Poststroke depression (PSD) is a common complication that can seriously affect patients' functional recovery and quality of life after a stroke. Various side effects have been found to be associated with the pharmacological therapies used for PSD. Studies have shown that Chinese herbal medicine (CHM) can effectively improve PSD-like behavior and neurological function in clinical and animal studies. The efficacy of CHM on PSD in animal models has not been systematically analyzed.The following electronic databases were searched for articles published up to September 2022: PubMed, Web of Science, the Cochrane Library, and Embase. Studies that reported the efficacy of CHM in animals with PSD and were written in English were included. Depression-like behavior and the neurological deficit score were assessed as measures of efficacy. The included studies assessed depression-like behavior using sucrose preference, open-field, forced swimming, and tail suspension tests, as well as body weight. The Review Manager version 5.4 and STATA version 13.1 software packages were used for the meta-analysis. The standardized mean difference (SMD) with 95% confidence intervals was used to assess all the outcomes. Subgroup analyses were performed to explore the sources of heterogeneity. The Egger's test and funnel plots were used to assess the potential publication bias. Sensitivity analyses were used to identify the stability of the results.A total of 14 studies, including 12 CHMs involving 442 rats, fulfilled the inclusion criteria for meta-analysis. The pooled results showed that CHM significantly alleviated neurological deficits  and was efficacious in improving the depression-like behavior of rats in the sucrose preference , open-field , forced swimming , and tail suspension tests .Our results suggest that CHM could significantly improve depression-like behavior and neurological function in animals with PSD. The current results should be interpreted with caution because only animal studies were included. Stroke remains the second-leading cause of death and the third-leading cause of death and disability combined (as expressed by disability-adjusted life-years lost) in the world . PoststrThe pathophysiology of PSD is multifactorial and unclear. It probably involves dysregulation of the hypothalamic-pituitary-adrenal axis, increased inflammatory factors, decreased levels of monoamines, glutamate-mediated excitotoxicity, and abnormal neurotrophic response . The treTraditional Chinese medicine, including Chinese herbal medicine (CHM), acupuncture, moxibustion, and Tuina, has long been used to manage various disorders in China . A serieWe performed this systematic review and meta-analysis in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines . The effStudies published up to 25 September 2022 were retrieved from the Web of Science, PubMed, Cochrane Library, and Embase databases. Studies that reported the efficacy of CHM in animals with PSD were included. The literature search was conducted using the terms (“depression”) AND (“stroke”) AND  AND . Only publications written in English were included in this review. All searches were performed by two independent researchers (TL and GS).The inclusion criteria were as follows: (1) studies that reported the efficacy of CHM in PSD animal models; (2) studies that used CHM, including Chinese herbal compounds, a single Chinese herbal extract (monomer), and compounds of several Chinese herbal extracts; (3) studies that mentioned the number of animals used; and (4) studies that were published in English.The exclusion criteria were as follows: (1) studies that included an agent that was a natural plant extract but not the main ingredient in traditional CHM; (2) studies where the data could not be obtained. Studies were independently screened by two researchers (LL and YG), and any disagreements were resolved through discussion.Two investigators (LL and BH) independently extracted data from the included studies. We extracted data on the outcome measures , publication details (author and year), treatments used (route and dose), and animals (species) from the studies. For each comparison, the sample size, mean value, and standard deviation (SD)/standard error for both the treatment and control groups were extracted. If multiple treatment groups shared the same control group, the sample size of the control group was divided by the number of treatment groups . If outcTwo researchers independently assessed the quality of the included studies using the Systematic Review Center for Laboratory Animal Experimentation Risk of Bias tool. The assessment covered 10 areas, and each item was scored as one point as follows: (1) sequence generation, (2) baseline characteristics, (3) allocation concealment, (4) random housing, (5) blinding , (6) random outcome assessment, (7) blinding , (8) incomplete outcome data, (9) selective outcome reporting, and (10) other sources of bias. Any disagreements arising from the evaluation were resolved through discussion until a consensus was reached.There were no significant differences in the subjects, modeling, and administration methods used in the included studies. While the CHM used differed, it could be grouped into monomers and compounds. We performed a subgroup analysis on the different types (according to name) and forms (compound or monomer) of CHM to evaluate the influence of the variables or research characteristics on the estimated effect size.Q statistic and I2 statistic  were found, 42 duplicate articles were excluded using Endnote X 9, and 44 articles were manually excluded according to the title and abstract. For the remaining 18 articles, four articles that did not meet the inclusion criteria were excluded after reading the full text. Therefore, 14 articles were finally included in our analysis. The specific screening procedure is shown in Altogether, 442 animals  were included in this meta-analysis. Twelve studies used Sprague Dawley rats, and two studies used Wistar rats. The weight of the rats varied between 155 and 320 g. Only four studies mentioned the age of the rats, which ranged from 7 to 24 weeks. There were five kinds of PSD models used in the included studies. Ten studies used middle cerebral artery occlusion combined with chronic unpredictable mild stress (CUMS). One study used unilateral carotid artery ligation combined with separate housing and a small dose of reserpine. One study used unilateral internal carotid artery ligation combined with solitary housing and CUMS. One study used carotid artery embolization combined with chronic sleep deprivation. One study used bilateral common carotid artery ligation combined with forced swimming.Regarding the intervention methods, there were 12 types of CHM, including eight compounds and four monomers. The monomers included Morinda officinalis oligosaccharides (MOOs), peoniflorin, Saikosaponin A (SSA), and Huperzine A (HupA). The intervention durations ranged from 14 to 56 days. Intraperitoneal administration was performed in 2 of the 14 studies, and gastrointestinal administration was performed in the other 12 studies. Among the 14 included studies, 13, 12, 7, and 3 performed the sucrose preference, open-field, forced swimming test, and tail suspension tests, respectively, and seven and four measured body weight and neurological deficit scores, respectively. Among the 12 studies that conducted an open-field test, the effect sizes of three studies were inconsistent with the other nine studies. Therefore, we chose to analyze only nine of them. The detailed study characteristics are listed in Among the 14 included studies, no study described the method used to generate the allocation sequence. All studies reported similar baseline characteristics between the groups. No study clarified whether the allocation of different groups was sufficiently blinded. The breeding conditions and environments of all experimental animals included in the studies were the same. Therefore, we considered that the animal placements complied with the principle of randomization. No study reported sufficient information regarding the blinding methods used by the caregivers or investigators. A total of 11 studies assessed the randomization of the outcomes. Seven studies described the blinding methods used for the outcome evaluation. All studies reported complete outcome data and preliminary reported results and had no other sources of bias. A complete quality assessment of the included studies is shown in P < 0.001; 23 comparisons), with substantial heterogeneity between studies . Subgroup analysis showed a significant correlation (P < 0.001). The effects of MOOs and SSA were similar to that of the control group . The effects of the other CHM on the sucrose preference test were more obvious than that of the control group (P < 0.05). The forest map showed that peoniflorin played a more prominent role.The sucrose preference test was used in 13 studies , which are included in the meta-analysis . The pooP < 0.001; 20 comparisons), with substantial heterogeneity between studies . Subgroup analysis did not show any effect of the type of CHM on the results (P = 0.1), and each drug improved the movement in the open-field test (P < 0.05).The open-field test was used in nine studies , which are included in the meta-analysis . The pooP < 0.00001; 11 comparisons), with moderate heterogeneity between studies . No statistical significance was found when the CHM type was analyzed as a subgroup (P = 0.07), and each drug reduced the immobility time of the forced swimming test (P < 0.05). The forest map showed that SSA played a more prominent role.As for the forced swimming test, seven studies  were included in the meta-analysis . The pooP < 0.001; 4 comparisons), with no heterogeneity between studies . Subgroup analysis did not show any effect of the type of CHM on the results (P = 0.47), and each drug reduced the immobility time in the tail suspension test (P < 0.05).The tail suspension test was used in three studies , which are included in the meta-analysis . The pooP < 0.001; 11 comparisons), with substantial heterogeneity between studies . Subgroup analysis showed a significant correlation between the type of CHM and effect size (P < 0.001). The effect of MOOs on body weight was similar to the control group (P = 0.1), but the remaining CHM were all associated with significantly increased body weight (P < 0.05). The forest map showed that the effect of SSA and peoniflorin remained more obvious.Body weight was assessed in seven studies , which are included in the meta-analysis . The pooP < 0.001; 6 comparisons), with substantial heterogeneity between studies . Subgroup analysis was performed according to the type of CHM and showed a significant correlation between the type of CHM and effect size (P = 0.002). The forest map showed that the effect of SSA and peoniflorin seemed