Infectious Inoculum Research Articles

Characterization of bacteria expectorated during forced salivation of the Phlebotomus papatasi: A neglected component of sand fly infectious inoculums.

The infectious inoculum of a sand fly, apart from its metacyclic promastigotes, is composed of factors derived from both the parasite and the vector. Vector-derived factors, including salivary proteins and the gut microbiota, are essential for the establishment and enhancement of infection. However, the type and the number of bacteria egested during salivation is unclear. In the present study, sand flies of Phlebotomus papatasi were gathered from three locations in hyperendemic focus of zoonotic cutaneous leishmaniasis (ZCL) in Isfahan Province, Iran. By using the forced salivation assay and targeting the 16S rRNA barcode gene, egested bacteria were characterized in 99 (44%) out of 224 sand flies. Culture-dependent and culture-independent methods identified the members of Enterobacter cloacae and Spiroplasma species as dominant taxa, respectively. Ten top genera of Spiroplasma, Ralstonia, Acinetobacter, Reyranella, Undibacterium, Bryobacter, Corynebacterium, Cutibacterium, Psychrobacter, and Wolbachia constituted >80% of the saliva microbiome. Phylogenetic analysis displayed the presence of only one bacterial species for the Spiroplasma, Ralstonia, Reyranella, Bryobacter and Wolbachia, two distinct species for Cutibacterium, three for Undibacterium and Psychrobacter, 16 for Acinetobacter, and 27 for Corynebacterium, in the saliva. The abundance of microbes in P. papatasi saliva was determined by incorporating the data on the read counts and the copy number of 16S rRNA gene, about 9,000 bacterial cells, per sand fly. Both microbiological and metagenomic data indicate that bacteria are constant companions of Leishmania, from the intestine of the vector to the vertebrate host. This is the first forced salivation experiment in a sand fly, addressing key questions on infectious bite and competent vectors.

Photosynthetic Costs and Impact on Epidemiological Parameters Associated with Ht Resistance Genes in Maize Lines Infected with Exserohilum turcicum.

Northern corn leaf blight, caused by Exserohilum turcicum, is mainly controlled by the use of resistant cultivars. Maize lines carrying individual resistance genes B37Ht1, B37Ht2, B37Ht3, and B37Htn1 express different defense symptoms having an impact on the photosynthetic activity, the accumulation of reactive oxygen species, and epidemiological parameters. Plants were inoculated with a race 0 isolate of E.turcicum conferring a compatible interaction with B37 and incompatible interactions with plants carrying resistance genes. Five days postinoculation (dpi), the resistant lines displayed a reduction in leaf CO2 assimilation of 30 to 80% compared with healthy plants. At 14 dpi, inoculated plants of B37Ht1 showed a significant decrease in leaf CO2 assimilation, similar to B37 (up to 94%). The instantaneous carboxylation efficiency was significantly reduced on inoculated plants of the lines B37Ht2, B37Ht3, and B37Htn1 (54 to 81%) at 5 dpi. Curiously, the reduction in carboxylation efficiency for B37 and B37Ht1 (up to 95%) was higher at 14 dpi than at 5 dpi (up to 81%). At 6 dpi, low levels of H2O2 were detected in B37Ht1, in contrast to B37Htn1, where a high H2O2 level and peroxidase activity were observed. The sporulation rate on B37Ht1, B37Ht3, and B37Htn1 decreased by 92% compared with the susceptible control, whereas strong sporulation occurred in lesions on line B37Ht2. The resistance in maize to E. turcicum conferred by Ht resistance genes is associated with photosynthetic costs and may have quite contrasting effects on host physiology and major epidemiological parameters, such as sporulation, which contributes inoculum for secondary infections.

