Atypical and dual biotypes variant of virulent SA-NAG-Vibrio cholerae: an evidence of emerging/evolving patho-significant strain in municipal domestic water sources

Introduction and purpose The recent cholera spread, new cases, and fatality continue to arouse concern in public health systems; however, interventions on control is at its peak yet statistics show continuous report. This study characterized atypical and patho-significant environmental Vibrio cholerae retrieved from ground/surface/domestic water in rural-urban-sub-urban locations of Amathole District municipality and Chris Hani District municipality, Eastern Cape Province, South Africa. Methods Domestic/surface water was sampled and 759 presumptive V. cholerae isolates were retrieved using standard microbiological methods. Virulence phenotypic test: toxin co-regulated pili (tcp), choleragen red, protease production, lecithinase production, and lipase test were conducted. Serotyping using polyvalent antisera (Bengal and Ogawa/Inaba/Hikojima) and molecular typing: 16SrRNA, OmpW, serogroup (Vc-O1/O139), biotype (tcpAClas/El Tor, HlyAClas/El Tor, rstRClas/El Tor, RS1, rtxA, rtxC), and virulence (ctxA, ctxB, zot, ace, cep, prt, toxR, hlyA) genes were targeted. Result Result of 16SrRNA typing confirmed 508 (66.9%) while OmpW detected/confirmed 61 (12.01%) V. cholerae strains. Phenotypic-biotyping scheme showed positive test to polymyxin B (68.9%), Voges proskauer (6.6%), and Bengal serology (11.5%). Whereas Vc-O1/O139 was negative, yet two of the isolates harbored the cholera toxin with a gene-type ctxB and hlyAClas: 2/61, revealing atypical/unusual/dual biotype phenotypic/genotypic features. Other potential atypical genotypes detected include rstR: 7/61, Cep: 15/61, ace: 20/61, hlyAElTor: 53/61, rtxA: 30/61, rtxC: 11/61, and prtV: 15/61 respectively. Conclusion Although additional patho-significant/virulent genotypes associated with epidemic/sporadic cholera cases were detected, an advanced, bioinformatics, and post-molecular evaluation is necessary. Such stride possesses potential to adequately minimize future cholera cases associated with dynamic/atypical environmental V. cholerae strains.

Dot blot immunochemical and infrared analyses of the adhesive layer applied to the painting Imago Pietatis by Domenico Morone

Purpose To investigate the nature of the materials used in the adhesive layer of the Imago Pietatis painting (end of the fifteenth century—beginning of the sixteenth century) by Domenico Morone as a prerequisite for its restoration. Methods Micro-FTIR spectra of the animal glue and a polished cross-section were acquired by a Jasco IRT3000 spectrometer, equipped with a 32× Cassegrain objective. A dot blot immunoassay was used to characterise a minor component of the adhesive layer. Results Micro-FTIR was used as an effective diagnostic tool to detect the major component of the adhesive layer and the binder of the paint. Despite the ageing, the complex matrix and the micro-size of the sample, using a dot blot immunoassay, it was possible to quantify 3.7 ± 2.0 ng of ovalbumin per microgram of sample (corresponding to 0.004 ± 0.002% of the weight). Conclusions The findings were in line with conservation practices described in the old treatises, confirming the correct interpretation of the adhesive layer compounds added to the painting and suggesting for the cleaning the use of an anionic water-soluble surfactant highly effective in the removing of proteinaceous materials.

Prospecting the plant growth–promoting activities of endophytic bacteria Cronobacter sp. YSD YN2 isolated from Cyperus esculentus L. var. sativus leaves

Purpose Plant growth–promoting (PGP) bacteria are an environment-friendly alternative to chemical fertilizers for promoting plant growth and development. We isolated and characterized a PGP endophyte, YSD YN2, from the leaves of Cyperus esculentus L. var. sativus. Methods Specific PGP characteristics of this strain, such as phosphate solubilization ability, potassium-dissolving ability, siderophore and indole-3-acetic acid (IAA) production, and salt tolerance, were determined in vitro. In addition, positive mutants were screened using the atmospheric and room temperature plasma (ARTP) technology, with IAA level and organic phosphate solubility as indices. Furthermore, the effect of the positive mutant on seed germination, biomass production, and antioxidant abilities of greengrocery seedling was evaluated, and the genome was mined to explore the underlying mechanisms. Results The strain YSD YN2 showed a good performance of PGP characteristics, such as the production of indole acetic acid and siderophores, solubilization ability of phosphate, and potassium-dissolving ability. It was recognized through 16S rRNA sequencing together with morphological and physiological tests and confirmed as Cronobacter sp. The strain exposed to a mutation time of 125 s by ARTP had the highest IAA and organic phosphate (lecithin) concentrations of 9.25 mg/L and 16.50 mg/L, 50.41% and 30.54% higher than those of the initial strain. Inoculation of mutant strain YSD YN2 significantly increased the seed germination, plant growth attributes, and the activities of peroxidase (POD) and superoxide dismutase (SOD), respectively, but decreased the content of malondialdehyde (MDA) significantly compared with the control. Furthermore, genome annotation and functional analysis were performed through whole-genome sequencing, and PGP-related genes were identified. Conclusion Our results indicated that the mutant strain YSD YN2 with PGP characteristics is a potential candidate for the development of biofertilizers.