to be more obvious, although each drug improved the neurological deficit score (P < 0.05).The neurological deficit score was assessed in four studies , which are included in the meta-analysis . The pooP < 0.001), with moderate heterogeneity . The effect of monomer CHM was similar to that of the control (P = 0.06), with considerable heterogeneity . However, there were no statistical differences between subgroups . For the open-field test, compound and monomer CHM were both associated with increased activity compared to the control group, but monomer CHM was better than compound CHM; this result was statistically significant . For the forced swimming test, tail suspension test, body weight, and neurological deficit score, the effects of both compound and monomer CHM were better than those of the control group. Still, there was no significant correlation between the form of CHM and effect size  . SubgrouP = 0.015, P = 0.014, and P < 0.001, respectively; P = 0.962; The Egger's test and funnel plots were performed to assess the potential publication bias. Funnel plots of all the outcomes are shown in A sensitivity analysis was performed to assess whether any individual study significantly affected the overall meta-analysis results by removing each study one by one . All effPoststroke depression, the most common psychiatric disorder following a stroke, seriously affects stroke survivors' quality of life and poststroke recovery and also increases their mortality risk . The patThe subgroup analysis according to the type of CHM showed that neurological deficit score, body weight, and sucrose preference test were statistically different among subgroups. Further analysis showed that each drug was effective in terms of the neurological deficit score. The difference among subgroups may be due to the more obvious effect of peoniflorin and SSA. For body weight, the difference among subgroups may have been due to the more obvious effect of peoniflorin, SSA, and the ineffectiveness of MOOs. For the sucrose preference test, the difference among subgroups may be due to the more obvious effect of peoniflorin and the ineffectiveness of MOOs and SSA. The form of CHM was also analyzed as a subgroup, which showed that monomer CHM was better than compound CHM in terms of the open-field test and neurological deficit score. The former was statistically significant, which could be related to the prominent effect of peoniflorin and SSA. We also found that monomers were not effective in terms of the sucrose preference test due to the negative results of MOOs.P-value was considered the critical value. Considering the measurement data error and the significant improvement of SSA on the other five indicators, especially the neurological deficit score and body weight, SSA can still be considered a potential drug for treating PSD. This study found that MOOs had no significant improvement in body weight and the sucrose preference test results with no significant heterogeneity. Still, MOOs significantly improved the other five indicators, implying that they may improve depressive symptoms other than anhedonia. We also found that HupA improved the neurological deficit score and sucrose consumption and decreased the immobility time in the forced swimming test. However, due to the small sample size and the small number of studies on monomers, this result should be considered with caution. More studies are needed to confirm this finding. For compound preparations, both the overall and subgroup analysis by drug type or form showed that the results of compound CHM were relatively stable and effective. This could be because there are more components in compounds and, thus, more corresponding targets. Our analysis presents new ideas for the clinical treatment of PSD.The subgroup analysis showed that monomers were more effective in improving neurological function and some depressive symptoms. Peoniflorin was more effective in improving all six indicators, especially the neurological deficit score, body weight, and sucrose preference test results, which were consistent with the results of the previous systematic analysis . In the There was heterogeneity in the results of some indicators. Unfortunately, due to the relatively small sample size and the lack of information provided in some studies, we did not conduct more subgroup analyses and did not identify the source of heterogeneity. Indeed, Egger's test results and funnel plots suggest that publication bias exists in the current study. The sensitivity analysis suggested that the results were stable and reliable, but our findings should be interpreted with caution due to the small number of studies included.This study has several limitations. First, we only included studies published in English, which may have led to publication bias. Second, only a limited number of studies were included, and the total sample size was small. Third, additional subgroup analyses could not be performed because of limited data. Fourth, we failed to get all the original data, and some data were measured using ImageJ. Therefore, some errors may have been introduced that could affect the accuracy of the results. Finally, the current studies only investigated the efficacy of CHM on PSD, and the safety of CHM has not been analyzed. In the future, more studies are needed to confirm the safety and efficacy of CHM in the treatment of PSD.Our results showed that CHM could significantly improve the depression-like behaviors and neurological function of animals with PSD. CHM could be potentially used in the treatment of PSD. However, this study is a meta-analysis based on animal studies. More high-quality preclinical trials and clinical studies are needed to verify our results before CHM can be used in clinical settings.The original contributions presented in the study are included in the article/LL and BH: conception and design, analysis and interpretation of data, drafting the manuscript, and statistical analysis. YuW, TL, and GS: searching the literature. LL and YG: screening of titles and abstracts and full-text data extraction. YaW, YZ, and TZ: critically revising the manuscript. All authors have read and agreed to the published version of the manuscript.


The COVID-19 pandemic has forced mental health professionals to adapt quickly. The pandemic has created multiple new tasks for the psychologist. In addition to the various stressors closely linked to the COVID-19 pandemic, psychologists were forced to make their services more flexible. Teleworking was a way of continuing to work.This study aimed to identify the impact of working pattern on the levels of burnout, depression, anxiety, and stress.This was a cross-sectional study based on an online questionnaire applied to eighty-three Portuguese psychologists. Data were collected from May 9 to June 8, 2020, a period comprising the declaration of a national calamity and then state of emergency, and the subsequent ease of lockdown measures. The Copenhagen Burnout Inventory Scale and Depression Anxiety and Stress Scale were used. Univariate multiple linear regression models were estimated for each mental health outcome.Significant differences were found between psychologists working in the workplace and in teleworking at the personal burnout, work-related burnout, client-related burnout, depression, and stress. In multiple linear regression, teleworking, not working, and being unmarried was significantly associated with higher levels of depression. Teleworking was significantly associated with higher stress scores and client-related and work burnout.This exceptional time of sudden, mandatory, and high-intensity teleworking, required rapid adaptation, giving rise to new stressors that might have been responsible for burnout levels in psychologists. The World Health Organization  declaredThis global outbreak created a significant set of challenges, notedly for the psychologists and other mental health professionals, as it affected mental health and aggravated previous psychopathologies . In factTeleworking or telecommuting “involves working at a remote location away from a central office”  (p. 44).The amount of research on the topic of telework during the COVID-19 pandemic is limited. However, pre-pandemic studies , ps, ps15)],Dealing with this set of demands can cause burnout in psychologists. Burnout is “a state of physical, emotional and mental exhaustion that results from long-term involvement in work situations that are emotionally demanding”  (p. 501)Despite this hypothesis, burnout research has focused on traditional workers, but those studies have been slow to extend their interest to teleworkers.In addition, under normal circumstances, teleworking in Portugal is not very common, with only 6.5% of workers working from home . Faced wThis study consisted of psychologists, who were Portuguese speakers, and were working or unemployed in Portugal at the time COVID-19 pandemic started. No other eligibility criteria existed.*Power version 3.1.9.7  participated in the present study. The power associated with the sample size obtained was computed using G 3.1.9.7 . For an n = 9) had their activity suspended, 2.4%; (n = 2) were in maternity leave or childcare; and 1.2% (n = 1) were unemployed. The demographics and professional characteristics of the 83 participating psychologists are summarized in Nearly all were women (84.3%), and 49.4% were married. Of the forty (48.2%) participants who had children, 72.5% had children aged 12 years old or less. A total of 16 (19.3%) participants were caregivers of older people or people with disabilities. Psychologists who were not working accounted for 14.5% of the sample, 10.8% (n = 78).Almost the entire sample indicated not working directly on diagnosis and/or treatment of persons infected with COVID-19 . To analyse psychological variables, the Copenhagen Burnout Inventory and the Depression Anxiety and Stress Scale were used.Copenhagen Burnout Inventory (CBI) —This is The Depression Anxiety and Stress Scale (DASS-21)  was usedvia social networks using a snowball technique, and supported by health care institutions and professional organizations. Data collection was performed on a Google Forms platform available between May 9 and June 8, 2020, a period included in the national calamity declaration and easing of lockdown measures that followed a state of national emergency (between March 18 and May 2). It was conducted in line with the Declaration of Helsinki and received approval from the Faculty of Medicine of University of Porto's Ethics Committee . All participants gave their online informed consent at the beginning of the survey; when accessing the link participants were presented with an introduction with the study purposes, duration of the survey, and guarantees of anonymity and confidentiality. If they agreed with the study procedures, they were asked to click on a confirmation