Predicting infection of strawberry fruit by Mucor and Rhizopus spp. under protected conditions

Postharvest spoilage of strawberry grown under protection, caused by Mucor spp. and Rhizopus spp., has recently become more common in the UK, but there is insufficient knowledge to develop and implement integrated management against Mucor and Rhizopus. Field sampling was conducted to obtain field data for developing models to predict the infection of Mucor and Rhizopus on strawberry fruit. Fruits were exposed to naturally occurring inoculum for a period of 24 hours before surface-sterilisation and incubation to estimate the level of infection by Mucor and Rhizopus. The observed incidence data, together with climatic conditions and inoculum trap counts, were used analysed firstly within the framework of (1) generalised linear model (GLM), and (2) classification tree. Field sampling confirmed previous research that ripening/ripe strawberry fruits are more susceptible to infection by Mucor and Rhizopus. Climatic variability, particularly in vapour pressure deficit, appears to be more important in influencing the rotting incidence of both Mucor and Rhizopus. However, the predictability of both Mucor and Rhizopus, whether as a continuous variable (incidence) in the GLM analysis or as a categorical risk in classification tree analysis, is too low to be of practical value based on those predictors used in the present study. Thus, current management may have to be based on scheduled preharvest application of alternative products to reduce infection and local pathogen inoculum as well as adopting management practice to minimise pathogen inoculum in the planting. Future research is needed to develop methods for rapid yet accurate in situ quantification of pathogen inoculum to improve disease risk predictions.

Systematic Review of Carbapenem Resistant Gram-negative Infective Endocarditis, Treatment Options and Outcomes

Background: Gram negative infective endocarditis is rare and usually associated with intravenous drug use or significant healthcare exposure and prosthetic cardiac valves or devices. Carbapenem resistance is a growing concern worldwide with limited drug options for these infections especially serious high inoculum infections such as endocarditis.
 Methods: A systematic review examining treatment options and outcomes was conducted with identification of 12 cases in the literature.
 Results: Pseudomonas aeruginosa was the most common organism with left sided valvular involvement being most common. 18 different antibiotic regimens were used with surgery occurring in 5 cases (42%). In hospital mortality was 33% which increased at 6 months post episode. Increased age (p= 0.056) and CCI (p=0.006) appeared to be associated with death. Microbiological cure was more common in patients who received combination therapy with 2 active agents (75% successful) and combination therapy including an active beta-lactam agent (100% successful) but these did not meet statistical significance.
 Conclusions: Recommendations for management of this rare condition based on this systematic review and other available evidence are summarised in this review. Management should generally involve multidisciplinary teams, combination therapy with at least 2 active agents including a beta-lactam agent where possible with consideration of surgery in all cases. Evidence is however limited with need for ongoing publication of cases and further research to guide therapy in the future.

Defining Parameters That Modulate Susceptibility and Protection to Respiratory Murine Coronavirus MHV1 Infection.

Patients infected with SARS-CoV-2 experience variable disease susceptibility, and patients with comorbidities such as sepsis are often hospitalized for COVID-19 complications. However, the extent to which initial infectious inoculum dose determines disease outcomes and whether this can be used for immunological priming in a genetically susceptible host has not been completely defined. We used an established SARS-like murine model in which responses to primary and/or secondary challenges with murine hepatitis virus type 1 (MHV-1) were analyzed. We compared the response to infection in genetically susceptible C3H/HeJ mice, genetically resistant C57BL/6J mice, and genetically diverse, variably susceptible outbred Swiss Webster mice. Although defined as genetically susceptible to MHV-1, C3H/HeJ mice displayed decreasing dose-dependent pathological changes in disease severity and lung infiltrate/edema, as well as lymphopenia. Importantly, an asymptomatic dose (500 PFU) was identified that yielded no measurable morbidity/mortality postinfection in C3H/HeJ mice. Polymicrobial sepsis induced via cecal ligation and puncture converted asymptomatic infections in C3H/HeJ and C57BL/6J mice to more pronounced disease, modeling the impact of sepsis as a comorbidity to β-coronavirus infection. We then used low-dose infection as an immunological priming event in C3H/HeJ mice, which provided neutralizing Ab-dependent, but not circulating CD4/CD8 T cell-dependent, protection against a high-dose MHV-1 early rechallenge. Together, these data define how infection dose, immunological status, and comorbidities modulate outcomes of primary and secondary β-coronavirus infections in hosts with variable susceptibility.

