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Mindfulness, a new therapeutic model proposed for the management of chronic stress, consists of an individual’s awareness of and attention to his or her symptoms of emotional distress experienced under chronic stress. This strategy facilitates therapies and enables the modifications necessary to improve lifestyle. This intervention is practiced by Manolete S. Moscoso at the University of South Florida. The purpose of this therapeutic intervention program is to instruct the individual in the relaxation response, reduce the level of chronic stress and change patterns of self-destructive behavior, obtaining an immune and neuroendocrine benefit that promotes the restoration of health and helps control the symptoms caused by medical treatments, which allows patients experiencing severe depression to counter the recurrence of this disease. Mindfulness allows focus and consciousness in the body through breathing, in the mind through thought, and the environment through the senses. The belief of “living on the run” from stress and emotional pain through daily life experiences contributes to fear, tension, anxiety, worry, anger and hostility. When behavior is modified in response to the difficulties of life and when it is understood that pain and pleasure are genuinely human experiences, an individual can achieve an adequate level of acceptance and peace [18, 62].
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| 99.75 |
Diseases are the result of an alteration at the bio-psycho-social level that can indicate lifestyle changes that should be made in addition to appropriate medical management and treatment. Emotions and stress significantly affect health and one’s susceptibility to a pathology, as well as one’s ability to recover from an illness. Psychoneuroimmunology should provide knowledge about the biological dynamics of conventional and alternative medicines for fighting disease. The psychoneuroimmunological axis comprises several disease-producing mechanisms in which different disciplines of medicine interact, implying the need for an integrative approach. The science of psychoneuroimmunology must go hand in hand with health education and the promotion of healthy lifestyles in order to attain patient health.
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| 99.7 |
Attitudes towards people with mental disorders refer to individual beliefs about what people with mental illness are like and how they should be treated [1, 2]. Previous studies have shown that these attitudes could vary from acceptance , tolerance , to stigma , and even fear . Researchers have proposed two different ways by which public attitudes might affect individuals with mental illness. The first relates to how the public might interact with, provide opportunities for, and help support a person with mental illness . In this case, the public refers to both the general public such as the community and institutions like hospitals, as well as co-workers, friends and family members of people with mental illness . When attitudes and beliefs are expressed positively, they can lead to supportive and inclusive behaviours (e.g. willingness to hire a person with mental illness); but when they are expressed negatively, they can cause avoidance, exclusion from daily activities, exploitation, and discrimination . The second frames how people with mental illness experience and express their own psychological problems and whether they are willing to disclose their symptoms and seek help [7, 8]. Rüsch et al. reported that after controlling for demographics, people’s intentions to seek help were positively associated with the tolerance and support for community care of mental illness.
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| 99.56 |
During the past few decades, there has been an increase in mental health literacy among the general public, especially regarding knowledge of the biological and genetic basis of mental illness [3, 10]. However, such increases have not lessened the stigmatization and discrimination towards people with mental illness . As a cluster of negative attitudes and beliefs, stigma is a concept which has received a lot of research attention [5, 7, 11]. Although there are different tools that can measure stigma [12, 13], public attitude is a much broader issue than these negative attitudes. Other attitudes like tolerance and acceptance are also part of it. Thus there is a need for multidimensional attitudes scales for assessing public attitudes to mental illness. The Attitudes to Mental Illness (AMI) questionnaire is a tool developed by the UK Department of Health, adapted from the 40-item Community Attitudes toward the Mentally Ill Scale . As a multidimensional instrument, it covers four aspects of public attitudes, namely fear and exclusion of people with mental illness, understanding and tolerance of mental illness, integrating people with mental illness into the community, and causes of mental illness and the need for special service . The UK Department of Health has used it to assess public attitudes among the UK general population for more than 20 years , and it showed good internal reliability (Cronbach’s alpha = 0.87 ). Another 2-factor structure was also identified, which included ‘prejudice and exclusion’ (Cronbach’s alpha = 0.83), and ‘tolerance and support for community care’ (Cronbach’s alpha = 0.77) . Given that these were based on the UK general population, it is therefore unknown whether these two different factor structures are applicable to the multi-ethnic population in Singapore.
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| 99.94 |
Previous studies suggested that socio-demographic characteristics could affect public attitudes. Lauber et al. found that demographic factors such as age, gender and the cultural background contributed to social distances, with the explained variance as 44.8%. Another study among Mexican Americans found that demographics such as age, gender and education were the primary variables associated with attitudes towards mental illness . Moreover, the direction of such relationships may vary across different cultures—a Swiss sample suggested that women tended to have more negative attitudes towards people who are mentally ill . While it was reversed among a sample from Germany, where female respondents were more accepting of people with mental illness . Locally, a study on general attitudes to mental illness found various socio-demographic correlates relating to attitudes including age and education; however, given this study was conducted over 10 year ago, there is a need for more up-to-date evidence. A more recent national study was conducted, which explored stigma (personal stigma and social distancing) and its correlates among the Singapore general population, specifically in relation to five mental disorders . This study also found that age, gender, ethnicity, education and income level were correlated with personal stigma towards people with mental illness.
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| 99.75 |
The current study aimed: 1) to explore the factor structure of the AMI questionnaire among the multi-ethnic general population in Singapore; and 2) to explore the socio-demographic correlates of each AMI factor and identify how these characteristics affect public attitudes to mental illness among this population.
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| 100.0 |
Data for the current study were from a nation-wide cross-sectional survey of mental health literacy conducted in Singapore from March 2014 to April 2015. It adopted a disproportionate stratified sampling design with 12 strata by age (18–34, 35–49, 50–65) and ethnicity groups (Chinese, Malay, Indian, and other ethnic groups). A probability sample was randomly selected via a registry that maintains the names and socio-demographic characteristics such as age, gender, ethnicity and household address of all residents in Singapore. To be included in this study, participants had to be Singapore residents (Citizens or Permanent Residents) aged between 18–65 years and living in Singapore during the recruitment period. Residents aged 50–65 years, Malays and Indians were over-sampled to ensure sufficient sample size for subgroup analysis. More detailed information on the sampling strategy is found in the paper by Chong et al. .
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| 100.0 |
Face-to-face interviews were conducted by trained interviewers; and the respondent could choose the language for the interview: English, Chinese, Malay or Tamil. Data was captured via iPad, which was programmed to display a dual language screen, allowing bi-lingual interviewers to translate terms or phrases as required (by the respondent) in a consistent way; this method minimized the potential for misinterpretation or ad hoc translations by interviewers. Individuals who were out of the country during the recruitment period, unable to be contacted due to incomplete or incorrect addresses, and unable to complete the interview in one of the specified languages were excluded from the current study. In total 4,231 people were contacted, of which 3,006 completed the survey which yielded an overall response rate of 71.1%.
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| 100.0 |
The study was approved by the National Healthcare Group Domain Specific Review Board in Singapore. Written informed consent was obtained from all participants aged 21 years and above as well as from parents or guardians of participants who were 18–20 years old.
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| 99.94 |
Twenty-six items of the original 27-item AMI questionnaire were used in the current study to measure public attitudes towards mental illness. The single item ‘most women who were once patients in a mental hospital can be trusted as babysitters’ was excluded due to its low loading (below 0.3) in a previous study . Items were rated on a 5-point Likert scale ranging from ‘1 = strongly agree’ to ‘5 = strongly disagree’. The original scale was proposed to cover 4 components: fear and exclusion of people with mental illness, understanding and tolerance of mental illness, integrating people with mental illness into the community, and causes of mental illness and the need for special service .
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| 100.0 |
The survey measures were translated into Mandarin, Malay and Tamil. To ensure the conceptual equivalence of the instruments in the different languages, the whole translation procedure followed a process that was adapted from guidelines of the World Health Organization, which had been used among two previous national studies in Singapore [22, 23]. This included 1) single forward translation by a professional firm; 2) review by an expert panel comprising the professional translators, content experts and a layperson to identify and resolve any inadequate expressions in the translation and discrepancies between the translated and original version; 3) pre-testing and cognitive interviews among individuals representing the target population in term of the age-groups, gender, ethnicities, and socio-demographics; and 4) development of the final version .
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| 99.94 |
The cognitive interviews refer to a common means of applying the cognitive model in a manner that may ultimately improve the quality of survey questions through the study of comprehension, retrieval, judgment, and response processes . During this process, respondents were interviewed by trained researchers who systematically probed on whether they could repeat the questions and what came to their mind when they heard a particular phrase or term and they were asked how they decided on their response. Respondents also reported any word they did not understand and any word or expression that they found offensive or unacceptable; and where alternative words or expressions exist for one item or expression, the respondent was asked which of the alternatives conforms better to their usual language. Minor changes were made to the questions based on the cognitive interview findings; this was to ensure the items of the instrument would be understood in the manner they were intended to be and to avoid potential misinterpretation. More information on this process is available in a previous publication .
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| 99.9 |
All estimates were weighted to adjust for over sampling and post-stratified for age and ethnicity distributions between the survey sample and the Singapore resident population in 2012. Descriptive analysis was conducted for the socio-demographic variables. Weighted mean and standard error (SE) were presented for continuous variables; while for categorical variables, they were presented as frequencies and percentages.
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| 99.94 |
The factor structure was determined by several steps. First of all, using confirmatory factor analysis (CFA), we tested the 4-factor and 2-factor structures of AMI from previous studies [9, 15]. However, the model fit indices suggested that these two models didn’t fit into our data. Therefore, following previous studies [13, 25], we randomly split the weighted dataset into 2 separate parts with equal number of observations for each (n = 1,503). Exploratory factor analysis (EFA) was conducted for the first half-dataset. Eigenvalues > 1.0, scree plot, pattern of loadings on each item (e.g. cross-loading), and the interpretability were used to determine the appropriate number of factors that should be extracted. To allow the correlation between factors, oblique promax rotation was used, and the factor loading cut-off was set as 0.4. This was followed by a CFA for the second half-dataset to confirm this factor structure derived from EFA. A good model was defined as 1) the comparative fit index (CFI) > 0.95; 2), the Tucker-Lewis index (TLI) >0.95, and 3) the root mean square error of approximation (RMSEA) <0.06 . All structural equation modelling analyses were performed on polychoric correlation matrix using Mplus version 7.0 with the weighted least squares with mean and variance adjusted estimator for categorical variables . The internal reliability (Cronbach’s alpha) for each factor was calculated as well.
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| 100.0 |
Multivariate linear regression was conducted to examine the socio-demographic correlates (i.e. age, gender, ethnicity, marital status, education level, employment status and personal income) for each of the AMI factor scores (dependent variables). A two-sided p-value below 0.05 was considered as statistically significant. The descriptive and the multivariate linear regression analyses were conducted using SAS 9.3.
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| 100.0 |
The results of the descriptive analysis are listed in Table 1. The participants had an average age of 40.9 (SE = 0.11), with almost equal percentage of both genders (male = 50.9%). Chinese made up about three quarters of the participants (74.7%), followed by Malays (12.8%) and Indians (9.1%). About 64% of the participants were married, compared to 31.4% who were never married. The education level of the participants tended to be moderately high; with 31.3% having a diploma and 29.6% having a university degree. Most of the participants were employed (77.6%), followed by housewives/homemakers (8.7%) and students (6.7%). The unemployment rate of the current sample was quite low, at around 4%. The majority of respondents had a monthly salary lower than SGD 6,000 (86.9%).