button to proceed to the survey.This was a cross-sectional quantitative web-based study applied to Portuguese psychologists, spread ®. Categorical variables were described by absolute and relative frequencies, n (%). Normally distributed quantitative variables were described by the mean and the respective standard deviation, M ± SD. Quantitative variables not normally distributed were described by the median and the interquartile interval, Med . The normality of quantitative variables was verified by observation of histograms. The comparison of quantitative variables between two groups was made by the Student t-test, if the variables were normally distributed or, otherwise, through the Mann-Whitney test. The comparison of quantitative variables between more than two groups was made by the One-way ANOVA test, if the variables were normally distributed or, otherwise, through the Kruskal-Wallis test; in both cases, if there were differences, multiple comparisons were performed using Bonferroni adjustments. For the psychological variables (outcomes) where there were significant differences between working patterns , a separated multiple linear regression was performed. To decide which independent variables to include in each multiple regression, simple linear regressions were performed with each of the following variables: years of professional experience , marital status , and children . All variables that correlated with the outcomes at p ≤ 0.05 in a simple regression were included in the multiple linear regressions. Only the significant variables were maintained in the final multiple models. The results of linear regressions were presented by the estimated coefficients (β), the respective 95% confidence interval (95% CI), and p-value. To evaluate the model, the F statistic of the overall model test, the respective p-value, and the value of the determination coefficient (R2) were presented. Assumptions of linear regressions were verified by visual observation of histograms, for normality of residuals and variance homogeneity of residuals were verified by visual inspection of scatter plots. Values of p ≤ 0.05 were considered significant.Data analysis was performed using SPSS v.26 (IBM SPSS Inc.)The average levels of burnout in psychologists were grouped according to high and low burnout. Among participants, 31 (37.3%) had high work-related burnout, 28 (33.7%) had high personal burnout, and 14 (16.9%) high client-related burnout.For depression, anxiety and stress, participants obtained a median Q1; Q3) score of 1 , 1 , and 5  score of, respectIt should be noted that, based on these results, the symptom of stress seems to be the most experienced by this sample, although it departs significantly from values considered worrying from the point of view of mental health.p = 0.022; see Regarding personal burnout and once this dimension has the objective of assessing the fatigue or exhaustion of individuals, regardless of their occupational status , a comparison was made between the three groups . Using the Kruskal Wallis test, results indicated significant differences between the three-working patterns (Work-related burnout and client-related burnout were compared only between the groups that were working. Psychologists in telework reported significantly higher levels of work-related burnout (Med = 42.9) and client-related burnout (Med = 29.2) than psychologists who were working in the workplace .p = 0.025 and p = 0.015, respectively). Again, psychologists in telework showed higher levels of depression (Med = 2) and stress (Med = 6) when compared to psychologists who were not working during the COVID-19 outbreak  and to those who were working in the workplace , depression , and stress , compared to psychologists who developed their activity in the workplace.Multiple comparisons were made for the scales in which there were differences between the three groups. ress see . The resn = 12). All variables that correlated with the outcomes at p ≤ 0.05 in a simple regression were included in the multiple linear regression , marital status , and children . Notice that for work-related burnout and client-related burnout, the psychologists who have not been working were excluded  in the average level of personal burnout in comparison with participants who worked in the workplace. Having children under 12 years was associated with higher personal burnout levels in comparison with not having or having children over 12 years old . In this model, the independent variables explained 14.1% of the variability of the personal burnout in the sample.Teleworking and having children under 12 years old were significantly associated with higher levels of personal burnout. Participants in teleworking had an 11-point rise . This variable explained 10.7% of the variability in the work-related burnout.Having children under 12 years was associated with increased levels of work-related burnout . This variable explained 17.4% of the variability in the client-related burnout in the sample.Teleworking was significantly associated with higher client-related burnout . Also, for psychologists, not working was associated with higher levels of depression . Single status showed significantly more depression when compared to marriage status/non-marital partnerships . In this model, the independent variables explained 15.5% of the variability of depression in the participants.Teleworking was associated with an increase in depression average levels . This variable explained 9.4% of the variability in stress.Teleworking was identified as significantly associated with higher stress levels  following COVID-19 restrictions on the levels of burnout, depression, anxiety, and stress in a sample of psychologists.According to Kristensen et al.  (p. 197)Regarding the remaining dimensions of burnout, the results indicate that 33.7% had high personal burnout, and 16.9% had high client-related burnout. These finds are in line with other studies e.g., , 26], wh, wh26)],Although the percentage of psychologists who showed burnout associated with the client was low 16.9%), teleworkers exhibited much higher levels of burnout compared to psychologists who were working in the workplace. The pattern of client-related burnout could be symptomatic of compassion fatigue (defined as secondary traumatic stress disorder) and compromising the psychotherapist's ability to experience the painful emotions of the client . Several.9%, teleThese results are worrying, whereas higher levels of burnout compromise the capacity of professionals to take care of themselves and their clients. Besides, burnout can also mitigate the ability to provide empathy, support, and guidance in their therapeutic work, thus compromising the client's progress and wellbeing .Being at telework and having children under 12 years old were associated with higher levels of personal burnout. If we consider these variables and the fact that most of our sample are women, we can hypothesize that achieving a balance between family and work is challenging. Recently, in the context of pandemic restrictions, the results of the survey conducted by Lean In and Survey Monkey  indicatePsychologists who had an infant or children under 12-year-old were more likely to experience personal burnout and work-related burnout. This is not surprising as, during the COVID-19 crisis, telework often must be combined with taking care of the children (due to the closure of schools and day-care facilities).Portuguese psychologists in teleworking and with suspended activity showed higher levels of depression. Under the assumption that teleworking, by definition, reduces physical and personal interaction , negativBeing not married was associated with higher levels of depression in comparison with married psychologists. It was not surprising that we did not find differences in burnout between married and unmarried people, since being married provides a support network that can be helpful in buffering the debilitating impact of occupational stress/ burnout .The identification of who might be more affected by COVID-19, not epidemiologically but simply by working patterns, has important implications for mental health professional practice and supervision. Our research insights can contribute to better organizational management of the challenges that psychologists face in teleworking, and consequently, they can help create preconditions for positive adaptation to the telecommuting work environment.It is essential, in the first instance, to recognize that teleworking involves not only merging the work and personal spaces, being far from the usual social and work dynamics, but also having to simultaneously manage unusual family dynamics .In the second instance, we must be aware that working remotely does not mean working alone. On the contrary, valuing teamwork becomes even more important in circumstances like this. That said, it is essential to share experiences, difficulties, and challenges between peers, establishing a connection and creating a feeling of mutual support. By strengthening this contact between peers, we will also be moving toward reducing social and professional isolation , which, Another crucial aspect that we want to highlight here is the importance of self-care. The term self-care refers to the “engagement in behaviors that maintain and promote physical and emotional wellbeing  (p. 56).This study has several limitations. First, it has a cross-sectional design, which does not allow for causal conclusions. Second, this was a cross-sectional online survey, which might have limited the accessibility of people less familiar or less prone to use the internet. Third, psychologists with high levels of burnout might have been less likely to participate in this study. Fourth, this study was unable to distinguish pre-existing mental health symptoms from new symptoms. Another limitation refers to the fact that the characteristics in which the telework was developed were not investigated, i.e., number of working hours per day in teleworking compared to usual; equipment used at home and working conditions, etc. We assumed, according to the Portuguese Labor Code, that the same rules were kept, i.e., up to 40 h per week. In this sense, in future studies variables associated with telework characteristics should be investigated.In summary, the present study, conducted during the first wave of the COVID-19 pandemic , found that 37% had high work-related burnout, 34% had high personal burnout, and 17% high client-related burnout. This exceptional time of sudden, mandatory, and high-intensity teleworking, required rapid adaptation, giving rise to new stressors that might have been responsible for burnout levels in psychologists. Emotion management strategies and self-care should be recommended. Mindfulness based interventions can be useful in the management of stress and anxiety.