Inoculum-Based Dosing: A Novel Concept for Combining Time with Concentration-Dependent Antibiotics to Optimize Clinical and Microbiological Outcomes in Severe Gram Negative Sepsis.

Certain classes of antibiotics show "concentration dependent" antimicrobial activity; higher concentrations result in increased bacterial killing rates, in contrast to "time dependent antibiotics", which show antimicrobial activity that depends on the time that antibiotic concentrations remain above the MIC. Aminoglycosides and fluoroquinolones are still widely used concentration-dependent antibiotics. These antibiotics are not hydrolyzed by beta-lactamases and are less sensitive to the inoculum effect, which can be defined as an increased MIC for the antibiotic in the presence of a relatively higher bacterial load (inoculum). In addition, they possess a relatively long Post-Antibiotic Effect (PAE), which can be defined as the absence of bacterial growth when antibiotic concentrations fall below the MIC. These characteristics make them interesting complementary antibiotics in the management of Multi-Drug Resistant (MDR) bacteria and/or (neutropenic) patients with severe sepsis. Global surveillance studies have shown that up to 90% of MDR Gram-negative bacteria still remain susceptible to aminoglycosides, depending on the susceptibility breakpoint (e.g., CLSI or EUCAST) being applied. This percentage is notably lower for fluoroquinolones but depends on the region, type of organism, and mechanism of resistance involved. Daily (high-dose) dosing of aminoglycosides for less than one week has been associated with significantly less nephro/oto toxicity and improved target attainment. Furthermore, higher-than-conventional dosing of fluoroquinolones has been linked to improved clinical outcomes. Beta-lactam antibiotics are the recommended backbone of therapy for severe sepsis. Since these antibiotics are time-dependent, the addition of a second concentration-dependent antibiotic could serve to quickly lower the bacterial inoculum, create PAE, and reduce Penicillin-Binding Protein (PBP) expression. Inadequate antibiotic levels at the site of infection, especially in the presence of high inoculum infections, have been shown to be important risk factors for inadequate resistance suppression and therapeutic failure. Therefore, in the early phase of severe sepsis, effort should be made to optimize the dose and quickly lower the inoculum. In this article, the authors propose a novel concept of "Inoculum Based Dosing" in which the decision for antibiotic dosing regimens and/or combination therapy is not only based on the PK parameters of the patient, but also on the presumed inoculum size. Once the inoculum has been lowered, indirectly reflected by clinical improvement, treatment simplification should be considered to further treat the infection.

Identification of Epicoccum poaceicola causing eggplant leaf spot and its cross-infection potential to other solanaceous vegetable crops

This study characterised and identified the causative agent of irregular necrotic leaf spots in eggplant. The culture characteristics and morphology of three fungal isolates indicate that they are from the Didymellaceae group. Using a polyphasic approach—combined morphological, cultural, pathogenicity, and molecular characterisation (concatenated sequences of three gene loci: ITS, TUB2, LSU), the fungal isolates MBELIQ03A and MBELIQ03D were identified as Epicoccum poaceicola. Isolate MBELIQ02 was identified as Epicoccum sp. due to a lack of clear grouping with a single Epicoccum species. In repeated trials, the three Epicoccum isolates were pathogenic to eggplant leaves in detached leaf assays. Similar fungi were consistently re-isolated from the inoculated leaves and thus, establishing Koch’s postulates. Inoculation of the three isolates on solanaceous leaves and fruits (tomato, eggplant, and pepper) revealed their varied pathogenicity. This is the first confirmed scientific report of Epicoccum poaceicola causing eggplant leaf spots. This is also the first record of this fungal species in the Philippines. Inoculum from leaf infection could serve as inoculum for fruit infection and other solanaceous vegetable crops grown nearby a field planted with eggplant.

