Transmission of antibiotic resistance through organic amendments in arable land: A 3-year field study with pigeonpea-wheat cropping system.

The worldwide emergence of antimicrobial resistance (AMR) poses a substantial risk to human health and environmental stability. In agriculture, organic amendments (derived from organic sources such as manure, and plant residues) are beneficial in restoring soil properties and providing essential nutrients to crops but raise concerns about harboring antibiotic resistance, which emphasizes the need for vigilant monitoring and strategic interventions in their application. The current study assessed the impact of farming practices (organic and conventional) in a three-year field experiment with pigeonpea-wheat cropping system, focusing on the transmission of AMR using culture-dependent and -independent approaches, and soil nutrient content. Markers for antibiotic resistance genes (ARGs) (aminoglycoside-aacA, ß-lactam-blaTEM, chloramphenicol-cmlA1, macrolide-ermB, sulfonamides-sul1, sul2, and tetracycline-tetO) and integrons (intl1 and intl2) were targeted using qPCR. Manure amendments, particularly FYM1, exhibited a higher abundance of copies of ARGs compared to the rhizospheric soil. Organic farming was associated with higher copies of intl2, sul1, blaTEM, and tetO genes, while conventional farming showed increased copies of sul2 and ermB genes in the rhizosphere. Significant positive correlations were observed among soil nutrient contents, ARGs, and MGEs. The notable prevalence of ARGs linked to manure amendments serves as a cautionary note, demanding responsible management practices.

Transplantation of soil from organic field confers disease suppressive ability to conducive soil.

Organic agriculture is a sustainable method of farming, and confers disease-suppressing abilities to disease-conducive soils via specialized soil microbiomes. This study aimed at transforming a disease-conducive soil from a conventional field into disease-suppressive soil by inoculating soil from an organic field previously established as "disease-suppressive". The effectiveness of the transformed soil was established with the model plant wheat (Triticum aestivum) grown under natural conditions, with regard to its potential in inhibiting fungal phytopathogens, Rhizoctonia solani and Fusarium oxysporum. The conducive soil inoculated with the disease-suppressive soil performed better than the control conducive soil in terms of reduced disease severity in plants, improved soil nutrient content, increased activity of hydrolytic enzymes, and increased abundance of structural and functional microbial markers. The study demonstrates the efficacy of the soil microbiome under long-term organic agriculture in transforming disease-conducive soil into disease-suppressive soils. Such practises are simple and easy to implement, and could greatly improve the sustainability and crop yield in developing countries.

Effectiveness of Injectable Platelet-Rich Fibrin in the Treatment of Oral Lichen Planus: A Systematic Review and Meta-Analysis.

Oral lichen planus (OLP) is a chronic inflammatory condition affecting the oral mucosa. The current review investigated the potential effectiveness of injectable platelet-rich fibrin (i-PRF) as a treatment for OLP when compared to other interventions. The current review adhered to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A comprehensive search strategy was implemented across databases such as PubMed, Embase, Scopus, Web of Science, CINAHL, and Google Scholar. The search utilized a combination of Boolean operators (AND, OR) and Medical Subject Headings (MeSH) terms to capture relevant studies. Comparative clinical studies focusing on i-PRF as a treatment for OLP and other interventions were included. Outcomes assessed were pain, surface area of lesions, and patient satisfaction. Review Manager 5.4 was used for statistical analysis. The Risk of Bias 2.0 (RoB 2.0) tool was used to assess the methodological quality of the studies. Three studies were included for the final analysis. The findings indicated that both the i-PRF and comparison treatment groups demonstrated reductions in pain and lesion size. The post-treatment Visual Analogue Scale (VAS) scores showed a decrease in pain intensity, and there was an improvement in lesion extension in the i-PRF-treated sites. The results also revealed increased patient satisfaction with i-PRF treatment. Adverse effects were not reported or specified in the included studies. Quantitative analysis for pain (VAS) showed a mean difference of 0.38 (95% CI: 0.63-1.40), but there was no significant difference between the i-PRF and control groups at p=0.46. Though intragroup differences showed statistically significant differences between pre and post intervention, intergroup differences were not significant for any of the assessed outcomes. The findings from this study suggest that i-PRF holds promise as a potential treatment for OLP. The use of i-PRF resulted in pain reduction, lesion size improvement, and increased patient satisfaction. However, it is important to consider the limitations of the included studies, such as variability in study designs, small sample sizes, and the limited number of studies.