Characterization of β-galactosidase and α-galactosidase activities from the halophilic bacterium Gracilibacillus dipsosauri

Purpose Gracilibacillus dipsosauri strain DD1 is a salt-tolerant Gram-positive bacterium that can hydrolyze the synthetic substrates o-nitrophenyl-β-d-galactopyranoside (β-ONP-galactose) and p-nitrophenyl-α-d-galactopyranoside (α-PNP-galactose). The goals of this project were to characterize the enzymes responsible for these activities and to identify the genes encoding them. Methods G. dipsosauri strain DD1 was grown in tryptic soy broth containing various carbohydrates at 37 °C with aeration. Enzyme activities in cell extracts and whole cells were measured colorimetrically by hydrolysis of synthetic substrates containing nitrophenyl moieties. Two enzymes with β-galactosidase activity and one with α-galactosidase activity were partially purified by ammonium sulfate fractionation, ion-exchange chromatography, and gel-filtration chromatography from G. dipsosauri. Coomassie Blue-stained bands corresponding to each activity were excised from nondenaturing polyacrylamide gels and subjected to peptide sequencing after trypsin digestion and HPLC/MS analysis. Result Formation of β-galactosidase and α-galactosidase activities was repressed by d-glucose and not induced by lactose or d-melibiose. β-Galactosidase I had hydrolytic and transgalactosylation activity with lactose as the substrate but β-galactosidase II showed no activity towards lactose. The α-galactosidase had hydrolytic and transgalactosylation activity with d-melibiose but not with d-raffinose. β-Galactosidase I had a lower Km with β-ONP-galactose as the substrate (0.693 mmol l−1) than β-galactosidase II (1.662 mmol l−1), was active at more alkaline pH, and was inhibited by the product d-galactose. β-Galactosidase II was active at more acidic pH, was partially inhibited by ammonium salts, and showed higher activity with α-PNP-arabinose as a substrate. The α-galactosidase had a low Km with α-PNP-galactose as the substrate (0.338 mmol l−1), a pH optimum of about 7, and was inhibited by chloride-containing salts. β-Galactosidase I activity was found to be due to the protein A0A317L6F0 (encoded by gene DLJ74_04930), β-galactosidase II activity to the protein A0A317KZG3 (encoded by gene DLJ74_12640), and the α-galactosidase activity to the protein A0A317KU47 (encoded by gene DLJ74_17745). Conclusions G. dipsosauri forms three intracellular enzymes with different physiological properties which are responsible for the hydrolysis of β-ONP-galactose and α-PNP-galactose. BLAST analysis indicated that similar β-galactosidases may be formed by G. ureilyticus, G. orientalis, and G. kekensis and similar α-galactosidases by these bacteria and G. halophilus.

Anthropogenic disturbances consistently favor the high-yield strategists of soil bacterial community in the Eurasian steppe

Purpose Multiple anthropogenic disturbances, such as climate warming and nitrogen deposition are affecting terrestrial ecosystems. Different disturbances may have some consistent effects on the soil microbial community, which remains largely unexplored. Methods We mimicked 16 anthropogenic disturbances in a steppe ecosystem, and measured the absolute abundance and taxonomic composition of soil bacterial communities with qPCR and amplicon sequencing, respectively. Results We found that while the absolute abundance of each of the four dominant bacterial phyla did not show a consistent response to these disturbances, that of the five subdominant phyla showed a consistent increase. Meanwhile, these disturbances consistently stimulated the relative abundances of metabolic functions for high-growth-yield, including the transport/metabolism of amino acids and carbohydrates. Stochastic processes (e.g., random birth) played more critical roles in structuring the subdominant than dominant phyla, and the disturbances promoted the stochastic processes. Conclusions Overall, the high-yield traits and stochasticity of subdominant phyla led to their positive responses to disturbances. Furthermore, our findings indicate that the intensifying human activities are likely to cause a high-yield-strategies-toward shift in soil microbial composition in the Eurasian steppe ecosystem.