Optimizing the Use of Beta-Lactam Antibiotics in Clinical Practice: A Test of Time.

Despite their limitations, the pharmacokinetics (PK) and pharmacodynamics (PD) indices form the basis for our current understanding regarding antibiotic development, selection, and dose optimization. Application of PK-PD in medicine has been associated with better clinical outcome, suppression of resistance, and optimization of antibiotic consumption. Beta-lactam antibiotics remain the cornerstone for empirical and directed therapy in many patients. The percentage of time of the dosing interval that the free (unbound) drug concentration remains above the minimal inhibitory concentration (MIC) (%fT > MIC) has been considered the PK-PD index that best predicts the relationship between antibiotic exposure and killing for the beta-lactam antibiotics. Time dependence of beta-lactam antibiotics has its origin in the acylation process of the serine active site of penicillin-binding proteins, which subsequently results in bacteriostatic and bactericidal effects during the dosing interval. To enhance the likelihood of target attainment, higher doses, and prolonged infusion strategies, with/or without loading doses, have been applied to compensate for subtherapeutic levels of antibiotics related to PK-PD changes, especially in the early phase of severe sepsis. To minimize resistance and maximize clinical outcome, empirical therapy with a meropenem loading dose followed by high-dose-prolonged infusion should be considered in patients with high inoculum infections presenting as severe (Gram negative) sepsis. Subsequent de-escalation and dosing of beta-lactam antibiotics should be considered as an individualized dynamic process that requires dose adjustments throughout the time course of the disease process mediated by clinical parameters that indirectly assess PK-PD alterations.

Seed Transmission of Begomoviruses: A Potential Threat for Bitter Gourd Cultivation.

Bitter gourd (Momordica charantia L.), one of the valued vegetable crops in India, is severely affected by yellow mosaic disease caused by two begomoviruses, tomato leaf curl New Delhi virus (ToLCNDV) and bitter gourd yellow mosaic virus (BgYMV). The symptoms are yellowing, distortion of leaf, puckering, and malformed fruits. Increased incidence of the disease and appearance of symptoms even in young emerging seedling stage were suggestive of seed transmission of the viruses, which was examined in detail. To study the seed transmission, two sources-seeds of elite hybrids H1, H2, H3, H4, and Co1 procured from a seed market; and seeds from infected plants in the farmer's field were tested. Detection of the virus by DAS-ELISA using polyclonal antibody indicated embryo infection up to 63%, 26%, 20%, and 10% in hybrids H1, H2, H3, and H4, respectively, for market-procured seeds. In PCR analysis with primers specific for ToLCNDV and BgYMV, infection by ToLCNDV was as high as 76% and mixed infection was 24%. In contrast, in seeds derived from field-infected plants, the percentage detection was less. Grow-out tests with market-procured seeds revealed no transmission for BgYMV compared with 5% transmission for ToLCNDV. Whether seed-borne inocula could serve as an inoculum for new infection in a field and further progress of the disease was investigated in a microplot study. The study clearly revealed variation in seed transmission between different sources, lots, cultivars, and viruses. The virus present in symptomatic and asymptomatic plants was easily transmitted by whitefly. In another microplot experiment, the potential of seed-borne virus as inoculum was proved. There was 43.3% initial seed transmission in the microplot, increasing to 70% after release of 60 whiteflies.