Organic amendments modulate the crop yield and rhizospheric bacterial community diversity: a 3-year field study with Cajanus cajan.

Excessive use of chemicals to enhance soil nutrient status and crop yield has resulted in a decline in soil health. Organic farming promotes organic amendments, which help to balance the ecosystem. Understanding the dynamic patterns of belowground microbial populations is essential for developing sustainable agricultural systems. Therefore, the study was designed to evaluate the effect of different agri-practices on rhizospheric bacterial diversity and crop yield in an Indian agricultural system. A 3-year field experiment was set up in a randomized block design using Cajanus cajan as a model crop, comparing conventional farming with organic practice (with animal manure and bio-compost as amendments). Plant and rhizospheric soil samples were collected at the harvest stage for assessing various growth attributes, and for characterizing rhizospheric bacterial diversity. Enhanced crop productivity was seen in conventional farming, with a 2.2-fold increase in grain yield over control. However, over the 3 years, an overall positive impact was observed in the bio-compost-based organic amendment, in terms of bacterial abundance, over other treatments. At the harvest stage of the third cropping season, the bacterial diversity in the organic treatments showed little similarity to the initial bacterial community composition of the amendment applied, indicating stabilization along the growth cycles. The study emphasizes the significance of the choice of the amendment for ushering in agricultural sustainability.

Seed biopriming as a promising approach for stress tolerance and enhancement of crop productivity: a review.

Chemicals are used extensively in agriculture to increase crop production to meet the nutritional needs of an expanding world population. However, their injudicious application adversely affects the soil's physical, chemical and biological properties, subsequently posing a substantial threat to human health and global food security. Beneficial microorganisms improve plant health and productivity with minimal impact on the environment; however, their efficacy greatly relies on the application technique. Biopriming is an advantageous technique that involves the treatment of seeds with beneficial biological agents. It exhibits immense potential in improving the physiological functioning of seeds, thereby playing a pivotal role in their uniform germination and vigor. Biopriming-mediated molecular and metabolic reprogramming imparts stress tolerance to plants, improves plant health, and enhances crop productivity. Furthermore, it is also associated with rehabilitating degraded land, and improving soil fertility, health and nutrient cycling. Although biopriming has vast applications in the agricultural system, its commercialization and utilization by farmers is still in its infancy. This review aims to critically analyze the recent studies based on biopriming-mediated stress mitigation by alteration in physiological, metabolic and molecular processes in plants. Additionally, considering the necessity of popularizing this technique, the major challenges and prospects linked to the commercialization and utilization of this technique in agricultural systems have also been discussed. © 2023 Society of Chemical Industry.

Factors affecting biofilm formation by bacteria on fabrics.

Fabrics act as fomites for microorganisms, thereby playing a significant role in infection transmission, especially in the healthcare and hospitality sectors. This study aimed to examine the biofilm formation ability of four nosocomial infection-causing bacteria (Acinetobacter calcoaceticus, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) on cotton, polyester, polyester-cotton blend, silk, wool, viscose, and nylon, used frequently in the healthcare sector, by qualitative and quantitative methods. The impact of temperature, pH, and relative humidity (RH) on biofilm formation was also assessed. P. aeruginosa and S. aureus were strong biofilm producers, while E. coli produced weak biofilm. Wool (maximum roughness) showed the highest bacterial load, while silk (lowest roughness) showed the least. P. aeruginosa exhibited a higher load on all fabrics, than other test bacteria. Extracellular polymeric substances were characterized by infrared spectroscopy. Roughness of biofilms was assessed by atomic force microscopy. For biofilm formation, optimum temperature, pH, and RH were 30 °C, 7.0, and 62%, respectively. MgCl2 and CaCl2 were the most effective in removing bacterial biofilm. In conclusion, biofilm formation was observed to be influenced by the type of fabric, bacteria, and environmental conditions. Implementing recommended guidelines for the effective disinfection of fabrics is crucial to curb the risk of nosocomial infections. In addition, designing modified healthcare fabrics that inhibit pathogen load could be an effective method to mitigate the transmission of infections.