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| 100.0 |
To explore the factor structure of the AMI, CFA was performed to confirm whether the 4-factor and 2-factor structures identified from previous studies [9, 15], were best suited to the current sample. However, results suggested that both of them had poor fit—for the 4-factor model , χ2(df) = 1325.167 (128), CFI = 0.802, TLI = 0.878, and RMSEA = 0.056; for the 2-factor model , χ2(df) = 1833.422 (129), CFI = 0.718, TLI = 0.828, and RMSEA = 0.066. In this case, EFA analysis was conducted for the first half-dataset (n = 1,503). The eigenvalues and scree plot on all 26 items of the questionnaire suggested that 4-, 5-, or 6-factor models were all potential solutions. During the analysis, factor loadings of each item in all 3 models were explored. Items were excluded based on the following priority: items have 1) consistently lowest loading across all models; 2) consistent cross-loading across all models; 3) lowest loading across different models; 4) lowest loading; and 5) cross-loading. After removing 2 items, the eigenvalues and scree plot suggested that the 6-factor model was no longer suitable; thus for the following steps, only the 4- and 5-factor models were considered. After removing another 2 items, the 5-factor model became unstable (one item had a factor loading above 1). In the end, a 4-factor structure comprising 20 items was selected, and it had a good model fit (χ2(df) = 213.808 (116), RMSEA = 0.024). The removed items are shown in S1 Appendix.
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| 100.0 |
The four extracted factors were subsequently reviewed and labelled—factor 1 (3 items) was named ‘social distancing’; factor 2 (9 items) was named ‘tolerance/support for community care’, since it shared almost all items with the same factor identified by Rüsch et al. (except the item ‘increased spending on mental health services is a waste of money’); factor 3 (3 items) as ‘social restrictiveness’; and factor 4 (5 items) as ‘prejudice and misconception’. Refer to Table 2 for more information on the 4-factor model. CFA was conducted to test the model fit of this 4-factor model among the second half-dataset (n = 1,503), and the result suggested that the model was acceptable (χ2(df) = 236.727 (80), CFI = 0.933, TLI = 0.955, and RMSEA = 0.036). The internal reliability statistics for the four factors were 0.707, 0.696, 0.709, and 0.665, respectively.
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| 100.0 |
To enable easier interpretation of the regression results, the selected items of AMI were reverse scored (changed to ‘1 = strongly disagree’ to ‘5 = strongly agree’), following Rüsch et al. . The total score of each factor was then summed up and used in the multivariate regression analyses. For factor 2, the scoring of the single item with positive factor loading was reverse scored again, and then added up with the scores of the remaining items within this factor. In this case, more positive attitudes towards people with mental illness were characterized as—lower ‘social distancing’, higher ‘tolerance/support for community care’, lower ‘social restrictiveness’, and lower ‘prejudice and misconception’ scores. The average factor score for ‘social distancing’ was 8.07(SE = 0.07, range 3 to15), 14.81 for ‘tolerance/support for community care’ (SE = 0.10, range 9 to 45), 7.21 for ‘social restrictiveness’ (SE = 0.07, range 3 to 15), and 15.36 for ‘prejudice and misconception’ (SE = 0.10, range 5 to 25).
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| 100.0 |
Multivariate linear regression analyses results suggested that age, gender, ethnicity, marital status, education level, employment status and personal income were all significantly associated with the AMI factors (Table 3). Those aged between 35–65 years, with relatively lower education (i.e. secondary education including O/N level, or below), and being a housewife/homemaker were significantly associated with higher ‘social distancing’; while any ethnicity other than Chinese was significantly associated with lower ‘social distancing’. Female gender, Indian ethnicity, and being unemployed were associated with more ‘tolerance/support for community care’; while lower education, and being a housewife/homemaker were negatively associated. ‘Social restrictiveness’ was positively associated with those aged between 35–65 years, education level lower than university, and being a housewife/homemaker; while negatively associated with being a female, Malay or Indian ethnicity, and being a student. Lastly, ‘prejudice and misconception’, was positively associated with those aged 50–65 years, Malay or Indian ethnicity, education level lower than university, and a monthly income less than SGD 2,000; on the other hand, it was negatively associated with female gender, never married, and being a student or unemployed.
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| 100.0 |
Unlike the two different factor structures of AMI from previous studies [9, 15], our results suggested a 4-factor structure with 20 items among the Singapore general population. The first factor is about ‘social distancing’–people’s intention to distance themselves from individuals with mental illness in their community. The second factor relates to understanding and tolerance towards the mentally ill and the intention of integrating mental health service into the community (i.e. ‘tolerance/support for community care’). The third factor is about limiting the societal roles and responsibilities of those with mental illness (i.e. ‘social restrictiveness’); while the last factor relates to public’s ‘prejudice and misconception’ over mental illness. CFA confirmed that this model had an acceptable fit, with only the CFI being slightly lower than the recommended cut-off of 0.95 . In this sense, the multidimensionality of public attitudes to mental illness was confirmed.
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| 100.0 |
Further analyses on the socio-demographic correlates of the four AMI factors suggested that the relationships between several socio-demographic characteristics and attitudes were quite consistent across different factors. Firstly, people belonging to the older age group (50–65 years) generally had more negative attitudes towards the mentally ill, and this applied for ‘social distancing’, ‘social restrictiveness’ and ‘prejudice and misconception’. A literature review on public attitudes towards mental illness suggested that out of the 33 studies, 32 reported positive associations between negative attitudes and age . Similar findings were also reported in local studies, with younger adults being more tolerant and less stigmatising . This might be due to improved public knowledge of mental illness , and the development of information technology which makes such knowledge easily accessible especially for young adults who are more familiar with the use of such technology. Gender was another factor affecting public attitudes. The relationship between gender and public attitudes could vary due to different reasons such as cultural differences [19, 20]. A previous local study on stigma suggested that female gender was likely to be associated with lower stigma among the general population in Singapore . A 2006 review also summarized that females reported more positive attitudes to mental illness among more than half of the studies which had explored this issue . This is similar to our findings, where females showed more tolerance and support for community care, less social restrictiveness, and less prejudice and misconception towards people with mental illness. Although some studies also indicated that females tended to desire less social distance than males [13, 29, 30]; this relationship was not evident among our sample. Potential reasons could be that items used to measure social distance in our instrument were about the distance in the neighbourhood or community; however, for other studies, they focused on social distance in terms of personal contact with individuals with mental illness [13, 30]. Lower education was also found to be consistently associated with more negative attitudes to mental illness across all four factors of AMI. This finding has been reported by other studies as well [13, 28, 31], and it suggests that individuals with higher education had greater knowledge relating to mental illness. Another explanation could be that people with higher education have more access to health information, or they have a better understanding of such information as a result of their higher education.
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study
| 99.9 |
An interesting finding lies in the correlation between ethnicity and AMI factors. In our study, as compared to Chinese, both Malays and Indians had higher prejudice and misconception towards mental illness; but they also had less social distancing and less social restrictiveness towards those who were mentally ill. Meanwhile, Indians tended to have higher tolerance and support for community care of people with mental illness. Prejudice and misconception is a factor highly correlated with people’s knowledge of mental illness. In this case, possessing less knowledge of mental illness didn’t necessarily mean that individuals would have more social distancing, less tolerance/support to community care, and more social restrictiveness towards people with mental illness; other factors such as cultural differences might also play an important role in this process. This could be particularly true for Malays. In Islam, the religion practiced by the large majority of Malays in Singapore, mental illness is perceived as a test from God [32, 33], and illness could be treated as an opportunity to remedy disconnection from God or resolve a lack of faith through regular prayer and a sense of self-responsibility [34, 35]. Thus they were more tolerant to mental illness, as reported in other local studies as well [13, 21]. Alternatively, such differences might also be caused by people’s misunderstanding on the scales items. In another study among the same study sample, by providing detailed vignettes on different mental health problems, Malays and Indians tended to view mental illness as weakness but not illness . In our measurement, the wording ‘mental illness’ might have misled the participants while they were answering the questionnaire. Although Malays and Indians scored higher on ‘prejudice and misconception’ in our study, they still tended to view mental illness as weakness subconsciously and thus showed more tolerance to those people. A previous study among rural Indian citizens suggested that Indians were generally willing to be a friend, neighbour or workmate of the mentally ill; and the belief that ‘agreeing mental problems were caused by personal weakness’ was positively associated with their intention to reduce social distance . These two explanations are a bit contradictory and hence, further studies are needed to test and clarify the differences, and to explore the underlying mechanisms. However, both explanations suggest that compared to Chinese, Malays and Indians in Singapore lack knowledge of mental illness, which indicates a need for future informational campaigns.
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| 99.94 |
Other predictors of public attitudes included marital status, employment status, and personal income. Being a housewife/homemaker was associated with more social distancing, less tolerance and support for community care, and higher social restrictiveness towards mentally ill. Being unemployed was associated with higher tolerance and support for community care; and being a student predicted less social restrictiveness towards people with mental illness. Being unmarried, unemployed, and being a student were associated with less prejudice and misconception towards those with mental illness; while having a monthly income less than SGD 2,000 predicted more prejudice and misconception. Marital status is usually related to age, with younger adults being less likely to be married ; this also applies to individuals who were students. In this sense, the association between being unmarried or a student and ‘prejudice and misconception’ might be simply caused by the effect of age. Further examination of our data suggested that about 88% of the housewives/homemakers in our study had a monthly income lower than SGD 2,000. In this case, being a housewife/homemaker or having a monthly income less than SGD 2,000 could be viewed as lower socio-economic status. This is consistent with findings that suggest people with low socio-economic status are much less tolerant of mentally ill patients [37, 38]. Our study also found that being unemployed was correlated with more positive attitudes towards mental illness. However, this is not consistent with findings from another study which found adults who were unemployed or unable to work to be more likely to show negative attitudes .
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study
| 99.94 |
There are some limitations to the study. Firstly, the cross-sectional design precluded us from drawing conclusions on causal-relationships. Secondly, although the interviewer-administered questionnaire could ensure the response quality, respondents might alter their response to avoid embarrassment in the presence of an interviewer or they may be reluctant to reveal beliefs unlikely to be endorsed by the interviewer i.e. there may have been some social desirability bias . Thirdly, previous studies suggested that people tended to have different attitudes towards different mental illnesses . However, in our study, the participants were asked to answer the questionnaire based on the general term, ‘mental illness’. As a result, their responses were highly dependent on how they interpreted this term, and thus might be inconsistent across the sample. Lastly, although several robust strategies were employed to ensure the conceptual equivalency of the assessment instruments in different languages, it is still possible that languages differences contributed to some of the significant findings in our study (e.g. difference by ethnicity groups).
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study
| 100.0 |
These limitations notwithstanding, this is the first study that has systematically studied AMI outside UK. Based on rigorous methodologies, a different factor structure was identified among the Singapore sample, which indicates potential cultural differences between Western and Asian populations in their perceptions of mental illness. It also has a large sample size with a good overall response rate (71.1%) which is representative of the general population. Lastly, before applying the questionnaire to the local population, necessary changes were made based on cognitive interviews to ensure its comprehension and local relevance.
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study
| 99.94 |
The current study confirmed the multidimensionality of the public’s attitudes to mental illness, and it also identified several key characteristics associated with negative attitudes to mental illness. Risk factors for negative attitudes include older age, male gender, Chinese ethnicity, lower education and lower socioeconomic status. For Indian and Malay ethnicities, although they tended to show more positive attitudes on social distancing, tolerance/support for community care, and social restrictiveness; they also had more prejudice and misconception towards the mentally ill. Tolerance or support to community care was positively associated with being female, Indian and with higher education level; while negatively associated with being a housewife or homemaker. The potential difference between Western and Asian population in how they perceived mental illness also suggested that there is need for well-planned and culturally sensitive public attitude campaigns. Future studies could explore cultural differences and how these differences might affect public attitudes.