Weed hosts and winter survival of Stemphylium vesicarium on onion in Ontario, Canada

Stemphylium leaf blight (SLB), caused by the fungal pathogen Stemphylium vesicarium, is an important disease of onion (Allium cepa). This pathogen also causes disease on a wide range of other plant species. SLB on onion is managed primarily through fungicide application. The source of primary inoculum and whether S. vesicarium can overwinter in the region were not known. Inoculation and subsequent re-isolation of the pathogen from common weed species present near onion fields demonstrated that many of these weedy species are hosts of S. vesicarium. Redroot pigweed (Amaranthus retroflexus), yellow nutsedge (Cyperus esculentus), yellow marshcress (Rorippa palustris), perennial sow thistle (Sonchus arvensis) and purslane (Portulaca oleracea) were identified as new hosts of S. vesicarium and bull thistle (Cirsium vulgare) was confirmed as a host. Sporulation on dead weed residue in spring may be a source of primary inoculum for infection of adjacent onion fields. Also, a study of pathogen survival in infected leaf pieces of onion buried in soil or left at the soil surface demonstrated that S. vesicarium can overwinter in infected onion leaf residue and produce pseudothecia and ascospores in early spring. These conclusions support existing management recommendations for the burial or removal of onion debris and managing weeds in and around onion fields.

A simple transwell-based infection system for obtaining pure populations of VZV-infected cells

Varicella-Zoster virus (VZV) is a human herpesvirus and causes chickenpox and shingles. Research into its molecular virology has been hampered by a lack of methods for generation of high-titre, cell-free infectious virus preparations. VZV propagation and infection in vitro are therefore commonly achieved by co-culture of uninfected ‘target’ cells with infected ‘inoculum’ cells. A major drawback of this approach is that it results in mixed cell populations after infection. To overcome this limitation we developed a transwell-based VZV infection system. Infected inoculum cells and uninfected target cells are spatially separated by a transwell membrane. While cell-cell contact and VZV spread can occur through membrane pores, the two cell populations do not mix. This simple protocol requires no special instrumentation or reagents. We successfully used this system for infection of a range of target cells and obtained pure populations for downstream analyses such as flow cytometry and RT-qPCR. In sum, we developed a broadly applicable approach to study the molecular and cellular biology as well as host-pathogen interactions of VZV.

A nested multiscale model to study paratuberculosis in ruminants

In this study, we present a nested multiscale model that integrates the within-host scale and the between-host scale disease dynamics for Paratuberculosis in ruminants (e.g., cattle, goats, and sheep), with the aim of ascertaining the influence of initial infective inoculum dose on its dynamics. Ruminant paratuberculosis is often characterized as an environmentally-transmitted disease and it is caused by bacteria called Mycobacterium avium subspecies paratuberculosis that can survive in the physical environment for a considerable period of time. In the context of nested multiscale models developed at host level, a key feature is that the within-host scale and the between-host scale disease dynamics influence each other in a reciprocal way, with the between-host scale influencing the within-host scale through initial infective inoculum dose which susceptible ruminants may consume from the environment. The numerical results of the nested multiscale model presented in this study demonstrate that once the minimum infectious dose is consumed, then the infection at the within-host scale is sustained more by pathogen replication than by super-infection. From these results we conclude that super-infection might have an insignificant effect on the dynamics of PTB in ruminants. However, at this stage we cannot precisely conclude if super-infection does not effect on the dynamics of the disease. This would be investigated further using an embedded multiscale model, which is more appropriate in giving us conclusive results. We further demonstrate the need to use nested multiscale models over single-scale modeling approach by estimating a key parameter for pathogen replication that cannot be estimated using single-scale models.

Biochemical Diversity, Pathogenicity and Phylogenetic Analysis of Pseudomonas viridiflava from Bean and Weeds in Northern Spain.