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Faculty of Medicine of University of Porto's Ethics Committee . The patients/participants provided their written informed consent to participate in this study.ID and CS contributed to conception and design of the study, project administration—supervision, and coordination. AT and LC organized the database and performed the statistical analysis. CS and AR wrote the first draft of the manuscript, manuscript preparation, manuscript revision, reviewing, editing, and manuscript final version approval. All authors were involved in the data collection, contributed to manuscript revision, read, and approved the submitted version.This work was supported by national funds through the FCT-Fundação para a Ciência e a Tecnologia, I.P., within CINTESIS, R&D Unit (reference UIDP/4255/2020) and FCT, under Grant . AR is supported by FCT through the Ph.D. grants: SFRH/BD/145253/2019.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cordia dodecandra is a deciduous tree used by Maya people for its fruit and wood, growing, to date, in remnant forest fragments and homegardens  in Yucatán. In this work, we evaluated the microbiota’s alpha and beta diversity per compartment (phyllosphere and rhizosphere) and per population (forest and homegarden) in the Northeast and Southwest Yucatán regions. Eight composite DNA samples (per compartment/population/region combination) were amplified for 16S-RNA (bacteria) and ITS1-2 (fungi) and sequenced by Illumina MiSeq. Bioinformatic analyses were performed with QIIME and phyloseq. For bacteria and fungi, from 107,947 and 128,786 assembled sequences, 618 and 1092 operating taxonomic units (OTUs) were assigned, respectively. The alpha diversity of bacteria and fungi was highly variable among samples and was similar among compartments and populations. A significant species turnover among populations and regions was observed in the rhizosphere. The core microbiota from the phyllosphere was similar among populations and regions. Forests and homegarden populations are reservoirs of the C. dodecandra phyllosphere core microbiome and significant rhizosphere biodiversity.During domestication, the selection of cultivated plants often reduces microbiota diversity compared with their wild ancestors. Microbiota in compartments such as the phyllosphere or rhizosphere can promote fruit tree health, growth, and development.  Since the early colonization of terrestrial ecosystems, plants have maintained a close relationship with diverse microbial communities, mainly bacteria and fungi, which comprise the microbiota. These communities can be found in different plant compartments, such as roots (rhizosphere), stems (caulosphere), leaves (phyllosphere), flowers (anthosphere), fruits (carposphere), or seeds (spermosphere) ,2. MicroCitrus genus [The composition and structure of the plant microbiota can present variations related, for example, to the genotype of the host and the geographic region where it develops ,5. Durinus genus .The rhizosphere is considered a complex habitat where the interactions between the edaphic microbiota and the host plant allow the development of a hyperdiverse microbiome ,8. PlantStudy of the microbiota structure has accelerated in the last decade through metagenomic studies using next-generation sequencing techniques and functional DNA analysis ,15. MetaCordia dodecandra A. DC. (Cordiaceae) is a native tree species from medium-sized forests of Mesoamerica and is a structural species of the Mayan homegarden, so it is frequently found in these agroforestry systems. However, forest populations in the area have decreased because of deforestation and fragmentation [C. dodecandra in the Northeast and Southwest regions of the Yucatán Peninsula. Results indicate that the trees have larger leaves and flowers in homegardens, characteristics which are associated with particular genotypic groups, and that there is a high genetic flow between both regions [entation . Fruits entation ,24. Prev regions . Homegar regions . The pop regions . Trees g regions . CollectC. dodecandra in wild and homegarden populations from the Northeast and Southwest regions of Yucatán. The amplicon MiSeq sequencing of 16S and ITS1-2 regions and the bioinformatic analysis from the sequences were used to evaluate whether the microbiota of bacteria and fungi has reduced structure and diversity in homegarden populations, because of the differential management that trees receive in these agroforestry systems, as well as to test whether microbial communities in the rhizosphere are more diverse than those in the phyllosphere. In this work we present the characterization of the microbiota of the rhizosphere and phyllosphere of In respect of bacteria, 281,587 representative sequences  were obtained, which, after exclusion of chloroplasts and mitochondria, totaled 107,946 . In respect of fungi, 128,786 representative sequences  were obtained. Due to variability in the number of representative sequences among samples, bacteria samples were normalized to 1000 and fungi to 3900 sequences, which are sufficient in order to infer differences in the microbiota diversity given that the rarefaction curves flatten out at a sequencing depth of 1000 for observed operating taxonomic units (OTUs)  and afteC. dodecandra microbiota accounts for a total of 618 and 1096 OTUs at the species level in the bacterial and fungal assignations, respectively. Taxonomic assignment was high (more than 80%) for taxa levels above family, less than 70% at genus and species level for bacteria, and less than 75% for all levels in fungi (The in fungi . The majoritarian classes (those with >2.5% relative abundance per sample) were the Actinobacteria, Rubrobacteria, and Thermoleophilia, of the phylum Actinobacteriota; Aphaproteobacteria and Gammaproteobacteria of the phylum Proteobacteriota; Vicinamibacteria and Blastocatellia of the phylum Acidobacteriota; and Bacilli of the phylum Firmicutes . The minAureimonas jathrophae, Methylobacterium hispanicum, Methylobacterium komagatae, and five unclassified OTUs from Aureimonas, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Sphingomonas, Sphingomonaceae, Rhizobiaceae, and Rhizobiales  and fungal microbiota . However, no differences in those values were found for forest and homegarden populations in the bacterial microbiota  or fungal microbiota . The bacterial microbiota was grouped separately in quadrants (Cartesian notation): quadrant I, for the rhizosphere of the forest and the SW homegarden populations; quadrant III, for the phyllosphere of all populations; and quadrant IV, for the rhizosphere of the forest and the NE homegarden populations  [Theobroma cacao growing in diversified homegardens in Africa [Citrus growing in organic orchards in Brazil [In this work, to our knowledge, we report for the first time the microbiota of t trees) , as wellt trees) ,29. The t trees) . Comparan Africa  and of Cn Brazil . In thesC. dodecandra since the relative abundance of the prevalent taxa among samples grouped by population was highly variable for the alpha-diversity indices. A similar pattern was found among the studied populations of T. cacao in Africa [In general, the microbiota of cultivated plants in agricultural systems shows a decrease in biodiversity compared to wild populations, associated with introducing a few host genotypes, clonal propagation, monoculture, and no-rotation practices ,17,32. Tn Africa . Mayan hn Africa . Therefon Africa . Our resAgave [As expected, phyllosphere and rhizosphere microbiota presented differences in relative abundance and differential abundance patterns, with only a few shared OTUs . It has been proposed that the rhizosphere results from the selective recruitment of the edaphic microbiota and that the secretion of metabolites by the roots facilitates chemical communication between the microbial and host plant communities, leading to the consolidation of a symbiotic relationship between them ,10,33. IAgave , maize [C. dodecandra in the first compartment. In the phyllosphere, endophytes constitute a core microbiome that promotes atmospheric nutrient capture, foliar health, and conversion of growth by-products in wild populations [Citrus fruit crops [Agave [C. dodecandra rhizosphere microbiota. To understand factors contributing to rhizosphere microbiota variation, further studies are required to analyze a more robust spectrum of samples from different populations and different edaphic and environmental conditions.The phyllosphere’s microbiota had a lower species turnover than the rhizosphere’s microbiota, which may be associated with the presence of a core microbiome for ulations ,37 and iit crops , as do tit crops ,39. The s [Agave . The spes [Agave . AlthougC. dodecandra were the genera Methylobacterium, Aureimonas, and Actinomycetospora. Methylobacterium fixes atmospheric nitrogen and uses methanol (CH₃OH) or methane (CH₄) of plant origin, facultatively, as a source of carbon and energy. The bacteria that inhabit the phyllosphere favor colonization and can promote the growth and development of plants [Actinomycetospora and Aureimonas genera are involved in carbon and nitrogen cycling [Allorhizobium–Neorhizobium–Parhizobium–Parhizobium–Rhizobium, and Sphingomonas, which are also considered diazotrophic and key organisms in plant growth [Bacillus arbutinivorans, which can solubilize phosphate and produce indole acetic acid in vitro, and when it is in a consortium with other Bacillus and Streptomyces it can increase the drought tolerance in poplar [Microlunatus and the Propionibacteriales order have dissimilatory nitrate reduction [C. dodecandra are related to beneficial bacterial species that confer a higher fitness to the host plant.As for annual and perennial horticultural species, fruit tree species maintain interactions with the rhizosphere and phyllosphere microbiome that impact their growth, development, and health ,2,9,40. f plants ,41,42. T cycling ,44. Othet growth . The bacn poplar . Some speduction  and may eduction . Most ofC. dodecandra. These phyla have been previously reported as components of the fungal communities in the phyllosphere from tropical forest and agroforestry systems, with the phylum Ascomycota being dominant [C. dodecandra phyllosphere microbiota. The majoritarian OTUs in the phyllosphere were the genera Strelitziana and Neostrelitziana, which cause leaf spots. The presence of these species in visually healthy trees suggests that they are latent saprotrophs, spreading once the tissue is dead, or that other species of bacteria or fungi associated with C. dodecandra may be antagonists to those pathogens. Further analysis of the functional networks of the microbiome of C. dodecandra may confirm that some bacteria may inhibit the growth of pathogens, as has been suggested [Citrus [Agave [C. dodecandra inhabits. The most abundant genera found