Polyclinic between January 2019 and March 2020. Identification of bacteria was performed by using the Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Using disk diffusion, antimicrobial susceptibility testing (AST) was conducted and interpreted according to the 2018 CLSI guidelines. Detection of ESBL and DEC genes was performed using Polymerase chain reaction (PCR).ResultsA total of 80.3% (98/122) Enterobacteriaceae was recovered from the patients in the study with an overall ESBL occurrence of 20.4% (20/98), predominantly among E. coli showed 13.2% (10/76), Klebsiella pneumoniae,35.7%(5/14) and Proteus mirabilis, 57.1%(4/7). Among the ESBL genes detected, blaTEM (n = 14) was common, followed by blaCTX-M (n = 13) and blaSHV (n = 4). Thirty-four E. coli isolates possessed the heat labile (Lt) gene of Enterotoxigenic E. coli (ETEC).ConclusionOur findings confirm the existence of DEC and the antimicrobial resistance patterns of ESBL-PE among stool isolates, limiting the options of commonly used drugs for diarrhoeal treatment in Ghana. Routine laboratory testing in health care facilities and strengthened surveillance systems among hospital networks are encouraged for a better understanding of their epidemiology and clinical implications.

Intradermal fractional-dose inactivated polio vaccine (fIPV) adjuvanted with double mutant Enterotoxigenic Escherichia coli heat labile toxin (dmLT) is well-tolerated and augments a systemic immune response to all three poliovirus serotypes in a randomized placebo-controlled trial

Eradication of poliomyelitis globally is constrained by fecal shedding of live polioviruses, both wild-type and vaccine-derived strains, into the environment. Although inactivated polio vaccines (IPV) effectively protect the recipient from clinical poliomyelitis, fecal shedding of live virus still occurs following infection with either wildtype or vaccine-derived strains of poliovirus. In the drive to eliminate the last cases of polio globally, improvements in both oral polio vaccines (OPV) (to prevent reversion to virulence) and injectable polio vaccines (to improve mucosal immunity and prevent viral shedding) are underway. The E. coli labile toxin with two or “double” attenuating mutations (dmLT) may boost immunologic responses to IPV, including at mucosal sites. We performed a double-blinded phase I controlled clinical trial to evaluate safety, tolerability, as well as systemic and mucosal immunogenicity of IPV adjuvanted with dmLT, given as a fractional (1/5th) dose intradermally (fIPV-dmLT). Twenty-nine volunteers with no past exposure to OPV were randomized to a single dose of fIPV-dmLT or fIPV alone. fIPV-dmLT was well tolerated, although three subjects had mild but persistent induration and hyperpigmentation at the injection site. A ≥ 4-fold rise in serotype-specific neutralizing antibody (SNA) titers to all three serotypes was seen in 84% of subjects receiving fIPV-dmLT vs. 50% of volunteers receiving IPV alone. SNA titers were higher in the dmLT-adjuvanted group, but only differences in serotype 1 were significant. Mucosal immune responses, as measured by polio serotype specific fecal IgA were minimal in both groups and differences were not seen. fIPV-dmLT may offer a benefit over IPV alone. Beyond NAB responses protecting the individual, studies demonstrating the ability of fIPV-dmLT to prevent viral shedding are necessary. Studies employing controlled human infection models, using monovalent OPV post-vaccine are ongoing. Studies specifically in children may also be necessary and additional biomarkers of mucosal immune responses in this population are needed.Clinicaltrials.gov Identifer: NCT03922061.

A short peptide derived from zebrafish AP‐2 complex subunit mu‐A AP2M1A354–382 has antimicrobial activity against multi‐drug resistant bacteria

AbstractOur previous study shows that the peptide consisting of 29 residues at positions 354–382 of zebrafish AP‐2 complex subunit mu‐A is an antimicrobial peptide (AMP) capable of inhibiting the growth of Escherichia coli and Staphylococcus aureus. Sequence analysis revealed that the N‐terminal 10 amino acids (designated AP10) of the peptide were highly conserved from nematode to humans. On this basis, we designed two AP10 analogs, AP10R and AP10W, by substituting the amino acids at selected positions of AP10 with tryptophan (W), isoleucine (I), and arginine (R). Both AP10 and AP10R as well as AP10W were found to possess the common features of AMPs: a high hydrophobic ratio, a net positive charge, and amphipathicity. Antibacterial activity assay showed that AP10W exhibited the strongest antimicrobial activity against Gram‐negative and Gram‐positive bacteria including MDR bacteria (with MICs ranging from 20 to 25 μg/ml) among the three peptides. In addition, AP10W was able to bind to lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN), cause depolarization of the bacterial plasma membrane, and stimulate intracellular reactive oxygen species (ROS) production, suggesting it executes antibacterial activity by a combined action of destabilization/destruction of bacterial cell wall through interaction with LPS, LTA, and PGN, disturbance of the usually polarized membrane through depolarization, and apoptosis/necrosis through intracellular ROS production. Importantly, AP10W is not toxic to mammalian cells, and shows tolerance to serum, salt, and pH variation though it is heat labile. These together suggest that AP10W is a promising template for future development of novel peptide antibiotics against MDR bacteria.