Pseudomonas viridiflava was originally reported as a bean pathogen, and subsequently as a wide-host range pathogen affecting numerous plants species. In addition, several authors have reported the epiphytic presence of this bacterium in “non-host plants”, which may act as reservoir of P. viridiflava and source of inoculum for crops. A new biotype of this bacterium, showing an atypical LOPAT profile, was found in Asturias, a Northern region of Spain, causing significant damage in beans, kiwifruit, lettuce, and Hebe. In order to investigate the involvement of weeds in bean disease, samples were collected from beans and weeds growing in the same fields. A total of 48 isolates of P. viridiflava were obtained, 39 from weeds and 9 from beans. 48% and 52% of them showed typical (L− O− P+ A− T+) and atypical (L+ O− P v A− T+) LOPAT profiles, and they displayed high biochemical diversity. Regarding virulence factors, the T-PAI and S-PAI pathogenicity islands were found in 29% and 70.8% of the isolates, 81.2% displayed pectinolytic activity on potato slices, and 59% of the weed isolates produced symptoms after inoculation on bean pods. A phylogenetic tree based on concatenated rpoD, gyrB, and gltA sequences separated the strains carrying S-PAI and T-PAI into different clusters, both containing isolates from beans and weeds, and pathogenic as well as non-pathogenic strains. Closely related strains were found in the two hosts, and more than half of the weed isolates proved to be pathogenic in beans. This is consistent with the role of weeds as a reservoir and source of inoculum for bean infection. Detection of P. viridiflava in weeds throughout the year further supports these roles.

On the relationship between Pathogenic Potential and Infective Inoculum.

Pathogenic Potential (PP) is a mathematical description of an individual microbe, virus, or parasite's ability to cause disease in a host, given the variables of inoculum, signs of disease, mortality, and in some instances, median survival time of the host. We investigated the relationship between pathogenic potential (PP) and infective inoculum (I) using two pathogenic fungi in the wax moth Galleria mellonella with mortality as the relevant outcome. Our analysis for C. neoformans infection revealed negative exponential relationship between PP and I. Plotting the log(I) versus the Fraction of animals with signs or symptoms (Fs) over median host survival time (T) revealed a linear relationship, with a slope that varied between the different fungi studied and a y-intercept corresponding to the inoculum that produced no signs of disease. The I vs Fs/T slope provided a measure of the pathogenicity of each microbial species, which we call the pathogenicity constant or kPath. The kPath provides a new parameter to quantitatively compare the relative virulence and pathogenicity of microbial species for a given host. In addition, we investigated the PP and Fs/T from values found in preexisting literature. Overall, the relationship between Fs/T and PP versus inoculum varied among microbial species and extrapolation to zero signs of disease allowed the calculation of the lowest pathogenic inoculum (LPI) of a microbe. Microbes tended to fall into two groups: those with positive linear relationships between PP and Fs/T vs I, and those that had a negative exponential PP vs I relationship with a positive logarithmic Fs/T vs I relationship. The microbes with linear relationships tended to be bacteria, whereas the exponential-based relationships tended to be fungi or higher order eukaryotes. Differences in the type and sign of the PP vs I and Fs/T vs I relationships for pathogenic microbes suggest fundamental differences in host-microbe interactions leading to disease.

Kinetics of the inflammatory response during experimental Babesia rossi infection of beagle dogs

Babesia rossi causes severe morbidity and mortality in dogs in sub-Saharan Africa, and the complications associated with this disease are likely caused by an unfocused, excessive inflammatory response. During this experimental B. rossi study we investigated inflammatory marker and cytokine kinetics during infection and after treatment. We aimed to determine whether infectious dose and treatment would influence the progression of the inflammatory response and clinical disease. Six healthy male beagle dogs formed the study population, one was used to raise the infectious inoculum, three were administered a high B. rossi infectious dose (HD group) and two a low infectious dose (LD group). Clinical examination, complete blood count (CBC) and C-reactive protein (CRP) were determined daily. Cytokines were quantified on stored plasma collected during the study, using a canine specific cytokine magnetic bead panel (Milliplex©). The experiment was terminated and treatment administered when predetermined experimental or humane endpoints were reached. Parasitemia occurred on day 1 and 3 in the HD and LD groups respectively. The rate of increase in parasitemia in the HD group was significantly faster than that seen in the LD group. Significant differences were found in heart rate, blood pressure, interferon gamma (INFγ), keratinocyte chemoattractant (KC), INFγ-induced protein 10 (IP10), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein 1 (MCP1), tumor necrosis factor alpha (TNFα), interleukin 2 (IL-2), IL-6, IL-7, IL-8, IL-10 IL-15, IL-18, CRP, neutrophils and monocytes between groups at multiple time points during the course of the infection. Our findings suggest that the initiation of inflammation occurs before the onset of clinical disease in B. rossi infection and infectious dose influences the onset of the inflammatory response. Treatment enhances the inflammatory response in the immediate post-treatment period which may contribute to disease associated complications. Finally, we found that there is an imbalance in pro/anti-inflammatory cytokine concentrations during infection which may promote parasite replication.