in this study were lignin and cellulose degraders, Fomitopsis, Trechispora [Peniophora [Lepiota [C. dodecandra rhizosphere. Several species of the genus Aspergillus have synergistic effects with mycorrhizae, which help promote plant growth [The phyla Ascomycota and Basidiomycota were the more abundant for the fungal microbiota of dominant ,34. Thisuggested ,50,51. Iuggested , and in  [Citrus  and Agavs [Agave , two tros [Agave . It has s [Agave . Therefochispora , Peniophniophora , and Lep[Lepiota . Togethet growth , even int growth . A greatCordia dodecandra is a native fruit tree that contributes to the food sovereignty of the Maya people when it is traditionally managed with other multipurpose species in the Mayan homegarden [C. dodecandra. However, further work is needed to understand whether the different microbiota communities recorded in this study are taxonomically diverse but functionally similar, and whether micro- and macro-environmental factors contribute to the large variability that this species has maintained during its domestication process. megarden ,22. The C. dodecandra populations analyzed in the Northeast region are in the Tizimin municipality, and those in the Southwest region in the municipality of Tzucacab, both in Yucatán, Mexico , and very warm and warm semi-dry (30.93%), with a mean annual temperature range of 24 to 26 °C and average precipitation of 600 to 1500 mm  . The prepurpurea . Cordia dodecandra in the homegarden are Brosimum alicastrum, Manilkara zapota, Swietenia macrophylla, and Cedrela odorata [In the Southwest region, the climate is warm sub-humid with summer rains, of lower humidity (97.54%), and warm sub-humid with summer rains, of average humidity (2.46%). The average temperature oscillates between 24 and 28 °C, and the average precipitation is 1000 to 1200 mm  . The pre odorata .Mature leaves were randomly collected from the canopies of trees. Samples in forest populations were obtained from 10 individuals in the Northeast region and 11 in the Southwest region. In the homegardens, samples from eight individuals in each region were obtained. The collected leaves were superficially washed with 70% ethanol, stored in sterile plastic bags, and transported on ice in a cooler to the facilities of El Colegio de la Frontera Sur Campeche unit’s facility, where they were stored at −80 °C for the subsequent extraction of metagenomic DNA.The rhizosphere soil was obtained from the fine roots of three individuals from each population type and region. These samples were not taken from the edges of extensive paths in the forest populations or heavily trafficked areas in the homegarden. Rhizospheres were extracted by vigorously shaking the fine roots until they had no more loose soil, and then obtaining the attached rhizosphere soil by gentle brushing. They were transported on ice in a cooler to the facilities of the Centro de Investigación Científica de Yucatán unit’s facility, where they were stored at −80 °C for the subsequent extraction of metagenomic DNA.The leaf surface was cleaned with 70% ethanol, and the tissue was macerated in liquid nitrogen. DNA extraction was performed with the ZymoBIOMICS™ DNA Miniprep kit  following the protocol proposed by the manufacturer. Leaf DNA was concentrated by precipitation with 10 M ammonium acetate and resuspended in DNA-free pure water. DNA concentration was quantified using a Thermo Scientific Multiskan GO model FI-01620 spectrometer  with μDrop plate and SkanIt version 4.1 software . Based on the concentration of each sample, aliquots were taken to make a composite mixture by population type and region . The rhizosphere soil of the three sampled trees was combined into a composite sample for each population type per region. DNA was extracted with the ZymoBIOMICS™ DNA Miniprep kit  following the manufacturer’s protocol. Following elution, DNA samples were concentrated by ethanol precipitation and resuspended in 100 mL of free DNAse water.The phyllosphere and rhizosphere composite samples were sent to RTL Genomics  for sequencing on the MiSeq Illumina platform  following the manufacturer’s protocol. For the bacterial microbiome, the universal primers 27F (5′-AGAGAGTTTGATCCTGGCTCAG-3′) and 338R (5′-GCTGCCTCCCGTAGGAGT-3′) were used for the 16S rRNA regions ,61 and fp value (Padj) with a PermANOVA. A bias-corrected microbiome composition analysis ANCOM-BC [The sequence processing was carried out using QIIME2 version 2022.2  to obtaiANCOM-BC  using thANCOM-BC  and the ANCOM-BC .C. dodecandra, is a key element for preserving the resilience of the forest and homegarden populations where they grow. Indirect effects of the management of the homegardens and the actual distribution of the species may contribute to the high species turnover in the rhizosphere microbiota, while genetic similarity and the beneficial roles of endophytes may explain the similarity among the phyllosphere microbiota. Future research should be focused on the key factors contributing to the high diversity of C. dodecandra microbiota and its bioprospection.The characterization of the microbiota associated with wild ancestors and traditional agroforestry systems represents a valuable source for further studies on functional microbiome diversity to understand domestication and attain sustainable agricultural systems ,45,75. T


Listeria monocytogenes, is characterized by the innate ability to survive in the environment and in food, even in adverse conditions and for long periods. Ready-to-eat food represents the category most at risk for contracting listeriosis. This study presents the result of an investigation carried out on a case of maternal-fetal transmission of listeriosis which occurred in 2020 in central Italy and which was linked, with a retrospective approach, to other cases residing in the same city of the pregnant woman. Thanks to the use of next-generation sequencing methodologies, it was possible to identify an outbreak of infection, linked to the consumption of ready-to-eat sliced products sold in a supermarket in the investigated city.Listeriosis is currently the fifth most common foodborne disease in Europe. Most cases are sporadic; however, outbreaks have also been reported. Compared to other foodborne infections, listeriosis has a modest incidence but can cause life-threatening complications, especially in elderly or immunocompromised people and pregnant women. In the latter case, the pathology can be the cause of premature birth or spontaneous abortion, especially if the fetus is affected during the first months of gestation. The causative agent of listeriosis,  Listeria monocytogenes is a facultative intracellular bacterium responsible for the food-borne disease listeriosis [The Gram-positive teriosis ,2,3, an L. monocytogenes occurs in stool of asymptomatic carriers probably due to a less diverse microbiota compared to non-carriers [Listeriosis has a wide spectrum of infections, from severe invasive listeriosis, non-invasive febrile gastroenteritis  to asympcarriers . The miccarriers . Invasivcarriers ,8.The incidence of infection ranged from 0.80 cases per 100,000 in North America in 1990 to 0.42 cases per 100,000 in Europe in 2020, mostly presenting as a sporadic disease, even if also food-borne outbreaks have been described . AlthougListeria infections occur in pregnant women [L. monocytogenes [L. monocytogenes shows both uterus and placenta tropism [Nosocomial outbreaks of listeriosis have been also reported, especially among newborns and immunocompromised patients ,13,14,15nt women ,17,18 annt women . During nt women ,21, makiytogenes ,23. The  tropism ,24.Sporadic cases of perinatal listeriosis, as well as epidemic cases, have been reported with a prevalence varying between 8.6 and 17.4/100,000 of live births, with a fatality rate as high as 20% ,26,27. TL. monocytogenes Operational Contact Point, has been assigned to the Istituto Superiore di Sanità .In Italy, listeriosis is a disease subject to mandatory notification, as required by the Ministerial Decree of 15 December 1990, which places it among the relevant diseases with high frequency or subject to control interventions. Starting from 2010, listeriosis has been part of the Enter-Net Italia surveillance network . SubsequIn Italy, listeriosis incidence rates significantly increased from 2014, reaching a value of 0.29 per 100,000 population in 2018 but still remaining lower when compared to the EU mean incidence rate of 0.47 per 100,000 in the same year . An ItalL. monocytogenes, which can be between two and ten weeks [Epidemiological links and sources of contamination are rarely demonstrated, probably due to the high frequency of asymptomatic clinical presentation and to long-term incubation of en weeks ,43,44.L. monocytogenes isolates. With a retrospective approach, the WGS analysis allowed us to identify a small outbreak that occurred in the same city as the pregnancy-related listeriosis.Herein is presented a case of a pregnancy-related invasive listeriosis occurring in central Italy at the beginning of 2020. Following the epidemiological investigation, a probable source of contamination was found in a local supermarket attended by the patient. A strong epidemiological link was demonstrated, partially supported by whole genome sequencing analysis of human, food and environmental 3 with 28,080/mm3 neutrophils), along with an increase in C-reactive protein (CRP) (90.1 mg/L). Blood cultures, serum Listeria IgM, screening for TORCH infections and group B Streptococcus analysis tested negative whereas no cerebrospinal fluid (CSF) examination was performed at that time. Ampicillin 2 gm intravenous (IV) q6h was promptly prescribed; the following day a cesarean section was performed and a 1540 gr female afebrile baby was born. Apgar at one minute was 7, than 8 at five minutes. The baby presented respiratory distress and she was diagnosed with hyaline membrane disease (HMD). Blood tests showed a noticeable increase in inflammatory indices , and the blood samples culture, collected few hours after the birth, documented the growth of L. monocytogenes. Antibiotic therapy with intravenous (IV) weight-adjusted ampicillin and gentamicin was promptly initiated. Lumbar puncture was also performed; cerebrospinal Fluid (CSF) was sterile, opalescent, with 100 white blood cells (WBC), mostly polymorphonuclear leukocytes, with normal glucose and protein levels.On 9 January 2020, a 28-year-old pregnant woman, residing in a city near Rome and without a significant medical history, was admitted in the 31st week of gestation to the Obstetrics and Gynecology Department of Casilino Hospital in Rome, due to premature rupture of membranes (PROM) and gastrointestinal symptoms. At the admission, neutrophilic leukocytosis was documented , dairy products as mozzarella and parmigiano in the two local supermarkets, and smoked salmon and tuna in the fish