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study
| 99.94 |
A holobiont is defined as a meta-organism consisting of a host (e.g., animal, plant, alga) and its associated microbiota. A healthy holobiont is considered to have a resilient microbiota, which may adapt and respond differently depending on external disturbances . The importance of the holobiont concept is increasingly coming into the focus of ecological and clinical research due to the major role of, for instance, digestive microbiota in host digestion, host health, and host development [2–5]. For instance, the ruminant gastro-intestinal tract contains specific bacterial groups able to degrade complex plant cell-wall polysaccharides such as cellulose, hemicelluloses, and pectins, allowing polymer assimilation during digestion and accounting for 70% of the host energy intake .
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| 99.8 |
In the marine environment, herbivores feed on macroalgae which live associated with an epiphytic microbiota. These play major roles in health and physiology of macroalgae , such as host adaptation to varying environmental conditions , bacteria-induced morphogenesis [9, 10], or via their antifouling activity . Reciprocally, algal biomass constitutes a large choice of carbon sources for heterotrophic bacteria, especially polysaccharides, which represent about 50% of the algal dry weight. Red, green, and brown algae each have a different polysaccharide composition and most of the macroalgal polysaccharides are absent from land plants [12, 13]. To feed on seaweeds, marine heterotrophic bacteria have evolved specific enzymes such as agarases, carrageenases, fucoidanases, or ulvan lyases [14, 15]. Therefore, the macroalgal microbiota is significantly enriched in algal polysaccharide-degrading bacteria (mainly Bacteroidetes and Gammaproteobacteria) in comparison to the water column . Notably, the macroalga-associated bacterial community differs between red, green, and brown algae [17–20].
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| 99.94 |
Most marine herbivores are generalists, meaning they are able to feed on the three macroalgal lineages, but only few studies have been performed on digestive microbiota in marine herbivores. Bacteria have been shown to help degrade macroalgal polysaccharides in the sea hare Aplysia spp., the snail Tegula funebralis, the limpet Patella pellucida, and the iguana Amblyrhynchus cristatus [21–24], but these studies considered neither different diet types nor seasonality. The abalone, a gastropod of high economic interest especially in China, Korea, South Africa, and Chile [25, 26], is also a generalist herbivore , but its digestive enzymes are not sufficient for macroalgal polysaccharide degradation . This makes the abalone an interesting model to investigate the composition, seasonality, and role of the digestive microbiota associated with several diet types.
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| 88.56 |
We selected four local monospecific macroalgal diets based on abalone food preferences, nutritional quality, and on their biochemical composition: the brown algae Saccharina latissima and Laminaria digitata, the red alga Palmaria palmata, and the green alga Ulva lactuca. We determined the composition of the microbiota from the abalone digestive gland, and we tested the following hypotheses: (i) the marine herbivore gastrointestinal tract offers a specific niche for specific marine bacteria, (ii) algal diet, host characteristics, and time influence microbiota composition and fluctuations, (iii) specific microbial groups are associated with the digestion of a monospecific macroalgal diet, allowing for the abalone’ polyphagy.
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| 100.0 |
Here, we investigate the impact of four monospecific algal diets on the abalone digestive microbial community. For each of the four algal diets, abalone reared in 3 independent cages were sampled at 6 time points over a year and the abalone digestive microbiota from the resulting 72 samples was further studied through a metabarcoding analysis.
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| 100.0 |
Operational taxonomic unit (OTU, defined at 97% sequence identity) richness and evenness of the digestive microbiota were investigated for each algal treatment and sampling date. When all sampling dates were pooled, alpha-diversity did not significantly vary between algal diets on average, but when algal diets were considered separately, alpha-diversity followed different seasonal patterns. In the case of the L. digitata diet, samples from colder months (February–April 2012, January 2013) presented significantly lower alpha-diversity indices on average than those from warmer months (Wilcoxon test, P < 0.001, Additional file 1: Figure S1, Additional file 2: Table S1). In the case of the S. latissima and U. lactuca diets, diversity increased over the year and diversity seemed stable with time for the P. palmata treatment.
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| 100.0 |
Similarly, when the structure of the bacterial community associated with the abalone digestive gland was investigated at the OTU level (Fig. 1), diet had only a minor impact on the bacterial community (ANOSIM, R = 0.05, P < 0.01, Additional file 3: Table S2). However, there was a clear grouping of samples according to season and date (R = 0.36 and R = 0.43, respectively, P < 0.01). When considering each monospecific diet individually, bacterial communities associated with P. palmata digestion were more different between sampling dates than between the cage triplicates (ANOSIM: R = 0.41–1). Bacterial communities associated with the U. lactuca diet showed noticeable differences between the beginning of the experiment (February–April 2012) and January 2013 (R = 0.83). Seasonal effects were stronger for the L. digitata diet than for the other diets, with samples from colder months separated from those from warmer months. For the microbial communities associated with the S. latissima diet, bacterial communities sampled in January 2013 were significantly different from other dates (R > 0.8, P < 0.001). Overall, diversity patterns of the abalone digestive microbiota showed seasonal patterns specific to a given monospecific diet.Fig. 1Community structure of the digestive microbiota of the abalone over 1 year. Samples were grouped a posteriori according to one of the four monospecific algal diets (a) or a sampling date (b). The Bray-Curtis dissimilarity index was calculated after Hellinger transformation of the metabarcoding data set at the OTU level. The low stress value of 18% validates the goodness-of-fit of the two-dimensional representation compared with the original matrix. Significance between groupings of samples according to algal diet, sampling date, or season was tested using ANOSIM (Additional file 3: Table S2)
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| 100.0 |
Community structure of the digestive microbiota of the abalone over 1 year. Samples were grouped a posteriori according to one of the four monospecific algal diets (a) or a sampling date (b). The Bray-Curtis dissimilarity index was calculated after Hellinger transformation of the metabarcoding data set at the OTU level. The low stress value of 18% validates the goodness-of-fit of the two-dimensional representation compared with the original matrix. Significance between groupings of samples according to algal diet, sampling date, or season was tested using ANOSIM (Additional file 3: Table S2)
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| 100.0 |
The distribution of OTUs linked to a monospecific diet was investigated by measuring proportions of OTUs shared between two consecutive sampling dates. For instance, in the digestive microbiota associated with the P. palmata diet, there were 29–40% OTUs common to abalone sampled in February 2012 and those sampled in April 2012, meaning that there were about 60–71% new OTUs after 2 months of monospecific diet (Additional file 1: Figure S2A). Over the year, the proportions of OTUs common to two consecutive sampling dates varied between 16–59%, 18–70%, 23–61%, and 10–71% for the P. palmata, U. lactuca, L. digitata, and S. latissima diets, respectively. These common OTUs, however, accounted for 40–99% of the sequence reads (Additional file 1: Figure S2). About 3% OTUs were present the whole year for each monospecific diet and corresponded to a large proportion of sequence reads (from 88–97% in each cage). Altogether, this indicates that the rarest OTUs fluctuate with time while the most abundant OTUs remain in the digestive gland during the year, indicating the presence of a dominant core microbial community associated with each algal diet.
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In February 2012, after abalone had not been fed for a week, the most abundant phyla were Fusobacteria, Tenericutes, and Gammaproteobacteria (average of 72, 14, and 9% of all sequence reads per cage, respectively, Additional file 1: Figure S3). The phylum Fusobacteria was mostly composed of the genus Psychrilyobacter (99.9% of sequences in the phylum; 31% to 94% of all sequence reads per cage) and the phylum Tenericutes of Mycoplasma (99.9% of sequences in the phylum; 3–32% of all sequence reads per cage). The third most abundant taxonomic group, the class Gammaproteobacteria, was composed mainly of Vibrio (68% of sequence reads in the phylum; 1–23% of all sequence reads per sample). Other genera present in all cages represented 2% or less of the sequence reads per cage (Fig. 2). These global patterns validate the dominance of few genera in the abalone digestive microbiota at the starting point of the experiment.Fig. 2Relative abundance and taxonomical composition of abalone digestive microbiota at the genus level. Each panel represents the digestive bacterial community associated with feeding with Palmaria palmata (a), Laminaria digitata (b), Ulva lactuca (c), and Saccharina latissima (d). The taxonomical composition is represented for each cage triplicate over 1 year. Water temperature is indicated on the right side of each panel (values varying from 10 °C (blue) to 15 °C (red)). Less abundant genera: total number of sequence reads of these 651 genera for clearer representation in the figure. In c, the sample from cage U1 in January 2013 is missing. For unclassified genera, genera were assigned to the lowest taxonomic level identified. Verruc., Verrucomicrobiales; BD7-8, BD7-8 marine group; JTB255, JTB255 marine benthic group; OM60_clade, Alteromonadaceae OM60_clade; Gammap., Gammaproteobacteria; unc, unclassified
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| 100.0 |
Relative abundance and taxonomical composition of abalone digestive microbiota at the genus level. Each panel represents the digestive bacterial community associated with feeding with Palmaria palmata (a), Laminaria digitata (b), Ulva lactuca (c), and Saccharina latissima (d). The taxonomical composition is represented for each cage triplicate over 1 year. Water temperature is indicated on the right side of each panel (values varying from 10 °C (blue) to 15 °C (red)). Less abundant genera: total number of sequence reads of these 651 genera for clearer representation in the figure. In c, the sample from cage U1 in January 2013 is missing. For unclassified genera, genera were assigned to the lowest taxonomic level identified. Verruc., Verrucomicrobiales; BD7-8, BD7-8 marine group; JTB255, JTB255 marine benthic group; OM60_clade, Alteromonadaceae OM60_clade; Gammap., Gammaproteobacteria; unc, unclassified
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Over a year, 10–15% OTUs were shared by the 4 monospecific diets. For all sampling dates pooled per algal diet, 22% of the OTUs of the whole community corresponded to a core community shared by the 4 diets (Fig. 3). To better understand the potential functioning of this core community, we focused on temporal fluctuations of these OTUs constantly found in the digestive microbiota. We considered as the core microbiota only the OTUs occurring in all samples, considering each of the four algal diets, for each cage triplicate and during the whole year. Six OTUs belonging to the classes Fusobacteriia, Gammaproteobacteria, and Mollicutes fit this definition: one Psychrilyobacter OTU, two Vibrio OTUs, two Mycoplasma OTUs, and an unclassified OTU. In terms of sequence read abundance, these six OTUs represented about 86% of the microbiota (Fig. 4), although relative contribution of each of these OTUs was variable (Fig. 2). Apart from these 3 dominating core genera, 5 to 8 additional genera belonged to a core microbiota specific to each algal diet and they represented lower relative abundances of OTUs, ranging from 0.001 to 24.5% of sequence reads per cage (Fig. 4). Some OTUs belonged to genera found with at least two monospecific diets: Lutimonas, Psychromonas, Roseobacter, and four unclassified genera. Other genera were specific to a given diet. Polaribacter and Pseudahrensia were associated with the P. palmata diet, Escherichia-Shigella to the U. lactuca diet, Roseibacillus to the L. digitata diet, and Ulvibacter was associated with the S. latissima diet (Fig. 4).Fig. 3Comparison of the microbial OTU composition associated with the four algal diets. Comparisons are described by proportions of shared or unique OTUs and were made for each sampling date from February 2012 to January 2013 and for the six sampling dates pooled according to diet. D, L. digitata; S, S. latissima; P, P. palmata; U, U. lactucaFig. 4Composition of the digestive core microbiota at the genus level associated with a monospecific diet. Pie charts on the left represent proportions of sequence read abundances, at the genus level, representative of the core microbiota of a monospecific diet. Pie charts on the right represent detailed proportions of less abundant genera representative of the core microbiota of a monospecific diet