Influence of blueberry tissue type, wounding and cultivar on susceptibility to infection by Neofusicoccum species.

AimBotryosphaeriaceae causing stem blight and dieback of blueberry are important pathogens limiting economic production worldwide. This study investigated the pathogenicity and relative virulence of isolates from the Neofusicoccum species commonly associated with blueberries in New Zealand on different tissues and cultivars of blueberries.Methods and ResultsBoth wounded and non‐wounded fruit and flower buds and wounded attached soft green and hard green shoots were susceptible to infection by conidia of Neofusicoccum australe, Neofusicoccum parvum and Neofusicoccum ribis. N. ribis was generally most virulent, followed by N. parvum and then N. australe. Inoculation of potting mixture with N. australe or N. ribis conidia showed that potting mixtures were not a source of inoculum for infection of blueberry roots. Wounded and non‐wounded leaf buds, fruit and wounded soft green shoots and hard green shoots of the different cultivars tested were susceptible to infection by N. parvum and N. ribis. Whilst the fruit of all cultivars were similarly infected, infection incidence in inoculated leaf buds was lowest in “Blue Bayou” and “Ocean Blue”. Cultivar susceptibility differed when tested on soft green shoots compared with hard green shoots, with shortest lesions developed on “Maru” on soft green shoots, and “Centra Blue” and “Ocean Blue” on hard green shoots.ConclusionsAll tested above‐ground blueberry tissues, including non‐wounded tissue, were susceptible to Neofusicoccum spp. All the cultivars assessed were susceptible to infection, although they varied in their relative susceptibility depending on the tissue assessed.Significance and impact of the studyThe potential for non‐wounded tissue to become infected indicate that fungicides may need to be applied to protect all tissue, not just wounds.

Uropathogenic Escherichia coli can cause cystitis at extremely low inocula in a pig model.

Urinary tract infection (UTI) is one of the most common bacterial infections worldwide. Experimental models that accurately reflect the high susceptibility to UTI in humans have, however, been lacking. This situation has limited detailed research into the early bladder colonization by uropathogens and the early innate defence mechanisms elicited to prevent this. We recently presented a model of urinary tract infection in pigs, animals that are naturally susceptible to UTI and have greater similarity to the physiology and anatomy of the human urinary tract than traditional rodent UTI models. In the current study, we used the pig model to investigate the minimal infectious inoculum of uropathogenic Escherichia coli, the most common cause of urinary tract infection. We show that in this animal a few individual bacteria that come into contact with the urothelium can give rise to fulminant cystitis, indicating the high infectious potential of uropathogenic E. coli.