Lactic acid bacterial bacteriocins and their bioactive properties against food-associated antibiotic-resistant bacteria

Purpose Incidence of foodborne diseases and growing resistance of pathogens to classical antibiotics is a major concern in the food industry. Consequently, there is increasing demand for safe foods with fewer chemical additives but natural products which are not harmful to the consumers. Bacteriocins, produced by lactic acid bacteria (LAB), is of interest because they are active in a nanomolar range, do not have toxic effects, and are readily available in fermented food products. Methods In this research, LAB were isolated from fufu, gari, kunu, nono, and ogi using De Mann, Rogosa, and Sharpe agar. Cell-free supernatants were prepared from 18-24 h LAB culture grown on MRS broth. Effect of organic acid was eliminated by adjusting the pH of the supernatants to 7.0 with 1M NaOH while the effect of hydrogen peroxide was eliminated by treating with Catalase enzyme. The supernatant was then filter-sterilized using a membrane filtration unit with a 0.2-μm pore size millipore filter and subjected to agar well diffusion assay against foodborne antibiotic-resistant bacteria. Result A total of 162 isolates were obtained from the food samples. The antimicrobial sensitivity test yielded positive results for 45 LAB isolates against Staphylococcus aureus ATCC 25923 while 52 LAB isolates inhibited Escherichia coli ATCC 25922. On confirmation of the bacteriocinogenic nature of the inhibitory substance, 4 of the LAB isolates displayed a remarkable degree of inhibition to Leuconostoc mesenteroides, Salmonella typhimurium, and Bacillus cereus. Agar well diffusion assay was also performed against antibiotic-resistant foodborne pathogens using the cell-free supernatant (CFS) obtained from Lactobacillus fermentum strain NBRC15885 (Limosilactobacillus fermentum), Lactobacillus fermentum strain CIP102980 (Limosilactobacillus fermentum), Lactobacillus plantarum strain JCM1149 (Lactiplantibacillus garii), and Lactobacillus natensis strain LP33 (Companilactobacillus nantensis). The foodborne pathogens exhibited a notable level of resistance to antibiotics, with B. cereus exhibiting a resistance profile of 40%, S. aureus (50%), K. pnuemoniae (70%), E. coli (60%), and S. typhi (40%). The (CFS) was able to inhibit the growth of B. cereus, Klebsiella pneumonia, S. typhimurium, S. aureus, and E. coli. Conclusion Therefore, it portends that the bacteriocins produced by the LAB isolated from these food products could act as probiotics for effective inhibition of the growth of antibiotic-resistant foodborne pathogens.

Assessing the viability of Legionella pneumophila in environmental samples: regarding the filter application of Ethidium Monoazide Bromide

Purpose Analyses of 34 water samples from 13 healthcare structures revealed how culture method and quantitative PCR (qPCR) often differ in the detection of Legionella pneumophila (Lp). With these considerations in hand, culture method, PCR and Ethidium Monoazide Bromide (EMA) qPCR have all been compared in order to detect Lp in water samples, identify a method able to speed up the procedures, detect the “viable but not cultivable” bacteria (VBNC) and exclude non-viable bacteria using a commercial kit for extraction and amplification as well as modification of the protocol. Methods Pure water samples artificially spiked with viable, non-viable and VBNC Lp ATCC 33152 were analyzed using a commercial kit for both qPCR and EMA-qPCR, while ISO 11731-2-2004 was used for culture method. Results Only 35% (12/34) of the environmental samples were positive in both culture and qPCR methods. With regard to EMA-qPCR, results showed the absence of dye toxicity on viable and VBNC strains and an incomplete effectiveness on the non-viable ones. In both viable and VBNC strains, a decrease of bacterial DNA amplification was recorded as a function of sample dilution but not of EMA concentration. Conclusions Discrepancies between culture method and EMA-qPCR were observed and may be due to different causes such as membrane-dye interactions, presence of interfering compounds and the sensitivity of the kit used. Study significance and impact In the presence of one or more suspected cases of nosocomial legionellosis, the application of a rapid molecular method able to identify only the viable and VBNC Lp would be useful in order to quickly identify the source of infection and to intervene with sanitation treatments. However, seeing that in our experience EMA pretreatment on the filter membrane did not come up with the expected results, it would be necessary to proceed with other experiments and/or different dyes. Graphical Abstract

Antifungal activity, identification and biosynthetic potential analysis of fungi against Rhizoctonia cerealis