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Comparison of the microbial OTU composition associated with the four algal diets. Comparisons are described by proportions of shared or unique OTUs and were made for each sampling date from February 2012 to January 2013 and for the six sampling dates pooled according to diet. D, L. digitata; S, S. latissima; P, P. palmata; U, U. lactuca
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| 100.0 |
Composition of the digestive core microbiota at the genus level associated with a monospecific diet. Pie charts on the left represent proportions of sequence read abundances, at the genus level, representative of the core microbiota of a monospecific diet. Pie charts on the right represent detailed proportions of less abundant genera representative of the core microbiota of a monospecific diet
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| 99.94 |
To further explore the potential role of Psychrilyobacter, Vibrio, and Mycoplasma in algal digestion, we compared their carbohydrate-active enzymes (CAZymes) and sulfatase contents to that of Zobellia galactanivorans DsijT, a model bacterium for the bioconversion of algal polysaccharides . This was done based on the public CAZy and SulfAtlas databases (Table 1, Additional file 4: Table S3 and Additional file 5: Table S4). This approach is of course limited by the availability of genomes of some genera in public databases. Still, similar results were found in several available genomes of a given genus and are likely to reflect at least some functions present in abalone digestive microbiota. As there was no Psychrilyobacter genome available in the CAZy and SulfAtlas databases, we manually annotated the CAZyme- and sulfatase-coding sequences in the only genome of the genus available to date in NCBI Genome , i.e., that of Psychrilyobacter atlanticus DSM 19335, a strictly anaerobic bacterium isolated from marine sediment . Compared to the genome of Z. galactanivorans DsijT, the genome of P. atlanticus DSM 19335, contains far fewer GHs and sulfatases as well as no polysaccharide lyases (Table 1, Additional file 5: Table S4). These genes are likely to enable the degradation of extracellular oligosaccharides, but not the degradation of complex algal polysaccharides (Additional file 5: Table S4). To evaluate the ability of Vibrio to degrade algal polysaccharides in the digestive gland, we examined the CAZyme and sulfatase content in the 127 Vibrio genomes available in the CAZy and SulfAtlas databases to date (Additional file 4: Table S3 and Additional file 5: Table S4). The 127 genomes present lower numbers of CAZyme and sulfatase families than the Z. galactanivorans genome (Table 1, Additional file 4: Table S3 and Additional file 5: Table S4). However, all examined Vibrio genomes contain at least one enzyme involved in chitin and/or cellulose metabolism (GH9, GH18, GH19, GH20, GH94 families) and at least one enzyme degrading extracellular oligosaccharides. Some Vibrio genomes such as V. crassostreae 9CS106, V. breoganii FF50, V. owensii XSBZ03, V. halioticoli NBRC 102217, three strains of V. alginolyticus (ATCC 33787, K08M4, and ATCC 17749), and V. harveyi also are able to degrade the red algal polysaccharide agarose (aga genes from families GH16, GH50), laminarin (lam genes family GH3), and/or alginate (alyA genes from families PL6, PL7, PL15, PL17) from brown algae (Additional file 4: Table S3, ). For Mycoplasma, we examined 111 genomes, which show poor capacities to degrade complex carbohydrates and no ability to degrade algal polysaccharides (Additional file 5: Table S4, ).Table 1Total number of putative glycoside hydrolase (GH), polysaccharide lyase (PL), and sulfatase genes in selected genomesGHaPLaSulfatasesReferencesPsychrilyobacter atlanticus DSM 1933513b23c0002127 Vibrio genomes9–2722–700–50–210–50–9[33, 34]111 Mycoplasma genomes0–60–120–10–100Z. galactanivorans DsijT441418151771This table also contains total number of GH, PL, and sulfatase families in selected genomes from the three genera of the core digestive microbiota of the abalone and of Zobellia galactanivorans DsijT, a model bacterium for algal polysaccharide degradation. For more details on the CAZyme and sulfatase content, see Additional file 4: Table S3 and Additional file 5: Table S4aCAZyme classbTotal number of CAZyme or sulfatase families in the given classcTotal number of CAZyme or sulfatase genes in the given classZ. galactanivorans Zobellia galactanivorans; GH glycoside hydrolase, PL polysaccharide lyase. Data retrieved from the CAZy and SulfAtlas databases (www.cazy.org, , http://abims.sb-roscoff.fr/sulfatlas/, )
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| 99.94 |
This table also contains total number of GH, PL, and sulfatase families in selected genomes from the three genera of the core digestive microbiota of the abalone and of Zobellia galactanivorans DsijT, a model bacterium for algal polysaccharide degradation. For more details on the CAZyme and sulfatase content, see Additional file 4: Table S3 and Additional file 5: Table S4
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| 99.94 |
Among the five to eight additional core genera per diet, we only found genomes of the genera Roseobacter, Psychromonas (two genomes each), and Polaribacter (six genome) in the CAZy and SulfAtlas databases, and we searched for their algal polysaccharide degradation abilities (Fig. 4, Additional file 4: Table S3). Roseobacter (a genus only found associated with the two brown algal diets) shows a low capacity to degrade algal polysaccharides with only some secreted exo-glycosidases. Psychromonas (associated with the L. digitata and P. palmata diets) is likely able to degrade brown algal polysaccharides with several enzymes acting on extracellular oligosaccharides and a laminarinase from family GH16, but there was no sign of enzymes acting on red algal polysaccharides. All six genomes of the genus Polaribacter, associated with the P. palmata diet, contain enzymes acting on red algal polysaccharides, such as agar or porphyran (e.g., enzymes from families GH16, GH86, or GH117), or more specifically on carrageenans (P. sp. KT25b contains 2 iota-carrageenases from family GH82). Most of these genomes also encode enzymes able to degrade major algal cell wall polymers such as cellulose and hemicelluloses (e.g., presence of an endo-1,4-β-xylanase from family GH10, a GH39 xylosidase, and enzymes from families GH43, GH51, GH53, GH115, GH127).
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| 100.0 |
Potential relationships between the structure of the digestive microbiota and contextual parameters were investigated by applying a multivariate variation partitioning approach. Biological variation of the digestive microbiota was significantly linked to the pure effect of diet composition (adjusted R2 = 5.4%, P < 0.05). Sampling dates, the combined effect of abalone and algal composition, the combined effect of algal composition and sampling dates, and the effect of the three explanatory variables together also explained the structuring of the microbial community (adj. R2 = 2.2%, 7.4%, 12.2%, 2.5%, respectively, Additional file 1: Figure S4). Overall, 30.8% of the biological variation of the microbial community structure was explained by abalone characteristics, algal composition, and sampling dates (P < 0.001, Additional file 1: Figure S4). The remaining 69.2% of the biological variation not explained by the selected model suggests the effect of additional variables not included in this analysis. Still, our analysis clearly demonstrates a relationship between algal diet composition, sampling dates, and the structure of the digestive microbiota.
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Pairwise comparisons between individual bacterial genera from the core digestive microbiota (Fig. 4) and contextual parameters showed specific ecological patterns. The genera Psychrilyobacter, an unclassified Flavobacteriaceae, and Ulvibacter followed similar patterns. They increased in relative OTU abundances with a decreasing content in mostly the same algal biochemical compounds and with increasing abalone digestive gland weight. Mycoplasma also showed similar patterns related to abalone characteristics. OTU proportions of Vibrio, Psychromonas, an unclassified Rhodobacteraceae, Polaribacter, and Pseudahrensia likely increased with increasing temperature and no significant relationships were found with other parameters (Fig. 5).Fig. 5Pairwise comparison between genera of the core microbiota and contextual parameters. Pairwise comparisons were measured by Spearman correlation. The core microbial genera were ordered according to sequence read abundance; with Psychrilyobacter being the most abundant group. The strength of the correlation is indicated by a heatmap colored from red to blue. The horizontal axis corresponds to sampling dates, temperature, abalone characteristics and algal diet composition. Only significant correlations after application of the Benjamini-Hochberg correction for multiple testing were represented. GDG, gonado-digestive gland; DG, digestive gland; G, gonad. Unc, unclassified
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| 100.0 |
Pairwise comparison between genera of the core microbiota and contextual parameters. Pairwise comparisons were measured by Spearman correlation. The core microbial genera were ordered according to sequence read abundance; with Psychrilyobacter being the most abundant group. The strength of the correlation is indicated by a heatmap colored from red to blue. The horizontal axis corresponds to sampling dates, temperature, abalone characteristics and algal diet composition. Only significant correlations after application of the Benjamini-Hochberg correction for multiple testing were represented. GDG, gonado-digestive gland; DG, digestive gland; G, gonad. Unc, unclassified
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In holobionts, the digestive enzymes of the host do not allow a complete degradation of complex polysaccharides which suggests the importance of a digestive microbiota. Marine generalist herbivores such as the abalone face the challenge of feeding on the three marine macroalgal lineages, which are composed of different combinations of neutral, carboxylated or carboxylic-bearing and sulfated polysaccharides . Here, we determine the taxonomic composition and the seasonal structure of the digestive microbiota of the abalone holobiont, a marine gastropod grown in natural conditions on four monospecific diets over a year. This long-term study revealed that (i) the marine herbivore gastrointestinal tract offers a specific niche for a dominant core microbiota, constitutive of the abalone holobiont, (ii) microbiota composition and fluctuations are correlated to seasonal variations of contextual parameters, and that (iii) few specific microbial groups were associated with a monospecific macroalgal diet.
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Three bacterial core genera, the Fusobacteria Psychrilyobacter, the Tenericutes Mycoplasma, and the Gammaproteobacteria Vibrio, dominated the abalone digestive microbiota for the whole year and for each of the monospecific algal diet. Bacteria from these genera are known as obligate (Psychrilyobacter) or facultative anaerobes (Mycoplasma and Vibrio) [31, 34, 35]. This unexpected dominance of a core microbiota through the entire year may not only be due to the initial diet composition but also to gut morphology and its physicochemical and biochemical conditions . The abalone gastrointestinal tract offers stable environmental conditions, which are microaerophilic or anaerobic, with an acidic pH of 5.3–6 in the crop and the digestive gland [36, 37]. It has been previously shown that an acidic pH together with the presence of volatile fatty acid in the rumen is toxic for some bacteria and thus likely to select for bacteria adapted to such conditions. Indeed, anaerobic bacteria from the classes Fusobacteriia and Mollicutes are only rarely found in coastal waters or associated with macroalgae [39–41]. However, these bacterial groups seem rather typical of digestive organs of marine holobionts as they are found in the sea squirt Ciona intestinalis, the limpet Patella pellucida, and marine carnivores [21, 42, 43]. Members of Vibrio are well known as pathogen of animals, and it is the most common bacterial genus in guts of marine animals such as the intestinal mucosa of marine fishes [35, 44–46]. Vibrio is also found associated with algae such as Ascophyllum nodosum, S. latissima, and decomposing fronds of Laminaria spp. [11, 16, 47]. Other gut microbiota also contain dominant core communities, e.g., the human gut microbiota is dominated by anaerobic bacteria belonging to Firmicutes and Bacteroidetes, representing more than 98% of 16S rRNA sequences, with few other anaerobes from other phyla and only a couple of aerobic Actinobacteria [6, 48, 49]. Gut microbiota of terrestrial gastropods are also dominated by a low diversity of anaerobes, including the Gammaproteobacteria Enterobacter, some Firmicutes, and Bacteroidetes [50, 51]. Therefore, the dominant digestive microbiota constitutive of the abalone holobiont comprises bacterial taxa typically found in marine environments and these taxa are not dominant in the gut of terrestrial holobionts.