Survival of pathogens after dormancy in apple tree twigs indicates potential risk as source of inoculum

Several diseases are difficult to control specially in subtropical regions and perennial hosts where the pathogen survive easily within the plant. The aim of this study was to identify which pathogens are surviving after end of one season and at the beginning of the next season to inform which pathogens represent potential risk as primary inoculum to the next season. Survival of pathogens on apple tree twigs was evaluated during dormancy and on beginning of vegetative growth in four orchards located in the two main apple production regions: Palmas and metropolitan region of Curitiba, in Paraná State, Brazil. For this purpose, 10 cm long asymptomatic twigs were collected from 10 randomly selected plants, 24 twigs per plant. Half of the twigs were left directly (without disinfection) in humid chambers for 30 days at 25ºC. The other half of the twigs were disinfected and kept in a freezer for 12 hours at -16ºC by the Over Night Freezing Incubation Technique (Onfit) and then kept in a humid chamber for 30 days at 25ºC. The fungi Colletotrichum sp., Botryosphaeria sp., Alternaria sp., and Fusarium sp. were detected in the two sampled dates and methodologies in all evaluated regions. Neonectria ditissima was only detected in both orchards from Palmas. The genera found surviving on twigs were confirmed molecularly by BLASTn and were pathogenic in wounded fruits from the cultivar ‘Gala’. Our results indicate that pathogens are surviving in orchards after winter treatment and throughout the apple season, being potential sources of inoculum for infections in flowers and fruits, where the pathogens detected cause important diseases as bitter rot, Neonectria fruit rot, white and black rots, and possibly cause Alternaria and Fusarium rots which have not been extensively studied in Brazil.

Prospective Assessment of Symptoms to Evaluate Asymptomatic SARS-CoV-2 Infections in a Cohort of Health Care Workers.

BackgroundThe frequency of asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is unclear and may be influenced by how symptoms are evaluated. In this study, we sought to determine the frequency of asymptomatic SARS-CoV-2 infections in a prospective cohort of health care workers (HCWs).MethodsA prospective cohort of HCWs, confirmed negative for SARS-CoV-2 exposure upon enrollment, were evaluated for SARS-CoV-2 infection by monthly analysis of SARS-CoV-2 antibodies as well as referral for polymerase chain reaction testing whenever they exhibited symptoms of coronavirus disease 2019 (COVID-19). Participants completed the standardized and validated FLU-PRO Plus symptom questionnaire scoring viral respiratory disease symptom intensity and frequency at least twice monthly during baseline periods of health and each day they had any symptoms that were different from their baseline.ResultsTwo hundred sixty-three participants were enrolled between August 25 and December 31, 2020. Through February 28, 2021, 12 participants were diagnosed with SARS-CoV-2 infection. Symptom analysis demonstrated that all 12 had at least mild symptoms of COVID-19, compared with baseline health, near or at time of infection.ConclusionsThese results suggest that asymptomatic SARS-CoV-2 infection in unvaccinated, immunocompetent adults is less common than previously reported. While infectious inoculum doses and patient factors may have played a role in the clinical manifestations of SARS-CoV-2 infections in this cohort, we suspect that the high rate of symptomatic disease was due primarily to participant attentiveness to symptoms and collection of symptoms in a standardized, prospective fashion. These results have implications for studies that estimate SARS-CoV-2 infection prevalence and for public health measures to control the spread of this virus.

Antibacterial properties of silver nanoparticles synthesized via nanosecond pulsed laser ablation in water

Ag nanoparticles were synthesized in pure water by a nanosecond pulsed laser ablation process and characterized using transmission electron microscopy and ultraviolet-visible spectroscopy. The as-synthesized nanoparticles were determined to have an average particle size of 10.17 ± 2.95 nm. Their absorption spectra were consistent with that of Ag nanoparticles produced through chemical methods. The antibacterial properties of Ag nanoparticles at concentrations of 100 and 250 μg/ml were studied against the gram-positive bacterium Staphylococcus aureus at two infectious inocula (103 and 105 CFU/ml). Over a 24-h test period, the samples containing 100 μg/ml exhibited early bacteriostatic activity but did not stop long-term bacterial growth, while the 250 μg/ml samples demonstrated bactericidal and bacteriostatic effects. These results highlight the use of Ag nanoparticles synthesized through laser ablation as green and effective antibacterial agents for biomedical applications.