Development of a simple, rapid, and sensitive diagnostic assay for enterotoxigenic E. coli and Shigella spp applicable to endemic countries.

Enterotoxigenic E. coli (ETEC) and Shigella spp (Shigella) are complex pathogens. The diagnostic assays currently used to detect these pathogens are elaborate or complicated, which make them difficult to apply in resource poor settings where these diseases are endemic. The culture methods used to detect Shigella are not sensitive, and the methods used to detect ETEC are only available in a few research labs. To address this gap, we developed a rapid and simple diagnostic assay–"Rapid LAMP based Diagnostic Test (RLDT)." The six minutes sample preparation method directly from the fecal samples with lyophilized reaction strips and using established Loop-mediated Isothermal Amplification (LAMP) platform, ETEC [heat labile toxin (LT) and heat stable toxins (STh, and STp) genes] and Shigella (ipaH gene) detection was made simple, rapid (<50 minutes), and inexpensive. This assay is cold chain and electricity free. Moreover, RLDT requires minimal equipment. To avoid any end user’s bias, a battery-operated, handheld reader was used to read the RLDT results. The results can be read as positive/negative or as real time amplification depending on the end user’s need. The performance specifications of the RLDT assay, including analytical sensitivity and specificity, were evaluated using fecal samples spiked with ETEC and Shigella strains. The limit of detection was ~105 CFU/gm of stool for LT, STh, and STp and ~104 CFU/gm of stool for the ipaH gene, which corresponds to about 23 CFU and 1 CFU respectively for ETEC and Shigella per 25uL reaction within 40 minutes. The RLDT assay from stool collection to result is simple, and rapid and at the same time sufficiently sensitive. RLDT has the potential to be applied in resource poor endemic settings for the rapid diagnosis of ETEC and Shigella.

Heat Treatment for Disinfecting Egg Transport Tools

HighlightsDisinfecting shell egg transport tools before their reuse is an integral biosecurity step.Avian influenza viruses are heat labile and it can be disinfected with high temperatures.Lab tests proved that egg transport flats and pallets can be heated to 54°C to 60°C in 7 to 9 h.Egg farm tests identified main factors for consideration in designing heat treatment system.Abstract.Disinfecting shell egg transport tools such as egg flats and pallets before their reuse has become an integral biosecurity step after the 2015 highly pathogenic avian influenza (HPAI) outbreaks in the United States. Lab-scale tests and field verification were conducted to investigate dynamic temperature changes and distributions inside stacks of egg pallet and flat at a surrounding air temperature of 54°C to 60°C (130ºF to 140ºF) in this study. The heat treatment tests were conducted in the lab first, and then verified in a newly built heat treatment room [12 m (L) × 4 m (W) × 3 m (H)] on a commercial laying hen farm in Iowa, the largest egg production state in the United States. Iowa lost more than 30 million hens during 2015 outbreak of HPAI. The lab tests proved that air circulation with fans could accelerate the heat exchange between air and stacks; blowing air from top and bottom at the same time plus arranging a 2.5 cm (1 in.) space between stacks could reduce the heat treatment time for the target temperature zone (i.e., 54ºC to 60ºC) from 26 to 60 h (without air circulation) to 7 to 9 h. For commercial egg farm tests, heat treatment results show that the room air and egg pallet stacks temperature reached the target zone (54ºC to 60ºC) in 30 min and 4 h after starting the heater, respectively. However, the temperature of egg flat stacks failed to reach the targeted zone after 8 h heating, because it had larger volume and heat transfer resistance as compared to egg pallet stacks. Further experiments such as extending heating period and installing mixing fans to accelerate heat exchange between the room air and the stacks are suggested for improving heat treatment system design on commercial farms. Keywords: Avian influenza, Disinfection, Egg flat and pallet, Egg production.