Temporal dynamics of endophytic bacterial and fungal communities during spike development in Piper longum L.

The female spikes (fruits) of Piper longum are widely used in Ayurvedic, Siddha and Unani medicine systems to treat respiratory and digestive disorders. The spikes are rich in piperine, a pharmacologically active amide alkaloid and a potent bioavailability enhancer, which accumulates to the highest level during the dark-green stage of spike development. Plant-associated microbiota influence the plant's fitness, response, and production of economically important metabolites. Considering the economic importance of piperine and other spike-derived alkaloids, understanding microbial community dynamics during spike development would be key to bioprospecting for economically important metabolites. In the present study, the structural diversity of microbial communities associated with early (SI), mid (SII), and late (SIII) stages of spike development in P. longum has been analysed by Illumina-based amplicon sequencing of 16S rRNA gene and ITS region. Results revealed that spike development significantly drives the diversity and abundance of spike-associated microbiota, especially bacterial communities. Cyanobacteria and Ascomycota constituted the most abundant bacterial and fungal phyla, respectively, across all stages of spike development. Interestingly, Halomonas, Kushneria and Haererehalobacter were found to be exclusively associated with SIII (corresponding to economically important) stage of spike development. Sphingomonas, Mortierella, Cladosporium and Vishniacozyma constituted the core microbiome of the spike. Besides, PICRUSt analysis revealed that amino acid metabolism was the most dominant metabolic function attributed to spike-associated bacterial communities. To the best of our knowledge, this is the first study to investigate the endomicrobiome dynamics during spike development in a medicinal plant species. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01352-2.

Composition of fungal communities upon multiple passaging of rhizosphere microbiome for salinity stress mitigation in Vigna radiata.

The top-down approach of microbiome-mediated rhizosphere engineering has emerged as an eco-friendly approach for mitigating stress and enhancing crop productivity. It has been established to mitigate salinity stress in Vigna radiata using multi-passaging approach. During the process of acclimatization under increasing levels of salinity stress, the structure of rhizospheric microbial community undergoes dynamic changes, while facilitating stress mitigation in plants. In this study, using ITS-based amplicon sequencing, the dynamics of rhizosphere fungal community was unravelled over successive passages under salinity stress in V. radiata. Clear shifts were evident among the fungal community members under stress and non-stress conditions, upon application of acclimatized rhizosphere microbiome in V. radiata across successive passages. These shifts correlated with enhanced plant biometrics and reduced stress marker levels in plant. Significant changes in the fungal community structure were witnessed in the rhizosphere across specific passaging cycles under salinity stress, which possibly facilitated stress mitigation in V. radiata.

Textiles as fomites in the healthcare system.

Nosocomial infections or healthcare-associated infections (HAIs) are acquired under medical care in healthcare facilities. In hospital environments, the transmission of infectious diseases through textiles such as white coats, bed linen, curtains, and towels are well documented. Textile hygiene and infection control measures have become more important in recent years due to the growing concerns about textiles as fomites in healthcare settings. However, systematic research in this area is lacking; the factors contributing to the transmission of infections through textiles needs to be better understood. The review aims to critically explore textiles as contaminants in healthcare systems, and to identify potential risks they may pose to patients and healthcare workers. It delineates different factors affecting bacterial adherence on fabrics, such as surface properties of bacteria and fabrics, and environmental factors. It also identifies areas that require further research to reduce the risk of HAIs and improve textile hygiene practices. Finally, the review elaborates on the strategies currently employed, and those that can be employed to limit the spread of nosocomial infections through fabrics. Implementing textile hygiene practices effectively in healthcare facilities requires a thorough analysis of factors affecting fabric-microbiome interactions, followed by designing newer fabrics that discourage pathogen load. KEY POINTS: • Healthcare textiles act as a potential reservoir of nosocomial pathogens • Survival of pathogens is affected by surface properties of fabric and bacteria • Guidelines required for fabrics that discourage microbial load, for hospital use.

Role of Fungi in Imparting General Disease Suppressiveness in Soil from Organic Field.