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| 97.25 |
Additionally, a less abundant core microbiota was specific to at least a monospecific diet and contained aerobic genera: the Flavobacteriia Lutimonas, Ulvibacter, and Polaribacter; the Alphaproteobacteria Roseobacter and Pseudahrensia; the Gammaproteobacteria Psychromonas and Escherichia-Shigella; and the Verrucomicrobia Roseibacillus [52–59]. Notably, all members of this diet-specific and less abundant core microbiota, with the exception of Pseudahrensia, were identified during a phytoplankton bloom in temperate coastal waters in the North Sea . This suggests that these bacteria may not only be part of the epiphytic algal diet microbiota but could also be part of the bacterioplankton and be ingested along with the alga. For instance, members of the genera, Roseobacter, Ulvibacter, and Roseibacillus, were previously isolated from green and brown algae, and Roseobacter was found associated with the red alga Gracilaria vermiculophylla [19, 53, 55, 58]. Strains of Polaribacter and Psychromonas were previously found associated with Laminaria sp. and S. latissima, and both genera have members that can degrade red algal polysaccharides [54, 57, 60, 61]. Lutimonas and Pseudahrensia, however, were found only in marine invertebrates or seawater but not associated with algae [52, 56, 62–64]. The occurrence and maintenance over time of few diet- or plankton-derived specific bacteria suggests a selectivity of some genera, which could participate to the global functioning of the digestive gland in abalone. The remaining OTUs that are not part of the core microbiota are transient bacteria and represent a smaller fraction of the digestive microbiota over the year (up to 14% of sequence reads per cage). These rarer bacteria are probably ingested along with the algal diet and have either (i) died and left DNA remains in the digestive gland or (ii) survived the selection pressure and may participate in the algal recycling in the abalone digestive gland .
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| 99.94 |
Such organization of the microbiota in abalone digestive gland consists of two groups of bacteria: one resident group comprising the dominant bacteria and another one comprising the less abundant diet-specific bacteria and rarer bacteria which are transient over the year. Dominance of few bacterial taxa in the digestive microbiota may contribute to holobiont health and, as proposed in the case of human gut microbiota, such stability may allow for a certain resiliency in response to invasive pathogens or other exogenous microbes [1, 3]. Further, the presence of less abundant taxa may also participate in abalone holobiont health. Previous studies on human lung- or plant-associated microbes showed that rare species were also involved in host protection against pathogens [66, 67]. On another hand, in a case of a disturbance strong enough to destabilize the core microbiota, these rarer OTUs may become more abundant and fill the niche previously occupied by the dominant bacterial community. They can either fill the same functions as the core microbiota and contribute to holobiont health or become pathogens [3, 68].
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| 99.9 |
Our experiment was carried out in an abalone farm located at the French Brittany coast where waters are impacted by seasonal variations specific to this permanently mixed ecosystem [69, 70]. These temperate coastal waters were coldest in February–April 2012 and January 2013 (10–11.5 °C) and warmest from June to October 2012 (12.5–15 °C, Additional file 6: Table S5). Several studies in temperate coastal areas showed seasonal succession and structuring of the bacterial community [39, 41, 71]. Accordingly, even if three genera were dominating for the entire year, the community structure of abalone digestive microbiota was impacted by seasonal variability (Fig. 1), and 17.6% of the biological variation of the microbiota was explained by the effect of sampling dates alone and by its combined effect with abalone characteristics and algal biochemical content. We found one study on seasonality of intestinal microbiota in a marine organism, the farmed Atlantic salmon, which showed highest number of bacteria in the gut with the highest temperature in August but a community composition that was quite stable over the year . More temporal studies were done in fresh water, where several examples of seasonality of fishes’ digestive microbiota could be observed either in terms of cell number or taxonomic composition . In our study, however, the biochemistry of the four algae showed significant seasonal variations through the year, as tested with their content in soluble carbohydrates, dry algal matter and protein (Friedman test with post hoc test after Nemenyi, Roussel, personal communication), which had a significant effect on the digestive microbiota, explaining 5.4% of the biological variation of the microbiota (Additional file 1: Figure S4). Also, individual microbial genera of the dominant core and the less abundant microbiota were differently correlated with temporal fluctuations of contextual parameters (Fig. 5). For instance, the presence of Vibrio, Psychromonas, an unclassified Rhodobacteraceae, Polaribacter, and Pseudahrensia was positively correlated with increasing water temperature (Fig. 5). Seasonal variations have been previously described for Vibrio and Polaribacter in the plankton and in particle-attached fractions in coastal waters [41, 74], but no study could be found on seasonality in coastal waters of Psychromonas and of Pseudahrensia. Seasonal fluctuations of Vibrio and the less abundant and diet-specific core microbiota may thus be not only influenced by algal diet biochemical content but also by the seasonality of the water column composition.
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| 100.0 |
A major factor that may explain part of the unexplained 69.2% of biological variation is microbial interactions. Following niche construction theory, microbiota play a role in modifying physicochemical conditions of the digestive gland and apply selection pressure to themselves, their descendants, and immigrating cells . Here, the selection of the holobiont microbiota may result in the cooperation between degraders of algal polysaccharides and fermenters of the resulting products in microaerophilic and acidic conditions. Indeed, aerobic bacteria ingested with the diet have been shown to rapidly consume all the available oxygen in the gastrointestinal tract, providing ideal conditions for fermentation of pyruvate from algal polysaccharide degradation [36, 76]. Genomes closely related to Psychrilyobacter, Mycoplasma, and Vibrio possess enzymes for the acetate pathway, allowing pyruvate fermentation to short chain fatty acids (SCFA) for further assimilation by the host (Additional file 5: Table S4, Additional file 8, Fig. 6). As hypothesized for gut colonization of the sea squirt Ciona intestinalis , these dominant fermenters, may be part of a resident anaerobic biofilm within the outer mucus layer at the epithelial cell surface and benefit from the host digestive properties such as the secretion of mucin-like glycoproteins .Fig. 6Proposed processes of algal polysaccharide degradation in the abalone digestive gland. For information, localization of the abalone digestive gland on an abalone without the shell (a). The schematic illustration recapitulates hypothetical processes of algal polysaccharide degradation in the case of a brown algal cell wall (b). The algal diet comes along with epiphytic bacteria, including (1) strictly aerobic bacteria which may die due to specific physiochemical conditions (low O2, low pH) and (2) facultative aerobic bacteria, which may act as primary degraders of complex algal polysaccharides. Primary degraders may (3) directly ferment polysaccharides to short-chain fatty acids (SCFA, e.g. Vibrio) or (4) transform polysaccharides into pyruvate or solubilized mono- and oligosaccharides (members of Flavobacteriia, Alpha-, and Gammaproteobacteria). These may then be (5) fermented by strictly or facultative anaerobic bacteria (e.g. Psychrilyobacter, Mycoplasma), which are probably localized in an anaerobic or microaerophilic part of the gland, such as the epithelial mucus. Resulting SCFA are then (6) assimilated by the host. Parts of the illustration are inspired from [6, 96]. Abalone picture: courtesy of Monique Ras
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| 98.56 |
Proposed processes of algal polysaccharide degradation in the abalone digestive gland. For information, localization of the abalone digestive gland on an abalone without the shell (a). The schematic illustration recapitulates hypothetical processes of algal polysaccharide degradation in the case of a brown algal cell wall (b). The algal diet comes along with epiphytic bacteria, including (1) strictly aerobic bacteria which may die due to specific physiochemical conditions (low O2, low pH) and (2) facultative aerobic bacteria, which may act as primary degraders of complex algal polysaccharides. Primary degraders may (3) directly ferment polysaccharides to short-chain fatty acids (SCFA, e.g. Vibrio) or (4) transform polysaccharides into pyruvate or solubilized mono- and oligosaccharides (members of Flavobacteriia, Alpha-, and Gammaproteobacteria). These may then be (5) fermented by strictly or facultative anaerobic bacteria (e.g. Psychrilyobacter, Mycoplasma), which are probably localized in an anaerobic or microaerophilic part of the gland, such as the epithelial mucus. Resulting SCFA are then (6) assimilated by the host. Parts of the illustration are inspired from [6, 96]. Abalone picture: courtesy of Monique Ras
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other
| 72.0 |
Regarding algal polysaccharide degradation in abalone digestive gland, two scenarios are possible, involving either (i) the core dominant community only, or (ii) the core dominant community together with the less abundant and the transient community (Fig. 6). The first scenario is based on putative lateral acquisitions of CAZymes by the three dominant genera of the abalone digestive microbiota, e.g., from algal epiphytic bacteria. Earlier studies have shown the possibility of horizontal transfer of CAZyme-genes (e.g., beta-porphyranases, GH117, alginate lyases) from algal epiphytic bacteria to intestinal bacteria from human or marine fishes in relation to the consumption of seaweeds [78–80]. In this scenario, the less abundant core microbiota and transient bacteria would play only a complementary role in algal degradation. The second scenario assumes that the dominant bacteria in the microbiota are largely incapable of degrading complex algal polysaccharides, as suggested by their low CAZyme and sulfatase content found in our survey. Only Vibrio may possess a limited capacity to degrade algal polysaccharides as suggested by the analysis of V. crassostreae and V. breoganii genome, which are often found in the gut of invertebrates, and genomes of the species V. crassostreae, V. harveyi, and V. halioticoli which were previously isolated from abalone gut and described as alginate degraders [44, 81]. Also, V. halioticoli isolated from abalone are able to produce SCFA such as acetate from alginate (Additional file 5: Table S4 and Additional file 8, [16, 81, 82]). The less abundant and diet-specific core microbiota may complement the capacities of Vibrio as they also have the potential to degrade algal polysaccharides, e.g., Polaribacter, Roseobacter, or Psychromonas [54, 55, 60, 81].
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| 100.0 |
In either of the two scenarios, the abalone holobiont seems to provide the ideal conditions in the digestive gland to maintain a core microbiota which probably plays a major role in producing assimilable products for the abalone from complex dietary compounds such as algal polysaccharides.
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| 99.9 |
The most striking finding here is that the abalone digestive microbiota seems to be already established during the first year of life of the abalone and that seasonal variation, feed type, and quality only account for a small proportion of the variability of the microbiota. This is in agreement with the existence of a relatively stable digestive microbiota, inherent part of the holobiont. In the case of a healthy abalone holobiont, digestive gland conditions may control the composition of the microbiota, and in return, this microbiota may be adapted to seasonal variations. In addition, transient and rare algal epiphytic bacteria may remain in the niche, depending on their ability to assist in algal polysaccharide degradation. In the abalone holobiont, two non-exclusive scenarios may occur during digestion: (i) the dominant fermenters have acquired polysaccharide degrading genes by lateral gene transfer from aerobic epiphytic algal microbiota, and degrade and ferment algal polysaccharides themselves, (ii) the aerobic epiphytic algal microbiota cooperate with the dominant fermenters to transform algal polysaccharides to SCFA. To test these hypotheses and to elucidate further details on the functional plasticity of the dominant fermenters or the aerobic microbiota, we would need to investigate microbial genes associated with abalone digestion, using metagenomic tools or trying to isolate candidates and further analyze their genomes.
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| 100.0 |
Altogether, these results on a marine generalist herbivore further highlight the specificity and durability of the holobiont association, between the host and its digestive microbiota, even in an open and mixing natural environment such as seawater. A better understanding of the functioning of this marine holobiont will contribute to increase our knowledge on abalone biology and to improve natural and sustainable cultivation methods.