market. She also claimed to have eaten once at a local restaurant and she denied any travel abroad during the past month.L. monocytogenes.According to the mother’s declarations, the local health authority’s attention was focused on the fresh horse meat purchased in the local butchery and cold cuts at the deli counter purchased in one of the two reported supermarkets. During the different inspections, food samples and environmental samples were collected to assess the presence of The patients’ clinical course and the events related to epidemiological investigation are summarized in L. monocytogenes isolated from clinical cases were found  residing in the same city, occurring from May 2019 to May 2020 and all belonging to the same serotype (see below). Where possible, patients were contacted by the competent authority to investigate any epidemiological links.After the notification of the neonatal listeriosis, additional inquiries were carried out to explore the occurrence of further listeriosis cases in the same city of the woman and her daughter (Case A). In the case series of samples collected by our regional center, three strains of L. monocytogenes meningitis was formulated. CSF cultures reported the growth of L. monocytogenes; antimicrobial susceptibility testing documented ampicillin, erythromycin and meropenem sensibility  and resistance to trimethoprim-sulfamethoxazole (0.094 µg/mL).The first case (B) occurred nine months before the above-mentioned episode and involved a patient admitted to the emergency department. A lumbar puncture was performed and a diagnosis of L. monocytogenes. Unfortunately, no data were available on the antimicrobial susceptibility profile.The second case (C) occurred three months later and a patient was hospitalized in April 2020. Blood cultures performed at the time of admission documented the growth of L. monocytogenes was isolated from both samples. The strain proved to be susceptible to ampicillin (≤0.25 mg/L), erythromycin (≤0.12 mg/L), meropenem (0.19 mg/L), penicillin (0.5 mg/L) and trimethoprim-sulfamethoxazole (0.012 mg/L).The third case (D) occurred four months after the above-described neonatal listeriosis case, in May 2020. Seven days after the admission, due to the occurrence of fever, the patient underwent two blood cultures and From epidemiological investigation and questionnaire collection it emerged that at least one patient (case B) confirmed their attendance at the same supermarket of the concerned neonatal listeriosis case. No further information could be obtained from the other patients.Listeria in the newborn were negative. Unfortunately, probably due to antibiotic therapy, it was not possible to isolate the bacterium responsible for the disease, and no more investigation could be carried out on this strain. Clinical information about the first diagnosis of listeriosis and the therapy carried out are untraceable. Blood cultures drawn by the newborn reported the growth of L. monocytogenes, passed from the mother, presumably by vertical uterus-placenta cell-to-cell transmission, before the delivery. In this case it was possible to isolate the strain for further analysis.Cervicovaginal swabs of the mother performed after identification of For all the clinical strains antimicrobial susceptibility testing (AST) was performed as minimum inhibitory concentration (MIC). All the strains were susceptible to ampicillin , meropenem  and erythromycin L. monocytogenes, meanwhile the mortadella samples from the first inspected supermarket were tested as positive, with a colony-forming unit less than 40 UFC/g. Subsequently, environmental samples were collected to identify the extent of contamination. Samples from the two meat-slicers  present in store were tested as positive for L. monocytogenes, as well as the cold food counter where the cold cuts were exposed to the public (H1–2). The worktable where the staff handled the meat (F), by contrast, was tested as negative for the contamination. All the positive strains were isolated for further analysis.The food samples of horse meat from the butcher shop were tested as negative for The isolates collected were assigned to the serotype 1/2b. Sequencing data confirmed serogroup IIb for the isolates and MLST analyses indicated that they all belonged to Sequence Type (ST) 5, Clonal Complex (CC) 5, lineage I.lin) with a new allele not described before, quinolone (norB), sulfonamides (sul), fosfomycin (fosX) antibiotics and cationic peptides that disrupt the cell membrane, including defensins (mprF). The virulence features were assessed and all the isolates shared the same virulence genes with the same alleles , the environmental samples  and food samples (M1–2), were found to bring the same genes for antibiotic resistance. The identified genes were those of resistance to the lincomycin  and chewBBACA (v2.8.5), described in  alleles A. The fi alleles B. The hu alleles .L. monocytogenes isolates from food samples among those received by our laboratory during routine activities. Food samples were found in the records concerning chicken meat (Food Sample 1), pork meat (2), smoked swordfish (3) and salmon (4). Representative strains were used to compare food isolates with the investigated cluster, following the workflow described in materials and methods. Results showed that there was no relation with the main cluster in the core genome, with 58–106 allelic distance calculated with cgMLSTFinder and 66–115 allelic distance with chewBBACA  and therefore probably to the same outbreak event.L. monocytogenes strain from mortadella (M) had two SNPs (in whole and core genome) differences with the environmental isolates. The human isolates from cases A and D differed 16–19 SNPs in whole genome (10–13 in core genome) from food and environmental isolates, and human strains from cases B and C. The SNPs’ matrix-based heatmap for whole genome and core genome are reported in The SNPs’ analysis confirmed the tight relationship of the strains considered in this investigation. The Herein is described a case of pregnancy-related invasive listeriosis occurring in central Italy, where the infection proved to be linked to contaminated food products and vertically transmitted from mother to baby.The mother’s infection manifested as asymptomatic form , with onL. monocytogenes from the mother, the effective differences between the mother’s and the child’s strains remain unknown, even if a previous study suggested that in such cases no differences occurred, given the vertical transmission of the pathogen [Since it was not possible to isolate the strain of pathogen .L. monocytogenes strains within seven allelic differences are considered to be linked to the same outbreak and are assigned to a unique cgMLST type (CT) [After a thorough completion of the epidemiological questionnaire by the mother, a strong epidemiological link with the mortadella sold in the supermarket was found, despite both the analyses of cgMLST showing allelic differences higher than the generally accepted limit to consider its inclusion in the same cluster. ype (CT) , althougype (CT) .https://github.com/B-UMMI/chewBBACA/issues (accessed on 11 February 2022).The use of different bioinformatics tools of analysis showed slightly different results. This could be due to the Prodigal gene prediction program, integrated in the chewBBACA pipeline, whose function is based on the identification of translation initiation sites  and, difL. monocytogenes all over the world, assigned the same CT7923 to all the strains involved in this investigation, suggesting a unique outbreak event. The overall view of their database suggests that the existence of other isolates, which act as a connection between groups, confirmed the existence of a single cluster.Even if chewBBACA found 9–11 allelic differences between the newborn isolate and the food and environmental samples, thus excluding the possibility of tracing the source of contamination, the cgMLSTFinder results, on the other hand, could put it into question, finding only eight differences, at the limit of the above-mentioned threshold value. Moreover, the Institute Pasteur, whose bioinformatics analysis platform is based on chewBBACA software and which collects and characterizes isolates of L. monocytogenes strains isolated from food samples during routine official control activities in the Lazio region by our laboratory, it seems to involve mainly pork meat (data not shown). These peculiar features, together with a strong epidemiological link found between the specific category of food, the geographical area and the habits of the mother of patient A during pregnancy and patient B, could support the hypothesis that all the reported episodes belong to the same outbreak event.Other considerations may support this hypothesis. First, the sequence type 5 (ST5) of the strains involved in this epidemiological investigation is very rare in human cases in Italy and almost limited to Lazio region  , the estL. monocytogenes core genome is estimated at 2.6 × 10−7 substitutions per site per year (one substitution every 2.5 years) [L. monocytogenes, significantly increase the generation time of the organism compared to the optimal growth temperature  [L. monocytogenes growth [In a recent work by Luo and colleagues in 2019, , it was 5 years) . Thus, a0–37 °C) . Furthers growth ,53.In addition to differences in generation time and variations in population sizes, the direction  and strength of natural selection could also contribute to differences in the rate of changes per site per year . Given tL. monocytogenes, not all transmitted or found to the newborn [Another hypothesis could be the occurrence of the analysis on a different portion or batch of the mortadella originally consumed, with a different strain than the original source of contamination, which could since have been completely sold by the supermarket and not available during the inspection. Alternatively, since it was not possible to isolate the maternal strain, it could have happened that the mother was originally infected with more than one strain of  newborn ,56,57.The analysis, through the use of WGS, showed that the three retrospectively identified human cases were closely linked with the newborn case A and with food and environmental samples. In After the correct sanitation of premises, sampling performed in self-control and by the local health authority tested negative. Consequently, the deli counter activity was reopened, according to current legislation. The contamination of mortadella was most likely secondary, since the sampled product was already open and no data relating to an intact package of the same batch were available.Based on that, it could be concluded that the source of contamination in the investigated area was eliminated, and that the actual food source of contamination remains to be investigated. Beyond the strictly sanitary