Purpose Wheat sheath blight mainly infected by Rhizoctonia cerealis is one of the soil-borne fungal diseases of wheat worldwide and prevalent in major wheat growing areas in China at present. This study aimed to evaluate the antifungal activity of 163 endophytic fungi on R. cerealis. Antifungal strains were identified and their biosynthetic potential was analysed. Methods The antifungal activity of the strains was evaluated via dual-culture antagonism assay. The antifungal strains were identified on the basis of morphological characteristics and internal transcribed spacer gene sequencing. The polyketide synthases (PKSs) and nonribosomal peptide synthetase (NRPS) genes in antifungal strains were detected via specific amplification of chromosomal DNA. Result Twelve out of 163 fungal strains, including seven strains with matrix competition and five strains with antibiosis, were obtained. The twelve antifungal strains belonged to four genera: Alternaria, Ascochyta, Botryosphaeria, and Talaromyces. The inhibition rate of the seven strains with matrix competition was greater than 50%, with that of Botryosphaeria dothidea S2-33 being the highest at 84.6%. The inhibition zone of Talaromyces assiutensis R-03 amongst the five strains with antibiosis was the widest at up to 7 mm. Among the twelve antifungal strains, the strain S2-16 contained all the genes tested, five B. dothidea strains contained PKS-II and NRPS genes, two Alternaria alternata strains only contained PKS-II gene and the remaining four strains did not contain any. Conclusion Results demonstrated twelve potential strains for the biocontrol of wheat sheath blight. In particular, T. assiutensis R-03 was determined as a promising agent. The active substances secreted by antifungal strains may be produced by other biosynthetic pathways.

Co-administration of vitamin D3 and Lacticaseibacillus paracasei DG increase 25-hydroxyvitamin D serum levels in mice

Purpose Subclinical vitamin D (vitD) deficiency enhances the predisposition to a myriad of acute and chronic pathologies in many people worldwide. Due to the scarcity of vitD-rich foods, the consumption of supplements or fortified foods can be required to maintain healthy serum levels of 25-hydroxyvitamin D [25(OH)D], and the major circulating form of vitD that is commonly measured in serum to determine the vitD status. Since the vitD absorption seems to resemble that of lipids, improved emulsification in the gut could favor vitD permeation through the enterocyte membrane. Contextually, we hypothesized that a microorganism with cholecalciferol (vitD3)-solubilization properties may potentially result in enhanced serum vitD levels. Methods and results Six probiotic strains were screened for their ability to create a stable suspension of vitD3 in water: Lacticaseibacillus paracasei DG, L. paracasei LPC-S01, L. paracasei Shirota, L. rhamnosus GG, Limosilactobacillus reuteri DSM 17938, and Lactobacillus acidophilus LA5. The DG strain displayed the strongest vitD3 solubilization ability and, consequently, were used in an in vivo trial where a commercial preparation of vitD3 in refined olive oil was administered by gavage to CD-1 mice with or without the concurrent administration of L. paracasei DG. ELISA measurements showed that the DG strain significantly increased the serum levels of 25(OH) D when administered once a day for 1 week in association with the vitD3 supplement. Conclusion This preliminary pre-clinical study suggests that the combined administration of L. paracasei DG with an oil-based cholecalciferol supplement could contribute to the maintenance of the adequate 25(OH) D serum levels in people at risk of vitD deficiency.

Accumulation of beneficial bacteria in the rhizosphere of maize (Zea mays L.) grown in a saline soil in responding to a consortium of plant growth promoting rhizobacteria

Purpose Salt stress reduces plant growth and is now becoming one of the most important factors restricting the agricultural productivity. Inoculation of plant growth-promoting rhizobacteria (PGPR) has been shown to confer plant tolerance against abiotic stress, but the detailed mechanisms of how this occurs remain unclear and the application effects in different reports are unstable. In order to obtain a favorite effect of PGPR inoculation and improve our knowledge about the related mechanism, we performed this study to analyze the mechanism of a PGPR consortium on improving the salt resistance of crops. Methods A region-specific (Saline land around Bohai Sea in China) PGPR consortium was selected that contains three strains (Pseudomonas sp. P8, Peribacillus sp. P10, and Streptomyces sp. X52) isolated from rhizosphere of Sonchus brachyotus DC. grown in a saline soil. By inoculation tests, their plant growth-promoting (PGP) traits and ability to improve the salt resistance of maize were investigated and shifting in rhizosphere bacterial community of the inoculated plants was analyzed using the high-throughput sequencing technology. Results The three selected strains were salt tolerant, presented several growth promoting properties, and inhibited several phytopathogenic fungi. The inoculation of this consortium promoted the growth of maize plant and enriched the beneficial bacteria in rhizosphere of maize in a saline soil, including the nitrogen fixing bacteria Azotobacter, Sinorhizobium, and Devosia, and the nitrification bacteria Candidatus Nitrososphaera, and Nitrosovibrio. Conclusions The bacterial consortium P8/P10/X52 could improve plant growth in a saline soil by both their PGP traits and regulating the rhizosphere bacterial community. The findings provided novel information about how the PGPR helped the plants in the view of microbiome.