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| 99.75 |
European abalone (Haliotis tuberculata Linnaeus, 1758), marine herbivorous gastropods, were bred at the sea-based abalone farm France Haliotis, on the coast of Brittany, France (48°36′46 N; 4°33′30 W). In summer 2010, juveniles were bred on land in nursery tanks for a year before being transferred to sea-based experimental cages and fed with a mixed algal diet composed of P. palmata, S. latissima, and L. digitata for about 6 months. Abalone were then not fed for a week before the beginning of the experiment (Additional file 1: Figure S5). The experiment started in February 2012, and three abalone were randomly sampled from each cage. Abalone were then fed ad libitum every month with one of the monospecific diets: P. palmata, U. lactuca, L. digitata, S. latissima, and this was replicated in three cages for each algal treatment. About every 2 months from February 2012 to January 2013, 3 abalone were taken randomly from each of the 12 cages and the resulting 216 abalone were frozen at − 80 °C until further analysis of the digestive bacterial microbiota (Additional file 1: Figure S6). On the same dates, additional abalone were sampled for measurements of growth and sexual maturation and the fresh algal diet was sampled before abalone feeding to subsequently analyze algal biochemical composition (see Additional file 8 for details).
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| 100.0 |
As it is closely surrounded by the gonads, the digestive gland could not be separated from the gonads during dissection. The gonado-digestive gland (simplified as the digestive gland throughout the manuscript) of 216 abalone was thus dissected as a whole and was freeze-dried before tissue grinding (Additional file 1: Figure S7). DNA was extracted from ground lyophilized glands and the V3-V4 variable region of the 16S rRNA gene was amplified using primers targeting Bacteria. Amplicons from three abalone of the same conditions (same cage and same sampling date) were then pooled before MiSeq paired-end sequencing (Illumina, San Diego, CA, USA), which resulted in about 10 million sequence reads. Sequence quality check, curation, alignment, and chimera removal were done using publicly available scripts (fastx_toolkit, http://hannonlab.cshl.edu/fastx_toolkit/; get_paired.py, http://abims.sb-roscoff.fr, mothur v.1.34.4, ). Sequences were taxonomically classified with the RDP classifier on the Silva release 119 and OTUs were clustered at 97% sequence identity using the average neighbor algorithm (see Additional file 8 for details, ).
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study
| 100.0 |
Shannon and Simpson indices were calculated from 1000 resamplings of the original table to the lowest number of sequence reads per sample (12,997 sequence reads). Dissimilarities in community structure were studied for all taxonomic levels using the Bray-Curtis dissimilarity index after Hellinger transformation of the data set . The analysis of similarity (ANOSIM) allowed testing the grouping of samples according to sampling date, season, or algal diet.
|
study
| 100.0 |
Contextual parameters were measured in a parallel study, and the best combinations of these to explain the variation in the microbial community matrix were determined by forward selection (Additional file 8 and Roussel, personal communication). Parameters were log10-transformed, and the community matrix was standardized by Hellinger transformation . Variation partitioning was then used on the community matrix to investigate the effects of the selected parameters (abalone characteristics, algal composition, sampling date) and of their covariation on the structure of the microbiota [88, 89].
|
study
| 100.0 |
Carbohydrate active enzymes (CAZymes) and sulfatases contents from 127 genomes of the genus Vibrio, 111 genomes of Mycoplasma, 6 genomes of Polaribacter, 2 genomes of Roseobacter, and 2 genomes of Psychromonas were retrieved from the CAZY and SulfAtlas websites [32, 91–93]. No Psychrilyobacter genome was available on the CAZY website, so the Psychrilyobacter atlanticus DSM 19335 genome was downloaded from NCBI, project accession PRJNA195804 as well as genomes from the fish-associated mycoplasma Mycoplasma mobile 163K as an example of marine Mycoplasma and Vibrio halioticoli, project accession BAUJ00000000 , a Vibrio isolated from abalone . CAZyme and sulfatase annotations of each sequence were performed manually by homology with sequences from the CAZy and SulfAtlas databases, from which fasta files can be downloaded, using a minimum sequence identity of 25%, and a minimum query and hit coverage of 70% between amino-acid sequences. To investigate pyruvate to acetate fermentation, a database including sequences of the enzymes involved in the according pathways was manually created to further annotate genes from P. atlanticus, M. mobile, and V. halioticoli by homology, following the same conditions as for CAZyme gene annotation (for details on the pyruvate to acetate fermentation database, see Additional file 6: Table S5, Additional file 7: Table S6 and Additional file 8). Gene annotations were carried out using the ngKLAST software (ngKLAST release 4.2, 2007–2013, Korilog SARL, France).
|
study
| 100.0 |
Additional file 1:Figure S1. Alpha-diversity of the digestive microbiota of abalone, as described by the Shannon (left panels) and Simpson (right panels) indices. Figure S2. Fluctuations of the digestive microbiota between consecutive dates over 1 year. Figure S3. Relative abundance and taxonomical composition of the digestive microbiota at the phylum level for abalone fed on Palmaria palmata (A), Laminaria digitata (B), Ulva lactuca (C), and Saccharina latissima (D). Figure S4. Ecological patterns of the digestive microbiota explained by a model of contextual parameters. Figure S5. Experimental site and set up. Figure S6. In situ sampling procedure of abalone for each algal treatment. Figure S7. Experimental procedure for DNA extraction and library preparation for studying the abalone digestive microbiota. (DOCX 1944 kb) Additional file 2:Table S1. Wilcoxon test to compare Shannon or Simpson indices between algal diets for the whole data set or between sampling dates or seasons for each algal diet (multiple comparisons were corrected using FDR). (XLSX 22 kb) Additional file 3:Table S2. R value of the ANOSIM calculated for the whole dataset and for each algal diet dataset separately between sample groups according to season, sampling date, or algal diet. Numbers in bold indicate the significance of the comparison after Bonferroni correction. (XLSX 15 kb) Additional file 4:Table S3. Number of annotated genes for each glycoside hydrolase, polysaccharide lyase, and sulfatase family in all Vibrio, Mycoplasma, Polaribacter, Roseobacter, and Psychromonas genomes available at http://www.cazy.org/ and/or at http://abims.sb-roscoff.fr/sulfatlas/ on 19 May 2017 and in Zobellia galactanivorans DsijT (Barbeyron et al. 2017). GH, Glycoside hydrolases; PL, Polysaccharide lyases, CAZyme, Carbohydrate active enzyme. Sulfatase gene counts are indicated when available in the SulfAtlas database. (XLSX 160 kb) Additional file 5:Table S4. Genes belonging to families of glycoside hydrolases, polysaccharide lyases, or sulfatases and genes putatively involved in the pyruvate fermentation to acetate pathway in the genomes of Psychrilyobacter atlanticus DSM19335 (A), Vibrio halioticoli NBRC 102217 (B), and Mycoplasma mobile 163 K (C), when present. Annotations include gene identifiers (locus tags), closest characterized homologs with their UniProtKB ID number when available, EC numbers, and information on their functioning. The percentage of amino-acid sequence identity is indicated in parentheses. (XLSX 66 kb) Additional file 6:Table S5. Measures of abalone characteristics and algal composition from April 2012 to January 2013. See supplementary text for details. GDG, gonado-digestive gland; DG, digestive gland. (XLSX 33 kb) Additional file 7:Table S6. Fasta sequences of the pyruvate to acetate formation pathways, I, II, and IV, commonly found in Bacteria according to MetaCyc (https://metacyc.org/META/NEW-IMAGE?object=Super-Pathways&detail-level=3). (DOCX 61 kb) Additional file 8:Supplementary text. Supplementary information on methods used and results analysed for the study. (DOCX 43 kb)
|
study
| 99.94 |
Figure S1. Alpha-diversity of the digestive microbiota of abalone, as described by the Shannon (left panels) and Simpson (right panels) indices. Figure S2. Fluctuations of the digestive microbiota between consecutive dates over 1 year. Figure S3. Relative abundance and taxonomical composition of the digestive microbiota at the phylum level for abalone fed on Palmaria palmata (A), Laminaria digitata (B), Ulva lactuca (C), and Saccharina latissima (D). Figure S4. Ecological patterns of the digestive microbiota explained by a model of contextual parameters. Figure S5. Experimental site and set up. Figure S6. In situ sampling procedure of abalone for each algal treatment. Figure S7. Experimental procedure for DNA extraction and library preparation for studying the abalone digestive microbiota. (DOCX 1944 kb)
|
study
| 100.0 |
Table S2. R value of the ANOSIM calculated for the whole dataset and for each algal diet dataset separately between sample groups according to season, sampling date, or algal diet. Numbers in bold indicate the significance of the comparison after Bonferroni correction. (XLSX 15 kb)
|
study
| 99.94 |
Table S3. Number of annotated genes for each glycoside hydrolase, polysaccharide lyase, and sulfatase family in all Vibrio, Mycoplasma, Polaribacter, Roseobacter, and Psychromonas genomes available at http://www.cazy.org/ and/or at http://abims.sb-roscoff.fr/sulfatlas/ on 19 May 2017 and in Zobellia galactanivorans DsijT (Barbeyron et al. 2017). GH, Glycoside hydrolases; PL, Polysaccharide lyases, CAZyme, Carbohydrate active enzyme. Sulfatase gene counts are indicated when available in the SulfAtlas database. (XLSX 160 kb)
|
study
| 99.94 |
Table S4. Genes belonging to families of glycoside hydrolases, polysaccharide lyases, or sulfatases and genes putatively involved in the pyruvate fermentation to acetate pathway in the genomes of Psychrilyobacter atlanticus DSM19335 (A), Vibrio halioticoli NBRC 102217 (B), and Mycoplasma mobile 163 K (C), when present. Annotations include gene identifiers (locus tags), closest characterized homologs with their UniProtKB ID number when available, EC numbers, and information on their functioning. The percentage of amino-acid sequence identity is indicated in parentheses. (XLSX 66 kb)
|
study
| 99.94 |
Spotted fever group rickettsioses (SFGR) have a wide global distribution and consist of more than 30 species or subspecies . Since the use of molecular tools for detection, human pathogenic species of SFGR are increasingly described . While most of these organisms are transmitted by ticks and in some cases by mites that generally feed on wild or domestic animals, the infection in human is accidental . Infections result in an acute febrile illness with undifferentiated clinical features. However, presence of a vasculitic rash that involves the body and the extremities during the course of illness together with a history of tick bites or exposure risk contributes to a clinical diagnosis . Patients can have various organ involvement such as pneumonitis, hepatitis, acute renal failure, encephalitis and when severe, may lead to multiple organ involvement carrying high mortality .
|
review
| 99.8 |
In Sri Lanka, rickettsial infections have been increasingly reported over the last 2-3 decades, and they include scrub typhus (ST) caused by Orientia tsutsugamushi and spotted fever group rickettsioses [5–7]. Although SFGR was described less commonly in the Western province of Sri Lanka, in a hospital based study where patient recruitment had been on strict inclusion criteria , with the establishment of Indirect Immunofluorescence antibody (IFA) based diagnostics it became apparent that SFGR are as common as scrub typhus (unpublished data). Furthermore, most cases of SFGR present sporadically throughout the year with no seasonal pattern compared to more seasonal outbreaks of ST (unpublished personal observation). Although the organisms causing SFGR in the Western Province of Sri Lanka are yet to be identified, dog ticks are suspected as the most likely vector transmitting the illness based on the reported experiences of patients.
|
study
| 99.8 |
Nonspecific clinical features and non-availability of early diagnostic facilities have resulted in delay in the diagnosis of rickettsial infections. Therefore, awareness of their prevalence and more importantly their clinical features would be help in the early diagnosis and institution of appropriate therapy.