Soil microbial communities are key players responsible for imparting suppressive potential to the soil against soil-borne phytopathogens. Fungi have an immense potential to inhibit soil-borne phytopathogens, but the fungal counterpart has been less explored in this context. We assessed the composition of fungal communities in soil under long-term organic and conventional farming practice, and control soil. The disease-suppressive potential of organic field was already established. A comparative analysis of the disease suppressiveness contributed by the fungal component of soil from conventional and organic farms was assessed using dual culture assays. The quantification of biocontrol markers and total fungi was done; the characterization of fungal community was carried out using ITS-based amplicon sequencing. Soil from organic field exhibited higher disease-suppressive potential than that from conventional farming, against the pathogens selected for the study. Higher levels of hydrolytic enzymes such as chitinase and cellulase, and siderophore production were observed in soil from the organic field compared to the conventional field. Differences in community composition were observed under conventional and organic farming, with soil from organic field exhibiting specific enrichment of key biocontrol fungal genera. The fungal alpha diversity was lower in soil from the organic field compared to the conventional field. Our results highlight the role of fungi in contributing to general disease-suppressive ability of the soil against phytopathogens. The identification of fungal taxa specifically associated with organic farming can aid in understanding the mechanism of disease suppression under such a practice, and can be exploited to induce general disease suppressiveness in otherwise conducive soil.

Nutrient concentrations affect the antimicrobial resistance profiles of cattle manures.

Antimicrobial resistance (AMR) in cattle is widespread because of the increased use of antibiotics to combat microbial diseases and enhance milk production. The cattle excreta released into the environment can be a potent source of contamination in spreading antibiotic resistance, especially upon its application in agriculture. However, the correlation of AMR profile of manure with other physico-chemical parameters is limited. Therefore, the study aimed to generate AMR profiles for manure samples collected from 25 different sites of two agriculturally important states in India, Madhya Pradesh and Uttar Pradesh. Samples were tested for physico-chemical parameters, viz., electrical conductivity, pH, total nitrogen (N), total phosphorus (P), and total potassium (K). Bacterial community analysis was done by culture-dependent and culture-independent methods. The influence of feeding practices, nutrient concentration, and bacterial abundance on antibiotic resistance profiles was observed in collected manure samples. Manures of intensive feeding animals harbored highly resistant profiles of bacteria as compared to natural grazing cattle.

Satellite hyperspectral imaging technology as a potential rapid pollution assessment tool for urban landfill sites: case study of Ghazipur and Okhla landfill sites in Delhi, India.

Hyperspectral imaging technology has been used for biochemical analysis of Earth's surface exploiting the spectral reflectance signatures of various materials. The new-generation Italian PRISMA (PRecursore IperSpettrale dellaMissione Applicativa) hyperspectral satellite launched by the Italian space agency (ASI) provides a unique opportunity to map various materials through spectral signature analysis for recourse management and sustainable development. In this study PRISMA hyperspectral satellite imagery-based multiple spectral indices were generated for rapid pollution assessment at Ghazipur and Okhla landfill sites in Delhi, India. It was found that the combined risk score for Okhla landfill site was higher than the Ghazipur landfill site. Various manmade materials identified, exploiting the hyperspectral imagery and spectral signature libraries, indicated presence of highly saline water, plastic (black, ABS, pipe, netting, etc.), asphalt tar, black tar paper, kerogen BK-Cornell, black paint and graphite, chalcocite minerals, etc. in large quantities in both the landfill sites. The methodology provides a rapid pollution assessment tool for municipal landfill sites.

Employing mutants to study the role of a three-membered bacterial consortium in growth promotion of Cajanus cajan.

Bacterial consortia, comprising plant growth promoting (PGP) rhizobacteria, are known to outcompete their impacts on plant attributes compared to their individual application. However, tracking of individual bacterial strains post application as consortium, remains challenging. The primary goal of this study was to develop an efficient method of tracking bioinoculants by generating spontaneous mutants of three different bacterial strains in an established consortium, using antibiotic-based screening, followed by their enumeration after application in Cajanus cajan. Mutants were generated for consortium members, viz. Azotobacter chroococcum (A), Priestia megaterium (formerly Bacillus megaterium) (B), and Pseudomonas sp. (P), against streptomycin, kanamycin and rifampicin, respectively. Those mutants having similar growth rates and PGP properties as compared to wild type bacterial strains were selected to test their efficacy in plant growth promotion. Selected mutant strains were applied as mono, dual and triple cultures to C. cajan grown hydroponically. Enumeration of mutant bacterial strains was carried out to check their viability. Bacterial colonization on roots was also analyzed. The application of triple (mutant) inoculants improved plant growth attributes significantly in comparison to mono and dual culture treatments and control. Cell enumeration revealed that the abundance of each bacterial strain increased till the 5th day of treatment. No significant change was observed later in their abundance for any treatment. The triple culture treatment showed greater abundance of bacterial mutant strains in comparison to mono- or dual cultures. To the best of our knowledge, this is the first mutant-based study to have reported the successful tracking and enumeration of bacterial consortium members, post their application in C. cajan.