purposes and given the molecular results that are not completely conclusive, further investigations on other probable contamination sources would probably have been useful, in order to assess if the contaminated food found in the inspections was the only one among the products consumed by the mother.L. monocytogenes and also suggest other factors, such as the frequency of the investigated sequence type and the presence of other strong epidemiological links.Since the used methodologies have highlighted tools-related results biases, the use of more than one method for analysing data related to an outbreak would be recommended, to avoid errors of underestimation or overestimation of the diversity between the involved strains. Our results also show the possibility that the cut-off of seven allelic differences represents a criterion that is sometimes too stringent for the definition of an epidemic cluster of Herein is described the first outbreak linked to the ST5 in Italy, which also involved a pregnancy-related case. This specific ST, based on national databases, is not common in Italy and seems to be limited in central Italy, in particular the Lazio region (source ISS database). Concerning Europe, sporadic occurrences of ST5 have been reported in literature. Several cases, mostly linked to maternal-fetal listeriosis, were reported during 1992–2003 in France . An inveL. monocytogenes in the past [Foods purchased from store delicatessen counters are a risk factor for sporadic listeriosis. Turkish delicatessen meat, pasteurized butter, and hot dogs have all caused outbreaks of the past . This exthe past .Finally, this study highlights the importance of a constant surveillance activity of foodborne diseases carried out by health authorities together with clinicians and laboratorians, from notification to investigation. The major aim is to identify and stop the source of contamination as soon as possible, to avoid the extension of a foodborne outbreak.After the assessment of the epidemiological questionnaire completed by the mother, the Veterinary Service of Hygiene for Foods of Animal Origin (SIAOA) of the local health authority conducted sampling at the butcher shop and the deli counter of the attended supermarket involved. Sampling of horse meat and mortadella was performed according to the Reg. (CE) 2073/2005. Briefly, by means of sterile scalpels four aliquots were aseptically collected, each consisting of five sample units of 120 g, and stored into sterile bags at +4 °C. Environmental sampling was performed by means of sterile swabs and containers. In particular, two meat slicers , the worktable for handling cold cuts (F), and a refrigerated counter for exposed and store-cold cuts (H) were sampled, each with more than one swab. All the collected samples and times of sampling are summarized in ® automated specimen processing  on Columbia Agar, Columbia CNA Agar, Mac Conkey II Agar, Mannitol Salt Agar, BBL CHROMagar Candida Medium and Chocolate Agar  and incubated at 37 °C for 24 h. Therefore, the multiplex PCR was performed on an aliquot of the positive sample . This technique resulted in the detection of 33 pathogens and 10 antibiotic resistance genes one hour after blood culture positivization. The identification of L. monocytogenes was also confirmed, with a score ≥2, by Maldi-TOF mass spectrometry  performed on plate-cultured colonies after 24 h of incubation.A blood sample of the newborn was collected, inoculated in Bactec Plus aerobic and anaerobic broth, and incubated in the automated culture system, Bactec 9420 . The positivity was assessed upon direct observation under the optical microscope of motile bacilli and stained with Gram dye. An aliquot of the sample was plated using WASPAll the isolated clinical strains were sent to the Regional Reference Centre for Pathogenic Enterobacteria (CREP) at the Food Microbiology Unit of IZSLT—central division of Rome.L. monocytogenes was performed by Real-time PCR using iQ-Check Listeria monocytogenes II PCR Detection Kit . Positive samples were cultured according to UNI EN ISO 11290-1:2017, using ALOA medium and LSM as the second selective medium. L. monocytogenes identification was confirmed by VITEK® 2 Compact System . The biochemical culture confirmation was carried out performing the beta-hemolysis test, the L-rhamnose test, and the D-xylose test.The food and environmental samples collected by LHA were sent to Food Microbiology Unit laboratories of IZSLT. The detection of Listeria ‘H’ and ‘O’ (Mast Group) according to the manufacturer’s instructions at CREP laboratory of IZLST. Antimicrobial susceptibility testing (AST) for all the clinical strains was performed as minimum inhibitory concentration (MIC) determination by broth microdilution , according to the European Committee on Antimicrobial Susceptibility Testing , using epidemiological cut-offs. The following drugs were tested: Meropenem, Ampicillin, Erythromycin. Streptococcus pneumoniae ATCC 49619 was used as a quality control strain.All the isolated strains were serotyped by using the Mast assure antiserum For whole genome sequencing analysis, one colony was selected for each food sample unit tested positive and one colony for each swab used for environmental sampling . Genomicprs (lmo0199) with BLAST (v2.11.0) [abcZ, blgA, cat, dapE, dat, ldh, and lhkA) retrieved from the BIGSdb-Lm [Raw reads quality was assessed with Fast QC (v0.11.5)  and low-v2.11.0) . In siliv2.11.0)  that useIGSdb-Lm .Listeria cgMLST database [The cgMLST analyses were performed using two pipelines. The first one was cgMLSTFinder (v1.1 developed by the Center for Genomic Epidemiology), that runs KMA  against database  to creatListeria cgMLST database [The second adopted pipeline was chewBBACA (v2.8.5)  based ondatabase . This sodatabase  to searcdatabase .The SNPs and core genome SNPs’ analysis was performed with the pipeline CSI phylogeny (v1.4) accessible from the Center for Genomic Epidemiology , startinListeria Genomic Islands) was assessed starting from the assemblies using BLAST against genes database available on the BIGSdb-Lm platform (accessed on 13 January 22). The same method was used to assess the presence for virulence and antimicrobial resistance genes of each strain.Identification of acquired antibiotic resistance genes and genes encoding for persistence (metal, detergent resistance, stress Survival Islands and


Mental wellbeing and life satisfaction play an important role in the development of adolescents, yet factors potentially influencing these states have not been sufficiently studied, especially in Vietnam. This study aims to fill the research gaps by exploring the associations of the parent-child relationship, self-esteem, and resilience on the mental wellbeing and satisfaction with life of adolescents.A cross-sectional study was conducted from June to July 2020 on 1,023 adolescents from 10 to 18 years old living in Vietnam. To assess the satisfaction with life as well as the mental wellbeing of participants, this study used the Satisfaction with life and The World Health Organization-Five Wellbeing Index scale.More than 70% of participants reported having conflicts with their parents (74.6% of those conflicted with their father ad 73.9% of those conflicted with their mother), while 26.3% stated dissatisfaction with life. The mean score of mental wellbeing was 61.5 (SD = 23.0). Higher academic performance, self-esteem, resilience, encounter loneliness and isolation within own family, and having support and sharing from family members had a positive effect on life satisfaction and mental wellbeing. Female participants had higher satisfaction with life score  but they had a lower mental wellbeing score  than male participants. High school students had lower both satisfaction with life and mental wellbeing scores than secondary students.The results highlight the importance of being aware of the influence that expectations (of higher grades) and bias  imposed by parents, teachers, and society on the mental wellbeing of youths, especially in Asian cultures. Strengthening the family bond and encouraging young people to share their feeling is also crucial to enhancing the mental health condition of adolescents. Mental wellbeing, as an integral part of our overall health, is considered a vital public health issue. It encompasses multiple dimensions, such as our performance in relationships, work, and entertainment activities in the face of adversity . Mental The most common factors associated with adolescents' life satisfaction include self-esteem, internal control, family, and peer relationships . AlthougIn Vietnam, existing literature suggested that adolescents are increasingly exposed to risk factors such as school violence, mental disorders, juvenile delinquency, and abortion . A recenWe conducted online cross-sectional research in four Vietnamese provinces  in Vietnam, from June to July 2020. These were Vietnam's major cities and provinces, all of which were undergoing rapid urbanization. Participation criteria were: (1) currently studying grades 6 to 12; (2) currently residing in Vietnam, and (3) willing to participate in this study by online informed consent. Participants who had major diseases or were unable to answer questions were excluded from the recruiting procedure. We used the convenience sampling approach in four steps as (1) the research team started with core groups of student union presidents at schools; (2) these core groups received a link to a website containing introductory information about the study and the questionnaire; (3) they prompted to forward it to others in their network by email, social media, or other online means; and (4) invitees continued to complete surveys and encourage others to do so as well. At the end of the data collection period, there were 1,023 students from grades 6 to 12 completed the survey.We developed a structured questionnaire and uploaded it onto the online survey platform SurveyMonkey (surveymonkey.com). This approach was adopted as it was economical, time-saving, user-friendly for teenagers, and effective in reaching a large sample. The questionnaire includes four major components: (1) general characteristics (of participants); (2) Satisfaction with Life among participants; (3) Relationship in the family of participants (4) Social support, the Protective Factors for Resilience scale (PFRS), and Self-esteem Scale. The survey was originally piloted with a small sample of students to confirm that the translated instruments in Vietnamese were cross-culturally valid. The updated questionnaire was then posted to the online survey platform. Data collection commenced after the online survey system was thoroughly tested to assure the accuracy of the survey contents and that no technical issues occurred.We asked participants to report information regarding their gender, education, living location, people