|
other
| 99.9 |
In SFG rickettsioses, subcutaneous necrosis and digital gangrene most likely related to small-vessel occlusion have been described in severe late stage infections caused by Rickettsia rickettsii (Rocky Mountain spotted fever) and Rickettsia conorii [8–11]. Such severe form of skin involvement is described as Purpura fulminans (PF) and include a heterogeneous group of disorders characterized by rapidly progressive purpuric lesions that may develop into extensive areas of skin necrosis, and peripheral gangrene. This rare disorder is associated with laboratory evidence of consumptive coagulopathy and is often fatal . PF is usually associated with many infections, most notably with meningococcal, staphylococcal, streptococcal infections in addition to SFG rickettsioses . Probable less severe or early form of PF; termed as “fern leaf pattern” skin rash has been previously reported in elderly patients with SFG rickettsiosis in the central hills of Sri Lanka [3, 6, 7, 12].
|
review
| 99.9 |
We present a sporadic case of SFGR infection from coastal belt of Sri Lanka who presented with an acute febrile illness and polyarthritis complicated with acute kidney injury and DIC. She probably developed early PF in the form of wide spread ‘fern-leaf’ pattern necrotic skin rash with evidence of vasculitis on the palms and soles and DIC on the 10th day of illness. We feel that clinicians should be aware of this unusual clinical presentation that could occur in association with SFGR infection. Such knowledge would not only benefit those who practice in tropics with limited diagnostic facilities but also would improve the management of acute febrile illness in returning travelers who visit endemic areas.
|
clinical case
| 99.94 |
A 39-year-old housewife, a mother of two children, presented with a history of high intermittent fever (101-103 °F), chills and rigors for eight days. Apart from mild intermittent asthma, she had no other co-morbidities. The intensity and frequency of fever had increased despite treatment with oral co-amoxyclav by a general practitioner. She had associated severe frontal headache, nausea and vomiting. However, the most striking feature was that she developed progressively severe pain in almost all large and small joints of the body with backache and neck pain limiting her movements. As a result, at the time of presentation she was bedbound. In addition, she had noted yellowish discoloration of the sclerae and dark urine around the third day of fever. She did not have any similar clinical manifestations in the past or any clinical signs suggestive of a connective tissue disease. She had an intrauterine contraceptive device for one year. Although menstrual periods were regular, she complained of intermittent offensive vaginal discharge for the past few months. She denied history of recent pregnancy or gynecological interventions.
|
clinical case
| 100.0 |
On examination she looked very ill, icteric, pale and dehydrated. She had severe neck stiffness and bilateral symmetrical large and small joint arthritis with distal interphalangeal joint sparing. Careful physical examination on admission did not reveal skin rash, oral ulcers or lymphadenopathy. Abdominal examination was unremarkable except for a non-tender enlarged liver 2 cm below the right costal margin. She had tachycardia of 100 beats/min and blood pressure of 110/60 mmHg with no cardiac murmurs. She was not tachypneic, and her lungs were clear. Vaginal examination was unremarkable.
|
clinical case
| 100.0 |
The differential diagnosis included leptospirosis, a palindromic rheumatism with systemic involvement or severe bacterial sepsis with a meningitic process. Admission investigations revealed a WBC count of 12.7 × 103/mL (Neutrophils 80% Lymphocytes 16%) and c-reactive protein (CRP) of 327 U/L (Normal < 5 U/L). Blood picture showed toxic granules in polymorphonuclear leukocytes together with a left shift suggesting a bacterial infection. Urinalysis on admission showed 2+ proteinuria with occasional leukocytes and red blood cells. Serum creatinine was 229 μmol/L (normal 45-90 micromol/L), aspartate transaminase (AST) 92 U/L(normal 0 – 35 U/L), alanine transaminase (ALT) 45 U/L (normal 0 – 35 U/L), bilirubin 95 μmol/L (normal 5.0–17.0 μmol/L), Direct 40 μmol/l. Serum alkaline phosphatase was 255 U/L(normal 50-100 U/L), her ESR was 72 mm/1sthr, and anti-nuclear antibodies and rheumatoid factor were negative. Urine culture was negative, and the blood culture were negative.
|
clinical case
| 99.94 |
However, despite the above treatment, she continued to deteriorate rapidly with worsening of symptoms. On the 2nd day of admission (10th day of illness) the patient rapidly developed a painful rash over the breasts (Fig. 1) which progressed to involve the rest of the body, mainly on the abdomen, upper limbs & hands (Fig. 2) and lower limbs (Fig. 3) with sparing of the face. Examination of the rash revealed multiple subcutaneous hemorrhagic skin lesions in a ‘fern leaf’ pattern together with a vasculitic rash over the soles (Fig. 4). She also had splinter hemorrhages, persistent tachycardia (120/min), tachypnea (33/min) and a high fever indicative of systemic inflammatory response syndrome.Fig. 1Patches of subcutaneous necrosis over the breasts Fig. 2Subcutaneous necrosis mainly involving the dorsal aspects of the joints of hands Fig. 3Areas of subcutaneous necrosis over the thighs and the knees Fig. 4Evidence of subcutaneous vasculitis over the soles of the feet
|
clinical case
| 100.0 |
At this stage, the blood picture showed fragmented red cells and thrombocytopenia suggesting early DIC. Her hemoglobin dropped to 8.6 g/dL from 10 g/dL and the platelet count was 76 × 103/mL, APTT 40 s with INR 1.3 and elevated D dimers of 1.2 ng/ml (normal < 0.5 ng/ml). However, she did not have active bleeding from elsewhere. The clinical picture together with the rash led to the clinical suspicion of infective endocarditis, meningococcal or gonococcal septicaemia, severe staphylococcal or streptococcal sepsis, acute flare of connective tissue disease with vasculitis, cryoglobulinemia, a hemorrhagic form of leptospirosis, or rickettsial infection.
|
clinical case
| 99.94 |
Transesophageal and transthoracic echocardiograms did not show evidence of infective endocarditis or myocarditis. ASOT titre was <200 Units. Chest radiograph was normal, and ultrasound scan of the abdomen revealed mild hepatomegaly. Intravenous vancomycin, oral doxycycline and azithromycin were added on the 3rd day of admission to cover severe gram positive sepsis and rickettsial infections. The IFA-IgG titre against Rickettsia conorii Ag was positive at a titre of 1: 8192 and leptospira antibodies were negative by both Microscopic Agglutination Test and ImmuneMed Leptospira Rapid Immuno-Chromatographic assay for qualitative detection of IgM, IgG antibodies to Leptospira. The patient showed gradual improvement with reduction in fever by the 2ndday of doxycycline (5th day of admission) and had a complete recovery from acute kidney and liver injury by the 4th treatment day. However, her skin rash and the joints became very painful over the 2nd-4th days of treatment. All these resolved completely within 5 days of treatment (8th day of admission), and she was discharged on the 11thday of admission. On review after one week of discharge, her rash had started to desiccate and peel off (Fig. 5). The timeline diagram of the evolution of illness is give in Fig. 6.Fig. 5Peeling of the skin over the areas of subcutaneous necrosis Fig. 6The timeline diagram of the evolution of illness
|
clinical case
| 99.94 |
This otherwise healthy female who presented with an acute febrile illness complicated by severe small joint and large joint arthritis, jaundice, acute kidney injury and DIC later developed a subcutaneous hemorrhagic and a vasulitic skin rash. Her acute illness was later confirmed as SFGR and had a complete recovery with anti-rickettsial antibiotics.
|
clinical case
| 100.0 |
At presentation, her clinical presentation mimicked palindromic rheumatoid arthritis or other connective tissue diseases with systemic involvement. However, exposure to floods suggested the possibility of acute leptospirosis. Neck stiffness and the haemorrhagic rash with rapid progressive illness suggested other agents of severe sepsis such as meningococci, gonococci, staphylococci or gram negative bacilli. However, the occurrence of the rash later in the illness and non-responsiveness to broad spectrum antibiotics suggested the possibility of rickettsial infection. Therefore, she was treated with anti-rickettsial antibiotics and had a rapid clinical response. She had a strong antibody response to SFGR demonstrated by IFA using R. conorii antigen, confirming the diagnosis. On specific questioning, she revealed that she had tick bites quite frequently when caring for the two dogs.
|
clinical case
| 99.94 |
The clinical spectrum of severity of rickettsial infections ranges from subclinical to fatal and presents with fever and may progress to various organ involvement such as encephalitis, interstitial pneumonia/ARDS, acute renal failure or with multiple organ failure. Furthermore, we have encountered patients with rickettsial illness, either SFGR or ST with illness mimicking acute rheumatic disease . Although most of the patients with SFG rickettsial disease have an unremarkable course, patients who develop complications related to organ involvement may suffer severe illness with mortality up to 2.5% among diagnosed cases . Classical risk factors for high mortality or severe forms of infection include advanced age, chronic alcoholism, glucose-6-phosphate-dehydrogenase deficiency, prior prescription of an inappropriate antibiotic, particularly sulfonamides, or delay in the treatment . Therefore, early clinical suspicion and empiric treatment with anti-rickettsial antibiotics are important in reducing duration of illness and mortality. It is important that rickettsial infections should always be considered in the differential diagnosis of acute febrile illness in endemic areas.
|
review
| 92.4 |
As there are no readily available rapid diagnostic tests for rickettsial illness, confirmation of the illness during its acute phase in most endemic settings is difficult. However, presence of clinical manifestations such as eschar or vasculitic rash favors the presumptive diagnosis and early institution of empirical anti-rickettsial antibiotics.
|
other
| 99.94 |
Typical cutaneous manifestations of SFGR include a discrete maculapapular rash which usually appears towards the end of the first week of illness and involves the trunk, limbs, palms and soles, however spares the face. The rash tends to be more prominent at the time the patient is having fever and fades in between fever spikes. Other rare forms of skin rash have been described, and involve a dusky erythematous hue, distributed mainly on the limbs, back of the chest, anterior abdomen and soles . Severe rashes associated with SFG rickettsioses include digital gangrene and extensive skin necrosis described as PF [9–11]. While the digital gangrene may result in auto-amputation of toes , the skin involvement in PF results in blackish discoloration of skin that desiccates and peels off with complete recovery or hypertrophic scar formation after healing .
|
review
| 99.9 |
The occurrence of PF or ‘fern leaf’ type of rash seems to be rare in rickettsial infections. While PF has been described mainly in the children [9–11], ‘fern leaf rash has been observed in several elderly patients with SFGR in the central province of Sri Lanka [3, 6, 7, 12]. However, this young female patient very rapidly developed a widespread ‘fern-leaf’ pattern rash over the 10th and 11th days of illness together with DIC suggesting early PF. It was noted predominantly over the breasts and proximal limbs with facial sparing. The rash was painful and tender. The tenderness increased during the first three days after commencement of specific treatment and later improved in parallel with the other clinical manifestations. She also had splinter hemorrhages and a vasculitic rash on the palms and soles. This patient did not have an eschar or marks of tick bites and eschars are an uncommon feature in patients with SFGR compared to scrub typhus in Sri Lanka .
|
clinical case
| 99.9 |
Today, the expansion of international and local travel, recreational activities, eco-tourism, animal transportation together with effects of global warming and migration of birds have resulted in vector borne diseases emerging as global threats. Non-availability of rapid diagnostic tests for most neglected tropical vector borne diseases has added to the clinical challenge in both the resource poor tropics as well as in the developed world. Until such time, as effective early diagnostic methods are available, the most important aspect in the management of such illnesses is the vigilance of clinicians. It is crucial to formulate the most likely differential diagnosis based on the clinical presentation of acute undifferentiated febrile illness and the epidemiological data. In order to achieve this goal, attention paid to the chronology of events such as progression of the symptoms or appearance of signs such as skin rash plays a vital role. Here we aim to highlight the unusual clinical presentation of this patient and the importance of the late onset skin rash that alerted the physicians to the possible diagnosis of SFGR.