Diagnostic and prognostic role of protein and ultrastructural alterations at cell-extracellular matrix junctions in neoplastic progression of human oral malignancy.

Despite advancements in technology and increase in favorable outcomes associated with oral cancer, early detection remains the most significant factor in limiting mortality. The current study aimed to develop early diagnostic and prognostic markers for oral tumorigenesis. Protein and ultrastructural alterations at cell-extracellular matrix (ECM) adhesion junctions were examined concurrently using immunohistochemistry (IHC) and transmission electron microscopy (TEM) on progressive grade of oral carcinomas (n = 285). The expression of hemidesmosome (HD) proteins-integrin ß4, BP180, and laminin-5 increased in hyperplasia as compared to normal, and significantly increased further, as the disease progressed. TEM analysis in parallel tissues revealed a significant decrease in HD number and increase in the length of basal lamina (BL) in hyperplasia. With cancer progression, the severity of ultrastructural alterations increased gradually and significantly. Overexpression of HD proteins, decrease in HD number and increase in BL length significantly correlated with nodal metastasis, local recurrence, and recurrence-free survival of patients. Concurrent use of IHC and TEM can add value to early recognition of neoplastic changes in primary carcinomas of oral cavity. In this regard, altered expression of integrin ß4 and laminin-5, loss of HDs, and increased BL length could offer criteria for early diagnosis and prognosis of oral malignancy.

Metabolomic insight into the synergistic mechanism of action of a bacterial consortium in plant growth promotion.

It has been established that a consortium consisting of compatible plant growth promoting rhizobacterial strains outperforms their individual impacts on plant attributes. While the phenomenon of synergism is extensively reported, the mechanism that underpins it is yet to be elucidated. In the present study the impact of three plant growth promoting bacteria, Azotobacter chroococcum (A), Priestia megaterium (formerly Bacillus megaterium) (B), and Pseudomonas sp. SK3 (P) was studied as a consortium on the growth attributes of pigeonpea. In addition, microbe-microbe interactions were investigated through metabolomic profiling to understand the mechanism of synergism. Plant growth experiments revealed that bacterial consortium A + B + P showed a significant increase in plant attributes such as shoot length, root length, fresh weight, and dry weight as compared to monocultures and two-membered consortia. Metabolomic profiling through high resolution liquid chromatograph mass spectrometer revealed the presence of a few bioactive compounds in the consortium that might play a potential role in the enhancement of biometric parameters of the plant. Several compounds, such as antipyrine, 6,6-dimethoxy-2,5,5-trimethyl-2-hexene, N-methyltryptamine, 2,2-dimethyl-3,4-bis(4-methoxyphenyl)-2H-1-benzopyran-7-ol acetate, N6-hydroxy-l-lysine, and l-furosin, were detected in the metabolome of the consortium, which was unique among all the treatments. The study also detected a few metabolites involved in sphingolipid biosynthesis (ketosphinganine and sphinganine) known for cell signaling in the consortium. This unravels the possible mechanism of synergism between bacterial strains in a consortium. The metabolomic profile would be helpful to strategically develop unique and more effective consortia that are tailored to the soil type.

Combating biotic stresses in plants by synthetic microbial communities: Principles, applications and challenges.