living together, academic performance, and the age of their father/mother. In terms of academic performance, in Vietnam, the academic performance of students was divided into four levels based on 10-scale: moderate and below (<7), fair (from 7 to <8), good (from 8 to <9), and excellent . Regarding level of education, we had three levels of education: Primary school (grade 1–5 correspond to 6–10 years old), secondary school (grade 6–9 correspond to 11–14 years old), and high school (grade 10–12 correspond to 15–17 years old). In this study, we defined a nuclear family as a family that had two generations such as parent and their child.The SWLS measures a person's overall happiness in life. This measure has five items, and participants respond to each question on a seven-point Likert scale ranging from one (strongly disagree) to seven (strongly agree) . The posThe WHO-5 is a brief self-reported assessment of present mental wellbeing developed by the WHO. This scale, which is appropriate for children aged 9 and up, has been proven to have sufficient validity in screening for depression and assessing outcomes in treatment studies . It consThree questions were used to access the relationship between parents and children such as “Do you have conflicts with your father,” and “Do you have conflicts with your mother” with 2 answer options (Yes/No), and “How many family members can you confide in.”We employed 8 questions to explore the connection with family members and the feeling of loneliness at home. These questions included:Encounter loneliness and isolation within own family:1) Do not feel the family member is a good companion.2) Feeling alone even at home.3) My family member is by my side but does not care about me.4) Feeling no connection to my family.5) Feeling family relationships have less meaning to me.Having support and sharing from a family member.6) Feeling part of the family.7) Family members are always willing to listen to and understand me.8) Family members are always ready to encourage and comfort me.Encounter loneliness and isolation within own family” and “Having support and sharing from a family member” was reversed from 1 to 10. Participants who had higher “Encounter loneliness and isolation within own family” scores indicated a stronger feeling of loneliness and isolation, by contrast, those who had higher “Having support and sharing from a family member” scores indicated better the connection with family members and the feeling of loneliness at home.Each item was marked from 1 (Never) to 5 (Always). The total score of “PFRS includes 15 categories in 3 domains: Personal Resource, Social Resources–Peers, and Social Resources–Family (5 items per domain) ( domain) . A 7-Lik domain) . The tot domain) . The CroRosenberg's self-esteem scale : This is a 10-item measure that assesses one's overall self-worth by assessing both positive and harmful feelings toward oneself . To clean data before analysis, we used the Listwise Deletion method, there were 1,222 participants recruited in this study, and 1,023 students from grades 6–12 completed the survey. Hence, the data of 199 people were deleted, and the response rate was 83.72%. The mean and standard deviation (SD) were used to describe continuous data, whereas frequencies and percentages were used to represent categorical variables. To see if there are any variations in participants' life satisfaction and mental wellbeing based on specific variables, Wilcoxon rank-sum test, Kruskal-Wallis test for continuous variables, and χ2 test for categorical variables were used.p < 0.2 as the threshold for included variables. The p-value (P)<0.05 was considered statistically significant.Potential covariates for full models of life satisfaction and mental wellbeing among Vietnamese students included individual characteristics, relationships in the family, and protective factors for resilience. Multivariate Tobit regression was conducted to determine factors related to life satisfaction as well as mental wellbeing among Vietnamese students. These models were then combined with the stepwise forward strategies to produce reduced models with The following were used in the goodness-of-fit tests for the hypothesis model: goodness-of-fit index (GFI), comparative fit index (CFI), root mean squared error of approximation (RMSEA), Tucker–Lewis index (TLI), and the standardized root mean squared residual (SRMR). The results showed that the structure had a good fit with RMSEA (0.049), CFI (0.994), and SRMR (0.014).All procedures performed in studies involving human participants were by the ethical standards of the Youth Research Institute, Vietnam, and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Online Informed consent was obtained from all participants. Informed consent was also obtained from the participants' parents.p < 0.05).The construct validity and reliability of the relationship in family participants were reported in p < 0.05). There was a statistically significant dissimilarity in the score for life satisfaction and mental wellbeing between people having conflict with their father, their mother, or their parent as well as the number of their family members that they can confide in. Participants who had no conflict with their father/mother/both parents or had people to confide in had higher scores for satisfaction with life and mental wellbeing than others. The value score of the life satisfaction scale and WHO-5 scale was about 23.9 points and 61.5 points, respectively.p-value of structural equation modeling analysis are presented in 2 = 0.427) of the life satisfaction variance and mental wellbeing variance.The path coefficients and p < 0.001) and mental wellbeing  via self-esteem. Through self-esteem and resilience, both two factors “having support and sharing” and “encounter loneliness and isolation” were positively associated with life satisfaction and mental wellbeing.Direct Effects: Significant direct effects were observed for conflict with both parents on satisfaction with life . Two constructs “Having support and sharing from family members,” and “Encounter loneliness and isolation within own family” influenced both satisfaction with life and mental wellbeing. In terms of the first construct, about 47.9 and 68.2% of the total effect on satisfaction with life and mental wellbeing respectively was indirect effect via the resilience scale, while these figures were 3.9 and 37.5% via self-esteem. Regarding the influence of the “Encounter loneliness and isolation” construct on life satisfaction and mental health, 4.7 and 28.0% of the total effect was indirect effect via the self-esteem scale, while 31.5 and 36.6% of the total effect was indirect effect via resilience scale.Total effects: Conflict with both parents was negatively associated with life satisfaction This study revealed that a high percentage of children had a conflict with their father or mother (>70%), and nearly 30% of participants were dissatisfied with life. Conflict with father was the negative factor that decrease life satisfaction and mental wellbeing score. Encounter loneliness and isolation within own family and protective factor for resilience from personal, peers, and family had a positive impact on life satisfaction and mental wellbeing.In terms of individual characteristics, our research reveals that male adolescents had higher life satisfaction scores than females. Gender differences in life satisfaction, though well-proven, are inconsistent. Research by Callahan et al. indicated that women tend to have a higher level of life satisfaction than men , while GThis study revealed encounter of loneliness and isolation within own family had negatively associated with satisfaction with life and mental wellbeing. This result was similar to the Çivitci et al. . LonelinIn terms of protective factors, individuals, families, and peers positively predict the resilience of participants, which increased the life satisfaction of adolescents. Previous studies have shown the correlation of different variables, such as individual characteristics , self-esteem, resilience, conflict with parents, loneliness, and isolation within their family, having supportive and sharing family members, with life satisfaction and mental wellbeing , 42–44. Our research once again highlights the impact of family on life satisfaction and the mental wellbeing of children. Not only at school, but at home, children should be encouraged to share their feelings and resolve conflicts with their parents. Our research lays the groundwork for multi-level analysis for simultaneous assessments of personal and interpersonal factors to address remaining problems and promote children's wellbeing and life satisfaction. From this ecological framework, interventions must focus on strengthening social support across all dimensions of the children's lives, community resources, and family relationships.Our study has certain limitations. Firstly, the cross-sectional study model prevented us from determining a causal relationship. Secondly, the information was collected from self-reported questionnaires, which may be subject to personal bias, such as academic performance may be subjective. Thirdly, although the Satisfaction with Life Scale (SWLS) is used worldwide, no previous studies have been conducted in Vietnam and therefore we could not provide a comparison of findings within the country. However, comparisons with global studies were conducted. Despite the above limitations, our research is among the pioneers in providing a comprehensive view of life satisfaction and mental wellbeing in adolescents by exploring individual and community-related factors, including social relationships.Our study investigates important factors associated with life satisfaction and mental wellbeing among adolescents, specifically parent-child relationships, self-esteem, and resilience. The results highlight the influences of academic expectations and gender bias on the mental wellbeing of youths, especially in Asian cultures. Strengthening the family bond and encouraging young people to share their feelings are crucial to enhancing adolescents' mental health status.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by all procedures performed in studies involving human participants were by the ethical standards of the Youth Research Institute, Vietnam, and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Online Informed consent was obtained from all participants. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.Survey conception: VT, HND, and TMT. Data collection: VT, TBT, KV, HMD, and NT. Project management: VT and TPT. Data analysis: TN and VT. Data interpretation: HND and TMT. Drafting of the manuscript: VT. Reviewing and editing the manuscript: PA, LB, GF, CL, CH, and RH. All authors revised the manuscript critically and gave final approval of the version to be published.