|
clinical case
| 99.94 |
We feel that clinicians should be aware of the unusual clinical presentations such as purpura fulminans and ‘fern-leaf’ pattern necrotic skin rash of SFGR infection. Such knowledge would not only benefit those who practice in tropics with limited diagnostic facilities but also would improve the management of acute febrile illness in returning travelers who visit endemic areas.
|
other
| 99.9 |
Clonorchis sinensis is an endemic trematode parasite that causes human clonorchiasis. It is estimated that approximately 35 million people are infected with this fluke in East Asia, including Korea, China and Vietnam . Individuals generally become infected with C. sinensis by the consumption of raw or undercooked freshwater fish containing metacercariae . The C. sinensis adult worms inhabit the bile ducts and cause a series of pathological changes, such as epithelial hyperplasia, periductal fibrosis, obstructive jaundice, dyspepsia and liver cirrhosis, in the infected hosts . Chronic clonorchiasis has been classified as a definite biological carcinogen that causes cholangiocarcinoma in humans by the World Health Organization .
|
review
| 99.7 |
The recommended treatment of clonorchiasis is the chemotherapeutic application of praziquantel, utilized for both the treatment and control of clonorchiasis . Praziquantel is also required for the treatment of other intestinal trematode infections. However, it has been reported that praziquantel exhibits low efficacy for the treatment of clonorchiasis in northern Vietnam . As dependency on a specific drug would be ineffective for any human trematode infections, novel anti-clonorchiasis compounds should therefore be developed to ensure continued or enhanced management of trematode-associated diseases.
|
other
| 99.2 |
Helminth tegumental proteins have raised interest as both a diagnostic and potentially druggable therapeutic target , as they are essential for establishing the host and parasite relationship. The tegument, which covers the entire surface of the worm, comprises a unique outer surface syncytium structure that is of crucial importance for nutrient uptake, excretion, osmoregulation, sensory and signal transduction, host response modulation and parasite survival . To date, five genes encoding C. sinensis tegumental proteins have been identified and characterized; additionally, their potential as diagnostic antigens for clonorchiasis has been evaluated . However, their druggability has yet to be assessed.
|
study
| 80.3 |
In the present study, we identified and characterized the tegumental protein of 20.6 kDa in C. sinensis (CsTegu20.6). Structure and functional analyses were carried out using combined three-dimensional (3D) modeling methods. Computer-aided drug discovery (CADD), such as virtual inhibitor screening and drug-likeness prediction, was used to identify a potent inhibitor of compound interactions with CsTegu20.6.
|
study
| 100.0 |
A cDNA clone representing the complete sequence CsTegu20.6 was isolated using the C. sinensis adult cDNA library . Sequence analysis of CsTegu20.6 indicated an open reading frame (ORF) of 528 nucleotides, and the deduced amino acid (aa) sequence revealed a protein of 175 residues with a calculated molecular mass of 20.57 kDa and a theoretical isoelectric point (pI) of 6.15. This protein was predicted to be localized in the cytoplasm and did not contain any signal peptides or transmembrane domains. A functional domain search indicated the presence of an EF-hand domain (PS00018) at aa46–58 and a dynein light chain domain (DLC) (PF01221) at the C-terminus (aa97–173) (Figure S1). This is in broad accordance with earlier work in which tegumental proteins were analyzed using bioinformatics tools and shown to be composed of one or two EF-hand domains and a DLC domain at the N- and C-termini, respectively .
|
study
| 100.0 |
When CsTegu20.6 was compared with six other tegumental proteins of C. sinensis, multiple sequences alignment of the amino acids revealed that CsTegu20.6 shares 39% identity with CsTegu21.1 (NCBI Accession No. ADZ13689.1) , 38% with CsTegu20.8 (ABC47326.1) , 29% with CsTegu21.6 (JF911532) , 38% with CsTegu_Ca_EF (ABZ82044), 32% with CsTegu22.3 (ABK60085.1) and 35% with CsTegu31.8 (ABK60086.1) (Figure 1). Both the consensus sequences (Asp46, Gly51, Ile53, Leu55, Cys59, Leu62 and Gly63, shaded in red) and the conserved helix-loop-helix (HLH) motif were observed in the calcium-binding EF-hand domain. The HLH structure is an important characteristic of EF-hand domains, of which residue mutation in the loop region can cause an inability of calcium-binding activity . The length of the EF-hand of CsTegu20.6 was almost similar to that of CsTegu21.6, which was conspicuously shorter than those of other C. sinensis tegumental proteins . The DLC domain was less conserved than the EF-hand domain. Although the functions of the DLC domain remain unclear, DLC appears to act as part of a large complex and to contribute to maintenance of the tegument .
|
study
| 100.0 |
We attempted to predict the 3D model of CsTegu20.6 based on homology modeling using Swiss-Model . However, the first attempt failed because the proper template structures were not found in the protein data bank (PDB) template library, as experimental structures of helminth tegumental proteins were not available. The search for templates showed a very low percentage of sequence identities (<25%) and coverages (<51%). We next attempted to predict a CsTegu20.6 model based on multiple-threading alignments using I-TASSER . The 3D model was constructed using 10 multiple templates and refined with energy minimization. However, the second attempt also was not successful because the Ramachandran plot for the model showed 64.8% residues in the most favored regions and 3.8% residues in the disallowed regions (Figure S2).
|
study
| 100.0 |
To improve the quality of the full-length CsTegu20.6 model, we then used combined 3D modeling methods and refinement as described in Section 3.2. (Figure 2). It should be noted that the disordered region was placed between Thr61 and Asp90 (Figure S3). This region can decrease the quality of the overall model, as it requires additional simulation time, and it interferes with the structural clustering process . These regions have been predicted representing tegumental protein linkers because they lack secondary structure in Schistosoma mansoni and Fasciola hepatica . To address this issue, we split the whole sequence into two domains, domain 1 (aa1–74) and domain 2 (aa75–175), based on the disordered region rather than removing the region. The model of each domain was constructed using I-TASSER based on iterative fragment assembly simulations. Then, the structures of the unaligned regions, as for the disordered region, were constructed by ab initio modeling based on replica-exchange Monte Carlo simulations . The structures of the two domains were finally combined into a full-length model using the C1 fragment aa50–99 as a bridge using TM-Scores . The initial model of CsTegu20.6 was obtained by refining the combined model using ModRefiner (Figure S4). This program is designed to improve the physical realism and structural quality of protein models via two-step atomic-level energy minimization. Because ModRefiner can build the coordinates of the unfolded regions, the program has been employed as a key method of the combined approach .
|
study
| 100.0 |
The final model of CsTegu20.6 was determined after the loops and both main- and side-chains of the initial model were further refined using GalaxyLoop and GalaxyRefine , respectively (Figure 3). The ab initio modeling method, hybrid-type energy function and an efficient search method were employed within GalaxyLoop. Then, both the backbone and side chain of the structure were refined using GalaxyRefine based on repeated perturbation and, hence, overall conformational relaxation by short molecular dynamics simulations. Through the refinement procedure of the initial model, the stereochemical quality of the final model was markedly improved over that of the initial model, especially in terms of the ERRAT values from 48.5% (Figure S3D) to 80.8% (Figure 3D). ERRAT calculates the overall quality score of non-bonded atomic interactions compared with the database of reliable experimental structures.
|
study
| 100.0 |
The Ramachandran plot , ProSA , QMEAN , ERRAT and ModFOLD6 methods were employed to validate the final model of CsTegu20.6. The Ramachandran plot ensured the quality of the final model, indicating that 85.5% of residues were in the favorable region and 11.9% in the additionally allowed region (Figure 3A). Furthermore, there were only 2.5% residues in the disallowed region, and 0% of the residues were located in the generously allowed region. Only Ile19, Ala47, Ala74 and Arg164 were found in the disallowed region of the plot. This plot was used to assess the constructed model for its main chain conformation, illustrating the Φ-Ψ torsion angles for all residues, except for Gly and Pro. The ProSA Z-score of −7.41 confirmed the final model as an extremely good model (Figure 3B), whereas the QMEAN Z-score of −2.33 suggested that the model might be a fairly good representation of the protein (Figure 3C). ProSA analyzed the overall quality score by calculating the atomic coordinates of the model, indicating the Z-score of the experimentally solved structures deposited in PDB , whereas the QMEAN Z-score is a measure of the “degree of nativeness” of a given structure and provides information regarding whether the model is of sufficient quality to be comparable to high-resolution crystal structures of similar size. The ERRAT overall quality score of 80.8 showed that the structure could be considered as a good model (Figure 3D). The ModFOLD6 global model quality score of 0.49 indicated that the final model was confidently folded, and the 3D cartoon of the model was colored by the per-residue error according to the B-factor values (Figure S5).
|
study
| 100.0 |
The results of the structure-based search for homologues using deconSTRUCT showed that CsTegu20.6 has the highest homology with several DLCs, followed by ribosomal proteins and calcium-binding proteins (Table 1). We superposed the structures of two DLCs (PDB IDs: 1YO3_A and 1RE6_A) (Figure 4B) and the calcium-binding protein (PDB ID: 1JFJ_A) (Figure 4A). The superposition of structures confirmed that (1) the proper templates were not available for whole structure and partial templates were aligned on the N- and C-termini; and (2) the structural and functional aspects of each domain can be interpreted because of high degrees of conservation.
|
study
| 100.0 |
The final model of CsTegu20.6 consisted of two domains: a N-terminal domain (calcium-binding domain) and a C-terminal domain (DLC domain) adopting a classic ferredoxin fold , which forms a four-stranded antiparallel β-sheet flanked on one side by two α-helices (Figure 5A,B). However, unlike a typical ferredoxin fold associated with the overall symmetry of β-α-β-β-α-β, the β1-α5-α6-β2-β3-β4 fold was present in the DLC domain of CsTegu20.6. The two α-helices, α6 and α7, were packed in an antiparallel hairpin structure against the β-sheet with four antiparallel β-strands, β1-β3-β4-β2. These findings are in line with structural data obtained from a DALI search that found similarities to numerous proteins with DLC1 (also known as DLC8), such as 5E0M and 3DVT, the structures of which represent the same fold . DLC-containing proteins can form homodimers through hydrogen bonds, backbone-side chain electrostatic interactions and van der Waals contacts between side chains . Previous studies have indicated that the homodimeric DLCs in S. mansoni and F. hepatica were composed of two asymmetric monomers through β-sheet interactions. The homodimer of CsTegu20.6 was constructed using GalaxyGemini with only the C chain of DLC1 (PDB ID: 4DS1_C), although the fifth β-strand (indicated with a dotted circles) as a monomeric counterpart was missed in the predicted structure (Figure 5C). This result suggests that CsTegu20.6 can dimerize, confirming that this feature is conserved between the DLCs.
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In a typical EF-hand, six residues are necessary for the coordination of the calcium ion. These are labeled as the X, Y, Z, −Y, −X and −Z residues, and consensus residues at these positions have been identified . Residues Asp46, Asp48, Ser50, Val52, Thr54 and Glu57 were predicted as constituting a potential calcium-binding site in the EF-hand of CsTegu20.6 using COACH (Figure 6). The consensus is mostly observed, with a different residue, Val52, at the −Y residue, which is rarely observed at this position. The orientation of the Val52 side chain is also more distant from the calcium-binding site compared to that of the Thr52 side chain in a typical EF-hand. Therefore, it is improbable that this EF-hand contacts calcium ions.
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