Presently, agriculture worldwide is facing the major challenge of feeding the increasing population sustainably. The conventional practices have not only failed to meet the projected needs, but also led to tremendous environmental consequences. Hence, to ensure a food-secure and environmentally sound future, the major thrust is on sustainable alternatives. Due to challenges associated with conventional means of application of biocontrol agents in the management of biotic stresses in agroecosystems, significant transformations in this context are needed. The crucial role played by soil microbiome in efficiently and sustainably managing the agricultural production has unfolded a newer approach of rhizosphere engineering that shows immense promise in mitigating biotic stresses in an eco-friendly manner. The strategy of generating synthetic microbial communities (SynComs), by integrating omics approaches with traditional techniques of enumeration and in-depth analysis of plant-microbe interactions, is encouraging. The review discusses the significance of the rhizospheric microbiome in plant's fitness, and its manipulation for enhancing plant attributes. The focus of the review is to critically analyse the potential tools for the design and utilization of SynComs as a sustainable approach for rhizosphere engineering to ameliorate biotic stresses in plants. Furthermore, based on the synthesis of reports in the area, we have put forth possible solutions to some of the critical issues that impair the large-scale application of SynComs in agriculture.

Bacterial inoculants as effective agents in minimizing the non-target impact of azadirachtin pesticide and promoting plant growth of Vigna radiata.

Microbes regulate soil health by negating ecological disturbances, and improve plant productivity in a sustainable manner. Indiscriminate application of pesticides creates a detrimental impact on the rhizospheric microbiota, thereby affecting soil health. Azadirachtin, earlier believed to be an environment-friendly alternative to chemical pesticides, exhibits a non-target impact on microbial communities. This study aimed to employ potent bacteria to promote the growth of mungbean plant (Vigna radiata), and mitigate the non-target impact of azadirachtin. Bacterial strains were isolated by enrichment from mungbean rhizosphere. A plant growth experiment was performed with mungbean, amended with azadirachtin to assess the impact of bacterial bioinoculants on the rhizospheric microbiota. The impact of azadirachtin on rhizospheric bacterial community was analyzed qualitatively and quantitatively by 16S rRNA PCR-DGGE and qPCR of various markers, respectively. Residual concentration of azadirachtin in the soil was estimated by HPLC. The bacterial inoculants used in combination significantly promoted plant growth and enhanced the diversity and abundance of total bacterial community in the presence of azadirachtin. Further, the abundance of specific bacterial groups (α-Proteobacteria, ß-Proteobacteria, Actinobacteria, Acidobacteria, and Firmicutes) were significantly boosted. Compared to the control, the isolates significantly facilitated the reduction in residual concentration of azadirachtin in the mungbean rhizosphere. Bacterial inoculants can serve a tripartite role in reducing the stress imparted by botanical pesticides, together with promoting plant growth and enriching the rhizospheric bacterial community structure.

COVID-19 infection, and reinfection, and vaccine effectiveness against symptomatic infection among health care workers in the setting of omicron variant transmission in New Delhi, India.

Background: Surge of SARS CoV-2 infections ascribed to omicron variant began in December 2021 in New Delhi. We determined the infection and reinfection density in a cohort of health care workers (HCWs) along with vaccine effectiveness (VE) against symptomatic infection within omicron transmission period (considered from December 01, 2021 to February 25, 2022. Methods: This is an observational study from the All India Institute of Medical Sciences, New Delhi. Data were collected telephonically. Person-time at risk was counted from November 30, 2021 till date of infection/ reinfection, or date of interview. Comparison of clinical features and severity was done with previous pandemic periods. VE was estimated using test-negative case-control design [matched pairs (for age and sex)]. Vaccination status was compared and adjusted odds ratios (OR) were computed by conditional logistic regression. VE was estimated as (1-adjusted OR)X100-. Findings: 11474 HCWs participated in this study. The mean age was 36⋅2 (±10⋅7) years. Complete vaccination with two doses were reported by 9522 (83%) HCWs [8394 (88%) Covaxin and 1072 Covishield (11%)]. The incidence density of all infections and reinfection during the omicron transmission period was 34⋅8 [95% Confidence Interval (CI): 33⋅5-36⋅2] and 45⋅6 [95% CI: 42⋅9-48⋅5] per 10000 person days respectively. The infection was milder as compared to previous periods. VE was 52⋅5% (95% CI: 3⋅9-76⋅5, p = 0⋅036) for those who were tested within 14-60 days of receiving second dose and beyond this period (61-180 days), modest effect was observed. Interpretation: Almost one-fifth of HCWs were infected with SARS CoV-2 during omicron transmission period, with predominant mild spectrum of COVID-19 disease. Waning effects of vaccine protection were noted with increase in time intervals since vaccination. Funding: None.